Plant Ecophysiology

Malcolm J. Hawkesford
Stanislav Kopriva
Luit J. De Kok
Editors

Nutrient Use
Efficiency in
Plants
Concepts and Approaches
Nutrient Use Efficiency in Plants
Plant Ecophysiology
Volume 10

Series Editors:

Luit J. De Kok
University of Groningen, The Netherlands

Malcolm J. Hawkesford
Rothamsted Research, United Kingdom

Aims & Scope:

The Springer Series in Plant Ecophysiology comprises a series of volumes that deals with
the impact of biotic and abiotic factors on plant functioning and physiological adaptation to
the environment. The aim of the Plant Ecophysiology series is to review and integrate the
present knowledge on the impact of the environment on plant functioning and adaptation at
various levels: from the molecular, biochemical and physiological to a whole plant level.
This series is of interest to scientists who like to be informed of new developments and
insights in plant ecophysiology, and can be used as advanced textbooks for biology students.

More information about this series at http://www.springer.com/series/6193

Malcolm J. Hawkesford • Stanislav Kopriva
Luit J. De Kok
Editors

Nutrient Use Efficiency

in Plants
Concepts and Approaches
Editors
Malcolm J. Hawkesford Stanislav Kopriva
Rothamsted Research Botanical Institute
Harpenden, Hertfordshire University of Cologne
United Kingdom Cologne, Germany

Luit J. De Kok
Laboratory of Plant Physiology
University of Groningen
Groningen, The Netherlands

ISSN 1572-5561 ISSN 2405-4321 (electronic)

ISBN 978-3-319-10634-2 ISBN 978-3-319-10635-9 (eBook)
DOI 10.1007/978-3-319-10635-9
Springer Cham Heidelberg New York Dordrecht London
Library of Congress Control Number: 2014951211

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Preface

Nutrient Use Efficiency in Plants: Concepts and Approaches is the ninth volume in
the Plant Ecophysiology series. It presents a broad overview of topics related to
improvement of nutrient use efficiency of crops.
Nutrient use efficiency (NUE) is a measure of how well plants use the available
mineral nutrients. It can be defined as yield (biomass) per unit input (fertiliser,
nutrient content). NUE is a complex trait: it depends on the ability to take up the
nutrients from the soil, but also on transport, storage, mobilization, usage within the
plant, and even on the environment. NUE is an important contributor to growth
control and yield; the same levels of nutrients may cause growth and yield penalty
in one species or variety but not in another one. NUE is of particular interest as a
major target for crop improvement. Improvement of NUE is an essential
pre-requisite for expansion of crop production into marginal lands with low nutrient
availability but also a way to reduce use of inorganic fertiliser. Aspects of NUE
have been covered in detail within the Plant Ecophysiology series, in the volumes
on nitrogen, phosphorus, sulfur, or root physiology. In this volume, the expanding
field of nutrient use efficiency is comprehensively discussed, with the aim to
present the current approaches, concepts, and ideas on how to better understand
the genetic control of NUE and how to use this knowledge for its improvement.
This volume, however, is special not only because of the new topic. It is also a
presentation of a Marie Curie Initial Training Network BIONUT-ITN (BIOchem-
ical and genetic dissection of control of plant NUTrition). BIONUT-ITN is a
network of eight research institutions and one company who came together to
provide state-of-the-art research training in different aspects of plant nutrient use
efficiency. The individual student’s projects have been linked to ensure that a fully
integrated approach is taken to get the whole picture of plant nutrition. This
integration is a key feature of the network, as it advances the science beyond
focusing on one mineral nutrient, such as nitrogen or sulfur, to look at the combined
nutritional needs of the plant using models and crops, in the laboratory as well as in
the field. BIONUT is also a hub for activities in plant nutrition field, organizing
conferences and fostering new collaborations. This volume is evidence of such
integration. The contributions of the eight students span a broad range of themes
v
vi Preface

within NUE. There are detailed reviews focused on single nutrients – sulfur,
phosphorus, iron, and boron. Thus, macronutrients are discussed alongside
micronutrients needed in small quantities, but still essential. But these reviews
stress also the importance of interaction between different nutrients and the need for
integrative view on plant nutrition. Other chapters bring overviews of large and
complex topics or approaches – natural variation, autophagy, or effects of elevated
CO2. Included are contributions targeting nutrient deficiency on a molecular level
as well as its monitoring in the field. Together with a thorough introduction into the
NUE topic, the book presents ten chapters that wrap up the theme of NUE and the
potential for crop improvement.
We hope that this book will find broad audience. It is not only an overview of an
interesting and important research area; it is also a snapshot of current activities in
the field and introduction of new generation of scientists from the BIONUT-ITN.
We believe that it will be of interest to graduate students and researchers in a wide
range of disciplines including plant nutrition, plant physiology, plant biochemistry,
and agriculture.

Harpenden, UK Malcolm J. Hawkesford

Cologne, Germany Stanislav Kopriva
Groningen, The Netherlands Luit J. De Kok
Acknowledgement

The editors thank Helen Jenkins for grammatical and technical editing of the
chapters.

vii
Contents

1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts,

Opportunities and Challenges for Its Improvement . . . . . . . . . . . . 1
Martin Reich, Tahereh Aghajanzadeh, and Luit J. De Kok
2 Natural Variation as a Tool to Investigate Nutrient Use
Efficiency in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Giorgiana Chietera and Fabien Chardon
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana . . . . 51
Patrycja Baraniecka and Stanislav Kopriva
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate
Uptake and Use Efficiency in Crops . . . . . . . . . . . . . . . . . . . . . . . . 93
Astrid Gruen, Martin R. Broadley, Peter Buchner,
and Malcolm J. Hawkesford
5 Micronutrient Use Efficiency – Cell Biology of Iron
and Its Metabolic Interactions in Plants . . . . . . . . . . . . . . . . . . . . . 133
Ilaria Forieri and Ruediger Hell
6 Boron: A Promising Nutrient for Increasing Growth
and Yield of Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Himanshu Bariya, Snehal Bagtharia, and Ashish Patel
7 Role of Autophagy in Plant Nutrient Deficiency . . . . . . . . . . . . . . . 171
Milagros Collados Rodrı́guez, Katarzyna Zientara-Rytter,
and Agnieszka Sirko
8 Mineral Nutrient Depletion Affects Plant Development
and Crop Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Sarah J. Whitcomb, Elmien Heyneke, Fayezeh Aarabi,
Mutsumi Watanabe, and Rainer Hoefgen

ix
x Contents

9 Nutrient Use and Nutrient Use Efficiency of Crops

in a High CO2 Atmosphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Sabine Tausz-Posch, Roger Armstrong, and Michael Tausz
10 Monitoring Plant Nutritional Status . . . . . . . . . . . . . . . . . . . . . . . . 253
Moez Maghrebi, Fabio Francesco Nocito, and Gian Attilio Sacchi

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Chapter 1
Physiological Basis of Plant Nutrient Use
Efficiency – Concepts, Opportunities
and Challenges for Its Improvement

Martin Reich, Tahereh Aghajanzadeh, and Luit J. De Kok

Abstract Knowledge on the underlying physiological processes and variables

which bias their contribution to nutrient use efficiency (NUE) is crucial to develop
strategies for improvement in agroecosystems. This chapter aims to contribute to
the understanding of the physiological basis of NUE to develop strategies for
improvement by modern breeding, but also conceive the challenges and current
limits to do so. General concepts will be summarized briefly and broken down to the
main components before, in the main part of this chapter, the involved physiolog-
ical processes are reviewed and discussed in their relation to NUE. This is followed
by an identification of the factors that make the individual contributions of these
processes to NUE so variable and impede one general concept for all crops,
environmental conditions and nutrients. The last part of the chapter is dedicated
to a critical analysis of the opportunities and challenges to improve NUE, which
arise from physiological interactions and trade-offs on a whole plant level.

Keywords NUE (nitrogen use efficiency) • Nitrogen • Agroecosystem • MRT

(mean residence time) • Crop yield • Oscillations • Acquisition efficiency • Utilization
efficiency

Introduction

Plants are principally, as are all living organisms, chemical compartments, which
are in thermodynamic disequilibrium with their environment. This is actively
maintained by the utilization of solar energy for driving selective chemical
exchange with the environment. In addition to carbon dioxide and water, which
provide the structural and metabolic backbone elements C, O and H, the complex
functioning of plants requires the uptake of at least 13 additional essential nutrients
from the soil. Nutrients can be classified into two very distinct groups depending on

M. Reich • T. Aghajanzadeh • L.J. De Kok (*)

Laboratory of Plant Physiology, University of Groningen, P.O. Box 11103, 9700 CC,
Groningen, The Netherlands
e-mail: [email protected]

© Springer International Publishing Switzerland 2014 1

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_1
2 M. Reich et al.

their concentration in plant tissues (macro- and micronutrients), and the specific
roles they fulfil in the plant’s metabolism are as diverse as their physiochemical
properties (Marschner 2012).
Plant NUE is a term, which describes a highly complex, multigenic trait with
various interconnected physiological processes involved and modified by numerous
factors. Consequently, there are numerous approaches to define, analyse and pos-
sibly improve NUE. For a long time it has been known that the ability of plants to
utilize nutrients can differ substantially between species and cultivars, and that this
could be the basis for further improvement through breeding (Gerloff 1963; Shea
et al. 1968; Siddiqi and Glass 1981). In order to develop a common framework for
NUE, scientists started to formulate concepts and definitions that should serve as a
basis for comparison and discussion of research. Since then countless studies in
various scientific disciplines dealing with different plant species, in different
contexts, under different conditions and focusing on different nutrients failed to
find one definition of NUE that describes all cases satisfactory but rather revealed
that the issue is too complex to do so.
In this chapter the definition of NUE will be discussed from a whole plant
perspective. It starts with the transfer of NUE from an ecological to an agronomical
context and the different levels of organization on which it can be discussed. This is
followed by a brief introduction into the conceptual framework of NUE, especially
for nitrogen (N) and equipped with these theoretical concepts the attention of the
reader will be drawn to the concrete physiological basis of NUE. These processes
are the targets of potential improvement of NUE for agricultural production by
modern breeding. However, how relevant a certain physiological process is in a
particular cropping system depends on three main variables: environment, plant and
nutrient, which influence the physiological basis of NUE. The last parts of this
chapter are dedicated to the need to improve NUE in modern agriculture and ends
with a critical review of the chances and challenges to improve plant NUE from a
whole plant perspective.

Nutrient Use Efficiency – Contexts and Concepts

A general definition of “efficiency” is: The achievement of an intended outcome

with a lowest possible input of costs. While the input in the concept of NUE
obviously is nutrients, the intended outcome needs to be further specified. This
can happen in different ways, which leads to many different versions of what NUE
actually means and how it can be improved. Very fundamental is the difference
between an ecological and agronomical context. Understanding this difference is
crucial to develop strategies of improving a plant with its complex ecophysiological
background in the straightforward input-output system of agriculture.
The environment of a plant is far from being a stationary equilibrium. Arising
from the way our planet turns around its own axis and follows its orbit around the
sun, all abiotic and biotic factors on its surface underlie oscillations over time and
all forms of living organisms are forced to adapt to the local oscillations in their
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 3

respective habitat in order to reach a lifespan of hours, days, weeks, months, years
or decades and finally produce successful offspring. This is especially true of plants
which being sessile, have evolved strategies to synchronize their internal processes
with the external oscillations of their environment to their best advantage. This
synchronization includes life cycle, developmental program, morphology, diurnal
physiological rhythms (Somers et al. 1998; McClung 2006) and also uptake and
assimilation of nutrients (Zhang et al. 1991; Bot and Kirkby 1992; Delhon
et al. 1995; Haydon et al. 2011) on different timescales. A well-adjusted synchro-
nization with the environment will increase the performance of a plant and increase
competiveness. From this ecological and evolutionary point of view plants can be
called “nutrient efficient”, if they use the temporal and spatial availability of
nutrients for an optimal and balanced vegetative and reproductive growth, which
is most suitable to survive and compete in their respective habitat and niche.
In an agricultural context, however, the quality of the intended outcome shifts.
Instead of offspring the plant produces a desired yield product, which can be
utilized for food production and other economically relevant purposes. With agri-
cultural practice and plant breeding to increase the production of this agronomic
intended outcome the plant is detached from its ecological and evolutionary con-
text. No longer exposed to the natural selection pressure but the artificial selection
by man, plants are reshaped for agriculture: development, morphology and fluxes of
resources are rerouted towards increased production of whatever yield is desired.
Even after thousands of years of breeding, plants still bear their ecological heritage,
which may conflict with agricultural interests and may limit the potential for
traditional plant breeding to improve NUE. Bringing these two contexts together
is one of the main tasks for plant scientists to understand the functioning of a plant
in the semi-natural system of agriculture. In this way, ecophysiological potentials of
plants might be further exploited for agricultural production and the limits for
improving plants with traditional breeding might be identified and overcome. A
profound understanding of the physiological background of NUE is the basis for
modern plant breeding using molecular techniques.
In an ecological context, NUE can be examined at the level of individuals,
populations, species, communities or entire ecosystems (Nardoto et al. 2006). NUE
in agronomy can also be discussed on several levels (Fig. 1.1). On each level, input
and output differ in kind, and different components have to be considered to
adequately calculate the NUE of the respective system. In scientific discussions it
is important to consider the same level to avoid confusion and misunderstanding.
During the past few decades, scientists have become increasingly aware that
agricultural systems can be regarded as ecosystems in which the role of soil
composition and fertility, the influences of biotic interactions as well as abiotic
environmental factors should not be underestimated. This is brought together in the
concept of agroecology (Gliessman 1990; Schnug and Haneklaus 1998; Francis
et al. 2003). In this holistic approach, not only the intended outcome but also the
input of costs becomes very complex, as negative impacts of fertilisation, pesticides
etc. have to be considered. In modern approaches many different benefits that an
intact ecosystem delivers to society are assessed. These “ecosystem services”
4 M. Reich et al.

Fig. 1.1 NUE can be analysed and discussed on different levels of organization. On each level
input and intended outcome differ in quality and a different terminology is used. Agroecologists
regard the agricultural system with all associated ecosystems and society as the entity with a
particular NUE, whereas agronomists put the field into focus. Plant physiologists deal with the
plant as a complex input-output system with an inherent NUE. To avoid confusion in comparative
research it helps to clarify on which level NUE is assessed

(Daily 1997) include delivery of agricultural goods as well as indirect beneficial

properties such as the protective role of an intact forest against flooding or its
capacity to purify water and bind carbon dioxide (Costanza et al. 1997; Daily
et al. 2000; Tilman et al. 2002). Although it is difficult to assess the actual monetary
value of all the components of an ecosystem, this concept is the only one that
adequately expresses the efficiency of an agricultural system for a society in the
long term by including all detrimental effects to the environment and to public
health in the input of costs side of the calculation.
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 5

From the less holistic perspective of agronomy, the field is usually the level of
choice for calculations of NUE. For farmers, the field represents an economic
entity, where the inputs are the costs for labour and materials while the output is
the harvest, usually measured by criteria such as “harvest index” (Donald and
Hamblin 1976). In addition to NUE the term “fertiliser efficiency” is often used
to describe how efficiently the applied fertiliser is used by the agronomic system:
how much yield is produced, the quantity of nutrients which remains in the soil and
how much is lost from the system by leaching and emission (Saurbeck and Helal
1990; Oenema et al. 2009). However, NUE can also be discussed at the plant level
where a single plant instead of a whole field is regarded as an input-output system
and in the present chapter we will deal with the individual plant and its NUE to
discuss all the other dimensions and factors in relation to it. Improvement of NUE at
the plant level also has the potential to improve NUE at higher levels and therefore
has strong agronomic and environmental implications.
A common conceptual framework ensures a consistent use of terminology and
definitions. As was noted above, NUE can be discussed in an ecological as well as
an agronomical context. One context again can be divided into a sub-set of different
levels of organization. By coming down to the level of an individual plant in an
agronomical context, much of the universality of the term has been reduced. As
already mentioned, the term efficiency implies the achievement of an intended
outcome with a lowest possible input of costs. Therefore a very simplified definition
for the efficiency of a given system can be expressed in the equation:

Efficiency ¼ Output=Input

If values for input and output are competitive, the maximum value of efficiency
is 1, in an ideal case where input equals output. Either decreasing the input or
increasing the output might achieve higher efficiency. Every economical concept,
which has to generate profit, in principle follows this simplified equation and
agronomy is no exception. In all sectors of an industrialized economy it is desirable
to make working processes less costly while maintaining or increasing the output.
Technical innovation leads to more sophisticated techniques and methods, which
also revolutionized the efficiency of agricultural practice at the field level. There is,
however, an essential difference in improving an inanimate machine or process,
which has been planned and constructed by man and improving a living organism
such as a plant whose functioning is still far from being fully understood.
From a whole plant perspective NUE consists of several components and by
regarding a plant as an input-output system, physiologists have established equa-
tions that put these components into context in relation to NUE. In the most
universal approach, NUE at a plant level can be divided into two main components:
the efficiency of nutrient acquisition (NAcE) and the efficiency with which the
nutrient is utilized to produce the desired yield (nutrient utilization efficiency,
NUtE):
6 M. Reich et al.

NUE ¼ NAcE  NUtE

While Chapin (1980) defined NUE simply as the inverse of the tissue nutrient
concentration, NUtE can be further sub-divided into nutrient productivity (NP) and
mean residence time, the period in which a certain nutrient can be used for
production (MRT; Berendse and Aerts 1987). In the 1980s, Vitousek and
co-workers (Vitousek 1982; Birk and Vitousek 1986) defined the nitrogen use
efficiency (NitUE) of perennials as the amount of organic matter, which is lost
from a plant or permanently stored in wood, divided by the amount of N lost or
permanently stored. It was shown that NitUE of Pinus taeda L. stands decreased
with increasing N availability. A more general definition was suggested by
Berendse and Aerts (1987), who identified MRT and nitrogen productivity (NitP)
as the main components of NitUE:

NitUE ¼ NitP  MRT

According to Berendse and Aerts (1987), NitP describes the instantaneous rate
of carbon fixation or biomass production per unit N present in the plant while MRT
is a measure for the period in which N can be used for carbon fixation. This concept
of MRT can theoretically be extended to other nutrients and plant species. In
fertilisation models NP can be used to calculate the nutrient flux density that is
necessary to maintain an optimal nutrient concentration in the plant (Ingestad 1988)
but this again refers to the field and not to NUE at the plant level.
It is well known that plant species and ecotypes, which naturally grow in
nutrient-poor soils possess mechanisms to increase the MRT of nutrients e.g.
slow growth, high accumulation of nutrients and efficient remobilization of such
storage capacities or a reduction of nutrient loss (Vázquez de Aldana and Berendse
1997). In soils where nutrients are available in excess or at least where nutrient
availability is not the limiting factor, there is less selective pressure on developing
such mechanisms. It is more important to have a high NP to grow fast and compete
with neighbouring individuals for space and light and one way to reach this might
be having a high nutrient throughput rather than a long MRT. Studies under
controlled conditions with plants from both soil types showed that in the short
term fast growing species were the better competitors in both optimal and limiting
N conditions while in the longer term, plants from nutrient-poor soils outcompeted
fast growing species under limiting conditions (Chapin 1980; Wedin and Tilman
1990; Berendse et al. 1992). It is considered that these differences in NUE between
plants, which originated from soils with different nutrient concentrations, are due to
differences in the underlying physiology, morphology and development. Van der
Werf et al. (1993) showed how important morphological traits are for adapting
NUE to the respective nutrient concentration in the soil. For instance, a high
investment in root mass served for the high NP of fast growing species, though it
should be noted that the majority of these studies on NUE dealt with wild species
and N.
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 7

Expression of NP as unit biomass produced per unit nutrient may not always be
the most suitable measure. The desired product in an agricultural system is not
always biomass, consisting of structural or non-structural carbohydrates, but more
often seeds that are rich in proteins or oil. It is thereby not only important how a
nutrient contributes to growth but also how it improves the yield and quality of the
desired product. Therefore, the respective nutrient can itself be a substrate for the
production (e.g. as N and sulfur (S) for proteins) or a facilitator of the production
(e.g. by being a component of an enzyme involved). Consequently NP on the basis
of biomass may not always be the best measure and a more general indicator may be
yield productivity (YP), which includes quantity and quality of the desired yield
product per unit nutrient in the plant tissue. However, in agriculture and in general
for all nutrients, NP (or YP) and MRT can be seen as sub-components of NUtE.

Physiological Processes Involved in NUE

After the derivation of a conceptual framework the key physiological processes

involved in the complex trait NUE will be briefly summarized (Fig. 1.2). As
described above, NAcE is one main component of NUE and consequently nutrient
uptake is one of the key processes involved. Although some nutrients can be
derived from the atmosphere (viz. N and S; Faller 1972; Stulen et al. 1998; De
Kok et al. 2007), the plant largely depends on mineral nutrients taken up from the
soil (Mengel and Kirkby 1987; Marschner 2012). These are either derived from
weathering of parental rock material or biological breakdown of organic matter and
the chemical availability to the plant depends on soil-specific properties which in
turn determine the proportion of nutrients dissolved in the soil water (usually less
than 0.2 %), bound to organic detritus (around 98 %) or adsorbed by soil colloids
(Larcher 1995).
There has been much discussion on the significance of NAcE in explaining
differences in NUE between plants. Most studies were focussed exclusively on N
and came to different conclusions. For corn (Zea mays L.) it has been concluded
from a study with different hybrids that NAcE is only relevant for differences in
NitUE if the outside N concentrations are high, while NUtE of accumulated N was
the driving variable if the supply was low (Moll et al. 1982). Whereas in pumpkin
NAcE was not a possible target to improve NUE at either high or low N concen-
trations (Swiader et al. 1994). However, recent studies have suggested that an
increased acidification capacity of the rhizosphere could be targeted to increase
nitrate uptake and improve NUE (Paez-Valencia et al. 2013). In addition, the NUE
of an agricultural system may be improved if plants could maintain internal nutrient
concentrations and optimal growth with a lower outside concentration in the soil.
Therefore understanding the response mechanisms of NAcE to nutrient deficiencies
may improve the ability of crops to tolerate lower nutrient concentrations in the soil
and thereby save fertiliser and reduce potential pollution.
8 M. Reich et al.

Fig. 1.2 Plant NUE has a

complex physiological basis
with interacting cellular and
whole plant processes. After
the acquisition of a nutrient
it contributes directly or
indirectly to the production
of biomass and the final
yield. Storage and
remobilization are
important processes that
buffer asynchronies in
nutrient demand and
availability and the efficient
reallocation of nutrients
between different plant
organs is a crucial process
during plant development.
Especially in cereals the
translocation of nutrients to
the finally harvested sink
organ, the grains, is of
particular importance.
Nutrient loss can happen in
several ways and displays a
general constraint for NUE

Nutrient storage is another process of importance and can be functionally

sub-divided into accumulation, reserve formation and recycling (Chapin
et al. 1990). Accumulation summarizes the increase of compounds that are not
directly related to growth. They accumulate simply because the availability exceeds
the demand of the plant metabolism for these compounds. Reserve formation in
contrast describes metabolically controlled storage in designated storage com-
pounds. In this way compounds that otherwise would promote growth are stored
in a form that does not. The formation of these storage compounds directly
competes with growth and other processes that would use the compound in its
original form as a substrate. In the process of recycling, compounds that originally
contributed to growth promotion or other physiological functions but which would
be lost are actively broken down to be used for future growth (Chapin et al. 1990).
The significance of nutrient storage and remobilization for NUE depends on
nutrient availability. A study with a number of hybrids of corn (Zea mays L.)
revealed that under low N supply differences in NitUE between hybrids are related
to variation in the utilization of stored N. However if N supply was high, acquisition
efficiency became more important (Moll et al. 1982). A low ability to remobilize N
leads to a lowered N harvest index in Brassica napus (Rossato et al. 2001). Sim-
ilarly for S the limits of storage capacity and remobilization efficiency of sulfate are
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 9

regarded as a constraint to NUE and a possible target for its improvement

(Hawkesford 2000).
For biomass production plants convert inorganic carbon dioxide from the atmo-
sphere to organic carbohydrates via photosynthesis. Fuelled by the energy of the
sun, this process is the primary generator of all biomass on earth. Although N is
most directly linked to photosynthesis, all the essential nutrients contribute in some
form to growth promotion i.e. biomass production. This can happen directly if the
nutrient is part of the carbon-assimilating apparatus or indirectly if it plays a role in
energy transfer, defence, homeostasis, tolerance and other processes that facilitate
optimal plant functioning. Consequently for every nutrient a respective NP can be
assigned which is a measure for the biomass produced per unit of the nutrient in the
plant. However, the mechanisms underlying this component of NUE and how the
NP (and consequently the NUtE) of a certain nutrient can be improved are manifold
and depend on the specific role that a nutrient plays in plant metabolism.
Again N is studied most intensively, and due to its direct link to photosynthesis
and biomass production, there is a clear cut correlation with the NP of N and (i) the
amount of total N invested in the photosynthetic tissue, (ii) the N efficiency of
photosynthesis and (iii) relatively low loss of carbon due to respiration (Ågren
1985; Poorter et al. 1990). The carbon-assimilating enzyme Rubisco is currently
one of the most prominent targets for possible genetic improvement of photosyn-
thesis (Loomis and Amthor 1999; Parry et al. 2011), largely due to its apparent
catalytic inefficiency in carboxylation and its consequent high abundance. The idea
is that a higher efficiency would lead to less Rubisco being needed to maintain the
same rate of photosynthesis and consequently, as this enzyme contains high
amounts of N, a higher NUE of N. One intriguing approach is the attempt to express
the Rubisco of some non-green algae, which have a greater specificity for CO2, into
higher plants (Whitney et al. 2001).
However, is the biochemical inefficiency of photosynthesis really the bottleneck
that hinders higher biomass production and NUE? Although photosynthetic effi-
ciency is in theory one of the key limiting factors for increasing biomass and crop
yields (Long et al. 2006; Parry et al. 2011), supportive correlations in practice are
not easy to assess and studies come to different conclusions. Studies on closely
related germplasm of wheat showed a correlation of photosynthetic rate and yield
(Watanabe et al. 1994), while comparisons of cultivated crops with their wild
ancestors showed that the latter have a higher photosynthetic rate (Evans and
Dunstone 1970). The potential limiting role of photosynthesis apparently depends
to a greater extent on other processes with negative feedback on photosynthesis. If
the capacity of the sink declines and the flux of photosynthates into sink products
stagnates, this results in a compensating down-regulation of photosynthesis. Con-
sequently the strength of the sink is just as important if not more so for yield as the
efficiency of the source (Zelitch 1982; Borrás et al. 2004; Reynolds et al. 2005).
According to these studies an increased sink capacity is required to increase
photosynthesis and not the other way around. However, field studies with C3
plant species under exposure to elevated levels of carbon dioxide (eCO2) suggested
10 M. Reich et al.

that an increase in yield is, indeed source-limited or at least that sink capacity is
stimulated by the increased source activity (higher net photosynthetic rate under
eCO2). These studies suggested that sink capacity is not necessarily a constraint to
increase yield production by means of improving photosynthesis (Kimball
et al. 2002; Ainsworth et al. 2004).
Apart from the question of how source and sink, or in other words supply and
demand, determine and influence each other, the duality of photosynthesis and
photorespiration makes the issue more complex. Up to one third of C fixed by the
carboxylation activity of Rubisco is again lost by photorespiration. While some
authors propose that photorespiration is of vital importance for plant functioning
(Kozaki and Takeba 1996), others see these functions as at least partially redundant
and suggest that a reduction of the C lost by this process would improve the
efficiency of photosynthesis and biomass production (Long et al. 2006; Peterhansel
and Maurino 2011). However, knowledge about the different roles of photorespi-
ration in plant metabolism and NUE is still limited.
Another important process involved in NUE is the reallocation of nutrients.
Re-use of nutrients from senescing leaves reduces nutrient loss and thereby
increases NUE. Once more N is tightly coupled to C gain and its efficient allocation
from one leaf to another contributes to optimal C fixation (Field 1983). In this
process older leaves with declining photosynthetic N efficiency are exploited as a
source for N, which is reallocated to young leaves to promote their growth. In this
way N is used efficiently for photosynthesis at a whole plant level (Westoby
et al. 2002; Escudero and Mediavilla 2003) and also NUE is increased, as loss is
reduced. Resorption of nutrients from senescing leaves has also been studied for P
(Lajtha 1987; Chapin and Moilanen 1991; Killingbeck 1996). It is generally
assumed that the costs of this process are very low for the plant (Givnish 2002),
which further supports reallocation of nutrients as a key process for the improve-
ment of NUE. However, nutrients, which are efficiently recycled within the plant
and thereby are not lost during senescence, will also not end up in the decomposi-
tion cycle in the soil. Whether this has negative feedback consequences for the plant
and NUE is not fully understood and much will depend on the particular system.
However, there are speculations about a general trade-off between efficient nutrient
re-sorption in plants and the decomposability of litter (Aerts 1997).
The translocation of nutrients to the harvestable yield organ follows the same
principles as the allocation to other plant organs. For obvious reasons it is, however,
the most crucial allocation process for yield production and therefore regarded as a
special case that is worthy of additional attention. Plant breeding has resulted in a
wide diversity of crops in which virtually any part of a plant might serve as a yield
organ: roots, stems, leaves, seeds, fruits. However, the six most important crops in
terms of worldwide food and feed production are all grain crops (corn, rice, wheat,
soybean, barley and sorghum) with seeds being the plant organ of interest and grain
filling as a crucial step for yield production (Borrás et al. 2004; Foulkes et al. 2011).
The reallocation of N from senescing leaves to the developing seeds is of particular
importance in determining the quality of the crop and thus increasing the efficiency
of reallocating N from leaves to grains is a potential target for improving NUE
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 11

(Barbottin et al. 2005). While N translocation to grain determines its quality, on

improving NUE of P could be achieved by decreasing its translocation to the grain
(Rose et al. 2010). Additionally it should be noticed that the efficient translocation
of nutrients (as well as C) to yield organs is not only determined by the efficiency of
the exploitation of the sink organs but also by the sink strength (e.g. for wheat:
Reynolds et al. 2005; for rice: Ntanos and Koutroubas 2002).
The loss of nutrients to the outside may be the most obvious constraint to NUE.
There are several paths by which nutrients can be lost by plants to their environ-
ment. Leaves lose nutrients by leaching, in some cases as gases or other volatile
compounds (Eichert and Fernández 2012) and finally by litter fall, i.e. senescence.
These processes of nutrient loss can either be a way for the plant to balance its
nutritional status or may be unavoidable, for example due to wash-off by rain or
evaporation due to a trade-off with stomatal conductance. In the latter case, a
reduction of nutrient loss from the plant to its environment before harvest may be
a target for the improvement of NUE, particularly in crop systems where litter and
its nutritional status play a minor role. Further research is required to more fully
understand the physiological significance of these losses in order to distinguish
between avoidable leaks and metabolic valves, which assure internal nutrient
balance and thereby optimal plant functioning.

Factors Affecting NUE

The complex physiological basis of NUE becomes even more complex in reality, as
the contribution of these processes is modified by numerous factors, which can be
categorized into plant, environment and nutrient (Fig. 1.3). Numerous studies and
reviews have pointed out that concrete definitions of NUE depend to a great extent
on plant species and growth type. Again it should be stated that NUE is an artificial
term based on a hypothetical input-output concept. The diversity of NUE in nature,
however, reflects the diversity of plant strategies to survive and produce successful
offspring in their very different niches. How they perform if we apply the agricul-
tural standard of NUE does not reflect their ecological and evolutionary fitness.
Despite breeding the strong influence of the respective phylogenetic background of
a cultivated plant on its performance and peculiarities in the field adds another
degree of difficulty when defining one general concept for NUE. Fundamental
differences between crops can be metabolic in nature, e.g. between C3 and C4
plants (Brown 1978) or arise from different growth forms such as trees which are
harvested after decades, and herbaceous crop species which produce their yield
within months. Processes such as nutrient storage and reallocation function very
differently and have different significance for NUE in annual and perennial species
(Aerts and Chapin 1999), as well as in deciduous and evergreens (Chapin and
Kedrowski 1983; Aerts 1990; Franklin et al. 2009). This variability in plant growth
strategy makes it hard to derive one concept and set of definitions for NUE and its
12 M. Reich et al.

Fig. 1.3 The complexity of NUE – In an agronomical context the plant is regarded as an input-
output system with an inherent efficiency that shall be improved (a). Numerous physiological
processes are determining the NUE of a plant (b) and the actual contribution of each process to the
NUE of the plant is biased by many factors of the three variables plant, environment and nutrient
(c). To develop strategies for the improvement of NUE in an agricultural system both the
physiological processes involved and the factors that influence their contribution to NUE have
to be considered

improvement for all species (and even cultivars). To identify the physiological
processes whose modification could increase the NUE of a respective crop, the
particular characteristics should be carefully taken into account and adequate
comparisons of NUE are often only possible within cultivars or strains of the
same species. However, the repertoire of physiological strategies in nature can
also serve as a pool of mechanistic possibilities to improve NUE, e.g. by transfer-
ring beneficial traits within distantly related species via transgenic methods.
Another plant-specific variable is the kind of yield the crop will produce. As
described in the introduction the intended outcome in agriculture is a maximum
quantity of yield. However, the specific NUE and the way to improve it will differ
fundamentally depending on the desired yield quality. The relevance of the phys-
iological processes described above for NUE shifts completely if the desired yield
is starch or sugar and not proteins or oil. While for the former efficient biomass
production and C storage will be important, for the latter allocation to the seeds
increases in relevance. Furthermore, virtually all morphological parts can be the
yield organ into which the desired compounds are allocated before it is finally
harvested.
Environment is the second variable that has an important impact on NUE.
According to Evans and Fischer (1999) yield potential (Yp) can be defined as
‘the yield of a cultivar when grown in environments to which it is adapted, with
nutrients and water non-limiting, and with pests, diseases, weeds, lodging and other
stresses effectively controlled’. The yield of a crop depends largely on the envi-
ronmental conditions during the growing period. Nutrients are not always the
limiting factor for plant growth and crop yield. Environmental factors such as
temperature, light and rain or soil-specific factors such as soil composition, pH or
pollution with salts or heavy metals may also be of great significance. If this is the
case, there are more urgent steps to be taken to increase productivity than increasing
NUE (Boyer 1982). Even if nutrient availability is the limiting factor, the
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 13

physiological processes that are involved in NUE may be modified by environmen-

tal conditions. For instance, drought has numerous implications for the mineral
nutrition of plants. As the uptake of many nutrients is depending on the mass flow of
water there is a direct relationship between water availability and the uptake of
mobile nutrients such as nitrate (Smika et al. 1965; Buljovcic and Engels 2001). In
turn, an optimal N supply was shown to alleviate detrimental effects of drought in
Zea mays (Zhang et al. 2007). However, not only the uptake but also the translo-
cation to the shoot via the xylem is impaired during drought stress, which may
affect all nutrients but which has been shown for P (Rasnick 1970). Even moderate
drought stress might cause P deficiency in crops, which is an explanation for the
often-observed positive effect of increased P fertilisation under dry conditions
(Turner 1985; Garg et al. 2004).
It is known that growth rate and the uptake of certain nutrients (e.g. P but not N)
may show a strong decline under low light compared to high light conditions, if the
nutrient supply is adequate (Bloom 1985; Chapin 1991). The same is true for both
low and high temperatures (Tindall et al. 1990). This dependency of growth and
nutrient uptake on temperature and light leads to a different relevance of these
processes for NUE in different patches of a field, during different seasons and in
different climatic regions of the world.
The mineralization and cycles of essential nutrients such as N are mainly driven
by the properties of the soil. Litter decomposition plays a crucial role in overall
nutrient cycling in an ecosystem as well as in an agroecosystem. Globally the extent
of litter decomposition depends mainly on temperature, while on a regional scale
the chemical composition of the litter becomes most important (Aerts and Chapin
1999). In this way the soil not only plays a crucial role in the NUE of a field or a
whole agroecosystem but also at plant level where NUE is partly governed by soil
specific factors. Nutrient uptake is the process, which is affected most obviously. In
natural ecosystems nutrient uptake is ultimately dependent on the nutrient supply
rate by the parental rock material. Its mineral composition, age and weathering rate
determine the nutritional status of a soil (Lambers et al. 2008). In an agricultural
system the nutrient composition of the soil is mainly controlled by fertilisation.
However, there are other factors with an impact on nutrient uptake, which are
usually under much less control. One is the pH of the rhizosphere, which has a
tremendous impact on the ability of the plant root to acquire different nutrients. In
addition the soil is much more than just part of the plant’s abiotic environment. It
hosts a still widely unknown diversity of microbial life. Interactions with arbuscular
mycorrhiza and rhizobacteria that increase the uptake surface of plant roots and
provide nutrients to it may alter the NUE of crops, increase yield significantly and
improve fertiliser management on a field scale (Smith et al. 1992; Adesemoye
et al. 2008; Adesemoye and Kloepper 2009).
While the below ground part of the plant is surrounded by soil, the above-ground
part is exposed to the atmosphere and although almost 100 % of its volume is equal
in composition around the globe, there are traces of gases which fluctuate in their
local concentrations (Kraus 2006). Some of these gases can have a significant
impact on plant metabolism. Most of these N- and S-containing gases are usually
14 M. Reich et al.

referred to as pollutants, as they originate to a great extent from anthropogenic

activities but also from volcanic activities. It has, however, been shown that some of
these gases have an ambivalent mode of action on plants, as they are not only toxic
if too high in concentration but can also serve as nutrients (De Kok et al. 2002,
2007). This can either happen by wet deposition, i.e. a deposition to the soil by rain,
or by dry deposition via the stomata of plants. This additional nutrition from the
atmosphere and predictions for changes in the concentration of these gases should
be included in future calculations of NUE and fertilisation regimes.
The third variable that complicates efforts to define one general concept for NUE
is the nutrient itself, as nutrients differ not only in their physiochemical properties
but also in their uptake by and function in the plant. For each of them, the relevance
of the underlying physiological processes for NUE differs and so do the necessary
strategies for the improvement of NUE. For one nutrient the capacity of storage
might be crucial, while for another the uptake or the allocation to the sink organ
limits production. For this reason, studies on NUE in which different nutrients are
used are often difficult to compare. More specific definitions and components have
to be developed to accommodate this diversity. A recent study suggested three
components for NitUE: N uptake efficiency, grain-specific N efficiency and grain N
concentration (Weih et al. 2011). S for example is involved in plant defence and
thereby using less S while maintaining the same defence status should increase S
use efficiency. Some micronutrients are co-factors of particular enzymes so conse-
quently a higher efficiency and decreased amount of these enzymes could increase
the NUE of these micronutrients. Furthermore, some nutrients are of nutritional
value, which again increases the relevance of translocation to the yield organ, e.g.
the grain, while other nutrients are not desired to be part of the yield but play a role
in its production. Conclusively the NUE of a given nutrient depends on its impor-
tance for yield production and its value for human nutrition, i.e. its concentration in
the yield. Each case needs a different set of strategies for an improvement of NUE.

Improvement of NUE

Although controlled by man, agriculture remains a semi-artificial environment,

which is still subject to oscillations, particularly from outside the agroecosystem.
These oscillations may be entirely different from those in the ancient, natural
habitat of the plant species and thus transferring it to an agricultural system may
lead to sub-optimal growth and yield. Since the beginning of agricultural practice
(ca. 11,000–13,000 years ago; Allard 1999) farmers have tried to solve these
problems with two different approaches:
(i) By reducing the amplitude of environmental oscillations either by growing
plants only in a certain season to avoid extremes in weather (e.g. cold winter, dry
summers or rainy seasons) or by creating more stable conditions in the field (e.g. by
using hedges as wind protection and digging moats to avoid flooding or, in modern
times, building partially closed and controlled environments such as greenhouses).
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 15

Another important factor is the nutrient content of the soil, which was controlled by
deploying manure before the revolutionary discovery of Liebig’s law of the min-
imum and the advent of modern fertilisers. (ii) By reducing all kinds of
unfavourable dynamics and traits in the plant’s phenotype, which are relics of
adaptation to its ancient habitat and/or part of its developmental program but no
longer needed in cultivation. This is done by breeding, which makes use of the same
mechanisms as evolution (variation and selection). Plants are selected for traits of
agricultural interest with a main focus on bigger sink organs and higher concentra-
tions of the compounds of interest at the expense of traits and adaptations that are no
longer necessary.
In theory a combination of (i) and (ii) could result in a stable farming system in
which environment and plant are under full control and no unfavourable oscilla-
tions and dynamics should occur anymore. Nutrient loss from the system would be
at a minimum resulting in an optimal input-output ratio. The result would be to
equal out all the variables that make NUE such a complicated trait: plant, environ-
ment and nutrient specific factors (see above). In reality, however, this is an ideal
scenario and is presently far from being achievable. First, the technological effort to
control all environmental factors and their respective oscillations is uneconomical
and second the complex ways in which plant functioning is still largely unknown.
Consequently the compromise that has developed over thousands of years of plant
domestication is the attempt to synchronize the oscillations of environment and
plant as well as possible. This is especially true for fertilisation because the
discrepancy between demand of the plant and availability of nutrients in the soil
is an important factor that can hinder optimal growth. Nutrient storage can buffer
this discrepancy only to some extent and matching nutrient supply by fertiliser
application to plant demand is regarded as one of the most promising ways to reach
higher fertiliser use efficiency (Cassman et al. 1993; Frink et al. 1999; Tilman
et al. 2002).
Without a deeper understanding of the biochemical and physiological processes
involved, traditional breeding managed for more than 10,000 years of agriculture to
develop plants with massive yield organs containing high protein, starch or oil
content, compared to their ancestors (Mazoyer and Roudart 2006). At the same time
major steps towards increasing output-input-ratios have been made by the progress
of agricultural practice, technology and science (Russell 1966; Thompson 2011).
With Liebig’s postulation of the “law of the minimum” (see review Browne
1942) and its resolution in terms of N supply by industrial production of relatively
cheap nitrogenous fertilisers (mainly by the Haber-Bosch process in which N and
hydrogen are directly converted to ammonia, see e.g. Tour 1920), the modern age of
agriculture began and led to a historical change of paradigms, also named the
“Green Revolution” (Borlaug 1972). Instead of trying to have an optimal input-
output ratio, as it is necessary if fertiliser is a rare commodity, the highest possible
output became the primary aim and remains so in present agricultural practice. The
increased growth of cereals due to super-optimal N supply led to another factor
becoming a major constraint for yield, namely the damage caused by lodging. The
solution was the breeding of “dwarf cultivars” of wheat and rice in the 1960s by
16 M. Reich et al.

deployment of dwarfing genes. This in turn was only possible with better weed
control through the development of herbicides so that the smaller cereals were not
overgrown by wild weeds. This innovative trinity of the Green Revolution made it
possible to neglect the input-output ratio and exclusively focus on maximum output
(Evans 1998). As a result, yield per hectare increased tremendously during the last
century and enabled an explosion of human population counting billions instead of
millions. However, it has become more and more apparent that this practice cannot
continue in the future. Parallel to a linear increase of global yields since the 1960s,
the NUE of agricultural system (measured as unit yield per unit fertiliser applied)
continuously declined. This “law of diminishing returns” implies that further
increases in fertiliser application will not lead to higher yields in the same propor-
tion as in the past (Tilman et al. 2002). High levels of nutrient input have resulted in
pollution of the environment on the one hand and anticipated shortages of
non-renewable resources such as inorganic P on the other. Furthermore, the benefits
of these high outputs are very unevenly distributed over the world and with some
production wasted in Europe and North America, spikes in food prices and hunger
crises are expected to occur more frequently in developing countries. The aware-
ness of this alarming trend led policymakers to put “food security” on the top of
political agendas and consequently also into scientific focus (Rosegrant and Cline
2003; Vitousek et al. 2009; Godfray et al. 2010; Hawkesford et al. 2013).
To some extent the agriculture of the future has to come back to the old paradigm
of requiring a more optimal input-output ratio. However, maintenance of the
ongoing trend of increasing output in form of yield is an imperative due to the
still-growing world population and no concept, which reduces the output, can be
realistically considered (Evans 1998). Consequently, modifications of the input-
output ratio have to concentrate on the input side of the equation. At field level
(Fig. 1.1) the approaches to improve the input-output ratio may be categorized in
two dimensions: time and space. This is again based on the general conception of
plant and environment as two non-stationary, heterogeneous and oscillating sys-
tems. The supply with nutrients (fertiliser) can be adapted to the need of the plant
for growth over time to lose fewer nutrients from the system in times of a low crop
demand and to avoid concentrations being too low if the demand is high. In
addition, nutrient supply and its demand in the field are not homogenously distrib-
uted in space but instead graduated or patchy. A homogenous application of
nutrients thereby leads to excess supply in some parts of the field, resulting in
nutrient loss, and to a sub-optimal supply in other parts, resulting in a non-optimal
growth and yield. For both dimensions technological innovations have been devel-
oped, commonly summarized with the term “precision agriculture” (Pierce and
Nowak 1999; Stafford 2000; Zhang et al. 2002). However, in addition there might
be ways to breed or genetically manipulate the plant in a way that it uses nutrients
more efficient and consequently produces the desired yield with less nutrient input.
While transgenic and genetic techniques offer the possibility for accelerated
improvement through genetic modifications and marker assisted breeding, whole
plant physiology provides the knowledge to use these tools in an effective way. To
practically improve the NUE of a plant in a particular agricultural system, the
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 17

relevance of the underlying physiological processes (Fig. 1.2) has to be analysed in

relation to the variables that modify it (Fig. 1.3). Once the limiting processes are
identified, breeding or transgenic methods might lead to further improvement of
NUE. For success, the physical and physiological trade-offs that limit an improve-
ment in NUE have to be identified. This will be the topic of the last part of this
chapter.

NUE – Challenges from a Whole Plant Perspective

As stated previously, NP and MRT can be seen as sub-components of NUtE.

Consequently both could be targeted to improve NUE in cropping systems.
Berendse and Aerts (1987) have pointed out the apparent ecological trade-off of
NitP and MRT. While in nutrient-poor soils a long MRT is favourable, a high NitP
gives advantages in nutrient-rich soils. Plant species in nutrient-rich soils generally
possess a larger photosynthetic apparatus and can thereby rapidly make use of
higher N availability, while species in nutrient-poor soils are able to use spare
nutrients more economically. Theoretically, generalist species should combine both
traits, but evidence suggests that they are competitive (Berendse et al. 1987). A later
study on species with different life forms in a sub-arctic environment confirmed this
negative relationship of NitP and MRT (Eckstein and Karlsson 1997), and others
followed (Yasumura et al. 2002; Silla and Escudero 2004). Within the same
species, this trade-off also appears to be consistent, with higher N supply leading
to higher NitP but lower MRT (Yuan et al. 2005) or the other way around (Yuan
et al. 2008). High nutrient supply, however, may even lead to a decline of both
components in certain circumstances. A decline in NP can be caused by cross- and
self-shading and an accompanied decrease in photosynthetic activity and a lower
MRT can be the result of enhanced litter production or leaching (Meuleman
et al. 2002).
As studies have almost exclusively focused on natural systems, perennials
and N, it is difficult to assess if a strict trade-off between NP and MRT exists in
agricultural crops and for all nutrients. As litter decomposability is not an issue in
annual crop production, a high NP and long MRT at the same time are desired to
maximize yield output and minimize nutrient input. The fact that traits, which lead
to either a high NP or a long MRT, are not co-occurring in nature does not
necessarily mean that breeding could not combine them. More integrated research
on the described underlying physiological processes and their possible trade-offs
are needed, including research with nutrients other than N.
In general, NUE is only studied for one single nutrient. There are few studies,
which address the question whether improving the NUE for one nutrient will affect
the NUE of others. Given the interactive and competitive nature of nutrients in
many physiological processes, it seems likely that increasing NUE for one nutrient
will also alter the NUE of others in a positive or negative manner.
18 M. Reich et al.

On the level of nutrient uptake from the soil there are manifold co-influences
known, but their whole extent is still far from being understood. Many of these are
caused by the charged nature of ions when dissolved in water. If, for example, N is
taken up predominantly as positive charged ammonium, the uptake of other cations
such as magnesium and calcium is impaired, probably in order to maintain a
balance of charge (Haynes and Goh 1978). On the other hand, plants that are
supplied with nitrate absorb less phosphate than plants supplied with ammonium
(Riley and Barber 1971). Similarly, do sulfate or nitrate show a higher accumula-
tion in the plant if the other is missing in the root medium (Steingröver et al. 1986;
Koralewska et al. 2009), either due to a replacement as an osmolyte in the vacuole
or to a balance of anion-cation uptake, i.e. a balance of charge. This is a hint that
increasing the storage capacity of the vacuole for one nutrient could decrease the
capacity for others of the same charge. Interactions of the uptake of nutrients with
different charge have been observed, for example for sulfate and iron (Paolacci
et al. 2013). Here a direct interaction on the level of uptake does not seem likely. In
contrast a higher uptake of sulfate under iron deficiency is suggested to serve for the
production of S-containing defence compounds and a coupling of both nutrients
could be due to their combination in Fe-S clusters (Forieri et al. 2013). Another
example of the interactive effects of the uptake of different nutrients has been
shown for sulfate, nitrate and ammonium (Clarkson et al. 1989) and there are many
more reported. The direct linkage between the uptake of different nutrients, how-
ever, is under critical discussion. Studies in which plants were exposed to an
atmospheric S source while deprivation occurred in the rhizosphere show an
apparent uncoupling of sulfate and nitrate uptake (Westerman et al. 2000, 2001;
Stulen and De Kok 2012).
Another potential conflict in the uptake of different nutrients arises if they use
the same uptake system. This may be true, for instance, for the transport of
phosphate and sulfate across the chloroplast membrane, which was reported to be
competitive (Gross et al. 1990). As well as Liebig’s “law of the minimum” states
that identifying and increasing the amount of the most limiting factor can increase
plant production, there is the far less popular but equally important “law of the
optimum” formulated by Liebscher (see review Browne 1942). It states that the
increase of such a limiting factor contributes more to the productivity of the system,
the closer all other factors are to their optimum. Liebscher studied N, P and K
nutrition of crops and was one of the first researchers to demonstrate the strong
interactive component of different nutrients and their contributions to yield. It does
not contradict Liebig’s “law of the minimum” but shows that reality is more
complex. Improvement of NUtE for any of the three nutrients N, P and K requires
a balanced supply with the other two (Janssen 1998).
An important question for the future improvement of plant NUE is whether
interferences with other efficiencies exist, especially if those turn out to be real
physical trade-offs. Such trade-offs usually arise from the involvement of physio-
logical mechanisms in several efficiencies. Studies on different tree species showed
intraspecific inverse relationships between water use efficiency (WUE) and NitUE
(Field et al. 1983; Reich et al. 1989), which appear to explain the spatial distribution
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 19

of species on either N-poor or water-deficient soils (Patterson et al. 1997) but also
have implications for the improvement of NUE in agriculture. This trade-off has
stomatal as well as non-stomatal components (Reich et al. 1989) depending on the
water and N status of the soil. How relevant it is for agricultural practice and crop
breeding towards an increased NUE has still to be clarified. It might help the case to
distinguish between the levels of NAcE and NUtE. If water is a limiting factor
during crop growth, NAcE usually increases in its relevance, as nutrient uptake is
physically coupled to water uptake. Climate change may mean seasonal droughts
appear more frequently in many regions of the world which further increases the
need for crops with a high WUE (Reynolds et al. 2011). For wheat, for example, it
has been shown that under water limiting conditions genotypes with a greater root
biomass produce more grains, probably due to both a high WUE and NAcE during
early growth due to enhanced ability to capture water and reduce nitrate leaching
(Ehdaie et al. 2010). Root traits are considered as a selection criterion especially
under drought conditions (Ren et al. 2012) but there are strong interactions with
NAcE that have to be considered. For example, the responses of root architecture to
P and N deficiency are very different. While P deficiency leads to a shallow root
system, foraging for the immobile phosphate, N deficiency causes a deep, scarcely
branched root system. The latter is also associated with an efficient capture of water
during periodical drought (White et al. 2013) implying that the NAcE for N and an
efficient water uptake are not in competition but share a similar morphological trait.
How the shallow root system that leads to a higher NAcE of P displays a constraint
to WUE needs to be further evaluated. However, there are also reports that show an
apparent negative interaction of N supply with WUE. High N supply leads to an
inhibition of the typical growth characteristics that lead to a higher WUE in
Sophora davidii while appropriate or low supply alleviated drought stress
(Wu et al. 2008).
NUtE, NitUE and WUE show strong interactions due to their correlative effects
on stomatal conductance, gas exchange and photosynthesis. In wheat, a higher N
supply is reported to lead to an increase in WUE but at the same time to a decreased
NitUE (Shangguan et al. 2000; Cabrera-Bosquet et al. 2007). This trade-off
between NUtE and WUE is particularly noticeable if the N-to-grain price ratio in
an agricultural system is high (Sadras and Rodriguez 2010). How the NUE of
nutrients other than N interferes with WUE is still an open question.
Other studies have revealed a trade-off between N and light use efficiency (LUE)
in canopies (Niinemets and Tenhunen 1997; Hirose and Bazzaz 1998). This can be
explained by the fact that a high concentration of N in leaves leads to a high LUE
but low NitUE. The impact of this trade-off again depends on the factors described
above. It becomes more relevant under shading conditions and for trees because the
build-up of the desired timber strongly depends on exploiting light efficiently
during the growing season.
Again, it should be noted that inverse relationships between certain plant traits
observed in nature do not necessarily display real physical trade-offs that cannot be
overcome in plant breeding. They might just reflect genetic adaptations to different
habitats or an ecological disadvantage of the combination of both traits (Veneklaas
20 M. Reich et al.

et al. 2012). If this is the case traits, which never occur together in nature may still
be combined in one crop.
Finding plant traits, which improve Yp and NUE is a major aim of modern
breeding programs. The genetic variability of crops, as well as of the model plant
Arabidopsis thaliana, may serve as sources of diversity and promising traits may
then be incorporated into transgenic crop plants. Additionally future techniques
might enable the transfer of physiological traits such as C4 metabolism (Leegood
2002) or N fixation (Charpentier and Oldroyd 2010; Beatty and Good 2011)
between distantly related species. In this way, ecological but non-physical trade-
offs could theoretically be overcome faster, and crop plants could be complemented
with physiological properties that they would never gain through breeding. The
diversity present in nature is potentially a rich source of traits that could improve
the NUE of crops. There may be plant species that evolved mechanisms to use
nutrients much more efficiently than crops but which science has yet to exploit. In
particular species from nutrient-poor habitats are very promising candidates. Thus
transgenics could be a useful tool to engineer crops whilst ecophysiology delivers
the ideas of how these modifications will look.

Conclusions
Plant NUE is as complex and multi-dimensional as the plant itself. Conse-
quently the greatest challenge, but also the greatest opportunity for modern
plant nutrition research, is the integration of various disciplines and
approaches. Use of state-of-the-art methods in transcriptomics,
metabolomics, and proteomics give researchers unprecedented opportunities
to obtain huge amounts of information with high resolution (see the other
chapters of this book), not least enabled by the exponential increase of
computing performance since its emergence (Moore 1975; Kurzweil 2001).
The growing understanding of complex molecular networks and their holistic
responses to alterations in nutrient availability contributes to uncovering the
genetic basis of NUE and shows how complex metabolic pathways are
interacting on a molecular level. However, the real power of molecular
techniques for future crop breeding can only unfold if the complexity of the
underlying physiological processes and their contribution to NUE is to be
further understood.
Additionally there are physiological trade-offs that challenge the improve-
ment of NUE. It might be possible to overcome some of them by modern
molecular breeding techniques; others might be physically determined and
display a real limit for the improvement of NUE. Further investigation is
necessary to determine the physiological basis of these trade-offs before tools
such as genetic manipulation may be used to overcome them. As studies on
NUE usually focus on only one nutrient (and usually only N or P), there is
little data available on possible trade-offs between the NUE of different

(continued)
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 21

nutrients. However, the compensatory and interactive nature of the uptake

and storage of different nutrients suggests that such inter-nutritional trade-
offs could exist. Adding to this complexity is the knowledge that all these
possible trade-offs are strongly influenced by environment, plant and nutrient
specific variables.
In conclusion, the physiological basis of NUE still displays a wide field for
future research and the complexity and plasticity of plant metabolism may yet
have many surprises in store in our attempts to “improve” it. More integrative
studies, connecting several scientific disciplines are required to understand
the complexity of all the variables that influence NUE resulting in physio-
logically relevant strategies for the improvement of plant NUE in agricultural
production.

References

Adesemoye AO, Kloepper JW (2009) Plant–microbes interactions in enhanced fertilizer-use

efficiency. Appl Microbiol Biotechnol 85:1–12
Adesemoye AO, Torbert HA, Kloepper JW (2008) Enhanced plant nutrient use efficiency with
PGPR and AMF in an integrated nutrient management system. Can J Microbiol 54:876–886
Aerts R (1990) Nutrient use efficiency in evergreen and deciduous species from heathlands.
Oecologia 84:391–397
Aerts R (1997) Nitrogen partitioning between resorption and decomposition pathways: a trade-off
between nitrogen use efficiency and litter decomposibility? Oikos 80:603–606
Aerts R, Chapin FS III (1999) The mineral nutrition of wild plants revisited: a re-evaluation of
processes and patterns. Adv Ecol Res 30:1–67
Ågren GI (1985) Theory for growth of plants derived from the nitrogen productivity concept.
Physiol Plant 64:17–28
Ainsworth EA, Rogers A, Nelson R, Long SP (2004) Testing the “source–sink” hypothesis of
down-regulation of photosynthesis in elevated [CO2] in the field with single gene substitutions
in Glycine max. Agric Forest Meteorol 122:85–94
Allard RW (1999) Principles of plant breeding. Wiley, New York
Barbottin A, Lecomte C, Bouchard C, Jeuffroy MH (2005) Nitrogen remobilization during grain
filling in wheat. Crop Sci 45:1141–1150
Beatty PH, Good AG (2011) Future prospects for cereals that fix nitrogen. Science 333:416–417
Berendse F, Aerts R (1987) Nitrogen-use-efficiency: a biologically meaningful definition? Funct
Ecol 1:293–296
Berendse F, Oudhof H, Bol J (1987) A comparative study on nutrient cycling in wet heathland
ecosystems. Oecologia 74:174–184
Berendse F, Elberse WT, Geerts RHME (1992) Competition and nitrogen loss from plants in
grassland ecosystems. Ecology 73:46–53
Birk EM, Vitousek PM (1986) Nitrogen availability and nitrogen use efficiency in loblolly pine
stands. Ecology 67:69–79
Bloom AJ (1985) Wild and cultivated barleys show similar affinities for mineral nitrogen.
Oecologia 65:555–557
Borlaug NE (1972) The green revolution, peace, and humanity. Speech delivered upon receipt of
the 1970 Nobel Peace Prize. CIMMYT reprint and translation series No. 3. Centro
Internacional de Mejoramiento de Maiz y Trigo, El Batan, Mexico
22 M. Reich et al.

Borrás L, Slafer GA, Otegui ME (2004) Seed dry weight response to source–sink manipulations in
wheat, maize and soybean: a quantitative reappraisal. Field Crop Res 86:131–146
Bot JL, Kirkby EA (1992) Diurnal uptake of nitrate and potassium during the vegetative growth of
tomato plants. J Plant Nutr 15:247–264
Boyer JS (1982) Plant productivity and environment. Science 218:443–448
Brown RH (1978) A difference in N use efficiency in C3 and C4 plants and its implications in
adaptation and evolution. Crop Sci 18:93–98
Browne CA (1942) Liebig and the law of the minimum. In: Moulton FA (ed) Liebig and after
Liebig. A century of progress in agricultural chemistry. American Association for the
Advancement of Science, Washington, DC, pp 71–82
Buljovcic Z, Engels C (2001) Nitrate uptake ability by maize roots during and after drought stress.
Plant Soil 229:125–135
Cabrera-Bosquet L, Molero G, Bort J, Nogués S, Araus JL (2007) The combined effect of constant
water deficit and nitrogen supply on WUE, NUE and Δ13C in durum wheat potted plants. Ann
Appl Biol 151:277–289
Cassman KG, Kropff MJ, Gaunt J, Peng S (1993) Nitrogen use efficiency of rice reconsidered:
what are the key constraints? Plant Soil 155:359–362
Chapin FS III (1980) The mineral nutrition of wild plants. Annu Rev Ecol Syst 11:233–260
Chapin FS III (1991) Integrated responses of plants to stress. Bioscience 41:29–36
Chapin FS III, Kedrowski RA (1983) Seasonal changes in nitrogen and phosphorus fractions and
autumn retranslocation in evergreen and deciduous taiga trees. Ecology 64:376–391
Chapin FS III, Moilanen L (1991) Nutritional controls over nitrogen and phosphorus resorption
from Alaskan birch leaves. Ecology 72:709–715
Chapin FS III, Schulze ED, Mooney HA (1990) The ecology and economics of storage in plants.
Annu Rev Ecol Syst 21:423–447
Charpentier M, Oldroyd G (2010) How close are we to nitrogen-fixing cereals? Curr Opin Plant
Biol 13:556–564
Clarkson DT, Saker LR, Purves JV (1989) Depression of nitrate and ammonium transport in barley
plants with diminished sulphate status. Evidence of co-regulation of nitrogen and sulphate
intake. J Exp Bot 40:953–963
Costanza R, d’Arge R, De Groot R, Farber S, Grasso M, Hannon B, Limburg K, Naeem S, O’Neill
RV, Paruelo J, Raskin RG, Sutton P, Van den Belt M (1997) The value of the world’s
ecosystem services and natural capital. Nature 387:253–260
Daily GC (1997) Nature’s services: societal dependence on natural ecosystems. Island,
Washington, DC
Daily GC, Söderqvist T, Aniyar S, Arrow K, Dasgupta P, Ehrlich PR, Folke C, Jansson A, Jansson
B-O, Kautsky N, Levin S, Lubchenco J, Mäler K-G, Simpson D, Starrett D, Tilman D, Walker
B (2000) Ecology. The value of nature and the nature of value. Science 289:395
De Kok LJ, Stuiver CEE, Westerman S, Stulen I (2002) Elevated levels of hydrogen sulfide in the
plant environment: nutrient or toxin. In: Omasa K, Saji H, Youssefian S, Kondo N (eds) Air
pollution and biotechnology in plants. Springer, Tokyo, pp 201–213
De Kok LJ, Durenkamp M, Yang L, Stulen I (2007) Atmospheric sulfur. In: Hawkesford MJ, De
Kok LJ (eds) Sulfur in plants – an ecological perspective. Springer, Dordrecht, pp 91–106
Delhon P, Gojon A, Tillard P, Passama L (1995) Diurnal regulation of NO3 uptake in soybean
plants I. Changes in NO3 influx, efflux, and N utilization in the plant during the day/night
cycle. J Exp Bot 46:1585–1594
Donald CM, Hamblin J (1976) The biological yield and harvest index of cereals as agronomic and
plant breeding criteria. Adv Agron 28:361–405
Eckstein RL, Karlsson PS (1997) Above-ground growth and nutrient use by plants in a subarctic
environment: effects of habitat, life-form and species. Oikos 79:311–324
Ehdaie B, Merhaut DJ, Ahmadian S, Hoops AC, Khuong T, Layne AP, Waines JG (2010) Root
system size influences water‐nutrient uptake and nitrate leaching potential in wheat. J Agron
Crop Sci 196:455–466
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 23

Eichert T, Fernández V (2012) Uptake and release of elements by leaves and other aerial plant
parts. In: Marschner P (ed) Marschner’s mineral nutrition of higher plants. Academic, Oxford,
UK, pp 71–84
Escudero A, Mediavilla S (2003) Decline in photosynthetic nitrogen use efficiency with leaf age
and nitrogen resorption as determinants of leaf life span. J Ecol 91:880–889
Evans LT (1998) Feeding the ten billion: plants and population growth. Cambridge University
Press, Cambridge, UK
Evans LT, Dunstone RL (1970) Some physiological aspects of evolution in wheat. Aust J Biol Sci
23:725–742
Evans LT, Fischer RA (1999) Yield potential: its definition, measurement, and significance. Crop
Sci 39:1544–1551
Faller N (1972) Schwefeldioxid, Schwefelwasserstoff, nitrose Gase und Ammoniak als
ausschließliche S- bzw. N-Quellen der höheren Pflanze. J Plant Nutr Soil Sci 131:120–130
Field C (1983) Allocating leaf nitrogen for the maximization of carbon gain: leaf age as a control
on the allocation program. Oecologia 56:341–347
Field C, Merino J, Mooney HA (1983) Compromises between water-use efficiency and nitrogen-
use efficiency in five species of California evergreens. Oecologia 60:384–389
Forieri I, Wirtz M, Hell R (2013) Towards new perspectives on the interaction of iron and sulfur
metabolism in Arabidopsis thaliana. Front Plant Sci 4:357
Foulkes MJ, Slafer GA, Davies WJ, Berry PM, Sylvester-Bradley R, Martre P, Calderini DF,
Griffiths R, Reynolds MP (2011) Raising yield potential of wheat. III. Optimizing partitioning
to grain while maintaining lodging resistance. J Exp Bot 62:469–486
Francis C, Lieblein G, Gliessman S, Breland TA, Creamer N, Harwood R, Salomonsson L,
Helenius J, Rickerl D, Salvador R, Wiedenhoeft M, Simmons S, Allen P, Altieri M, Flora C,
Poincelot R (2003) Agroecology: the ecology of food systems. J Sustain Agric 22:99–118
Franklin O, McMurtie ROSS, Iversen CM, Crous KY, Finzi AC, Tissue DT, Ellsworth DS,
Oren R, Norby RJ (2009) Forest fine‐root production and nitrogen use under elevated CO2:
contrasting responses in evergreen and deciduous trees explained by a common principle. Glob
Change Biol 15:132–144
Frink CR, Waggoner PE, Ausubel JH (1999) Nitrogen fertilizer: retrospect and prospect. Proc Natl
Acad Sci U S A 96:1175–1180
Garg BK, Burman U, Kathju S (2004) The influence of phosphorus nutrition on the physiological
response of moth bean genotypes to drought. J Plant Nutr Soil Sci 167:503–508
Gerloff GC (1963) Comparative mineral nutrition of plants. Annu Rev Plant Physiol 14:107–124
Givnish TJ (2002) Adaptive significance of evergreen vs. deciduous leaves: solving the triple
paradox. Silva Fenn 36:703–743
Gliessman SR (1990) Agroecology: researching the ecological basis for sustainable agriculture.
Springer, New York
Godfray HCJ, Beddington JR, Crute IR, Haddad L, Lawrence D, Muir JF, Pretty J, Robinson S,
Thomas SM, Toulmin C (2010) Food security: the challenge of feeding 9 billion people.
Science 327:812–818
Gross A, Brückner G, Heldt HW, Flügge UI (1990) Comparison of the kinetic properties,
inhibition and labelling of the phosphate translocators from maize and spinach mesophyll
chloroplasts. Planta 180:262–271
Hawkesford MJ (2000) Plant responses to sulphur deficiency and the genetic manipulation of
sulphate transporters to improve S-utilization efficiency. J Exp Bot 51:131–138
Hawkesford MJ, Araus JL, Park R, Calderini D, Miralles D, Shen T, Zhang J, Parry MA (2013)
Prospects of doubling global wheat yields. Food Energy Secur 2:34–48
Haydon MJ, Bell LJ, Webb AA (2011) Interactions between plant circadian clocks and solute
transport. J Exp Bot 62:2333–2348
Haynes RJ, Goh KM (1978) Ammonium and nitrate nutrition of plants. Biol Rev 53:465–510
Hirose T, Bazzaz FA (1998) Trade-off between light-and nitrogen-use efficiency in canopy
photosynthesis. Ann Bot 82:195–202
24 M. Reich et al.

Ingestad T (1988) A fertilization model based on the concepts of nutrient flux density and nutrient
productivity. Scand J Forest Res 3:157–173
Janssen BH (1998) Efficient use of nutrients: an art of balancing. Field Crop Res 56:197–201
Killingbeck KT (1996) Nutrients in senesced leaves: keys to the search for potential resorption and
resorption proficiency. Ecology 77:1716–1727
Kimball BA, Kobayashi K, Bindi M (2002) Responses of agricultural crops to free-air CO2
enrichment. Adv Agron 77:293–368
Koralewska A, Buchner P, Stuiver CEE, Posthumus FS, Kopriva S, Hawkesford MJ, De Kok LJ
(2009) Expression and activity of sulfate transporters and APS reductase in curly kale in
response to sulfate deprivation and re-supply. J Plant Physiol 166:168–179
Kozaki A, Takeba G (1996) Photorespiration protects C3 plants from photooxidation. Nature
384:557–560
Kraus H (2006) Die Atmosphäre der Erde: Eine Einführung in die Meteorologie. (The atmosphere
of the earth: an introduction to meteorology). Springer, Berlin/Heidelberg, p 23
Kurzweil R (2001) The law of accelerating returns. Retrieved from www.kurzweilai.net in 2013
Lajtha K (1987) Nutrient reabsorption efficiency and the response to phosphorus fertilization in the
desert shrub Larrea tridentata (DC.) Cov. Biogeochemistry 4:265–276
Lambers H, Chapin FS III, Pons TL (2008) Plant physiological ecology, 2nd edn. Springer, Berlin
Larcher W (1995) Plant physiological ecology, 3rd edn. Springer, Berlin
Leegood RC (2002) C4 photosynthesis: principles of CO2 concentration and prospects for its
introduction into C3 plants. J Exp Bot 53:581–590
Long SP, Zhu XG, Naidu SL, Ort DR (2006) Can improvement in photosynthesis increase crop
yields? Plant Cell Environ 29:315–330
Loomis RS, Amthor JS (1999) Yield potential, plant assimilatory capacity, and metabolic effi-
ciencies. Crop Sci 39:1584–1596
Marschner H (2012) In: Marschner P (ed) Marschner’s mineral nutrition of higher plants.
Academic, Oxford, UK
Mazoyer M, Roudart L (2006) A history of world agriculture: from the neolithic age to the current
crisis. Monthly Review Press, New York
McClung CR (2006) Plant circadian rhythms. Plant Cell 18:792–803
Mengel K, Kirkby EA (1987) Principles of plant nutrition, 4th edn. International Potash Institute,
Bern
Meuleman AF, Beekman JHP, Verhoeven JT (2002) Nutrient retention and nutrient-use efficiency
in Phragmites australis stands after waster water application. Wetlands 22:712–721
Moll RH, Kamprath EJ, Jackson WA (1982) Analysis and interpretation of factors which contrib-
ute to efficiency of nitrogen utilization. Agron J 74:562–564
Moore GE (1975) Progress in digital integrated electronics. IEEE, international electron devices
meeting, IEDM Tech. Digest 1975, pp 11–13
Nardoto GB, da Cunha Bustamante MM, Pinto AS, Klink CA (2006) Nutrient use efficiency at
ecosystem and species level in savanna areas of Central Brazil and impacts of fire. J Trop Ecol
22:191–201
Niinemets Ü, Tenhunen JD (1997) A model separating leaf structural and physiological effects on
carbon gain along light gradients for the shade‐tolerant species Acer saccharum. Plant Cell
Environ 20:845–866
Ntanos DA, Koutroubas SD (2002) Dry matter and N accumulation and translocation for Indica
and Japonica rice under Mediterranean conditions. Field Crop Res 74:93–101
Oenema O, Witzke HP, Klimont Z, Lesschen JP, Velthof GL (2009) Integrated assessment of
promising measures to decrease nitrogen losses from agriculture in EU-27. Agric Ecosyst
Environ 133:280–288
Paez-Valencia J, Sanchez-Lares J, Marsh E, Dorneles LT, Santos MP, Sanchez D, Winter A,
Murphy S, Cox J, Trzaska M, Metler J, Kozic A, Facanha AR, Schachtman D, Sanchez C,
Gaxiola RA (2013) Enhanced proton translocating pyrophosphatase activity improves nitrogen
use efficiency in romaine lettuce. Plant Physiol 161:1557–1569
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 25

Paolacci AR, Celletti S, Catarcione G, Hawkesford MJ, Astolfi S, Ciaffi M (2013) Iron deprivation
results in a rapid but not sustained increase of the expression of genes involved in iron
metabolism and sulfate uptake in tomato (Solanum lycopersicum L.) seedlings. J Integr Plant
Biol 56. Early View Online Version
Parry MA, Reynolds M, Salvucci ME, Raines C, Andralojc PJ, Zhu XG, Price GD, Condon AG,
Furbank RT (2011) Raising yield potential of wheat. II. Increasing photosynthetic capacity and
efficiency. J Exp Bot 62:453–467
Patterson TB, Guy RD, Dang QL (1997) Whole-plant nitrogen-and water-relations traits, and their
associated trade-offs, in adjacent muskeg and upland boreal spruce species. Oecologia
110:160–168
Peterhansel C, Maurino VG (2011) Photorespiration redesigned. Plant Physiol 155:49–55
Pierce FJ, Nowak P (1999) Aspects of precision agriculture. Adv Agron 67:1–85
Poorter H, Remkes C, Lambers H (1990) Carbon and nitrogen economy of 24 wild species
differing in relative growth rate. Plant Physiol 94:621–627
Rasnick M (1970) Effect of mannitol and polyethylene glycol on phosphorus uptake by maize
plants. Ann Bot 34:497–502
Reich PB, Walters MB, Tabone TJ (1989) Response of Ulmus americana seedlings to varying
nitrogen and water status. 2 Water and nitrogen use efficiency in photosynthesis. Tree Physiol
5:173–184
Ren Y, He X, Liu D, Li J, Zhao X, Li B, Tong Y, Zhang A, Li Z (2012) Major quantitative trait loci
for seminal root morphology of wheat seedlings. Mol Breed 30:139–148
Reynolds MP, Pellegrineschi A, Skovmand B (2005) Sink limitation to yield and biomass: a
summary of some investigations in spring wheat. Ann Appl Biol 146:39–49
Reynolds MP, Manes Y, Izanloo A, Langridge P (2011) Raising yield potential of wheat.
I. Overview of a consortium approach and breeding strategies. J Exp Bot 62:439–452
Riley D, Barber SA (1971) Effect of ammonium and nitrate fertilization on phosphorus uptake as
related to root-induced pH changes at the root-soil interface. Soil Sci Soc Am J 35:301–306
Rose TJ, Pariasca-Tanaka J, Rose MT, Fukuta Y, Wissuwa M (2010) Genotypic variation in grain
phosphorus concentration, and opportunities to improve P-use efficiency in rice. Field Crop
Res 119:154–160
Rosegrant MW, Cline SA (2003) Global food security: challenges and policies. Science
302:1917–1919
Rossato L, Laine P, Ourry A (2001) Nitrogen storage and remobilization in Brassica napus
L. during the growth cycle: nitrogen fluxes within the plant and changes in soluble protein
patterns. J Exp Bot 52:1655–1663
Russell EJ (1966) A history of agricultural science in great Britain 1620–1954. George Allen &
Unwin, London
Sadras VO, Rodriguez D (2010) Modelling the nitrogen-driven trade-off between nitrogen
utilisation efficiency and water use efficiency of wheat in eastern Australia. Field Crop Res
118:297–305
Saurbeck DC, Helal HM (1990) Factors affecting the nutritional efficiency of plants. In: Bassam
NEL, Dambroth M, Loughman BC (eds) Genetic aspects of plant mineral nutrition. Martinus
Nijhoff, Dordrecht, pp 361–372
Schnug E, Haneklaus S (1998) Diagnosis of sulphur nutrition. In: Schnug E (ed) Sulphur in
agroecosystems. Springer, Dordrecht, pp 1–38
Shangguan ZP, Shao MA, Dyckmans J (2000) Nitrogen nutrition and water stress effects on leaf
photosynthetic gas exchange and water use efficiency in winter wheat. Environ Exp Bot
44:141–149
Shea PF, Gerloff GC, Gabelman WH (1968) Differing efficiencies of potassium utilization in
strains of Snapbeans, Phaseolus vulgaris L. Plant Soil 28:337–346
Siddiqi MY, Glass AD (1981) Utilization index: a modified approach to the estimation and
comparison of nutrient utilization efficiency in plants. J Plant Nutr 4:289–302
26 M. Reich et al.

Silla F, Escudero A (2004) Nitrogen-use efficiency: trade-offs between N productivity and mean
residence time at organ, plant and population levels. Funct Ecol 18:511–521
Smika D, Haas H, Power W (1965) Effects of moisture and nitrogen fertilizer on growth and water
use by native grass. Agron J 57:483–486
Smith SE, Robson AD, Abbott LK (1992) The involvement of mycorrhizas in assessment of
genetically dependent efficiency of nutrient uptake and use. Plant Soil 146:169–179
Somers DE, Devlin PF, Kay SA (1998) Phytochromes and cryptochromes in the entrainment of the
Arabidopsis circadian clock. Science 282:1488–1490
Stafford JV (2000) Implementing precision agriculture in the 21st century. J Agric Eng Res
76:267–275
Steingröver E, Woldendorp J, Sijtsma L (1986) Nitrate accumulation and its relation to leaf
elongation in spinach leaves. J Exp Bot 181:1093–1102
Stulen I, De Kok LJ (2012) Exploring interactions between sulfate and nitrate uptake at a whole
plant level. In: De Kok LJ, Tausz M, Hawkesford MJ, Hoefgen R, McManus MT, Norton RM,
Rennenberg H, Saito K, Schnug E, Tabe L (eds) Sulfur metabolism in plants: mechanisms and
application to food security, and responses to climate change. Springer, Dordrecht, pp 1–8
Stulen I, Perez-Soba M, De Kok LJ, Van der Eerden L (1998) Impact of gaseous nitrogen
deposition on plant functioning. New Phytol 139:61–70
Swiader JM, Chyan Y, Freiji FG (1994) Genotypic differences in nitrate uptake and utilization
efficiency in pumpkin hybrids. J Plant Nutr 17:1687–1699
Thompson RP (2011) Agro-technology: a philosophical introduction. Cambridge University Press,
Cambridge, UK
Tilman D, Cassman KG, Matson PA, Naylor R, Polasky S (2002) Agricultural sustainability and
intensive production practices. Nature 418:671–677
Tindall JA, Mills HA, Radcliffe DE (1990) The effect of root zone temperature on nutrient uptake
of tomato. J Plant Nutr 13:939–956
Tour RS (1920) The direct synthetic ammonia process. Ind Eng Chem Res 12:844–852
Turner LB (1985) Changes in the phosphorus content of Capsicum annuum leaves during water-
stress. J Plant Physiol 121:429–439
Van der Werf A, Van Nuenen M, Visser AJ, Lambers H (1993) Contribution of physiological and
morphological plant traits to a species’ competitive ability at high and low nitrogen supply.
Oecologia 94:434–440
Vázquez de Aldana BR, Berendse F (1997) Nitrogen-use efficiency in six perennial grasses from
contrasting habitats. Funct Ecol 11:619–626
Veneklaas EJ, Lambers H, Bragg J, Finnegan PM, Lovelock CE, Plaxton WC, Price CA, Scheible
W-A, Shane MW, White PJ, Raven JA (2012) Opportunities for improving phosphorus-use
efficiency in crop plants. New Phytol 195:306–320
Vitousek P (1982) Nutrient cycling and nutrient use efficiency. Am Nat 119:553–572
Vitousek PM, Naylor R, Crews T, David MB, Drinkwater LE, Holland E, Johnes PJ,
Katzenberger J, Martinelli LA, Matson PA, Nziguheba G, Ojima D, Palm CA, Robertson
GP, Sanchez PA, Townsend AR, Zhang FS (2009) Nutrient imbalances in agricultural devel-
opment. Science 324:1519
Watanabe N, Evans JR, Chow WS (1994) Changes in the photosynthetic properties of Australian
wheat cultivars over the last century. Funct Plant Biol 21:169–183
Wedin DA, Tilman D (1990) Species effects on nitrogen cycling: a test with perennial grasses.
Oecologia 84:433–441
Weih M, Asplund L, Bergkvist G (2011) Assessment of nutrient use in annual and perennial crops:
a functional concept for analyzing nitrogen use efficiency. Plant Soil 339:513–520
Westerman S, De Kok LJ, Stuiver CEE, Stulen I (2000) Interaction between metabolism of
atmospheric H2S in the shoot and sulfate uptake by the roots of curly kale (Brassica oleracea).
Physiol Plant 109:443–449
1 Physiological Basis of Plant Nutrient Use Efficiency – Concepts. . . 27

Westerman S, Stulen I, Suter M, Brunold C, De Kok LJ (2001) Atmospheric H2S as sulphur source
for Brassica oleracea: consequences for the activity of the enzymes of the assimilatory
sulphate reduction pathway. Plant Physiol Biochem 39:425–432
Westoby M, Falster DS, Moles AT, Vesk PA, Wright IJ (2002) Plant ecological strategies: some
leading dimensions of variation between species. Annu Rev Ecol Syst 33:125–159
White PJ, George TS, Gregory PJ, Bengough AG, Hallett PD, McKenzie BM (2013) Matching
roots to their environment. Ann Bot 112:207–222
Wu F, Bao W, Li F, Wu N (2008) Effects of drought stress and N supply on the growth,
biomass partitioning and water-use efficiency of Sophora davidii seedlings. Environ Exp Bot
63:248–255
Yasumura Y, Hikosaka K, Matsui K, Hirose T (2002) Leaf-level nitrogen-use efficiency of canopy
and understorey species in a beech forest. Funct Ecol 16:826–834
Yuan ZY, Li LH, Huang JH, Han XG, Wam SQ (2005) Effect of nitrogen supply on the nitrogen
use efficiency of an annual herb, Helianthus annuus L. J Integr Plant Biol 47:539–548
Yuan ZY, Chen HY, Li LH (2008) Nitrogen use efficiency: does a trade-off exist between the N
productivity and the mean residence time within Species? Aust J Bot 56:272–277
Zelitch I (1982) The close relationship between net photosynthesis and crop yield. Bioscience
32:796–802
Zhang FS, Römheld V, Marschner H (1991) Diurnal rhythm of release of phytosiderophores and
uptake rate of zinc in iron-deficient wheat. Soil Sci Plant Nutr 37:671–678
Zhang N, Wang M, Wang N (2002) Precision agriculture – a worldwide overview. Comput
Electron Agric 36:113–132
Zhang LX, Li SX, Zhang H, Liang ZS (2007) Nitrogen rates and water stress effects on production,
lipid peroxidation and antioxidative enzyme activities in two maize (Zea mays L.) genotypes.
J Agron Crop Sci 193:387–397
Chapter 2
Natural Variation as a Tool to Investigate
Nutrient Use Efficiency in Plants

Giorgiana Chietera and Fabien Chardon

Abstract A huge natural variation exists between individuals within a given plant
species. Most of the responses of growth-related traits to different environmental
scenarios are genotype dependent. Hence, natural variation in plants provides an
interesting and valuable source of genetic diversity to study plant responses to
environmental factors. The identification of genes that underlie phenotypic varia-
tion has an enormous practical implication by providing a means to improve crop
yield and quality. The approach based on natural variation aims to use naturally
occurring differences to improve our knowledge about complex physiological
responses of plants to their environment, including nutrition efficiency. An over-
view of different approaches currently used in plant research aimed at dissecting
complex quantitative traits is presented here, with a special focus on those related to
Nutrient Use Efficiency, to explain strategies based on QTL mapping in segregating
populations and association mapping in wild populations. Some case studies
regarding each of the investigative strategies described are detailed.

Keywords NUE (nitrogen use efficiency) • Nitrogen • Assimilation efficiency

• Remobilization efficiency • QTL • MAGIC populations • GWAS • Nutrient
limitation • Candidate genes • Arabidopsis • Natural variation

Introduction

Plants are considered adapted to variable and sub-optimal environments when they
show the ability to successfully grow and reproduce in them. In order to deal with
changing and challenging environmental conditions, plants exhibit a wide range of
integrated responses, which usually display complex quantitative variation. Plant
adaptation interests a large community of scientists, from ecologists and molecular
geneticists working on fundamental mechanisms of adaptation, to crop breeders
looking for natural variants which could optimise environmental resources and
provide targets for breeding programs (Trontin et al. 2011). The identification of
genes that underlie phenotypic variation can have enormous practical implications

G. Chietera • F. Chardon (*)

Institute Jean-Pierre Bourgin INRA, Versailles, France
e-mail: [email protected]

© Springer International Publishing Switzerland 2014 29

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_2
30 G. Chietera and F. Chardon

by providing a means to increase crop yield and quality in an agricultural context

(Bergelson and Roux 2010). Many elements in the soil serve as mineral nutrients
for plants. Among them, nitrogen (N), potassium (K), phosphorus (P) and sulfur
(S) are required in relatively large amounts for plant growth. Therefore, deficiency
in any one of these four elements in the field affects plant metabolism, and shows a
dramatic impact on yield, nutritional quality and taste as well as pathogen and pest
resistance in crops (Laegreid et al. 1999). In developed countries, plentiful amounts
of fertilisers are applied, resulting in abundant inorganic nutrients in the ecosystem
that may cause disturbance in normal bio-geochemical cycles of nutrients. How-
ever, this trend is being challenged by the current emphasis on developing more
efficient cultivars for sustainable, low-input agriculture, fuelled by increasing cost
of fertilisers, restrictions to minimise environmental impact and the increased use
of poor quality land (Rengel and Damon 2008). Some plant species and genotypes
within species have a capacity to grow and yield well on soils with a low level of
available nutrients; these species and genotypes are considered as tolerant to
nutrient deficiency and with high nutrient use efficiency (NUE; Good et al. 2004).
The NUE can be described as the proportion of potential yield that can be achieved
under that mineral deficiency availability. It is the product of nutrient uptake
efficiency (NUpE) and nutrient utilisation efficiency (NUtE), which is the optimal
combination between nutrient assimilation efficiency (NAE) and nutrient
remobilisation efficiency (NRE) (Masclaux-Daubresse et al. 2010). Improving
each crop individually requires a global knowledge of the different mechanisms
that control all the steps involved in nutrient management in plants, and also a good
knowledge of the specificities of each plant species in terms of metabolism
(Chardon 2012).

Investigation of Natural Variation in Plants Reveals

Different Strategies of Response to Nutrient Limitation

A huge natural variation exists between individuals within a given plant species,
affecting for example the colour or the shape of leaves, grain composition, seed
dormancy, flowering date or maturity date. These variations could have an impor-
tant impact on yield or quality of their products in crops, fruit trees and forestry
management species (Saisho et al. 2011; Arikita et al. 2013; Eduardo et al. 2010;
Łata et al. 2005; Robinson et al. 2012). Some developmental traits, such as
flowering time or seed dormancy, have drawn particular attention, partly because
they are of applied interest to crop breeding, and partly because they are easy to
investigate (Shindo et al. 2007). Natural variation involves not only the morphology
of plants but also their behaviour when facing contrasted environments. The
responses or growth-related traits to different environmental scenarios are genotype
dependent. Hence, natural variation in plants provides an interesting and valuable
source of genetic diversity to study plant responses to environmental factors. The
plant’s capacity to adapt to environmental constraints is called plant plasticity. It
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 31

reflects both plant age and adaptation to environment (e.g. light intensity, photo-
period, temperature and nutrient availability). The approach based on natural
variation aims to use naturally occurring differences to improve our knowledge
about complex physiological responses of plants to their environment, including
nutrition efficiency.
Plant morphology and physiology are complex quantitative traits, implying that
they are genetically controlled but also influenced by the environment. The mea-
surements of such traits show differences of averages between genotypes as well as
variations due to chance and technical inaccuracies. Therefore, the investigation of
such complex traits needs statistical tools for taking into account fluctuations due to
chance in order to correctly understand the genetics behind them.
Investigation of natural variation is of interest from two general points of view.
First, analysing this variation makes it possible to identify the function of individual
genes. Despite the fact that mutant approaches have been very powerful for
functional analysis, the small number of genetic backgrounds analysed limits the
definition of gene functions using these procedures. Ultimately, the sort of mutant
phenotypes that can be identified depends on the wild type genotype (Koornneef
et al. 2004). There are specific alleles in nature that would not be easily recognised
in mutant screens because they require very specific amino acid changes and
therefore appear at an extremely low frequency (El-Din El-Assal et al. 2001).
Second, analysis of natural variation has an increasing interest from an ecological
and evolutionary perspective since diversity of physiologies and evolved responses
in nature result from millions of generations of evolution (Ungerer et al. 2008).
While much has been learned from bringing organisms into the laboratory to study
elements of their biology in isolation, ignoring the ecological context in which these
elements arose and persist runs the risk of a suboptimal understanding of particular
biological responses and processes. Thus, the patterns of phenotypic and molecular
variation observed are analysed to elucidate the mechanisms generating and
maintaining this variation, and to identify which allelic variants are adaptive
under specific environmental conditions.
Several recent papers demonstrated that natural variation exists for the different
steps of plant nutrition: nutrient uptake and roots to shoot translocation, their
assimilation in leaves, as well as their recycling and remobilisation for seed filling.
The uptake efficiency is dependent on the root system architecture (Dunbabin
et al. 2004), and specific software have been developed for analysis (Armengaud
et al. 2009; Ristova et al. 2013; Galkovskyi et al. 2012). It has long been known that
root architecture and plasticity reveal a response of plants to scarce nutrients, and
natural variation exists for these traits in different species, related to potassium
(Kellermeier et al. 2013; Jia et al. 2008), nitrogen (De Pessemier et al. 2013), and
phosphorus availability (Wang et al. 2010). Natural variation for NUpE has been
shown directly by measuring a specific mineral content in different genotypes, as
Burns et al. demonstrated for nitrate in different varieties of lettuce. Otherwise, a
complementary approach aimed at studying the activity of enzymes involved in
mineral assimilation in roots can be used. For example, Blair et al. (2010) identified
important differences between varieties of beans for their ability to reduce iron
32 G. Chietera and F. Chardon

when grown at various hydroponic iron concentrations, ranging from 0 to 20 μM

Fe. Interestingly, these differences were more evident in plants grown at low Fe
concentrations (iron limiting conditions) than at high iron concentrations (suffi-
ciency conditions), revealing a genotype x environment interaction. Finally, the use
of isotope labelling has been used to evaluate nutrient uptake capacity. In maize,
nitrogen-15 labelling was used to study NUpE (Coque et al. 2008), showing that
28.3 % of whole-plant nitrogen was taken up after silking, and 93 % of this post-
silking nitrogen uptake was allocated to kernels.
A similar approach could be used to analyse the roots to shoot translocation of
nutrients. As an example, in order to understand why Noccaea caerulescens has
good properties for phytoremediation, Xing et al. (2008) investigated the root-to-
shoot translocation of Cd and Zn in different genotypes. The percentages of Cd and
Zn transported to shoots within 24 h exposure varied widely among the 11 acces-
sions analysed. Interestingly, the translocation efficiency did not correlate with the
uptake for either metal, suggesting independent variation in uptake and transloca-
tion among different accessions of Noccaea.
Post-genomic studies integrating all “omics” sciences can depict precise pictures
of nutrient assimilation in plants (Hirai et al. 2004). Sulpice et al. (2013) studied the
response of 97 accessions of Arabidopis to different nitrogen and carbon condi-
tions, an important number of traits showed significant natural variation between
accessions. For example, biomass differed between the 97 accessions by 3.1-fold
and 2.8-fold in high and low nitrogen, respectively, relative to the accession with
the lowest biomass in that growth regime. The impact of low nitrogen and low
carbon differed between accessions, with some accessions showing a greater than
70 % decrease in biomass and others showing no decrease. Moreover, accessions
that maintained a relatively high biomass in low nitrogen tended to show only a
small increase in biomass in high nitrogen, whereas accessions that showed a
relatively small biomass in low nitrogen showed a large (greater than 3-fold)
increase in biomass in high nitrogen. The total nitrogen concentration in the rosette
was unrelated to the biomass difference between low and high nitrogen The
nitrogen content (mg/N per rosette) was strongly related to the response of an
accession to nitrogen; accessions that maintained biomass in low nitrogen
contained more nitrogen in the rosette than accessions that showed a large gain in
biomass in high nitrogen. These results imply that accessions differ in the extent to
which they can acquire nitrogen from low nitrogen soil and that this is far more
important for the response of biomass to nitrogen supply than changes in the
nitrogen content of the rosette.
As for uptake efficiency, natural variation for remobilisation efficiency can be
directly investigated by measuring nutrient content in the seeds. For instance, Khan
et al. (2012) revealed differences in oil content in the seeds of various Acacia
species, revealing some species as a novel source of edible vegetable fat. As for the
studies conducted on N uptake, an isotope labelling technique is suitable to inves-
tigate the remobilisation efficiency. This was shown in the work of Coque
et al. (2008), who investigated N remobilisation in maize with the aim of mapping
and characterising loci involved in the variation of quantitative traits (QTL) related
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 33

to NUpE, grain N yield, N remobilisation and post-silking N uptake. They stated

that QTLs for remobilisation mainly coincided in clusters with loci for leaf senes-
cence, underlying the role of a “stay-green” phenotype in favoring N uptake
capacity, and thus grain yield and N grain yield. Similarly, in Arabidopsis a range
of variation for Harvest Index (HI) and Nitrogen Harvest Index (NHI) has been
found when 20 accessions were cultivated with a limited or ample supply of
nitrogen (Masclaux-Daubresse and Chardon 2011). It was observed that the range
of variation among the 20 accessions was conserved between the two nitrogen
levels. Globally HI is similar at high and low nitrogen, while NHI, which defines the
resource allocation, is twice as high at low nitrogen compared to high nitrogen,
indicating that grain NUE is higher when nitrogen fertiliser is limited.
Several studies have been conducted by investigating natural variation in crop
species, which were focused mainly on yield improvement (Shewry et al. 2013) and
grain composition (Li et al. 2011), but only a few have been done evaluating the
interaction of those traits with fluctuation of nutrient content in the environment.
This kind of study requires highly controlled conditions in order to clearly elucidate
the impact of the genotype and the environment on the variance of traits. This is
why such studies have been up until now conducted mainly in Arabidopsis, as
shown in the review of Chardon et al. 2012. Variation between Arabidopsis
accessions can be explored to discover ideotypes that can match with different
crop specifications: four groups, corresponding to different agronomic indicators
such as grain yield, vegetative biomass and composition of the grains at low or high
nitrogen supply, can be defined. On the basis of their physiological performance for
nitrogen uptake and remobilisation, some Arabidopsis accessions are presented as
good models for the investigation of the agronomic performances required to fit
with crops specifications. Some other examples come from the investigations of
Arabidopsis accessions grown in limiting nutrition (Reymond et al. 2006; North
et al. 2009) or complete nitrogen starvation (Richard-Molard et al. 2008; Ikram
et al. 2012). The aim of these studies was to investigate the extent of variation of
growth responses to nitrate limitation and starvation in Arabidopsis to identify
accessions showing contrasted responses and eventually different growth adaptive
strategies.

Uncovering Genes Involved in NUE Quantitative Loci by

QTL Mapping in Segregating Populations

Principles of QTL

The establishment of the genetic basis of quantitative traits is commonly referred to

as quantitative trait locus (QTL) mapping and has been hampered by their
multigenic inheritance and the often strong interaction with the environment. The
principle of QTL mapping in segregating populations is based on the genotyping of
34 G. Chietera and F. Chardon

progenies derived from a cross of distinct genotypes for the trait under study.
Phenotypic values for the quantitative traits are then compared with the molecular
marker genotypes of the progeny to search for particular genomic regions showing
statistically significant associations between polymorphism and the trait variation,
which are then called QTL. QTL analysis makes use of the natural variation present
within species. Once genetic variation is found among accessions, the aim is to
identify how many loci account for it and where they are located in the genome
(Koornneef et al. 2004).
Identifying the number and genome position of the segregating QTL in an
experimental population requires the following steps: (a) the generation of an
experimental mapping population; (b) its genotyping with markers throughout the
genome and the phenotyping for the trait of interest; (c) the association analysis
between phenotypic values of the trait and genotypic classes of the polymorphic
markers. Thus, the number and genetic position of loci that control the trait
variation in that population, their relative additive effect, the contribution of genetic
interactions between loci (epistasis) and the mode of action of each QTL (domi-
nance effects) are calculated depending on the population type (Koornneef
et al. 2004). The number of loci identified per analysis varies from 1 to >10,
depending on the complexity of the genetic variation under study, including
parameters such as the true number of loci segregating, the relative additive effect
of each QTL, and the effect of genetic interactions. In addition, this number
depends on the heritability of the trait in the assay performed, i.e. the control of
the environmental uniformity, the quality and density of genotypic data, the statis-
tical method used to map QTL, and the size of the mapping population.

Mapping Populations

In plants, the use of “immortal” mapping populations consisting of homozygous

individuals is preferred because it allows performance of replications and multiple
analyses of the same population. Such populations known as recombinant inbred
lines (RILs) or introgression lines (ILs), also referred to as near isogenic lines
(NILs), are practically homozygous and therefore phenotypic values can be based
on multiple replicates, reducing the environmental effects and increasing the power
to detect QTL. They can be analysed in multiple environments without the need for
further genotyping, and thus, the effects of each QTL in different environments can
be precisely estimated and tested for QTL
environment interactions (Koornneef
et al. 2004). Homozygous populations can be obtained by repeated selfing, as for
RILs, but also by induced chromosomal doubling of haploids. In contrast, NILs
consist of lines containing a single fragment or a small number of genomic
introgression fragments from a donor parent into an otherwise homogeneous
genetic background, which increases the power to detect a small-effect QTL. In
plants, RILs and NILs are the most common types of experimental populations used
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 35

for the analysis of quantitative traits. In both cases the accuracy of QTL
localisation, referred to as mapping resolution, depends on population size. The
mapping of QTL in segregating populations has limited resolution since loci
associated with the expression of a quantitative trait can be mapped with a precision
of about 5–20 cM depending on its relative effect and the quality of the QTL
mapping assay (Keurentjes et al. 2007). The choice of one mapping population over
another depends on the plant species and the specific parents of interest. In cases
where different cultivars or wild accessions are studied, preference is often given to
RILs. However, when different species or wild and cultivated germplasm are
combined, NILs are preferred. In Arabidopsis for example, the ease with which
fertile RIL populations with complete genome coverage can be generated, due to its
fast generation time, has led to their extensive use in mapping quantitative traits. An
overview of the steps undertaken to generate a set of RILs is represented in Fig. 2.1.
The population derived from a European accession and a genetically distant one
from central Asia, Bay-0 and Shahdara, is an example of a novel RIL population
suitable for the investigation of traits such as the response to nitrogen availability,
root architecture, seed germination, drought tolerance and virus resistance (Loudet
et al. 2002). The phenotypic variation resulting from such a cross is expected to
reflect the adaptation to the specific habitat and the genetic distance between the
parental accessions. The first extensive study of N metabolism in Arabidopsis using
QTL mapping was conducted by Loudet on 415 RILs derived from
Bay-0
Shahdara population (Loudet et al. 2003) to describe whole plant N
physiology and growth at a vegetative stage. The study, conducted in controlled
growth conditions, aimed at comparing two different N environments (10 mM and
3 mM nitrate) and identified several loci explaining the variability of growth and
total N, nitrate, and free amino acid contents.
Other approaches involve the use of multiple parents, as in the multiple
advanced generation intercross (MAGIC) and Arabidopsis multiparent RIL
(AMPRIL) populations (Kover et al. 2009; Huang et al. 2011). The MAGIC design
is more elaborate and generates more recombination events per line than the
AMPRIL strategy, but the founder genomes are less evenly represented in the
final lines. Mapping in either population is more complex than with RILs, but
with a sufficiently high density of intermediate frequency markers, one can infer the
most likely local founder genotype. Some of the advantages of using RIL-type
populations will continue to apply in the future.
Maize is the crop species, which has traditionally been involved in QTL map-
ping, and numerous QTL studies for NUE are now available. Zhang et al. (2010)
has published recently a study on QTL mapping for several enzyme activities. They
detected 73 QTLs for the activity of 10 enzymes involved in carbon and nitrogen
metabolism and eight QTLs for biomass in an intermating RIL population devel-
oped by randomly intermating plants for four generations following the F2, prior to
the derivation of mapping progeny. A RIL population of rice has also been tested
for tolerance to salinity, measuring the amount of Na+ and K+ ions in shoots
and roots in three environmental conditions of 0, 100 and 120 mM NaCl
36 G. Chietera and F. Chardon

Fig. 2.1 Generation of RILs by successive selfings: two parental lines are crossed to produce an
F1. The F1 is then selfed to obtain an F2. The selfing process continues until a certain level of
homozygosity is reached. The end product consists of a set of RILs, each of wich is a fixed
recombinant of the parental lines. HIFs individuals are derived from RILs in wich a small portion
of the genome is still heterozygous (shown in red rectangle). Selfing such a RIL, it is possible to
obtain individuals fixed for parental’s 1 or parental’s 2 allele. Only one chromosome pair is shown
for each individual

(Wang et al. 2012). It is known that plants, which tolerate salinity are able to
maintain a flux of Na+ between shoots and roots in order to keep the ratio Na+/K+ as
low as possible. The authors identified several major QTLs for salt tolerance and
one of them, named qSNC11, will be suitable for use in marker-assisted selection
when developing new salinity-tolerant cultivars.
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 37

QTL Fine Mapping

NILs have been used in various studies to confirm and fine map QTLs previously
mapped in RIL populations (Koornneef and Smeekens 2005; Edwards et al. 2005)
for which heterogeneous inbred families (HIFs) have also been used (Tuinstra
et al. 1997). To construct an HIF-type NIL, a RIL is chosen that is still heterozygous
around the QTL of interest but homozygous elsewhere. In fact, even after six
generations of inbreeding, which is customary for RILs, a small percentage of the
genome remains heterozygous. This RIL is then selfed and genotyped so that each
homozygous genotype at the region of interest can be identified and studied in
detail. HIFs should not to be compared with the reference parental genotype, but
with one another within the descendants (family) of the chosen RIL. In contrast to
“conventional” NILs, the genetic background of an HIF line is not homogeneous,
but a mix of both parental genomes since these lines originate from one RIL of the
population. The ease with which NILs can be extracted from a large population of
HIFs may also allow a QTL mapped at low significance thresholds to be confirmed
by subsequent examination of NILs. Second, NILs extracted from segregating HIFs
are useful for the fine mapping of QTLs. Each segregating HIF is independent and
contains unique recombination events in genomic regions flanking the QTL. An
example of fine mapping using HIFs comes from the investigation of the four loci
identified in the Arabidopsis population Bay-0
Shahdara in low and high nitrogen
environments by Loudet et al. (2003). The production of homogeneous plant
material for a large number of lines was certainly the most challenging and limiting
step of this work, but the authors also remarked that uncontrolled environmental
effects can affect the evaluation of the quantitative traits and that environmental
heterogeneity can occur between two different cultivation repetitions (even in the
same growth chamber), as well as within one single growth chamber during a
repetition. For these reasons, it is recommended (a) to always study all of the lines
in the same cultivation repetition and (b) to compare different N environments in
the same cultivation repetition. One of the limitations of HIF analysis for the
evaluation of QTL is that the genetic background of NILs derived from HIFs are
unique and cannot be easily replicated. NILs are not easily developed for evaluating
the effects of more than one QTL in a single genetic background or for comparing
the effects of QTL identified in different populations.

Candidate Genes

Co-localisation with candidate genes sometimes allows rapid molecular identifica-

tion of QTL. This was the case in a study by Loudet et al. (2007) where QTL
mapping for sulfate content in Arabidopsis leaves resulted in the identification of
several minor QTLs and one major QTL on chromosome 1 in two contrasted N
levels (3 mM and 10 mM). The major QTL co-localised with the gene APR2 coding
38 G. Chietera and F. Chardon

for the adenosine-50 -phosphosulfate reductase, a key enzyme for assimilatory

sulfate reduction pathway. The authors showed that the difference in sulfate
contents between the two parental lines was not due to a different expression of
the gene in the two genotypes, but to a change of alanine into glutamic acid in the
APR2 protein. However, it is possible for there to be no overlap between the QTL
and the candidate genes, as happened in the study on common bean conducted by
Blair et al. (2010). A QTL for iron reductase activity in roots was identified under
iron sufficiency (15 μg Fe), but it was mapped on a different chromosome from the
one found under iron-limited growth (1 μM). Therefore, it was postulated that iron
reductase activity was influenced by more than one locus, with a first iron
reductase-related locus on chromosome b02, which contributed to the trait in
iron-limited plants, and a second iron reductase-related locus present on chromo-
some b11 contributing in iron-sufficient plants. The authors also mapped loci for
the FRO genes (iron-reductase homologues) but since there is no co-localisation
with the QTL they concluded that some other gene may control iron reductase
activity. Another resource for finding candidate genes is analysing gene expression
in the vicinity of the QTL. This can be done using standard assays for a limited
number of candidate genes, or using high-throughput genome-wide techniques,
such as microarrays (Borevitz and Nordborg 2003). When the functional allelic
variation results in gene expression differences, this may clearly indicate the
candidate gene. Identifying an artificially induced mutant showing phenotypic
effect in the trait of interest provides a unique functional argument to select a
candidate gene. The availability of T-DNA insertion mutants for almost any
Arabidopsis gene and the efficiency of TILLING (Targeting Induced Local Lesions
in Genomes) procedures to identify mutations in numerous candidate genes provide
efficient strategies to analyse knock-out phenotypes of (nearly) all genes in a QTL
region. Nevertheless, most collections of mutants are in the laboratory backgrounds
Ler (Landsberg erecta) and Col (Columbia), which do not necessarily carry func-
tional alleles at the gene of interest and, consequently, will not always show a
distinct phenotype when mutated. Therefore, loss-of-function mutants of particular
lines, such as NILs, carrying alleles different from the common laboratory acces-
sions, can also be induced by mutagenesis with standard chemical or physical
agents. This approach is especially useful when identifying novel alleles that are
dominant over laboratory backgrounds (Koornneef et al. 2004). Ultimately, the
proof for the identification of a QTL gene should come from complementation
experiments by plant transformation.

QTL Validation

The presence of a QTL is validated when allelic variation of that QTL area has an
effect on the studied trait. The search for polymorphic region-specific markers is
crucial to fine map a QTL. NILs and HIFs are screened with molecular markers that
are contrasted at the target region in parents. Finally, combining the analysis of a
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 39

large number of segregating recombinants and the use of new polymorphic markers
in the QTL area makes it possible to define a candidate region of less than 50 kb
which will contain about 10 ORFs (open reading frames). If the region size is not
sufficiently small for further analysis, new screening of rHIFs (possible
recombinants within a heterozygous region of a QTL) is performed. When the
desired region size contains only few genes, the QTL is considered as fine mapped
and genes within it are examined for potential clues (Ikram and Chardon 2010). In
addition to fine mapping, several functional strategies are available for plants whose
complete genome sequence is available in order to select relevant candidate genes
for the QTL. The knowledge of the complete genome sequence allows the search of
such candidates on the bases of the predicted gene functions. Nevertheless, the
function of many ORFs remains unknown at the cellular and/or phenotypic level
and, therefore, it is not always possible to find obvious candidates from the genome
sequence (Koornneef et al. 2004).
QTL cloning is a very efficient way to verify a new gene without a priori. For
instance, Calenge et al. (2006) were interested in two genotypes, which accumu-
lated soluble sugars at different rates in 10 and 3 mM nitrate nutrition. QTL analysis
resulted in a major QTL for fructose content in leaves. The fine mapping restricted
the QTL area to a 3 kb interval enclosing the single gene SWEET17 (Chardon
et al. 2013). The authors showed by functional analysis that variation in fructose
content is uncoupled from further metabolic pathways, which result from seques-
tration of fructose into the vacuole, the main compartment for soluble sugars. They
demonstrated that SWEET17 is a new vacuolar transporter of fructose in plant.
In contrast, simply resequencing a region with dozens or more genes is, on its
own, not generally informative because of the high number of polymorphisms that
distinguish an arbitrary pair of accessions, about 1 in every 200 bp (Weigel 2012).
Fortunately, compared to other multicellular organisms in which natural variation is
studied, Arabidopsis has the enormous advantage that almost all accessions are
quite easily transformed by dipping flowering plants into a suspension of
Agrobacterium tumefaciens containing a T-DNA vector with the transgene of
interest.

QTL Meta-analysis

With data on multiple populations, it useful to know whether QTL identified for a
given trait in one population correspond to those detected in other populations, or
whether QTL locations identified in one species correspond to QTL or other types
of loci detected in corresponding regions in other plant species. With this aim, a
method has been developed by Goffinet and Gerber (2000) to estimate the mini-
mum number of loci giving the observed QTL in individual studies and to combine
the available information to precisely give the position of each individual QTL.
Such an approach is called ‘meta-analysis’ and its usefulness is to pool information
when raw data are not available. Comparative analysis of QTL between species
40 G. Chietera and F. Chardon

reveals the existence of homologous QTL for traits involved in domestication, such
as plant height and maturity, as well as tolerance to abiotic stress, within the cereals
(Chardon et al. 2005; Hanocq et al. 2007; Li et al. 2013; Swamy et al. 2011). A
recent example of meta-QTL analysis of a nutrition use efficiency-related trait
comes from the dissection of an ortho-metaQTL in bread wheat by Quraishi
et al. (2011). The authors identified a major NUE ortho-metaQTL conserved at
orthologous positions in wheat, rice, sorghum and maize. Starting from three
independent studies reporting QTL detection for traits related to NUE components
in wheat, the authors proposed that a glutamate synthase (GoGAT) gene is con-
served structurally and functionally at orthologous positions in rice, sorghum and
maize genomes, and it that may contribute to NUE in wheat and other cereals.

Exploiting Genetic Variation in Wild Populations to Reveal

NUE Genes by Association Mapping

First Results Obtained on Arabidopsis to Reveal NUE Genes

Over the past 10 years, traditional QTL mapping has led to the identification of
sequence variants that modulate a range of physiological and developmental traits.
Prior knowledge of the biological function of the affected genes was often helpful
in identifying them, but increasingly the responsible locus is found to encode a
protein without known biochemical function (Lempe et al. 2005). Apart from
alleles that alter expression levels or protein function, a surprising number of drastic
mutations such as deletions and stop codons underlie phenotypic variation. Some of
these changes are found in many accessions (Weigel and Mott 2009) suggesting
that they are adaptive. Nevertheless, despite some success stories, the number of
known alleles responsible for phenotypic variation among accessions remains
limited, mostly because fine mapping and dissection of QTLs are time consuming.
Arabidopsis thaliana was the first plant species for which a genome sequence
became available (Arabidopsis Initiative 2000). This initial sequence was from a
single, high quality, inbred strain (accession) with each chromosome represented by
only two contigs, one for each arm. In addition to functional analyses, the 120 Mb
reference sequence of the Columbia (Col-0) accession proved to be a boon for
evolutionary and ecological researches. A particular advantage in this respect is that
the species is mostly self-fertilising, and most strains collected from the wild are
homozygous throughout the genome (Weigel and Mott 2009). This distinguishes
Arabidopsis from other model organisms such as the mouse or the fruit fly. In these
systems, inbred strains have been derived, but they do not represent any individual
actually found in nature. Numerous plants genomes have been completely
sequenced and released recently.
Natural Arabidopsis accessions show tremendous genetic and phenotypic diver-
sity. Thus far, significant natural variation has been reported for every phenotypic
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 41

trait investigated (Koornneef et al. 2004). Moreover, assays of metabolite profiles

by large-scale unbiased metabolomics methods have uncovered natural variation at
the level of small molecules, suggesting that they reflect physiological phenotypes
that could be under selection in nature (Keurentjes et al. 2006). Efforts to accelerate
the discovery of functionally important variants began with a large-scale study in
which some 1,000 fragments across the genomes of 96 accessions of Arabidopsis
thaliana gathered from all over the world were compared by dideoxy sequencing
(Rosenberg et al. 2005). A major conclusion from this work was that there has been
considerable global gene flow, so that most sequence variants are found worldwide,
although genotypes are not entirely random. There is isolation by distance, and even
though population structure (which is a division of the population into distinct
subgroups related by kinship) is relatively moderate, it can easily be a confounding
factor in association studies. From this first set of 96 strains, 20 maximally diverse
strains were chosen for much denser polymorphism discovery using array-based
resequencing (Clark et al. 2007). This led to the identification of approximately one
single nucleotide polymorphism (SNP) for every 200 base pairs of the genome,
constituting one quarter or so of all SNPs estimated to be present. In addition,
regions that are missing or highly divergent in at least one accession encompass
about a quarter of the reference genome. For this reason it is becoming increasingly
clear that it is inappropriate to think about ‘the’ genome of a species, even though
this is what the initial sequencing papers stated in their titles just a few years ago
(Weigel and Mott 2009). The previous emphasis on relatively minor changes
between individuals, such as SNPs, was largely due to the fact that sequence
variation had overwhelmingly been studied by PCR-based methods or hybridisation
to known sequences. It is now known that Arabidopsis accessions can vary in
hundreds of genes. Of particular importance is the observation that some genes
with fundamental effects on life history traits such as flowering are not even
functional in their reference accession, and thus could not have been discovered
on the basis of the first genome sequence alone. Whilst knowledge about the origin
and phenotypic effects of sequence polymorphisms is central to understanding how
species adapt to their natural environment, most studies of genetic variation in
Arabidopsis have probably been motivated by the desire to identify regulatory and
other genes that are not present in the common laboratory accessions (Weigel
2012). A project begun in 2009 aimed to sequence the genome of 1001 accessions
of A. thaliana (Weigel and Mott 2009), and the task is almost complete now. The
main motivation for the 1001 Genomes project is, however, to enable genome-wide
association studies (GWA) in this species. The seeds from the 1001 accessions are
freely available from the Arabidopsis stock centres and each accession can be
grown and phenotyped by scientists from all over the world (Weigel and Mott
2009). Importantly, because an unlimited supply of genetically identical individuals
will be available for each accession, even subtle phenotypes and ones that are
highly sensitive to the microenvironment, which is often difficult to control, can be
measured with a high degree of confidence. The phenotypes can include morpho-
logical analyses, such as plant stature, growth and flowering; investigations of plant
content, such as metabolites and ions; responses to the abiotic environment, such as
42 G. Chietera and F. Chardon

resistance to drought or salt stress and to N deficiency; or resistance to disease

caused by a host of prokaryotic and eukaryotic pathogens, from microbes to insects
and nematodes.

Candidate Gene Association and Genome-Wide Association

Studies

An explanation of how association mapping refers to the analysis of statistical

association between genotypes (usually individual SNPs or SNP haplotypes, deter-
mined in a collection of individuals), and the phenotypes (traits) of the same
individuals is undertaken here. Until recently, genetic mapping was usually done
in purpose-created populations, such as progeny of parents chosen on the basis of
the difference between them for the trait(s) of interest, or in defined pedigrees
(families) (Rafalski 2010). By contrast, genetic association mapping involves using
a collection of individuals, such as those derived from wild populations, germplasm
collections or subsets of breeding germplasm. Consequently, at each locus several
alleles may be simultaneously evaluated for association in a diverse population,
while only two alleles segregate in any biparental population. Two association
mapping methodologies are in use: Candidate Gene Association and Whole
Genome Scan, also called Genome-Wide Association Study. In the candidate
gene approach, the hypothesis that there is a correlation between DNA polymor-
phisms in gene A and the trait of interest is tested. For example, it is possible to test
if in a diverse germplasm collection there is a correlation between DNA sequence
alleles of phytoene synthase (or any other gene involved in carotenoid biosynthesis)
and carotenoid content of seeds (Palaisa et al. 2003; Pozniak et al. 2007). This
approach assumes good understanding of the biochemistry and genetics of the trait,
but many genes may escape attention. Therefore, in the absence of detailed knowl-
edge of the biochemical pathway of interest, including regulatory genes, whole
genome scan (described below) is a better choice (Rafalski 2010). Genome scan
involves testing most of the segments of the genome for association by genotyping
densely distributed genetic marker loci over all chromosomes. The simple hypoth-
esis that one of the genetic loci being considered is either causal for the trait or in
linkage disequilibrium (LD, defined as association between genetic loci) with the
causal locus is under consideration. The choice of population for association
mapping, and of the appropriate marker density, are crucial decisions. One of the
sources of false positives in association mapping is population structure. Complex
population structure could be expected in crop species that were subject to a severe
domestication bottlenecks followed by breeders’ selection. Pronounced differences
in the germplasm used in different regions of the world and maturity-related sets of
allele frequencies for many genes may also be expected. Examples include the
division of maize germplasm into heterotic groups (Reif et al. 2005) and a severe
post-domestication bottleneck associated with adoption of soybean in North
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 43

America (Hyten et al. 2006). Before choosing the appropriate number of genetic
markers (usually SNPs) for a genome scan, it is necessary to have some under-
standing of the LD in the population selected for the study. In general, LD decreases
with distance between marker loci, more slowly in inbreds (soybean), faster in
outbred species (maize), although breeding practices have a large impact (Flint-
Garcia et al. 2003). LD is, however, very non-uniform across the genome, with both
general trends (more LD in centromeric regions) and pronounced local fluctuation
(Rafalski 2010). For instance, LD in Arabidopsis extends for roughly 10 kb, which
is a nearly ideal distance for mapping since it extends up to the gene level
(Bergelson and Roux 2010). Genetic resolution of any mapping methodology
ultimately depends on the amount of recombination available in the experimental
population, as measured by the rate of decay of LD (Rafalski 2010). In collections
of distantly related individuals many generations have passed and much recombi-
nation occurred since the last common ancestor, therefore resolution of association
mapping will, in general, be considerably higher than in simple biparental
populations. The power of association mapping is strongly dependent upon the
quality of phenotypic data. It is important to stress that in most cases it is necessary
to use well-controlled environmental conditions, including, when possible, use of
growth chambers, especially for the collection of samples for metabolomic or
biochemical phenotypes. Relevance of such phenotypes for field performance
will have to be separately established (Rafalski 2010). High throughput methods
to precision phenotyping, frequently referred to as phenomics, are developing
rapidly and automated facilities for high precision phenotyping are being
established (E. Finkel 2009). The use of such a facility has been recently presented
as a powerful tool to investigate plant response to drought stress by Tisné
et al. (2013), allowing the precise control of watering condition of more than
700 plants. In this way, the environmental variance was strongly reduced allowing
the identification of QTLs for complex traits related to drought response.

Validation and Applications

Validation of the hypotheses generated by association mapping constitutes an

integral part of the experiment (Rafalski 2010). In one approach, NILs differing
in the alleles at the candidate locus are constructed by repeated backcrossing into a
reference genetic background (Vlad et al. 2010). The resulting NILs are then
phenotyped side by side, and the amount of phenotypic variation ascribed to the
presence of introgressed segment is estimated. Biparental populations segregating
for the relevant alleles at the associated locus may also be used (Beló et al. 2008).
Alternatively, the association experiment could be expanded by the inclusion of
additional individuals in the expectation that the strength of the association should
improve if the association hypothesis is correct. Association mapping is usually
performed with the objective of applying the results for genotype-based selection of
superior individuals in plant breeding, or as a step toward positional cloning
44 G. Chietera and F. Chardon

(Rafalski 2010). In marker assisted recurrent selection, breeders identify desirable

alleles at one or more loci, basing on the outcome of a mapping experiment, and
then use closely linked genetic markers for selecting individuals in breeding
populations (Collard and Mackill 2008; Ribaut et al. 2010). This approach results
in fixing the desirable allele(s) in the population(s) of interest.

Limitations

The detection power of association mapping greatly depends not only on the
magnitude of the effect that can be ascribed to a locus, relative to other loci present
in the population, but also on the allele frequency distribution. Rare alleles cannot
be detected with good confidence, unless their effect is very large. Therefore,
segregating biparental populations are more appropriate for the mapping of alleles
rare in the germplasm pool of interest (Rafalski 2010). Genetic association mapping
enriches the repertoire of tools available for the dissection of trait architecture in
crop plants and model species. As high-density genotyping becomes increasingly
accessible, this approach will gain power to identify with high-resolution genetic
loci and in some cases causal polymorphism affecting agronomic and end-use traits
in crop plants, as long as relevant alleles are present at high frequency. Mapping in
defined biparental populations will remain the method of choice for rare alleles,
especially those with moderate effects, and for the study of epistatic interactions.
Independent validation of the associations found by both approach and evaluation
of their effects in different genetic backgrounds remains an essential, even though
sometimes neglected, aspect of a genetic experiment. Improvement in phenotyping
remains a major challenge for mapping many agronomical important traits such as
NUE or drought tolerance.

Future Perspectives

In addition to visually obvious phenotypes, natural variation has also been observed
in genetic mechanisms such as cytosine methylation (Riddle and Richards 2002). A
recent interest concerns the exploitation of natural variation in gene expression,
leading to the first studies using expression QTL mapping (eQTL) proposed as a
valuable approach to dissect the genetic basis of transcript variation, one of the
prime causes of natural phenotypic variation. A recent study using eQTL conducted
on 191-individual pseudo-F1 progeny of grape to dissect the genetic basis of berry
colour formation (Huang et al. 2013), led to the identification of two major QTL
explaining 20 % of genotypic variance and co-locating with a key enzyme for
anthocyanin synthesis. With available genomic tools such as the whole genome
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 45

Fig. 2.2 A scheme of two possible strategies to be followed in the investigation of nutrient use
efficiency related traits using natural variation in plants

sequence of many species, further investigation through genome-wide eQTL stud-

ies should bring a valuable contribution to understanding the molecular basis of
traits of interest and should offer the opportunity for knowledge transfer to horti-
cultural plants. In Fig. 2.2, a graphical overview of the possible strategies to
investigate plant mineral use efficiency is displayed.
46 G. Chietera and F. Chardon

Conclusions
In light of scarce resources, increasing fertiliser production costs and the
demand for greater crop production, the development of nutrient-efficient
varieties is increasingly important. Both nutrient uptake and metabolic path-
ways are under the control of a complex regulatory network involving many
genes. The identification of large-effect QTL/genes is therefore a challenge
(Vinod and Heuer 2012). With the experiences gained in QTL mapping and
the rapid development of genome-sequencing and molecular-marker technol-
ogies, more high-impact, large-effect QTL will surely be identified in the
future. These efforts require expertise in different disciplines and, therefore,
modern breeding is being implemented more and more in multidisciplinary
teams involving breeders, physiologists and molecular biologists/geneticists.
With the advances in molecular breeding technologies, breeders now have
access to genes from wild species and unadapted genotypes that are difficult
to use in breeding programmes due to crossing barriers and their poor
agronomic performance. Molecular breeding therefore provides an exciting
opportunity to use these gene pools effectively for the development of
well-adapted and nutrient-efficient plants.

References

Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant
Arabidopsis thaliana. Nature 408:796–815
Arikita FN, Azevedo MS, Scotton DC, Pinto MDS, Figueira A, Peres LEP (2013) Novel natural
genetic variation controlling the competence to form adventitious roots and shoots from the
tomato wild relative Solanum pennellii. Plant Sci 199/200:121–130
Armengaud P, Zambaux K, Hills A, Sulpice R, Pattison RJ, Blatt MR, Amtmann A (2009)
EZ-Rhizo: integrated software for the fast and accurate measurement of root system architec-
ture. Plant J 57:945–956
Beló A, Zheng P, Luck S, Shen B, Meyer DJ, Li B, Tingey S, Rafalski A (2008) Whole genome
scan detects an allelic variant of fad2 associated with increased oleic acid levels in maize. Mol
Genet Genomics 279:1–10
Bergelson J, Roux F (2010) Towards identifying genes underlying ecologically relevant traits in
Arabidopsis thaliana. Nat Rev Genet 11:867–879
Blair MW, Knewtson SJ, Astudillo C, Li CM, Fernandez AC, Grusak MA (2010) Variation and
inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L.) and
association with seed iron accumulation QTL. BMC Plant Biol 10:215
Borevitz JO, Nordborg M (2003) Update on genomics and natural variation in Arabidopsis. The
impact of genomics on the study of natural variation in Arabidopsis. Plant Physiol 132:718–
725
Calenge F, Saliba-Colombani V, Mahieu S, Loudet O, Daniel-Vedele F, Krapp A (2006) Natural
variation for carbohydrate content in Arabidopsis. Interaction with complex traits dissected by
quantitative genetics. Plant Physiol 141(4):1630–1643
Chardon F (2012) Exploring NUE in crops and in Arabidopsis ideotypes to improve yield and seed
quality. J Exp Bot 63:3401–3412
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 47

Chardon F, Hourcade D, Combes V (2005) Mapping of a spontaneous mutation for early flowering
time in maize highlights contrasting allelic series at two-linked QTL on chromosome 8. Theor
Appl Genet 112:1–11
Chardon F, Noël V, Masclaux-Daubresse C (2012) Exploring NUE in crops and in Arabidopsis
ideotypes to improve yield and seed quality. J Exp Bot 63(9):3401–3412. doi:10.1093/jxb/
err353
Chardon F, Bedu M, Calenge F, Klemens PAW, Spinner L, Clement G, Chietera G, Léran S,
Ferrand M, Lacombe B, Loudet O, Dinant S, Bellini C, Neuhaus HE, Daniel-Vedele F, Krapp
A (2013) Leaf fructose content is controlled by the vacuolar transporter SWEET17 in
Arabidopsis. Curr Biol 23:697–702
Clark RM, Schweikert G, Toomajian C, Ossowski S, Zeller G, Shinn P, Warthmann N, Hu TT,
Fu G, Hinds DA, Chen H, Frazer KA, Huson DH, Schölkopf B, Nordborg M, Rätsch G, Ecker
JR, Weigel D (2007) Common sequence polymorphisms shaping genetic diversity in
Arabidopsis thaliana. Science 317:338–342
Collard BCY, Mackill DJ (2008) Marker-assisted selection: an approach for precision plant
breeding in the twenty-first century. Philos Trans R Soc Lond B Biol Sci 363:557–572
Coque M, Martin A, Veyrieras JB, Hirel B, Gallais A (2008) Genetic variation for
N-remobilization and postsilking N-uptake in a set of maize recombinant inbred lines.
3. QTL detection and coincidences. Theor Appl Genet 117:729–747
De Pessemier J, Chardon F, Juraniec M, Delaplace P, Hermans C (2013) Natural variation of the
root morphological response to nitrate supply in Arabidopsis thaliana. Mech Dev 130:45–53
Dunbabin V, Rengel Z, Diggle AJ (2004) Simulating form and function of root systems: efficiency
of nitrate uptake is dependent on root system architecture and the spatial and temporal
variability of nitrate supply. Funct Ecol 18:204–211
Eduardo I, Chietera G, Bassi D, Rossini L, Vecchietti A (2010) Identification of key odor volatile
compounds in the essential oil of nine peach accessions. J Sci Food Agric 90:1146–1154
Edwards KD, Lynn JR, Gyula P, Nagy F, Millar AJ (2005) Natural allelic variation in the
temperature-compensation mechanisms of the Arabidopsis thaliana circadian clock. Genetics
170:387–400
El-Din El-Assal S, Alonso-Blanco C, Peeters AJ, Raz V, Koornneef M (2001) A QTL for
flowering time in Arabidopsis reveals a novel allele of CRY2. Nat Genet 29:435–440
Finkel E (2009) With ‘phenomics’ plant scientists hope to shift breeding into overdrive. Science
325:380–381
Flint-Garcia SA, Thornsberry JM, Buckler ES (2003) Structure of linkage disequilibrium in plants.
Annu Rev Plant Biol 54:357–374
Galkovskyi T, Mileyko Y, Bucksch A, Moore B, Symonova O, Price C, Topp CN, Iyer-Pascuzzi
AS, Zurek PR, Fang S, Harer J, Benfey PN, Weitz JS (2012) GiA roots: software for the high
throughput analysis of plant root system architecture. BMC Plant Biol 12:116
Goffinet B, Gerber S (2000) Quantitative trait loci: a meta-analysis. Genetics 155(1):463–473
Good AG, Shrawat AK, Muench DG (2004) Can less yield more? Is reducing nutrient input into
the environment compatible with maintaining crop production? Trends Plant Sci 9:597–605
Hanocq E, Laperche A, Jaminon O, Lainé AL, Le Gouis J (2007) Most significant genome regions
involved in the control of earliness traits in bread wheat, as revealed by QTL meta-analysis.
Theor Appl Genetic 114:569–584
Hirai MY, Yano M, Goodenowe DB, Kanaya S, Kimura T, Awazuhara M, Arita M, Fujiwara T,
Saito K (2004) Integration of transcriptomics and metabolomics for understanding of global
responses to nutritional stresses in Arabidopsis thaliana. Proc Natl Acad Sci U S A 101:10205–
10210
Huang X, Paulo MJ, Boer M, Effgen S, Keizer P, Koornneef M, van Eeuwijk FA (2011) Analysis
of natural allelic variation in Arabidopsis using a multiparent recombinant inbred line popu-
lation. Proc Natl Acad Sci U S A 108:4488–4493
48 G. Chietera and F. Chardon

Huang YF, Bertrand Y, Guiraud JL, Vialet S, Launay A, Cheynier V, Terrier N, This P (2013)
Expression QTL mapping in grapevine-revisiting the genetic determinism of grape skin colour.
Plant Sci 207:18–24
Hyten DL, Song Q, Zhu Y, Choi IY, Nelson RL, Costa JM, Specht JE, Shoemaker RC, Cregan PB
(2006) Impacts of genetic bottlenecks on soybean genome diversity. Proc Natl Acad Sci U S A
103:16666–16671
Ikram S, Chardon F (2010) Plant quantitative traits. In: Encyclopedia of life sciences (ELS). John
Wiley, Chichester
Ikram S, Bedu M, Daniel-Vedele F, Chaillou S, Chardon F (2012) Natural variation of Arabidopsis
response to nitrogen availability. J Exp Bot 63:91–105
Jia Y, Yang X, Feng Y, Jilani G (2008) Differential response of root morphology to potassium
deficient stress among rice genotypes varying in potassium efficiency. J Zhejiang Univ Sci B
9:427–434
Kellermeier F, Chardon F, Amtmann A (2013) Natural variation of Arabidopsis root architecture
reveals complementing adaptive strategies to potassium starvation. Plant Physiol 161:1421–
1432
Keurentjes JJB, Fu J, de Vos CHR, Lommen A, Hal LRD, Bino RJ, van der Plas LHW, Jansen RC,
Vreugdenhil D, Koornneef M (2006) The genetics of plant metabolism. Nat Genet 38:842–849
Keurentjes JJB, Bentsink L, Alonso-Blanco C, Hanhart CJ, Blankestijn-De Vries H, Effgen S,
Vreugdenhil D, Koornneef M (2007) Development of a near-isogenic line population of
Arabidopsis thaliana and comparison of mapping power with a recombinant inbred line
population. Genetics 175:891–905
Khan R, Srivastava R, Khan MA, Alam P, Abdin MZ, Mahmooduzzafar (2012) Variation in oil
content and fatty acid composition of the seed oil of Acacia species collected from the
northwest zone of India. J Sci Food Agric 92:2310–2315
Koornneef M, Smeekens S (2005) Sucrose-specific induction of anthocyanin biosynthesis in
Arabidopsis requires the MYB75/PAP1 gene. Plant Physiol 139:1840–1852
Koornneef M, Alonso-Blanco C, Vreugdenhil D (2004) Naturally occurring genetic variation in
Arabidopsis thaliana. Annu Rev Plant Biol 55:141–172
Kover PX, Valdar W, Trakalo J, Scarcelli N, Ehrenreich IM, Purugganan MD, Durrant C, Mott R
(2009) A multiparent advanced generation inter-cross to fine-map quantitative traits in
Arabidopsis thaliana. PLoS Genet 5:e1000551
Laegreid M, Bockman O, Kaarstad O (1999) Agriculture, fertilizers and the environment. CABI
Publishing in association with Norsk Hydro ASA, New York
Łata B, Przeradzka M, Bińkowska M (2005) Great differences in antioxidant properties exist
between 56 apple cultivars and vegetation seasons. J Agric Food Chem 53:8970–8978
Lempe J, Balasubramanian S, Sureshkumar S, Singh A, Schmid M, Weigel D (2005) Diversity of
flowering responses in wild Arabidopsis thaliana strains. PLoS Genet 1:109–118
Li Y, Fan C, Xing Y, Jiang Y, Luo L, Sun L, Shao D, Xu C, Li X, Xiao J, He Y, Zhang Q (2011)
Natural variation in GS5 plays an important role in regulating grain size and yield in rice. Nat
Genet 43:1266–1269
Li WT, Liu CJ, Liu YX, Pu ZE, Dai SF, Wang JR, Lan XJ, Zheng YL, Wei YM (2013) Meta-
analysis of QTL associated with tolerance to abiotic stresses in barley. Euphytica 189:31–49
Loudet O, Chaillou S, Camilleri C, Bouchez D, Daniel-Vedele F (2002) Bay-0
Shahdara
recombinant inbred line population: a powerful tool for the genetic dissection of complex
traits in Arabidopsis. Theor Appl Genet 104:1173–1184
Loudet O, Chaillou S, Merigout P (2003) Quantitative trait loci analysis of nitrogen use efficiency
in Arabidopsis. Plant Physiol 131:345–358
Loudet O, Saliba-Colombani V, Camilleri C, Calenge F, Gaudon V, Koprivova A, North KA,
Kopriva S, Daniel-Vedele F (2007) Natural variation for sulfate content in Arabidopsis
thaliana is highly controlled by APR2. Nat Genet 39:896–900
Masclaux-Daubresse C, Chardon F (2011) Exploring nitrogen remobilization for seed filling using
natural variation in Arabidopsis thaliana. J Exp Bot 62:2131–2142
2 Natural Variation as a Tool to Investigate Nutrient Use Efficiency in Plants 49

Masclaux-Daubresse C, Daniel-Vedele F, Dechorgnat J, Chardon F, Gaufichon L, Suzuki A (2010)

Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and
productive agriculture. Ann Bot 105:1141–1157
North KA, Ehlting B, Koprivova A, Rennenberg H, Kopriva S (2009) Natural variation
in Arabidopsis adaptation to growth at low nitrogen conditions. Plant Physiol Biochem
47:912–918
Palaisa KA, Morgante M, Williams M, Rafalski A (2003) Contrasting effects of selection on
sequence diversity and linkage disequilibrium at two phytoene synthase loci. Plant Cell
15:1795–1806
Pozniak CJ, Knox RE, Clarke FR, Clarke JM (2007) Identification of QTL and association of a
phytoene synthase gene with endosperm colour in durum wheat. Theor Appl Genet 114:525–
537
Quraishi UM, Abrouk M, Murat F, Pont C, Foucrier S, Desmaizieres G, Confolent C, Rivière N,
Charmet G, Paux E, Murigneux A, Guerreiro L, Lafarge S, Le Gouis J, Feuillet C, Salse J
(2011) Cross-genome map based dissection of a nitrogen use efficiency ortho-metaQTL in
bread wheat unravels concerted cereal genome evolution. Plant J 65:745–756
Rafalski JA (2010) Association genetics in crop improvement. Curr Opin Plant Biol 13:174–180
Reif JC, Hamrit S, Heckenberger M, Schipprack W, Maurer HP, Bohn M, Melchinger AE (2005)
Trends in genetic diversity among European maize cultivars and their parental components
during the past 50 years. Theor Appl Genet 111:838–845
Rengel Z, Damon PM (2008) Crops and genotypes differ in efficiency of potassium uptake and
use. Physiol Plant 133:624–636
Reymond M, Svistoonoff S, Loudet O, Nussaume L, Desnos T (2006) Identification of QTL
controlling root growth response to phosphate starvation in Arabidopsis thaliana. Plant Cell
Environ 29:115–125
Ribaut JM, de Vicente MC, Delannay X (2010) Molecular breeding in developing countries:
challenges and perspectives. Curr Opin Plant Biol 13:213–218
Richard-Molard C, Krapp A, Brun F, Ney B, Daniel-Vedele F, Chaillou S (2008) Plant response to
nitrate starvation is determined by N storage capacity matched by nitrate uptake capacity in
two Arabidopsis genotypes. J Exp Bot 59:779–791
Riddle NC, Richards EJ (2002) The control of natural variation in cytosine methylation in
Arabidopsis. Genetics 162:355–363
Ristova D, Rosas U, Krouk G, Ruffel S, Birnbaum KD, Coruzzi GM (2013) RootScape: a
landmark-based system for rapid screening of root architecture in Arabidopsis. Plant Physiol
161(3):1086–1096. doi:10.1104/pp. 112.210872
Robinson KM, Ingvarsson PK, Jansson S, Albrectsen BR (2012) Genetic variation in functional
traits influences arthropod community composition in aspen (Populus tremula L.). PLoS One
7:e37679
Rosenberg NA, Shah C, Wall JD, Wang J, Zhao K, Kalbfleisch T, Schulz V, Kreitman M,
Bergelson J (2005) The pattern of polymorphism in Arabidopsis thaliana. PLoS Biol 3:e196
Saisho D, Ishii M, Hori K, Sato K (2011) Natural variation of barley vernalization requirements:
implication of quantitative variation of winter growth habit as an adaptive trait in east Asia.
Plant Cell Physiol 52:775–784
Shewry PR, Hawkesford MJ, Piironen V, Lampi AM, Gebruers K, Boros D, Andersson AAM,
Aman P, Rakszegi M, Bedo Z, Ward JL (2013) Natural variation in grain composition of wheat
and related cereals. J Agric Food Chem. doi:dx.doi.org/10.1021/jf3054092
Shindo C, Bernasconi G, Hardtke CS (2007) Natural genetic variation in Arabidopsis: tools, traits
and prospects for evolutionary ecology. Ann Bot 99:1043–1054
Sulpice R, Nikoloski Z, Tschoep H, Antonio C, Kleessen S, Larhlimi A, Selbig J, Ishihara H,
Gibon Y, Fernie AR, Stitt M (2013) Impact of the carbon and nitrogen supply on relationships
and connectivity between metabolism and biomass in a broad panel of Arabidopsis accessions.
Plant Physiol 162(1):347–363. doi:10.1104/pp. 112.210104
50 G. Chietera and F. Chardon

Swamy BP, Vikram P, Dixit S, Ahmed HU, Kamar A (2011) Meta-analysis of grain yield QTL
identified during agricultural drought in grasses showed consensus. BMC Genomics 12:319
Tisné S, Serrand Y, Bach L, Gilbault E, Ben Ameur R, Balasse H, Voisin R, Bouchez D, Durand-
Tardif M, Guerche P, Chareyron G, Da Rugna J, Camilleri C, Loudet O (2013) Phenoscope: an
automated large-scale phenotyping platform offering high spatial homogeneity. Plant J
74:534–544
Trontin C, Tisné S, Bach L, Loudet O (2011) What does Arabidopsis natural variation teach us
(and does not teach us) about adaptation in plants? Curr Opin Plant Biol 14:225–231
Tuinstra MR, Ejeta G, Goldsbrough PB (1997) Heterogeneous inbred family (HIF) analysis: a
method for developing near-isogenic lines that differ at quantitative trait loci. Theor Appl
Genet 95:1005–1011
Ungerer MC, Johnson LC, Herman MA (2008) Ecological genomics: understanding gene and
genome function in the natural environment. Heredity 100:178–183
Vinod KK, Heuer S (2012) Approaches towards nitrogen- and phosphorus-efficient rice. AoB
Plants 2012: pls028
Vlad D, Rappaport F, Simon M, Loudet O (2010) Gene transposition causing natural variation for
growth in Arabidopsis thaliana. PLoS Genet 6(5):e1000945
Wang X, Yan X, Liao H (2010) Genetic improvement for phosphorus efficiency in soybean : a
radical approach. Ann Bot 106:215–222
Wang Z, Chen Z, Cheng J, Lai Y, Wang J, Bao Y, Huang J, Zhang H (2012) QTL analysis of Na
+ and K + concentrations in roots and shoots under different levels of NaCl stress in rice (Oryza
sativa L.). PLoS One 7:e51202
Weigel D (2012) Natural variation in Arabidopsis: from molecular genetics to ecological geno-
mics. Plant Physiol 158:2–22
Weigel D, Mott R (2009) The 1001 genomes project for Arabidopsis thaliana. Genome Biol
10:107
Xing JP, Jiang RF, Ueno D, Ma JF, Schat H, McGrath SP, Zhao FJ (2008) Variation in root-to-
shoot translocation of cadmium and zinc among different accessions of the hyperaccumulators
Thlaspi caerulescens and Thlaspi praecox. New Phytol 178:315–325
Zhang N, Gibon Y, Gur A, Chen C, Lepak N, Höhne M, Zhang Z, Kroon D, Tschoep H, Stitt M,
Buckler E (2010) Fine quantitative trait loci mapping of carbon and nitrogen metabolism
enzyme activities and seedling biomass in the maize IBM mapping population. Plant Physiol
154:1753–1765
Chapter 3
Macronutrient Use Efficiency – Sulfur
in Arabidopsis thaliana

Patrycja Baraniecka and Stanislav Kopriva

Abstract Sulfur is an essential macronutrient required for proper growth of not

only plants but also fungi and prokaryotes. It is present in a wide variety of
metabolites such as amino acids: cysteine and methionine, coenzymes, vitamins
and many others having distinctive biological functions. Plants take up sulfur from
the soil in the form of sulfate via sulfate transporters. It is then reduced and
assimilated in bioorganic compounds where cysteine is the first stable product.
This process is very well described on both biochemical and molecular levels. Both
reduction and assimilation are tightly regulated in demand-driven manner. The
pathway has been extensively studied over last years because of important func-
tions of sulfur in plant metabolism and stress defence. Here we summarise the up-
to-date knowledge about the pathway and its regulation based mainly on the study
on model plant Arabidopsis thaliana. We also emphasize areas in which little is
known including the interconnection of sulfate metabolism with other nutrients.

Keywords Sulfur • Regulation • Arabidopsis • Sulfate transporters • Assimilation

• Metabolism • APS reductase • Cysteine biosynthesis • OAS-TL • Glutathione
• Methionine • Glucosinolates

Introduction

Apart from oxygen, carbon dioxide and water, plants require at least 14 mineral
elements for sufficient nutrition. Six of them: nitrogen, phosphorus, potassium,
calcium, magnesium and sulfur are required in large amounts and these are called
macronutrients. The requirements for the rest, chlorine, boron, iron, manganese,
copper, zinc, nickel, and molybdenum, are much lower and these are called
micronutrients. Essential nutrients can be supplied to plants as fertilisers in case of
deficiency to increase plant yield and quality. This applies mainly to crop production

P. Baraniecka
Department of Metabolic Biology, John Innes Centre, Norwich, UK
e-mail: [email protected]
S. Kopriva (*)
Botanical Institute, University of Cologne, Cologne, Germany
e-mail: [email protected]

© Springer International Publishing Switzerland 2014 51

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_3
52 P. Baraniecka and S. Kopriva

(White and Brown 2010). However, this practice needs to be used optimally in
agriculture as too high concentration of some of these elements in the soil solution
may inhibit plant growth and reduce crop yield (White and Brown 2010).
Adequate sulfur supply is required for proper growth and fitness of all living
organisms. Sulfur is present in a wide variety of metabolites with specific biological
functions. Sulfur is cycled in the global ecosystem and can be converted to its
organic compounds by photosynthetic organisms and microorganisms. The most
common form of sulfur in nature is sulfate (SO42
), which is the most oxidised
form, in which sulfur is in the + VI redox state. Sulfate is taken up from the soil and
reduced to sulfide in an energy-dependent reduction pathway. Sulfide can be
incorporated into O-acetylserine to form cysteine, which is the first stable form of
bound reduced sulfur in plants. Apart from cysteine, sulfur is present in methionine,
sulfolipids, and cell walls. It also is contained in thiols, which are involved in redox
control in plant cells and in vitamins and cofactors such as coenzyme A, thiamine
and biotin. Noteworthy are the sulfur-containing secondary metabolites such as
alliins and glucosinolates. Their breakdown products are responsible for character-
istic smell and taste of many vegetables but also deter plant pathogens. Both of
these classes of natural products are also very beneficial for human health. Alliins,
found in large amounts in garlic, have antimicrobial properties, and glucosinolate
degradation products induce enzymes that prevent tumour formation.
Sulfur availability in agricultural soils has been decreasing in many areas of
Europe during the last two decades (McGrath et al. 1996; Zhao et al. 1996). The
main reasons are: (i) reduction of sulfur dioxide emissions and subsequent reduc-
tion in sulfur depositions and (ii) changes in fertiliser practice, i.e. higher definition
fertilisers without sulfate contamination (Blake-Kalff et al. 2001). Additionally, it
was suggested that the demand for sulfur in many crops has increased due to
intensive agriculture and optimisation during plant breeding programmes (Abdallah
et al. 2010). The crop’s requirement for sulfur varies in different species. Generally,
wheat requires approximately 2–3 kg of sulfur for each tonne of grain produced
(Zhao et al. 1999) whereas the production of 1 t of oilseed rape seeds requires about
16 kg of sulfur (McGrath and Zhao 1996). This high demand for sulfur of oilseed
rape is probably due to an ineffective xylem-to-phloem transport mechanism in this
species. A large amount of sulfate is accumulated in the vacuoles of mature oilseed
rape leaves in sulfur sufficient conditions. This sulfate is not easily available for
redistribution if sulfur is limited (Blake-Kalff et al. 1998). Therefore oilseed rape is
particularly sensitive to sulfur deficiency or limitation which reduce both seed
quality and yield (Malhi et al. 2007). Sulfur deficiency in crop plants has been
recognised as a limiting factor not only for crop growth and seed yield but also for
poor quality of products which is particularly important in the case of wheat and the
maintenance of baking quality (Shahsavani and Gholami 2008). The protein frac-
tion is known to play an essential role in the bread-making quality of wheat. The
gluten proteins, gliadins and glutenins, represent about 80–85 % of total flour
protein. These are responsible for elasticity and extensibility that are essential for
functionality of wheat flours (Hussain et al. 2012; Kuktaite et al. 2004). However, in
sulfur-limited conditions the synthesis of sulfur-poor storage proteins such as
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 53

ω-gliadin and the high molecular weight subunits of glutenin is favoured at the
expense of sulfur-rich proteins which may cause unpredictable and unwanted
variations in wheat quality (Flæte et al. 2005; Moss et al. 1981). Another problem,
discovered relatively recently, is the formation of acrylamide during high-
temperature processing of potato and wheat products (Tareke et al. 2002). Acryl-
amide is potentially carcinogenic to human. It also has negative neurological and
reproductive system effects (Friedman 2003). The major determinant of acrylamide
forming potential is the concentration of free asparagine (Curtis et al. 2010). The
accumulation of free asparagine in wheat grain in severe sulfur deprivation may
increase to 30-fold higher levels compared to sulfur-sufficient conditions, which
makes asparagine up to 50 % of the total free amino acid pool. For that reason, even
very small amounts of such grain entering the food chain could have a significant
effect on acrylamide formation, which makes the application of sulfur fertilisers
over entire fields very important (Halford et al. 2012).
As sulfur deficiency in Europe appeared relatively recently, research on sulfur
use efficiency still lags behind that on the other major nutrients. Therefore, explor-
ing sulfur assimilation and the regulation of sulfur metabolism is of great interest
for agriculture and plant science, because it is important to understand the process
in order to optimise it for commercial use. However, there are still many gaps in our
knowledge in this area. Studies on model plants provide a great tool for further
exploration of the sulfur metabolic pathway mainly because of their rapid life cycle.
The knowledge obtained from research on model plants can subsequently be
transferred to crops and used for improving crop-breeding strategies. For these
reasons this review is focused on the sulfur metabolism of the model plant
Arabidopsis thaliana.

Sulfate Transport

Sulfate is the major form of sulfur in plants. Sulfate uptake from the soil is the first
stage of plant sulfate metabolism. After entry into the plants, sulfate needs to be
delivered to the plastids for assimilation or to the vacuoles for storage. Cell-to-cell
transport as well as long-distance transport between organs required to fulfil the
source/sink demands during plant growth, involve specific sulfate transporter pro-
teins (Buchner et al. 2004b). Genes encoding these proteins belong to the sulfate
transporter gene family and are divided into five groups. Members of different
groups vary in kinetics of transport and in patterns of expression indicating different
functions in the process of sulfate uptake and distribution. In general, high-affinity
transporters are responsible for the initial uptake of sulfate by the root epidermis
and cortex cells whereas low-affinity transporters are involved in the vascular
transport. Sulfate transporters may be expressed constitutively or depending on
sulfur availability. Decreased intracellular content of sulfate, cysteine and glutathi-
one (GSH) results in increased transporter activity (Smith et al. 1997). The Group
3 sulfate transporters are not influenced by the sulfur status of the plant (Kataoka
54 P. Baraniecka and S. Kopriva

et al. 2004a; Takahashi et al. 2000). Sulfate transporters from Groups 1, 2, and
4 undergo a dual pattern of regulation: semi-constitutive and inducible. Under
sufficient sulfur conditions and saturable uptake kinetics of the high- and
low-affinity transporters, a semi-constitutive expression of one high-affinity trans-
porter from Group 1, one low-affinity Group 2 transporter, undefined Group 3 trans-
porters, and vacuolar sulfate efflux transporter from Group 4 are necessary for
proper sulfate uptake and distribution (Smith et al. 1997; Takahashi et al. 2000).
Under excess sulfur nutrition the expression of inducible sulfate transporters is
repressed in order to prevent toxic internal sulfate accumulation. Feedback loops
involving key metabolites from the sulfate reduction pathway are a common
regulatory mechanism for expression of sulfate transporters (see further parts of
this chapter).
The influx of sulfate through the plasma membrane is well characterised at the
physiological and functional level. Plants essentially use a proton/sulfate
co-transport system to mediate sulfate flux (Lass and Ullrich-Eberius 1984). A
proton gradient is generated by plasma membrane proton ATPase (Saito 2004) and
the transport process is pH dependent with 3H+/sulfate stoichiometry (Hawkesford
et al. 1993; Smith et al. 1995a). Products encoded by sulfate transporter genes
possess 12 membrane-spanning domains and belong to a large family of cation/
solute co-transporters (Saito 2000). Additionally, the analysis of the C-terminal
region of these transporters revealed the presence of STAS (sulfate transporters and
antisigma factor antagonists) domains (Aravind and Koonin 2000). It was shown
that deletion of the STAS domain affects the localisation of transporters to the
membrane (Shibagaki and Grossman 2004) and results in a loss of sulfate transport
activity (Rouached et al. 2005). These results indicate that STAS domains have a
regulatory function in sulfate uptake and flux.

Functions of Sulfate Transporters in Arabidopsis thaliana

The first sulfate transporters were identified by functional complementation of a

sulfate transporter-deficient yeast mutant (Smith et al. 1995a, b). Subsequently,
yeast deletion mutants became the main tool for functional characterisation of
sulfate transporters. To date, a number of sulfate transporters from a variety of
plant species have been described (Buchner et al. 2004a; Howarth et al. 2003;
Vidmar et al. 2000; Yoshimoto et al. 2002, 2003). The availability of fully
sequenced genomes for Arabidopsis thaliana and rice has enabled analyses,
which led to identification of 14 putative sulfate transporter genes in each genome.
Based on phylogenetic analysis they were subdivided into four closely related
groups, and a fifth group containing two smaller proteins lacking the STAS domain
(Hawkesford 2003).
The analysis of the first isolated sulfate transporters identified high- and
low-affinity transport characteristics (Smith et al. 1995a) and more detailed analysis
revealed that the high-affinity components facilitate uptake of sulfate into the plant
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 55

root (Barberon et al. 2008; Yoshimoto et al. 2007). The high-affinity sulfate trans-
porters are very well studied. In Arabidopsis thaliana they comprise three genes
(SULTR1;1–3) and belong to the clade which forms Group 1 of the sulfate trans-
porters (Fig. 3.2). The primary sulfate acquisition in roots is mediated by two
transporters: SULTR1;1 and SULTR1;2 (Fig. 3.1). Various studies confirmed the
expression of these two genes in root hairs, root epidermal and cortical cells (Rae
and Smith 2002; Takahashi et al. 2000; Yoshimoto et al. 2002) suggesting the
capacity of these tissues for a high-affinity sulfate influx into the symplast.
SULTR1;3 appears to be an exception in this group with specificity of localisation
to the phloem in both roots and cotyledons (Yoshimoto et al. 2003). Sulfur

Fig. 3.1 Sulfate transport system in Arabidopsis thaliana. Blue shapes and labels indicate sulfate
transporters (SULTRs) mediating transport across plasma membranes. Dashed arrows indicate yet
unknown transport pathways
56 P. Baraniecka and S. Kopriva

deficiency massively increased sulfate uptake (Clarkson et al. 1983). SULTR1;2

mediates sulfate uptake in normal conditions and in sulfur deficiency, and its
expression is relatively independent of sulfate supply. In contrast, SULTR1;1 is
strongly inducible by sulfate limitation but almost absent in normal sulfur condi-
tions (Howarth et al. 2003; Yoshimoto et al. 2002). Moreover, another study
showed that in mutants deficient in SULTR1;2, the expression of SULTR1;1 is
slightly up-regulated. However, reduced growth suggests that SULTR1;1 is not able
to compensate for the missing SULTR1;2. This might indicate that SULTR1;2 is
the major component for sulfate acquisition (Maruyama-Nakashita et al. 2003).
Sulfate absorbed into the epidermis needs to be transferred across the root cells
to the xylem. This step is necessary for delivery to the target cells in shoot organs
for reduction or storage into the vacuole. The horizontal sulfate transfer from the
epidermis to the central cylinder cells may occur via plasmodesmata. This is the
likely strategy to cross the barrier of the Casparian strip at the endodermal cell
layers. During this process sulfate may leak from the symplast to apoplast. This
mechanism seems to be passive but has not yet been identified (Takahashi
et al. 2011). The efflux of sulfate into the xylem vessels is still unknown. There is
no evidence that sulfate transporters may act in the reverse direction (Buchner
et al. 2004b). However, the expression patterns of Arabidopsis Group 2 low-affinity
sulfate transporters in the central cylinder cells suggest they may contribute to long
distance sulfate transport (Fig. 3.1). In roots SULTR2;1 is expressed in the xylem
parenchyma and pericycle cells whereas SULTR2;2 is restricted to the root phloem.
In contrast, in leaves SULTR2;1 is expressed in xylem parenchyma and phloem
cells, and SULTR2;2 in the cells surrounding the xylem vessels (Takahashi
et al. 2000). Additionally, SULTR2;1 was assumed to be involved in sulfate
transport into developing seeds (Awazuhara et al. 2005). Localisation of
SULTR2;2 suggests a role in sulfate transport via the phloem. The efflux of sulfate
to the apoplast of the root vascular tissue leads to a high sulfate concentration.
SULTR2;1 expressed in the xylem parenchyma cells can reabsorb this sulfate, thus
regulating the amount of sulfate which is transported to the shoots. Induction of the
SULTR2;1 gene during sulfur starvation strongly supports this strategy. In the leaf
the expression of SULTR2;2 in the closest cells to the xylem vessels suggests its
role in sulfate uptake from the vessels, most likely at millimolar concentrations.
Subsequently sulfate is probably transferred to cells where it will be assimilated.
The expression of SULTR2;1 in the phloem suggests its role in sulfate transfer to
other organs, and in xylem parenchyma – reabsorption for further xylem transport
(Buchner et al. 2004b). Moreover, it was shown that SULTR3;5 from the Group
3 sulfate transporters is co-expressed with SULTR2;1 and involved in the sulfate
influx to the xylem parenchyma cells in the roots. However, it does not work as a
sulfate transporter by itself. It has been suggested that formation of heterodimer is
required for the activity of SULTR3;5 and maximum activity of SULTR2;1
(Kataoka et al. 2004a). Taken together, it seems that Group 2 sulfate transporters
are involved in the balancing of the sulfate flux through the plant in various sulfate
supply (Takahashi et al. 2000). It was also suggested that not only low- but also
high-affinity sulfate transporters from Group 1 SULTR1;3 are involved in long
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 57

distance sulfate transport. SULTR1;3 is expressed in phloem in all the Arabidopsis

organs tested (Yoshimoto et al. 2003). Additionally, the analysis of a tomato
homologue of this transporter showed that it is expressed under sulfur stress
conditions (Howarth et al. 2003). These results indicate that plants are able to
maintain balanced distribution of sulfate during limitation by inducing additional
genes (Yoshimoto et al. 2003).
Plastids are the final destination for sulfate where it is assimilated. Alternatively,
it can also be transported to the vacuoles, which play the role of sulfate reservoir in
cells. It was shown that vacuoles isolated from the Arabidopsis sultr4;1/sultr4;2
double mutant contain more sulfate than the wild type, suggesting that the efflux of
sulfate from the vacuole is mediated by Group 4 sulfate transporters (Fig. 3.1).
Moreover, enhanced expression of these two genes under sulfur stress conditions
indicates that sulfur is released from the vacuole in response to sulfur demands in
the cells (Kataoka et al. 2004b). The vacuole influx transporters have not yet been
identified. A chloroplast sulfate transporter SULTR3;1 has been characterised only
recently (Cao et al. 2013); chloroplast subcellular localisation was confirmed with
the analysis of SULTR3;1-GFP constructs and the sulfate transport functionality
with a validated in organello assay. However, the affinity of chloroplast for sulfate
did not change despite the disruption of SULTR3;1 suggesting the existence of other
chloroplast sulfate transporters. The analysis of other members of the SULTR3
subfamily revealed that SULTR3;2, SULTR3;3, SULTR3;4 but not SULTR3;5 might
be also chloroplast sulfate transporters (Cao et al. 2013). These results are consis-
tent with those of Kataoka et al. (2004a) who demonstrated an essential role for
SULTR3;5 in the vascular root-to-shoot transport.
As mentioned above, the members of Group 5 differ significantly from the other
groups (Fig. 3.2). This group contains two isoforms, which are also dissimilar to
each other. SULTR5;2 was described as involved in molybdenum transport (Baxter
AtSULTR11

AtS
AtS

UL
UL

TR
TR

AtS
2

AtS UL
1
13

UL TR
AtS TR 22
ULT 12
R52
TR33
AtSUL
AtS
51 UL
TR TR
34
AtSUL
At
AtSU

AtSU

AtS SU
ULT LT
AtS

R42
L

R
L
TR3

35
TR3
LT U

2
R4

0.2
1

Fig. 3.2 Unrooted phylogenetic tree of the Arabidopsis thaliana members of the sulfate trans-
porter family. Different colours represent different subfamilies. The tree was drawn using
MEGA5.1 software
58 P. Baraniecka and S. Kopriva

et al. 2008; Tomatsu et al. 2007) and renamed the MOT1 transporter. It is clear that
MOT1 is essential for molybdenum accumulation. However, the exact subcellular
localisation of this protein remains unclear. Tomatsu et al. (2007) localised this
transporter to endomembrane system and plasma membrane whereas Baxter
et al. (2008) suggested its localisation in the mitochondrial membrane. The expla-
nation for these two predicted locations may be found in the different sites of GFP
fusion, either to the N-terminal end of MOT1 (Tomatsu et al. 2007) or to its
C-terminus (Baxter et al. 2008). Nevertheless, the exact location of MOT1 requires
further investigation. SULTR5;1 was shown to be expressed in most plant tissues
but it was not affected by sulfate supply (Shinmachi et al. 2010). Only recently its
function as a vacuolar molybdate export protein was shown and it was renamed for
MOT2 (Gasber et al. 2011). As there are no reports indicating a sulfate transport
function for these two transporters and because of the absence of the STAS domain,
which is present in all other sulfate transporters, these two genes could reasonably
be excluded from the sulfate transporter family.

Regulation of Sulfate Transport at the Whole Plant Level

Growing plants require sulfate to synthesise amino acids, sulfolipids, thiols and
many other sulfur-containing compounds. The demand for sulfur differs depending
on the tissues, organs and developmental stage. Sulfate uptake and distribution is
regulated in response to plant demand and changing environment. Specific patterns
of expression of sulfate transporters genes in particular cells indicate the impor-
tance of proper sulfate distribution. Additionally, sulfate may be redistributed
during development from mature leaves to roots, younger leaves or seeds. Redis-
tribution requires changes in expression patterns. For example the up-regulation of
SULTR2;2 and SULTR1;3 in leaves during sulfate starvation indicates that these
transporters play an important role in sulfate allocation to other tissues (Yoshimoto
et al. 2003). It is an important process that allows interconnection of different
nutrients and that keeps a balanced system even during large environmental fluc-
tuations. The main reservoir of sulfate is in the vacuoles of mature leaves. Redis-
tribution of sulfate from vacuoles is particularly important during sulfur limitation.
Accumulation of high levels of sulfate metabolism products such as cysteine and
GHS decreases sulfate uptake and transport whereas sulfur stress conditions
increase expression of sulfate transporters and activities of key enzymes in the
sulfate metabolism pathway. Regulation of sulfate transport is sulfate nutrition-
dependent. Many studies confirmed that the regulation occurs primarily at the level
of mRNA (Smith et al. 1997; Takahashi et al. 2000; Yoshimoto et al. 2002). In
addition, post-transcriptional regulation and protein-protein interactions of sulfate
transporters with O-acetylserine (thiol)lyase were described (Shibagaki and
Grossman 2010; Yoshimoto et al. 2007). Their contribution to the overall control
of sulfate uptake is, however, not clarified yet. Changes in expression patterns of
sulfate transporter genes follow complex regulation responses. Firstly, tissue/organ-
specific expression of transporters from Group 3 is not regulated by sulfur nutrition.
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 59

Secondly, cell-specific expression of some transporters from Groups 1, 2, and 4 is

modulated by sulfur limitation. Finally, cell/tissue-specific, sulfur deficiency-
related transporters from Groups 1 and 2 are derepressed (Buchner et al. 2004b).
The question of how plants sense sulfate deficiency and how the signal is trans-
duced to the transporter gene promoters still needs to be answered. Lejay
et al. (2003) studying the regulation of ion transporter in roots by photosynthesis,
concluded that some sulfate transporters (but also nitrate, phosphate, potassium and
other metal transporters) show diurnal changes in expression and might be induced
by sucrose, linking thus sulfate uptake with general primary metabolism.
In conclusion, plants are able to adjust sulfate transport to environmental
changes and to the availability of the other nutrients. It is worth noting that these
adaptations vary between species similarly to demands in sulfate metabolism.
Different molecular regulation mechanisms are described in the following sections
of this chapter.

Sulfate Assimilation and Metabolism in Arabidopsis thaliana

In nature, sulfur occurs in many oxidation states in inorganic, organic and

bioorganic compounds. Various organisms such as algae, bacteria, plants and
fungi are able to reduce sulfate and incorporate it into amino acids in the assimi-
latory sulfate reduction pathway. This process is very well described on both
biochemical and molecular levels. In photosynthetic organisms it occurs in plastids
(Brunold and Suter 1989). The only exception is Euglena gracilis where sulfate
reduction takes place in mitochondria (Brunold and Schiff 1976). Since sulfate is
chemically very stable, it requires activation before reduction. This occurs by
adenylation to adenosine 50 -phosphosulfate (APS), which is catalysed by ATP
sulfurylase (ATPS; EC: 2.7.7.4). ATPS in plants appears to be a homotetramer
composed from 52 to 54 kDa polypeptides (Murillo and Leustek 1995). Most of
total ATPS is plastid localised where it is responsible for sulfate activation for
further reduction. However, activity was also detected in the cytosol (Lunn
et al. 1990; Renosto et al. 1993; Rotte and Leustek 2000). During plant growth
activity in chloroplasts declines, whereas it increases in the cytosol. ATPS is
encoded by a small multigene family. Most plant species possess two ATPS
isoforms. However, four different isoforms were isolated from Arabidopsis,
which may indicate some level of genetic redundancy (Kopriva et al. 2009). Sur-
prisingly all contain a chloroplast transit peptide (Hatzfeld et al. 2000a; Murillo and
Leustek 1995). The most likely explanation for the existence of cytosolic ATPS
isoforms is the use of a different translational start codons (Hatzfeld et al. 2000a).
However, this hypothesis remains to be investigated.
APS forms a branching point in the sulfate reduction pathway (Fig. 3.3). APS
can be directly reduced to sulfite by APS reductase (APR; EC: 1.8.99.2) or
phosphorylated by APS kinase (APK; EC: 2.7.1.25) to form 30 -phosphoadenosine
60 P. Baraniecka and S. Kopriva

Fig. 3.3 Cellular organization of sulfate metabolism in Arabidopsis thaliana. Enzymes and
transporters are indicated in purple characters. Abbreviations of enzymes and transporters:
ATPS ATP sulfurylase, APK APS kinase, APR APS reductase, SiR sulfite reductase, OAS-TL
OAS(thiol)lyase, SAT serine acetyltransferase, CGS cystathionine γ-synthase, CBL cystathionine
β-lyase, TS threonine synthase, MS methionine synthase, SAM S-adenosylmethionine synthetase,
γ-ECS γ-glutamylcysteine synthetase, GSHS glutathione synthetase, CLT thiol transporter, GST
glutathione-S-transferase, MRP multidrug resistance-associated protein, GGT
γ-glutamyltransferase, SOT sulfotransferase, SULTR sulfate transporter, PAPST1 plastidic PAPS
transporter. Abbreviations of metabolites: APS adenosine 50 -phosphosulfate, Cys cysteine, Cyst
cystathionine, Hcy homocysteine, OPH O-phosphohomoserine, Thr threonine, Met methionine,
SAM S-adenosylmethionine, SAH S-adenosylhomocysteine, γ-GluCys γ-glutamylcysteine, GSH
glutathione, GS-X glutathione conjugate, Glu glutamate, X-CysGyl cysteinylglycine conjugate, Ser
serine, OAS O-acetylserine, PAPS 30 -phosphoadenosine 50 -phosphosulfate, R-OH hydroxylated
precursor

50 -phosphosulfate (PAPS) which serves as a donor of activated sulfate for various

cellular reactions modifying proteins, saccharides or synthesis of secondary metab-
olites including glucosinolates. This process is called sulfation and it is important in
regulating plant growth and development. However, sulfate reduction is a dominant
route for assimilation (Leustek et al. 2000) and is fulfilled in two steps. In the first
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 61

step APR transfers two electrons to APS to produce sulfite; the electrons are derived
from GSH (Bick et al. 1998). Subsequently, sulfite is reduced to sulfide by
ferredoxin-dependent sulfite reductase (SiR; EC: 1.8.7.1); this reaction requires a
transfer of six electrons from ferredoxin to sulfite. Sulfide is then incorporated into
the amino acid skeleton of O-acetylserine (OAS) to form cysteine. This reaction is
catalysed by OAS thiol-lyase (OAS-TL; EC: 2.5.1.47) (Kopriva 2006; Leustek
et al. 2000; Takahashi et al. 2011).
APR is a key enzyme of the sulfur assimilation pathway. It is regulated by
various environmental factors and signalling molecules (Kopriva 2006; Koprivova
et al. 2008). Similarly to ATPS, APR is encoded by a small multigene family and
three isoforms are known in Arabidopsis. It was shown in various studies that APR1
and APR3 are co-regulated and share the highest sequence similarity. However,
APR2 responds differently to hormone treatments (Koprivova et al. 2008) which
indicates specific functions of particular APR isoforms. The amino acid sequence of
APR suggests a multi-domain structure. Its precursor is synthesised with an
N-terminal plastid transit peptide. In the mature protein the N-terminal domain is
similar in predicted amino acid sequence to PAPS reductase (PAPR) from bacteria,
and the C-terminal domain with thioredoxin (Trx). Due to the lack of the C-terminal
domain, PAPR requires thioredoxin or glutaredoxin as a cofactor (Kopriva
et al. 2007). This may suggest the role of C-terminal domain as redox cofactor
(Leustek et al. 2000). Arabidopsis APR is a dimer of 45 kDa subunits (Kopriva and
Koprivova 2004) binding a [Fe4S4] iron-sulfur cluster (Kopriva et al. 2001). PAPR
consist of two 28 kDa subunits without any prostetic groups. A single conserved
cysteine residue is responsible for its activity and dimerization.
APK catalyses the transfer of phosphate from ATP to APS to form PAPS which
is a sulfate donor for sulfotransferases. Four genes coding APK are found in the
Arabidopsis genome, all located on different chromosomes and with a high level of
similarity. Three of the isoforms contain chloroplast transit peptides at the
N-termini and these have been confirmed to be localised in plastids (Mugford
et al. 2009). The APK3 isoform does not contain the N-terminal extension and it
is likely to be responsible for cytosolic activity. Little is known about the biochem-
istry and the functions of the individual plant APKs. However, they have a
significant effect on sulfur metabolism. It was shown that apk1apk2 double mutant
has a dramatically low glucosinolate level and also substantially higher levels of
cysteine and GHS than wild type plants (Mugford et al. 2009). This suggests a
compensation of low glucosinolate level by increases in cysteine and GSH. This
subsequently may indicate that primary sulfate metabolism is up-regulated in this
mutant, implying an important role of APK in controlling sulfur distribution in
plants. Plants with APK1 as the only active APK isoform showed the wild type
phenotype suggesting the major contribution of this isoform to total enzyme
activity (Mugford et al. 2010). The analysis of mutants lacking various APR
isoforms and differences in tissue-specific expression between the isoforms indicate
specific roles of particular isoforms in plant sulfur metabolism (Kopriva et al. 2012;
Mugford et al. 2009).
62 P. Baraniecka and S. Kopriva

The six electron reduction of sulfite to sulfide is catalysed by SiR in plastids.

Plant SiR is a 65 kDa monomer and requires the presence of a siroheme and an FeS
cluster as cofactors, and ferredoxin as an electron donor (Nakayama et al. 2000). In
contrast to other enzymes of the sulfur metabolism pathway, SiR is encoded by
single gene in Arabidopsis. The amino acid sequence and protein structure are very
similar to nitrite reductase (NiR) which catalyses a six electron reduction of nitrite
to ammonia in nitrate assimilation pathway. The 19 % amino acid sequence identity
suggests that these two enzymes have the same evolutionary origin.
Only recently a new enzyme in the sulfate assimilation pathway was identified
(Eilers et al. 2001): Arabidopsis sulfite oxidase (SO; EC: 1.8.3.1) catalyses the
oxidation of sulfite to sulfate in a two electron reaction. It is localised in peroxi-
somes (Nowak et al. 2004). Plant SO lacks the haem domain, which is known from
animal SO’s but it contains a molybdenum cofactor-binding domain. It was shown
that the terminal electron acceptor for plant SO is molecular oxygen, the reaction
converting O2 into hydrogen peroxide. Hänsch et al. (2006) also provided evidence
that hydrogen peroxide can oxidise sulfite non-enzymatically. It was shown that SO
can protect plants from an excess of sulfite which is toxic in high concentrations,
and SO2 gas in the atmosphere (Brychkova et al. 2007). However, the exact role of
SO in sulfur primary metabolism is still not clear.

Cysteine Biosynthesis

Cysteine is the key sulfur-containing compound in plants. It is synthesised by

incorporation of sulfide into the β-position of the serine carbon skeleton in the
terminal step of sulfur assimilation (Saito 2004). Before the incorporation of
sulfide, serine needs to be activated to O-acetylserine (OAS). This process occurs
by acetyl transfer from acetyl coenzyme A, which is catalysed by serine
acetyltransferase (SAT; Serat; EC 2.1.3.30). Subsequently, OAS and sulfide are
the substrates for O-acetylserine (thiol) lyase (OAS-TL; EC: 2.5.1.47) which
catalyses the β-replacement reaction (Hell and Wirtz 2011; Takahashi
et al. 2011). The enzymes involved in the cysteine biosynthesis process, SAT and
OAS-TL, are localised in plastids, mitochondria and cytosol (Saito 2000; Fig. 3.3).
Additionally, β-cyanoalanine synthase was identified in mitochondria, which has a
similar catalytic activity to OAS-TL, and was previously identified as its mitochon-
drial form (Hatzfeld et al. 2000b). This enzyme is important for detoxification of
cyanide produced e.g. during ethylene synthesis (Garcia et al. 2010). The analysis
of whole plant protein extracts showed that entire SAT activity is always associated
with OAS-TL, and that an excess of free active OAS-TL is present (Hell and Wirtz
2011). This and other results indicate that these two enzymes are associated and
form a hetero-oligomeric cysteine synthase complex (Hell and Wirtz 2008, 2011;
Saito 2004; Takahashi et al. 2011; Wirtz et al. 2001; Wirtz and Hell 2006). The
binding of OAS-TL to SAT stabilises SAT. In the complex, SAT is the only active
enzyme. The product OAS causes the release of OAS-TL from the complex which
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 63

enables the conversion of OAS to cysteine by the free enzyme, and in a negative
feedback, reduces the rate of OAS formation (Hesse et al. 2004b). Therefore,
formation of the cysteine synthase complex appears to be a main regulatory step
in cysteine synthesis (see the “Regulation of cysteine synthesis – protein-protein
interactions” section in this chapter).
The crystallisation of SAT protein revealed that it is a hexamer composed from
29 kDa subunits which are folded in a left handed parallel β-helix, characteristic for
this protein family (Olsen et al. 2004). The SAT gene family includes five members
in Arabidopsis, two of which were recognised only recently (Hell and Wirtz 2008;
Kawashima et al. 2005). Among the five SAT proteins three of them, SAT2, 4, and
5 (Serat 3.1, 3.2, and 1.1, respectively) are located in the cytosol whereas SAT1
(Serat 2.1) was found in plastids and SAT3 (Serat 2.2) in mitochondria. Addition-
ally, because of the very low substrate affinity of SAT2 and 4 compared to SAT1,
3, and 5, it was suggested that these isoenzymes may actually process different
substrates in vivo (Kawashima et al. 2005; Krueger et al. 2009). However, the
analysis of multiple knock-out mutants for different SAT isoforms revealed that all
of Arabidopsis SAT are able to complement, at least partially, for the loss of other
isoforms (Watanabe et al. 2008).
OAS-TL belongs to the β-replacement enzyme family which requires pyridoxal-
50 -phosphate as a cofactor. The protein is a homodimer composed from 35 kDa
subunits. The Arabidopsis gene family contains nine members which encode eight
functionally transcribed proteins (Hell and Wirtz 2008). Three of them, called
OAS-TL A, B, and C, are thought to be the main OAS-TL proteins in plant cells.
Similarly to SAT, they are localised in the cytosol, plastids and mitochondria,
respectively (Wirtz et al. 2004). OAS-TL proteins seem to have wide range of
functions. It is likely that apart from cysteine synthesis they are also responsible for
other processes such as sulfide and cyanide detoxification in mitochondria (Alvarez
et al. 2012) or determination of antioxidative capacity in cytosol (López-Martı́n
et al. 2008). They also seem to be involved in the synthesis of secondary metabo-
lites in various species.
Recent studies of SAT and OAS-TL mutants suggest the cytosol as the main cell
compartment for cysteine production and mitochondria as the main place for OAS
synthesis (Haas et al. 2008; Krueger et al. 2009; Watanabe et al. 2008). Plants with
decreased mitochondrial SAT activity show strongly reduced OAS levels and
reduced flux into cysteine and GHS (Haas et al. 2008). The analyses of OAS
content and SAT activity in non-aqueous gradients showed the largest amount of
OAS in mitochondria and the smallest in plastids, whereas the OAS-TL activity was
localised in the cytosol and plastids (Krueger et al. 2009). However, OAS can be
transferred between all three compartments. Additionally, the analysis of
compartment-specific OAS-TL mutants revealed reduced cysteine content only in
a mutant lacking the cytosolic OAS-TL isoform (Haas et al. 2008; Watanabe
et al. 2008). Consequently, OAS has to be transported from mitochondria to the
cytosol for efficient cysteine biosynthesis. Taking into account that plastids are the
main compartment for sulfide production, the presence of sulfide in cytosol also
requires sulfide transport across the chloroplast envelope membrane. Taken
64 P. Baraniecka and S. Kopriva

together, very low SAT activity in plastids, the presence of sulfide in the cytosol and
the cytosolic localisation of cysteine strongly suggest the cytosol as the main
cellular compartment for cysteine biosynthesis in Arabidopsis (Krueger
et al. 2009).

Glutathione Biosynthesis and Functions

Formation of cysteine is the terminal step of sulfate assimilation pathway and the
starting point for production of methionine, GHS and many other sulfur-containing
compounds (Fig. 3.3). GSH is the main thiol-containing molecule in plant cells and
is present in much higher concentrations than cysteine. It has a broad range of
functions, which include removal of reactive oxygen species (ROS), detoxification
of heavy metals and xenobiotics, sulfur donation, transport and storage (in catalytic
reactions), redox signalling and many others. It is synthesised from glutamate,
cysteine, and glycine by two enzymes: γ-glutamylcysteine synthetase (γ-ECS)
and glutathione synthetase (GSHS). The reaction consumes two ATP molecules.
γ-ECS is redox sensitive; only its oxidised form has high activity, whereas in
reduced form the activity is much lower. The increase in γ-ECS transcript level
in response to various environmental changes suggests its role as regulatory factor
(Xiang and Oliver 1998). γ-ECS is also inhibited by higher concentrations of GSH.
Synthesis of GSH is regulated by cysteine availability (Noctor et al. 2002). In
Arabidopsis thaliana GSH is synthesised in cytosol and plastids, whereas γ-ECS is
localised to plastids only and GSHS activity is distributed between plastids and
cytosol. GSH from leaves is transported to roots, seeds and fruits via the phloem
(Leustek et al. 2000), which supports an important role as a sulfur donor. In
addition, GSH degradation is an important process in plants, however, the mecha-
nism is not well understood. The main enzymes responsible for GSH degradation
include glutathione reductase, γ-glutamyltransferase (GGT), glutathione
S-transferase (GST), and glutaredoxin. It seems that GSH turnover in cells is
maintained mainly by GGT activities (Takahashi et al. 2011). However, the intra-
cellular degradation of GSH is independent of GGT and it is initiated by
γ-glutamylcyclotransferase (Ohkama-Ohtsu et al. 2008).

Methionine Biosynthesis

Methionine is a sulfur-containing amino acid belonging to the aspartate family of

amino acids together with lysine, threonine, leucine and isoleucine. It is essential
amino acid for mammals and must be obtained entirely from the diet. Plants are able
to synthesise it de novo from cysteine or homocysteine. Methionine plays important
roles in plant metabolism. First of all it is a protein component. It is also involved in
initiation of translation. S-adenosylmethionine (SAM), which is produced from
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 65

methionine is a methyl-group donor and precursor of important secondary metab-

olites. It was shown that 80 % of methionine is used for SAM synthesis whereas
20 % is incorporated into proteins (Giovanelli et al. 1985). Synthesis of methionine
requires products of three metabolic pathways: the carbon skeleton originates from
aspartate, the sulfur atom from cysteine, and the methyl group from serine (Ravanel
et al. 1998). In higher plants methionine synthesis starts from a γ-replacement
reaction catalysed by cystathionine γ-synthase (CGS) leading to formation of
cystathionine from cysteine and O-phosphohomoserine (OPH; Fig. 3.3).
Cystathionine is converted to homocysteine by α,β-elimination catalysed by
cystathionine β-lyase. The final step includes the transfer of the methyl group
from N5-methyl-tetrahydrofolate to homocysteine, which in plants is catalysed by
cobalamin-independent methionine synthase (MS; Hesse et al. 2004a; Ravanel
et al. 1998). In Arabidopsis three MS isoforms are known: two of them are in
cytosol and a third one is localised to the plastids (Ravanel et al. 2004). Subse-
quently methionine is converted to SAM by SAM synthetase which requires ATP.
It was shown that accumulation of SAM inhibits the enzyme activity (Ravanel
et al. 1998). When SAM is used for synthesis of ethylene or polyamines,
methylthioadenosine (MTA) is produced as intermediate. MTA can be used for
synthesis of another methionine molecule increasing SAM availability as methyl-
group donor (Burstenbinder et al. 2007).
Methionine synthesis undergoes a complex regulation. The pathway is regulated
by feedback inhibition of aspartate kinase: the carbon flux inhibits the synthesis of
methionine (Hesse et al. 2004a). It was also shown that CGS is involved in the
control of the pathway on the level of sulfur and carbon-nitrogen skeleton delivery
to the pathway (Hesse et al. 2004a). OPH is a common substrate for CGS and
threonine synthase (TS). TS affinity for OPH is significantly higher than CGS
affinity. TS is also positively regulated by SAM. Sufficient concentration of SAM
enhances the activity of TS and affinity for OPH (Curien et al. 1996). Carbon
skeletons are thus delivered to methionine biosynthetic pathway only when the
SAM concentration decreases (see also “Control of methionine biosynthesis –
posttranscriptional regulation” in the following sections of this chapter).

Sulfur-Containing Secondary Metabolites: Glucosinolates

and Phytosulfokines

Sulfur is also present in plant metabolites as sulfo-group-modifying carbohydrates,

proteins, and many natural products. Many sulfated metabolites play distinct roles
in plant defence against biotic and abiotic stresses. Some of the best known groups
of sulfated compounds are glucosinolates, which play an important role in protec-
tion against herbivores. They are also responsible for taste and flavour of many
Brassica vegetables (e.g. cabbage, broccoli). Products of glucosinolate degradation,
isothiocyanates, possess an anticarcinogenic activity in mammalian cells (Mithen
66 P. Baraniecka and S. Kopriva

Fig. 3.4 Schematic representation of glucosinolate hydrolysis (middle) including examples of

aliphatic, aromatic and indolic glucosinolates (left) and most common degradation products (right)

et al. 2003). In general, glucosinolates are synthesised in a three-block pathway.

The elongation of certain amino acids, which are the precursors of aliphatic or
aromatic glucosinolates, by sequential insertions of few (up to nine) methylene
groups into the side chain is the first phase of the process. Subsequently, the amino
acid moiety, elongated or not, is converted to form the core structure, which is
common to all glucosinolates. The final block concerns various modifications, such
as hydroxylation, O-methylation, desaturation, acylation and others, which leads to
the formation of a broad range of structures (Halkier and Gershenzon 2006). To
date, more than 140 different glucosinolates have been described (Fahey
et al. 2001). In Arabidopsis nearly 30 different glucosinolates have been found in
most organs, at various developmental stages (Brown et al. 2003). The chemical
structure of most of them consists of a β-D-thioglucose group linked to a (Z)-N-
hydroximinosulfate ester via a single sulfur atom (Halkier and Gershenzon 2006).
Although knowledge about the glucosinolate biosynthesis is important, it is the
degradation products that are responsible for all their biological functions (Fig. 3.4).
The process of hydrolysis begins with breakdown of the thioglucoside bond, which
leads to the formation of glucose and unstable aglycone. The latter may then
isomerise to different products depending on the structure of side chain and
availability of various cofactors. The initiation process is catalysed by myrosinase
(EC: 3.2.3.1) and has been investigated in a number of biochemical and molecular
studies. Myrosinase is separated from glucosinolates in idioblast cells to avoid
unnecessary glucosinolate hydrolysis. These two components, however, mix very
quickly after the loss of cellular integrity as a result of wounding or insect or
pathogen attack, to activate the binary glucosinolate – myrosinase system leading to
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 67

the generation of glucosinolate hydrolysis products, which serve as plant defence

molecules. The system, however, seems to work both ways: it deters usual patho-
gens but it may also attract some specialised herbivores (Renwick 2001). Many of
them use glucosinolates as a food or use glucosinolates-containing plants for
oviposit (Halkier and Gershenzon 2006).
Phytosulfokines (PSK) are another sulfur-containing metabolite of importance
to plant growth and fitness, which promote various stages of plant growth such as
somatic embryogenesis, adventitious bud and root formation and pollen germina-
tion (Chen et al. 2000; Kobayashi et al. 1999; Yamakawa et al. 1998). PSK-α is the
first sulfated peptide found in plants. Its unique signal strongly promotes cell
proliferation in plant cells at low concentrations. PSK-α is universally distributed
in the plant kingdom (Yang et al. 2000) and is synthesised from an ~80 amino acid
precursor peptide which has a secretion signal at its N-terminus (Yang et al. 1999).
In Arabidopsis five genes encoding the precursor peptide have been identified
(Yang et al. 2001). The expression of PSK genes in Arabidopsis is not limited to
tissues characterised by active cell division and differentiation. It has been detected
in most plant organs including mature leaves, stems, roots, and calluses, which
indicate that it is not a simple mitogen or differentiation initiator. PSK precursor
over-expression causes no apparent changes in plant growth or development under
normal growth conditions (Matsubayashi and Sakagami 2006). It acts by binding to
the specific binding sites which have been identified on the surface of suspension-
cultured cells and in plasma membrane-enriched fractions of various plant species
(Matsubayashi and Sakagami 1999). Based on the internal sequence of the
PSK-binding protein, the component of the functional PSK receptor, LRR-RLK,
was identified (Matsubayashi et al. 2002) and LRR-RLK Arabidopsis knock-out
mutants are used to study the in vivo function of PSK (Matsubayashi and Sakagami
2006). The same receptor is used by another sulfated peptide called PSY1 which is
also involved in the promotion of cell proliferation and expansion at nanomolar
concentrations (Amano et al. 2007). Recently discovered RGF family peptides
seem to be involved in root development (Matsuzaki et al. 2010; Meng
et al. 2012), however, the exact role of these proteins and its sulfation remains
unclear.

Regulation of Sulfate Assimilation

Understanding the tight regulation of the sulfate assimilation pathway is extremely

important for two reasons: the essential role of sulfur for plant growth and quality,
and the potential cytotoxicity of sulfite and sulfide, which are intermediates in
sulfate assimilation. Various stages of the assimilatory pathway are regulated in
both positive and negative feedback mechanisms in a demand-driven manner
(Fig. 3.5).
68 P. Baraniecka and S. Kopriva

Fig. 3.5 Current understanding of the regulatory network of sulfate metabolism in Arabidopsis
thaliana. Black arrows indicate positive regulation (induction) and red arrows indicate negative
regulation (repression). The grey circles correspond to the key regulatory factors of the pathway,
grey boxes correspond to all the other components involved in regulation. Blue colour indicates
processes, red – genes and proteins, green – metabolites. Abbreviations: APR APS reductase, MYB
MYB factors, APK APS kinase, SLIM1 Sulfur Limiting factor 1, SULTRs sulfate transporters, SAT
serine acetyltransferase, ATPS ATP sulfurylase, OAS-TL OAS(thiol)lyase, OAS O-acetylserine,
CSC cysteine synthase complex

Regulation on the Level of Sulfate Uptake and Transport

The efficient acquisition of sulfate from the soil and its distribution in the plant is of
great importance especially under sulfur limiting conditions. In a number of studies
it was shown that the rate of sulfate transport during low sulfate supply is driven
mainly by the regulation of the two high-affinity sulfate transporter genes,
SULTR1;1 and SULTR1;2 (Shibagaki et al. 2002; Takahashi et al. 2000; Vidmar
et al. 2000; Yoshimoto et al. 2002). The study of promoter-reporter constructs
indicated that both are regulated in response to sulfate nutrition (Maruyama-
Nakashita et al. 2004a). Further studies led to the identification of a transcription
factor, SLIM1 (sulfur limitation 1), which is responsible for the regulation of sulfate
uptake and metabolism during insufficient sulfur supply (Maruyama-Nakashita
et al. 2006) (Fig. 3.5). Slim1 mutants showed about 30 % reduction in root length
and a 60 % decrease of sulfate uptake rates in sulfur-limiting conditions. The
SLIM1 transcription factor belongs to the family of ethylene insensitive-like
(EIL) transcription factors, from which EIL3 has a specific function in the
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 69

regulation of sulfate uptake and metabolism (Maruyama-Nakashita et al. 2006).

EIL3 was the only protein from the EIL-family able to restore the wild type
phenotype of slim1 mutants and was therefore renamed SLIM1. SLIM1 regulates
the expression of the majority of sulfate-limitation responsive genes in the pathway,
which suggests a hub-like function in the regulation system. Interestingly, APR, the
key enzyme of sulfur metabolism, seems not to be subjected to the control of
SLIM1, which suggests that it is not the only factor involved in the regulation
process.
Most sulfur responsive genes are co-ordinately regulated for metabolism and
stress reduction. A sulfur-responsive cis-acting element (SURE) was identified first
in the sequence of the promoter of SULTR1;1 sulfate transporter (Maruyama-
Nakashita et al. 2005). SURE is a 7 nucleotide long specific sequence localised in
the 50 -region of SULTR1;1. This sequence contains the core sequence of the auxin
response factor (ARF) (Hagen and Guilfoyle 2002). However, SURE is sulfur-
specific and is not relevant to the auxin response. The analysis of sulfur limitation
inducible expression of SULTR1;1 revealed that the SURE element was an essential
target for the sulfur limitation response in Arabidopsis roots. Interestingly, SURE
was identified in SULTR1;1 but not in SULTR1;2, suggesting different mechanisms
of regulation of these two transporters (Maruyama-Nakashita et al. 2005). Indeed,
SULTR1;1 is known to be controlled more specifically by sulfur limitation whereas
the control of SULTR1;2 is more dependent on metabolic demand and cellular
status (Rouached et al. 2008). The in silico promoter analysis with GeneChip
microarrays of 15 genes, which are known to be up-regulated by sulfur limitation,
revealed the presence of a SURE core sequence (GAGAC or GTCTC) in the
promoters of all of these genes. Similar sequences are present in the NIT3 nitrilase
(Kutz et al. 2002) and β-conglycinin β-subunit (Awazuhara et al. 2002). Therefore it
was concluded that the SURE core sequences are conserved in sulfur-limiting
inducible promoters and may play a key role in sulfur limitation induction
(Maruyama-Nakashita et al. 2005). However, a number of genes regulated by sulfur
starvation do not have a SURE element. Despite the important role of SURE
elements in the sulfur limitation-inducible response, there are still many gaps in
our knowledge. Many important questions, such as specific SURE-binding candi-
dates, still require further investigation.
In addition to transcriptional regulation, sulfur metabolism genes are also con-
trolled post-transcriptionally. The main player in post-transcriptional regulation is
microRNA395 (miR395; Fig. 3.5). MiRNAs are a group of small RNAs, which are
formed from noncoding double-stranded RNA precursors. They are able to nega-
tively regulate their target genes by cleavage or by binding to the complementary
sequences in the target genes and repressing the translation (Bartel 2004). Analysis
of the Arabidopsis genome revealed the low-affinity sulfate transporter SULTR2;1
and ATPS1 and ATPS4 isoforms of ATPS as target genes for miR395 (Jones-
Rhoades and Bartel 2004). The accumulation of miR395 increases during sulfate
starvation and this process is dependent on SLIM1 (Kawashima et al. 2009).
Further studies revealed that miR395 regulates sulfate accumulation in shoots by
cleavage of ATPS isoforms 1 and 4 during sulfate starvation. MiR395 was also
70 P. Baraniecka and S. Kopriva

shown to play a role in sulfate translocation from old to young leaves by targeting
SULTR2;1 (Liang et al. 2010). Subsequently Kawashima et al. (2011) in experi-
ments on SLIM1 and miR395-dependent regulation, reported an increase in
SULTR2;1 mRNA levels, a decrease in ATPS4 levels and no changes in ATPS1
mRNA levels in sulfate-limited conditions, suggesting three different mechanisms
of miR395-mediated regulation. The increased expression of SULTR2;1 in roots
during sulfate starvation is limited to xylem parenchyma cells (Kawashima
et al. 2009). The decrease in mRNA levels of ATPS4 following induction of
miR395 suggests a canonical regulation of ATPS4 by miR395. The lack of response
in ATPS1 transcripts is in contrast to results obtained by Jones-Rhoades and Bartel
(2004) who observed a decrease in ATPS1 mRNA, however, these differences may
be caused by different experimental setups. The increase in SULTR2;1 expression
in the xylem and reduction of flux through sulfate assimilation in the roots caused
by miR395 indicate the importance of the SLIM-1-dependent induction of miR395
for the increased translocation of sulfate to the shoots when sulfate is limited. This
results in an increase in the efficiency of sulfate assimilation in leaves (Kawashima
et al. 2011). Very recently Matthewman and co-workers (2012) have shown that the
complex regulation of miR395 is linked not only to SLIM1-dependent regulation
during sulfate starvation but also to GHS and more generally thiol levels and/or cell
redox state. Increased expression of miR395 in fou8 and sultr1;2 mutants affected
in sulfate accumulation, suggests that miRNA395 is regulated by the internal
sulfate level irrespective of external sulfate availability. All these results confirm
that miR395 is an integral component of the sulfate assimilation regulatory network
in a complex regulation mechanism.

Regulation of Sulfate Assimilation

Sulfate assimilation is tightly regulated in response to sulfate demand and environ-

mental changes. The control mechanisms appear on different steps of the pathway
and involve regulation of specific enzymes. Additionally, the whole pathway may
be controlled as a process. Experiments focused on the first step of the pathway
revealed the regulation of ATPS by sulfate availability (Logan et al. 1996). It was
also shown that ATPS activity is inhibited by GSH. However, a number of studies
revealed that the key regulatory step is sulfate reduction by APR (Fig. 3.5), as it is
strongly affected by various treatments and environmental changes (Hesse
et al. 2003; Koprivova et al. 2008; Vauclare et al. 2002). Vauclare et al. (2002) in
the GSH feeding experiment showed that an increase of GSH concentration in the
medium decreases APR expression and activity, indicating its regulation by GSH
rather than cysteine. They also estimated the flux control coefficient for APR as
0.57, considering that the coefficient of all enzymes in the sulfate uptake and
reduction pathway is up to 1, confirming the importance of APR in control of flux
through sulfate assimilation pathway. The analysis of natural variation in sulfate
content between two wild Arabidopsis accessions Bay-0 and Shahdara (Sha),
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 71

performed to identify the genes controlling sulfate level, also revealed APR as a key
control step in sulfate reduction pathway. The analysis of Bay-0 x Sha recombinant
inbred lines (RILs) led to the identification of a single nucleotide polymorphism in
an APR2 isoform of APR. The substitution of alanine with glutamate in a conserved
domain of protein resulted in significant differences in enzyme activity leading to
sulfate accumulation (Loudet et al. 2007). Additionally, reduction of APR activity
and mRNA accumulation in low nitrogen availability shown in this study confirmed
an interconnection of the two assimilatory pathways. Furthermore, APR over-
expression leads to accumulation of sulfite and thiosulfate which are toxic for
plants and strongly affect plant fitness (Martin et al. 2005). Such an effect is not
observed when over-expressing other enzymes in the pathway suggesting that APR
regulation affects the entire pathway. It was also shown that APR activity increases
during the day and decreases during the night, which shows that it has a diurnal
rhythm (Kopriva et al. 1999). A recently discovered transcription factor, Long
Hypocotyl 5 (HY5), seems to be responsible for APR regulation by light. In dark
adapted Arabidopsis seedlings, a rapid increase in the transcript levels of all three
APR isoforms was observed to different extents, the highest being for the APR2
isoform where it reached a 12-fold higher level after 90 min of illumination,
compared to the control plants kept in the dark. However, in hy5 mutant seedlings
no light induction was observed for APR1, and APR2 induction was lower. Further
analysis revealed that HY5 is involved in APR regulation by OAS and nitrogen
deficiency which alter the demand for reduced sulfur (Lee et al. 2011). Interest-
ingly, the analysis of the sir1-1 mutant revealed the downregulation of ATPS4,
APR2, and SULTR2;1 in the mutant (Khan et al. 2010). The most likely reason for
the downregulation of these genes, especially ATPS4 and APR2, is to avoid the
accumulation of sulfite which cannot be incorporated into cysteine as a result of
reduced SiR activity in the mutant. These results suggest that SiR can contribute to
the control of sulfate reduction pathway (Khan et al. 2010).

Regulation of Cysteine Synthesis – Protein-Protein

Interactions

Cysteine synthesis plays an important role in regulation of sulfate metabolism. The

regulation of SAT and OAS-TL, the main enzymes in cysteine biosynthesis, is
mainly due to a protein-protein interaction in the cysteine synthase complex (CSC;
Fig. 3.5). As mentioned above, SAT is strongly activated by OAS-TL which is
inactive in the complex and has only a regulatory role (Droux et al. 1998). Forma-
tion of the complex is strongly dependent on the availability of OAS and sulfide.
When SAT is bound to OAS-TL, the access of OAS to the complex is strongly
inhibited (Francois et al. 2006). OAS, which cannot be bound to the complex, is
released and metabolised by free OAS-TL dimers. During sulfur deficiency there is
not enough sulfide for cysteine synthesis and therefore OAS accumulates in cells.
72 P. Baraniecka and S. Kopriva

Accumulated OAS dissociates from the CSC complex, which rapidly decreases
SAT activity (Hell and Wirtz 2008). High OAS concentration increases the expres-
sion of sulfate transporter genes, APR, SAT and OAS-TL. This leads to increased
sulfate uptake and reduction and equilibrates the system (Hopkins et al. 2005;
Koprivova et al. 2000; Smith et al. 1997). Hubberten et al. (2012) have shown
recently that OAS may serve as a signalling molecule and change the transcription
levels of specific genes irrespective of the sulfur status in the plant. The feedback
inhibition of the cytosolic isoform of SAT by cysteine content serves as another
form of regulation in Arabidopsis. It is important to note that in A. thaliana,
plastidial and mitochondrial isoforms of SAT remain mostly insensitive to the
changes of cysteine content (Noji et al. 1998). Cytosolic SAT activity may be
considered as important for control of OAS concentration. The cysteine insensitive
SAT isoforms in organelles may allow independent formation of cysteine (Noji
et al. 1998). Although the regulation of cysteine biosynthesis is very important for
sulfur homeostasis in plants and therefore it has been intensively studied over the
past few years, many important aspects still require further investigation.

Control of Methionine Biosynthesis – Post-transcriptional

Regulation

Methionine biosynthesis is subject to complex regulation. The whole pathway is

controlled by feedback inhibition of aspartate kinase by lysine, threonine or lysine
together with SAM. It seems that the competition between CGS and TS for their
common substrate, O-phosphohomoserine, has an important regulatory role in the
flux of carbon into methionine, which can be a limiting factor in the process (Hesse
et al. 2004a). A recent study of transgenic Arabidopsis plants provided evidence
that the regulation of methionine biosynthesis also occurs at the posttranscriptional
level (Chiba et al. 1999, 2003). The analysis of this process is focused on the MTO1
region in exon 1 of CGS (Suzuki et al. 2001). The MTO1 mRNA region may act in
cis and destabilise CGS mRNA in response to high concentrations of methionine or
SAM (Chiba et al. 1999, 2003; Lambein et al. 2003). Computational analysis
revealed the possible formation of a stable stem-loop structure in the MTO1 region
which could support the posttranscriptional mechanism of regulation (Amir
et al. 2002). More recently the presence of a truncated form of CGS transcript in
Arabidopsis was demonstrated (Hacham et al. 2006). This transcript lacks about
90 nucleotides from the first exon. Over-expression of this CGS transcript causes
even higher levels of methionine than the over-expression of full length CGS. This
may suggest that this truncated transcript is not subject to feedback regulation by
methionine (Hacham et al. 2006).
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 73

Control of Glucosinolate Biosynthesis – MYB Transcription

Factors

Glucosinolate biosynthesis is highly regulated by a network of transcription factors

in response to biotic and abiotic stress (Gigolashvili et al. 2007, 2008; Sønderby
et al. 2007). They belong to two groups of the R2R3-MYB family of transcription
factors. The first group consists of three members: MYB28, MYB76 and MYB29,
and is involved in the control of aliphatic glucosinolate biosynthesis (Gigolashvili
et al. 2008). The second group is responsible for control of biosynthesis of indolic
glucosinolates and includes MYB51, MYB122, and MYB34 (Gigolashvili
et al. 2007). Glucosinolates controlled by members of these two groups can be
alternatively called high aliphatic glucosinolates (1–3) and high indolic
glucosinolates (1–3), respectively (Fig. 3.5). T-DNA insertions, RNAi or over-
expression of these factors affects the expression of glucosinolate biosynthesis
genes and the level of glucosinolates. MYB34, MYB51 and MYB122 have differ-
ent functions in the regulation of the pathway (Gigolashvili et al. 2007). Although
all of them up-regulate the genes from the biosynthesis pathway, MYB34 and
MYB122 also function as stimulators of auxin biosynthesis, whereas MYB51
additionally activates the glucosinolate biosynthesis pathway (Gigolashvili
et al. 2007). Studies on MYB28, MYB29 and MYB 76 showed that they are able
to transactivate each other in control of biosynthesis of aliphatic glucosinolates.
They also downregulate the expression of enzymes involved in the synthesis of
indolic glucosinolates (Gigolashvili et al. 2008). Analysis of MYB28 and MYB29
revealed that MYB28 is essential for the synthesis of aliphatic glucosinolates
whereas MYB29 induces biosynthetic genes in response to plant hormone methyl
jasmonate (Hirai et al. 2007). Maruyama-Nakashita and co-workers (2006) showed
that mutation of SLIM1 additionally affects the expression of MYB34, suggesting
that this factor may be negatively controlled by SLIM1 in response to sulfur
deficiency. There is some evidence that SLIM1 also might affect the expression
of MYB28 and MYB29, however the effect is not completely clear (Hirai
et al. 2007). More recently, it was suggested that MYB factors are not only involved
in the regulation of the glucosinolate biosynthesis genes, but also other genes in the
sulfur reduction pathway. Yatusevich et al. (2010) concluded that APR1 and APR3
are under control of all MYB factors whereas APR2 is regulated by only some of
them. They suggested also that APK1 and APK2 are a part of the glucosinolate
synthesis network controlled by MYB factors, whereas APK3 and APK4 have much
lower input to the network. Glucosinolate biosynthesis may be also regulated by the
DNA-binding-with-one-finger (DOF) transcription factor, OBP2, which is induced
by herbivory and methyl jasmonate (Skirycz et al. 2006). Additionally, changes in
expression of calmodulin binding IQD protein cause the variation in glucosinolate
composition (Levy et al. 2005).
74 P. Baraniecka and S. Kopriva

Regulation by Hormone Signals

Metabolic regulation is not the only means of controlling metabolic pathways. Plant
hormones play very important roles in many developmental processes. Recent
studies show involvement of plant hormones in regulation of different nutrient
metabolic pathways (Fig. 3.5).
Cytokinins are adenine-derived plant hormones, which are responsible for reg-
ulation of cell division and differentiation in plants together with auxin. The best
known example of regulation of the sulfate reduction pathway by cytokinin is the
downregulation of high-affinity transporters SULTR1;1 and SULTR1;2
(Maruyama-Nakashita et al. 2004b). Addition of cytokinins to wild type plants
decreases sulfate uptake and mRNA levels for the two transporters. It is interesting
that SULTR1;2 is much more responsive to the cytokinins than SULTR1;1. It was
suggested that in this regulation, the cytokinin response 1 (CRE1)/wooden leg
(WOL)/Arabidopsis histidine kinase 4 (AHK4) cytokinin receptor is involved.
The Arabidopsis cre1-1 mutant was unable to regulate the high-affinity sulfate
transporters in response to cytokinins (Maruyama-Nakashita et al. 2004b)
suggesting the independent regulation of high-affinity sulfate transporters by cyto-
kinin (repression) and by sulfate (induction). In contrast, Ohkama and co-workers
(2002) have shown that sulfur responsive genes APR1 and SULTR2;2 were
up-regulated by cytokinin. They have concluded that this induction is mediated
by an increase in sucrose content.
Auxin-dependent signalling in the regulation of sulfate assimilation may be
connected to the response to sulfate deficiency by indole glucosinolate hydrolysis
(Kutz et al. 2002). During sulfate deficiency aglycon is released from indole
glucosinolates and indole acetic acid (IAA) is generated from the remaining indole
acetonitrile, catalysed by nitrilase NIT3. IAA may stimulate root growth, and
indeed an increase in length and the numbers of lateral roots is a common pheno-
type of sulfur-deficient plants (Hell and Hillebrand 2001). Additionally, various
studies indicate positive regulation of auxin-responsive genes during sulfate star-
vation (Maruyama-Nakashita et al. 2003; Nikiforova et al. 2003). Decrease in
cysteine production leads to accumulation of OAS and its precursor serine and
sulfate deficiency induces tryptophan synthase. Both of these events result in
increase in tryptophan biosynthesis as in plants it is synthesised from indole and
serine through the activity of the tryptophan synthase β-subunit. In consequence,
increased biosynthesis of tryptophan increases production of auxin (Nikiforova
et al. 2003).
Jasmonic acid (JA) is another plant hormone, which participates in regulation of
sulfate metabolism. JA is involved in response to oxidative stress and synthesis of
defence molecules. The cellular GSH content rapidly decreases during sulfur
deficiency and this may lead to oxidative stress in cells. It was shown, however,
that JA increases the expression of GSH synthesis pathway enzymes (Xiang and
Oliver 1998). Additionally, microarray studies showed induced expression of JA
genes during sulfur limitation and in sultr1;2 mutants (Maruyama-Nakashita
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 75

et al. 2003; Nikiforova et al. 2003). JA was also shown to induce the expression of
the genes involved in glucosinolate biosynthesis (Brader et al. 2001; Doughty
et al. 1995). JA-deficient mutants showed normal responses to sulfur limitation,
similar to those when sulfate transporters and APR are induced and glucosinolate
biosynthesis genes are repressed under sulfur starvation (Takahashi and Saito
2008). JA significantly induced expression and activity of APR, mainly isoforms
1 and 3 (Koprivova et al. 2008).
To summarise, JA acts positively for synthesis of both antioxidants and
glucosinolates and has an important general role in plant pathogen defence and
detoxification. It also co-ordinately induces multiple genes of sulfate assimilation
suggesting its positive effect on sulfur homeostasis in plants (Jost et al. 2005).

Interconnection of Sulfate Assimilation with Other Nutrients

The sulfate assimilation pathway provides a range of sulfur-containing metabolites

which play essential roles in a number of cellular processes. Many reports have
shown that this pathway is very well co-ordinated with the assimilation of other
nutrients such as nitrate, phosphate or carbon. This interconnection is crucial in
dealing with the deficiency of one or more nutrients. It leads to activation or
deactivation of important regulatory components. Such cross-talks provide flexi-
bility in the adaptation to fluctuating environmental conditions, which helps plants
to maintain nutrient homeostasis and thus complete the life cycle.

Regulation of Sulfate Assimilation by Nitrogen

The regulatory interactions between sulfate and nitrate assimilation in plants have
been confirmed in a number of studies (Kim et al. 1999; Koprivova et al. 2000;
Takahashi and Saito 1996). These two pathways are coordinated in the way that
deficiency of one element represses the assimilation of the other. Studies on Lemna
minor revealed that the activities of ATPS, APR and OAS-TL decreased during
nitrogen deficiency (Brunold and Suter 1984). Addition of nitrate or ammonia to
nitrate-deficient medium restored the activity of these enzymes very quickly.
Additionally, when a normal nutrient solution was supplemented with ammonia
or amino acids such as arginine, glutamate or aspartate, APR activity increased
about 50–110 %. These nitrogen compounds also increased the flux through the
sulfate assimilation pathway (Brunold and Suter 1984). Koprivova et al. (2000)
have shown that after 72 h of nitrate deficiency in Arabidopsis, the activity of APR
decreased about 30 % in leaves and 50 % in roots. This is correlated with the
decrease of APR mRNA and protein accumulation, which suggests that nitrogen
availability regulates sulfate assimilation on the transcriptional level. This finding is
supported by the results of Yamaguchi and co-workers (1999) who have shown that
76 P. Baraniecka and S. Kopriva

nitrogen deficiency affects the mRNA accumulation of several other sulfate metab-
olism genes. Interestingly, the concentration of cysteine and GSH in both roots and
leaves does not change significantly. Addition of ammonia or glutamate increased
the sulfate flux through the Arabidopsis plants (Koprivova et al. 2000). Analysis of
the hy5 mutant revealed that the decrease in APR activity in nitrate deficiency was
much more rapid in Col-0 than in the mutant suggesting HY5 involvement in
regulation of APR in nitrate deficiency (Lee et al. 2011). Although this regulatory
mechanism seems to play an important role in sulfate homeostasis, the molecular
basis still requires elucidation. Koprivova and co-workers (2000) used feeding
experiments to demonstrate a strong positive effect of nitrogen-containing com-
pounds on the flux of sulfate through the pathway. Moreover, the closer the
metabolic relationship between nitrogen source and OAS, the higher was the
incorporation of 35S into proteins. OAS also caused the accumulation of APR
mRNA level and proteins (Koprivova et al. 2000). Therefore, it was proposed to
play a signalling role in the co-ordination of nitrate and sulfate metabolic pathways.
Other suggested signalling molecules are cytokinins: biosynthesis of cytokinins
seems to be related to nitrate availability and they are known to affect nitrogen
nutrition. As mentioned above, Ohkama et al. (2002) have shown that the APR1 and
SULTR2;2 were up-regulated by cytokinins. Additionally, Wang et al. (2003) dem-
onstrated that adding nitrate to plants grown on ammonium as the main source of
nitrogen increased the accumulation of mRNA for high-affinity transporters and
APR. Therefore, the nitrate effect on sulfate metabolism genes could be mediated
by cytokinins (Kopriva and Rennenberg 2004). On the other hand, it was shown that
sulfate deficiency decreased nitrate uptake and the activity of nitrate reductase and
amino acid accumulation (Prosser et al. 2001). Abdallah et al. (2010) have shown
that oilseed rape, which is known to have a high demand for sulfate, is able to
maintain growth during sulfate deficiency by an optimisation of nitrogen uptake
and remobilising sulfate from internal reservoirs. In field experiments with oilseed
rape it was demonstrated that sulfur deficiency reduces nitrogen use efficiency and
nitrogen deficiency reduces sulfate use efficiency (Fismes et al. 2000). This sug-
gests the importance of further investigation of regulatory processes of nutrient
metabolism in plants in order to improve crops yield and quality.

Interaction of Sulfate Assimilation with Carbon Metabolism

Carbohydrates provide the acceptor of sulfide for cysteine biosynthesis and serve as
a source of reductants for sulfate reduction in non-photosynthetic tissues (Kopriva
and Rennenberg 2004). However, despite this crucial role very little is known about
regulation of sulfate assimilation by carbon. Kopriva et al. (1999) have shown that
APR can be induced by light, suggesting a diurnal rhythm of regulation. However,
when plants were subjected to continuous light or darkness the diurnal rhythm
disappeared. This indicates that the light is not the direct signal for the changes in
APR expression and the enzyme undergoes regulation by carbohydrates
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 77

synthesised during photosynthesis. Furthermore, light induction of APR expression

could be mimicked by addition of sucrose during the dark period (Kopriva
et al. 1999). Subsequently, Hesse and co-workers (2003) have confirmed similar
effect of glucose on APR activity. In addition, they have shown that sorbitol,
mannitol and 2-deoxyglucose do not affect APR activity. Sugars seem to act in
the same way as OAS: feeding plant with glucose and OAS simultaneously resulted
in greater induction of APR activity than the sum of levels induced by individual
treatments. Additionally, APR was also induced by glucose in plants subjected to
nitrogen deficiency. Hesse et al. (2003) concluded that signals from nitrogen and
carbon metabolism act synergistically on sulfate assimilation. Moreover, positive
signals from sugars can exceed negative signals from nitrate assimilation. The
reduction in APR mRNA level and activity was also observed in a study on
Lemna minor grown in an atmosphere without CO2 (Kopriva et al. 2002). The
addition of sucrose prevented the decrease in APR activity indicating that this
regulation is not dependent on CO2 fixation. Additionally, it was shown that
incubation in an atmosphere without CO2 strongly inhibited sulfate uptake and
the flux through the pathway, which also could be restored by addition of sucrose.
However, sucrose was not as effective in restoring enzyme activities as normal air,
suggesting that although it is the final product of carbon assimilation, it probably is
not the molecular signal connecting the sulfate and carbon metabolism (Kopriva
et al. 2002).

Coordination of Sulfate and Phosphate Homeostasis

Plants have evolved coordinated mechanisms to maintain the homeostasis of sulfur

and phosphorus in response to changing environmental conditions, which confirms
the importance of these two macroelements. It was shown that during sulfate
limitation, sulfolipids are rapidly replaced by phospholipids and vice versa
(Essigmann et al. 1998). Additionally, phosphate deficiency leads to an increase
of GSH levels (Kandlbinder et al. 2004). It is interesting to note the similarity
between the topology and regulatory mechanisms in the two pathways (Rouached
2011). In addition to the similar expression patterns of the main uptake transporters
and the regulation of both of the pathways by cytokinins, the involvement of
microRNAs in the regulation of both pathways seems to be particularly noteworthy
in terms of cross-talk. As mentioned above, sulfate assimilation is regulated by
miR395, whereas miR399 is known to be involved in regulation of phosphate
uptake and translocation (Chiou et al. 2006; Fujii et al. 2005; Lin et al. 2008). It
has been shown that miRNAs and the Phosphate Response 1 (PHR1) transcription
factor are involved in the interconnection of sulfate and phosphate assimilation
pathways (Rouached 2011). Additionally, it was suggested that miR395 might be
suppressed in phosphate deficient plants (Hsieh et al. 2009), however, this obser-
vation requires further investigation. Recently Rouached et al. (2011) have shown
that SULTR1;3, SULTR2;1 and SULTR3;4 sulfate transporters are up-regulated by
78 P. Baraniecka and S. Kopriva

phosphate deficiency. Induction of SULTR1;3 and SULTR3;4 was repressed by

phosphite which is known to be involved in local phosphate sensing and long-
distance signalling (Varadarajan et al. 2002). In contrast, SULTR2;1 was shown to
belong to the class of phosphate-inducible genes not responding to phosphite
(Rouached et al. 2011). Additionally, they have shown that PHR1 plays an impor-
tant role in sulfate homeostasis in plants grown under phosphate deficient condi-
tions by stimulating the expression of SULTR1;3 whereas SULTR2;1 and
SULTR3;4 seem to be repressed by PHR1 in shoots only. These results suggest
an important effect of phosphate as a signaling factor in maintaining source-sink
sulfate distribution (Rouached et al. 2011).
To maintain sulfur homeostasis, plants need to cope with rapid environmental
changes, which cause disturbances in levels of pathway intermediates and products.
Therefore, a tight regulation of sulfate uptake and assimilation is required
according to the demand for reduced sulfur. To ensure the precise modulation of
sulfur metabolism, sulfate uptake and assimilation have to be coordinated. Indeed,
sulfur limitation increases sulfate uptake and the expression and activity of key
enzymes of the pathway. Other enzymes such as SiR and SAT are also involved in
the control of sulfur metabolism, as disturbances in their activity cause growth
defects. Sulfur uptake and assimilation are regulated on transcription, post-
transcriptional and post-translational levels. However, some aspects of the control
are specific only for uptake, such as regulation of sulfate transporters by SLIM1,
and some only for assimilation. This suggests different mechanisms of achieving
efficient control and maintaining system homeostasis. However, there are still gaps
in the understanding of regulation of uptake, assimilation and distribution of sulfate
at the whole plant level, the examination of which is a great challenge for the future
research. Better understanding of molecular basis of control mechanisms will allow
the production of engineered plants with improved sulfur use efficiency, with
modulated amount of desirable sulfur-containing metabolites or with better resis-
tance to low sulfur availability, drought and many other stresses.

Dealing with Sulfur Deficiency

As it was mentioned at the beginning of this chapter the reduction of sulfur dioxide
emissions and changes in fertiliser practises have resulted in a widespread increase
in the occurrence of sulfur deficiency in crops (McGrath et al. 1996). The applica-
tion of fertilisers may be helpful in dealing with deficiency in many instances.
However, the costs of fertilisers together with the possible negative effects of
improper use with respect to timing and type of sulfur application in terms of its
availability to the plant have led to the growing interest in looking for new
solutions. Very often a substantial seasonal variation in sulfur availability occurs.
Therefore, modern methods should be focused on uptake maximisation when sulfur
is abundant in order to increase the tolerance to low sulfur availability periods
(Hawkesford 2000).
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 79

Sulfur deficiency may not be easily recognised in the field as the symptoms are
not obvious, except in severely deficient plants. In general, sulfur deficiency results
in uniform pale green chlorosis through the plant. A considerable reduction in
growth may be suffered without the appearance of any other visible symptoms.
Clear symptoms are associated with severe stunting, reduced leaf size and activity
of axillary buds, which results in less branching. Physiologically in wheat plants,
sulfur deficiency affects CO2 assimilation rates and Rubisco enzyme activity as
well as protein abundance which results in general inhibition of de novo synthesis
of the photosynthetic apparatus (Gilbert et al. 1997). Additionally, depression of
root hydraulic conductivity was observed in sulfur-deficient barley plants. It was
suggested that this response can have a role in signalling nutrient starvation from
shoots to roots (Karmoker et al. 1991). Sulfur deficiency is dependent on the soil
type and predominant climatic conditions and does not occur uniformly. Therefore,
a reliable field based test is required to determine where it is likely to occur (Blake-
Kalff et al. 2000). Soil testing can provide information about the amount of sulfur
available to plants. However, there is no direct correlation between the content of
sulfate in the soil solution and plant yield in field conditions (Zhao and McGrath
1994). Thus, the analysis of sulfur content in plant tissue might be a better indicator
of sulfur available for the plant at the time of sampling. However the determination
of a reliable diagnostic indicator is problematic because of constantly changing
environmental conditions, variation in sulfur metabolism between species and plant
demands (Blake-Kalff et al. 2000).
Genetically engineered plants are one of the most promising approaches to
achieve an enhancement of sulfate use efficiency. Possible targets for genetic
engineering might be focused on improved resource capture or efficient utilisation
of increased uptake (Hawkesford 2000). The first goal could be achieved by
modulation of transport systems. It is known that the expression of sulfate trans-
porters is controlled by sulfur availability. During sulfur limitation the expression
of sulfate transporter genes increases significantly, but it decreases when sulfate is
abundant. Overriding this control might be achieved by expressing transporter
genes under the control of an appropriate constitutive promoter. However, the
control mechanisms would have to be removed only for sulfate transport and not
for other steps of the pathway in order to prevent for example accumulation of
sulfide, which is toxic for plants in high concentration. Alternatively, root structure
and proliferation could be targeted. The second goal might be achieved by improv-
ing the mobilisation of vacuolar reserves or introducing an increased demand for
sulfate to stimulate further sulfate uptake (Hawkesford 2000). In order to find the
most efficient targets, further exploration of sulfate metabolism pathway and the
regulatory mechanisms is required.
80 P. Baraniecka and S. Kopriva

Natural Variation in Model Plants as a Tool to Improve

Crop Quality

Restrictive regulations for commercial breeding of genetically modified plants have

persuaded breeders and scientists to look for alternative methods to improve crop
yield and quality. The result is a growing interest in naturally occurring genotypic
variation. The enhancement of crop productivity is fundamental for human society.
Therefore, exploring natural variation is of rapidly increasing importance to
improve quality aspects that are associated with the chemical composition of
agricultural products. The potential of wild species in the analysis of the molecular
genetic basis of plant nutrition for crop improvement was recognised a century ago
(Bessey 1906), however this area of research started to expand only recently (Fernie
et al. 2006; McCouch 2004; Zamir 2001).
To investigate natural variation in model plants and in crops two main
approaches are used. The first, the more traditional and still most popular way, is
quantitative trait loci (QTL) analysis (Koornneef et al. 2004). It is based on the
mapping of segregating populations such as a RILs derived from the crosses
between two or more parental accessions. This leads to the localisation in the
genome of loci exerting a control of a complex phenotypic trait in a given envi-
ronment. A second approach is to use Arabidopsis natural accessions to identify
polymorphisms associated with adaptation by exploiting a genome-wide associa-
tion mapping (GWAS) also called linkage disequilibrium (LD) (Atwell et al. 2010).
In contrast to QTL, in GWAS there are no crosses or pedigrees required, which
makes it easier to collect the data. It also has advantages over QTL analysis because
there are usually more alleles in the mixed population than in the two parents of the
cross (Myles et al. 2009).
Mineral use efficiency, root uptake, translocation of nutrients from the roots to
the shoots, and their accumulation in the seeds are the traits that, with many others,
undergo a substantial natural variation. To date the QTL studies in crops have been
focused mainly on seed or leaf mineral concentration in rice, wheat, barley,
soybean, and Brassica species (Alonso-Blanco et al. 2009). Natural variation pro-
vides a complementary resource to discover novel gene functions as well as the
variants of alleles that interact specifically with the genetic background or environ-
mental changes, which is of great interest for the study focused on plant adaptation
(Benfey and Mitchell-Olds 2008). This approach also allows creation of correlation
networks between different nutrients that provide an overview of plant demands in
various environments (Buescher et al. 2010). For example, a recent QTL study has
identified a group of six genes which are involved in complex control of the mineral
homeostasis in Arabidopsis thaliana (Ghandilyan et al. 2009). These results illus-
trate that the study of natural variation has significantly expanded our understanding
of plant mineral nutrition, its regulation, and plant adaptation to environmental
changes. The wide genetic variation allows the modification of quantitative traits
that often could not be achieved through mutagenesis or transgenic approaches.
They should therefore be of major interest for commercial agriculture. More detailed
descriptions of natural variation approaches can be found in Chap. 2 of this book.
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 81

References

Abdallah M, Dubousset L, Meuriot F, Etienne P, Avice JC, Ourry A (2010) Effect of mineral
sulphur availability on nitrogen and sulphur uptake and remobilization during the vegetative
growth of Brassica napus L. J Exp Bot 61:2635–2646
Alonso-Blanco C, Aarts MG, Bentsink L, Keurentjes JJ, Reymond M, Vreugdenhil D, Koornneef
M (2009) What has natural variation taught us about plant development, physiology, and
adaptation? Plant Cell 21:1877–1896
Alvarez C, Garcia I, Romero LC, Gotor C (2012) Mitochondrial sulfide detoxification requires a
functional isoform O-acetylserine(thiol)lyase C in Arabidopsis thaliana. Mol Plant
5:1217–1226
Amano Y, Tsubouchi H, Shinohara H, Ogawa M, Matsubayashi Y (2007) Tyrosine-sulfated
glycopeptide involved in cellular proliferation and expansion in Arabidopsis. Proc Natl Acad
Sci U S A 104:18333–18338
Amir R, Hacham Y, Galili G (2002) Cystathionine gamma-synthase and threonine synthase
operate in concert to regulate carbon flow towards methionine in plants. Trends Plant Sci
7:153–156
Aravind L, Koonin EV (2000) The STAS domain – a link between anion transporters and
antisigma-factor antagonists. Curr Biol 10:R53–R55
Atwell S, Huang YS, Vilhjalmsson BJ, Willems G, Horton M, Li Y, Meng D, Platt A, Tarone AM,
Hu TT, Jiang R, Muliyati NW, Zhang X, Amer MA, Baxter I, Brachi B, Chory J, Dean C,
Debieu M, de Meaux J, Ecker JR, Faure N, Kniskern JM, Jones JD, Michael T, Nemri A,
Roux F, Salt DE, Tang C, Todesco M, Traw MB, Weigel D, Marjoram P, Borevitz JO,
Bergelson J, Nordborg M (2010) Genome-wide association study of 107 phenotypes in
Arabidopsis thaliana inbred lines. Nature 465:627–631
Awazuhara M, Kim H, Goto DB, Matsui A, Hayashi H, Chino M, Kim S-G, Naito S, Fujiwara T
(2002) A 235-bp region from a nutritionally regulated soybean seed-specific gene promoter can
confer its sulfur and nitrogen response to a constitutive promoter in aerial tissues of
Arabidopsis thaliana. Plant Sci 163:75–82
Awazuhara M, Fujiwara T, Hayashi H, Watanabe-Takahashi A, Takahashi H, Saito K (2005)
The function of SULTR2;1 sulfate transporter during seed development in Arabidopsis
thaliana. Physiol Plant 125:95–105
Barberon M, Berthomieu P, Clairotte M, Shibagaki N, Davidian JC, Gosti F (2008) Unequal
functional redundancy between the two Arabidopsis thaliana high-affinity sulphate
transporters SULTR1;1 and SULTR1;2. New Phytol 180:608–619
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Baxter I, Muthukumar B, Park HC, Buchner P, Lahner B, Danku J, Zhao K, Lee J, Hawkesford MJ,
Guerinot ML, Salt DE (2008) Variation in molybdenum content across broadly distributed
populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter
(MOT1). PLoS Genet 4(2):e1000004. doi:10.1371/journal.pgen.1000004
Benfey PN, Mitchell-Olds T (2008) From genotype to phenotype: systems biology meets natural
variation. Science 320:495–497
Bessey CE (1906) Crop improvement by utilizing wild species. J Hered 2:112–118
Bick JA, Aslund F, Chen Y, Leustek T (1998) Glutaredoxin function for the carboxyl-terminal
domain of the plant-type 50 -adenylylsulfate reductase. Proc Natl Acad Sci U S A 95:8404–8409
Blake-Kalff MM, Harrison KR, Hawkesford MJ, Zhao FJ, McGrath SP (1998) Distribution of
sulfur within oilseed rape leaves in response to sulfur deficiency during vegetative growth.
Plant Physiol 118:1337–1344
Blake-Kalff MMA, Hawkesford MJ, Zhao FJ, McGrath SP (2000) Diagnosing sulfur deficiency in
field-grown oilseed rape (Brassica napus L.) and wheat (Triticum aestivum L.). Plant Soil
225:95–107
Blake-Kalff MMA, Zhao FJ, Hawkesford MJ, McGrath SP (2001) Using plant analysis to predict
yield losses caused by sulphur deficiency. Ann Appl Biol 138:123–127
82 P. Baraniecka and S. Kopriva

Brader G, Tas E, Palva ET (2001) Jasmonate-dependent induction of indole glucosinolates in

Arabidopsis by culture filtrates of the nonspecific pathogen Erwinia carotovora. Plant Physiol
126:849–860
Brown PD, Tokuhisa JG, Reichelt M, Gershenzon J (2003) Variation of glucosinolate accumula-
tion among different organs and developmental stages of Arabidopsis thaliana. Phytochemis-
try 62:471–481
Brunold C, Schiff JA (1976) Studies of sulfate utilization of algae: 15. Enzymes of assimilatory
sulfate reduction in euglena and their cellular localization. Plant Physiol 57:430–436
Brunold C, Suter M (1984) Regulation of sulfate assimilation by nitrogen nutrition in the
duckweed Lemna minor L. Plant Physiol 76:579–583
Brunold C, Suter M (1989) Localization of enzymes of assimilatory sulfate reduction in pea roots.
Planta 179:228–234
Brychkova G, Xia Z, Yang G, Yesbergenova Z, Zhang Z, Davydov O, Fluhr R, Sagi M (2007)
Sulfite oxidase protects plants against sulfur dioxide toxicity. Plant J 50:696–709
Buchner P, Prosser IM, Hawkesford MJ (2004a) Phylogeny and expression of paralogous and
orthologous sulphate transporter genes in diploid and hexaploid wheats. [Research Support,
Non-U S Gov’t]. Genome 47:526–534
Buchner P, Takahashi H, Hawkesford MJ (2004b) Plant sulphate transporters: co-ordination of
uptake, intracellular and long-distance transport. J Exp Bot 55:1765–1773
Buescher E, Achberger T, Amusan I, Giannini A, Ochsenfeld C, Rus A, Lahner B, Hoekenga O,
Yakubova E, Harper JF, Guerinot ML, Zhang M, Salt DE, Baxter IR (2010) Natural genetic
variation in selected populations of Arabidopsis thaliana is associated with ionomic differ-
ences. PLoS One 5:e11081
Burstenbinder K, Rzewuski G, Wirtz M, Hell R, Sauter M (2007) The role of methionine recycling
for ethylene synthesis in Arabidopsis. Plant J 49:238–249
Cao MJ, Wang Z, Wirtz M, Hell R, Oliver DJ, Xiang CB (2013) SULTR3;1 is a chloroplast-
localized sulfate transporter in Arabidopsis thaliana. Plant J 73:607–616
Chen YF, Matsubayashi Y, Sakagami Y (2000) Peptide growth factor phytosulfokine-alpha
contributes to the pollen population effect. Planta 211:752–755
Chiba Y, Ishikawa M, Kijima F, Tyson RH, Kim J, Yamamoto A, Nambara E, Leustek T,
Wallsgrove RM, Naito S (1999) Evidence for autoregulation of cystathionine gamma-synthase
mRNA stability in Arabidopsis. Science 286:1371–1374
Chiba Y, Sakurai R, Yoshino M, Ominato K, Ishikawa M, Onouchi H, Naito S (2003) S-adenosyl-
L-methionine is an effector in the posttranscriptional autoregulation of the cystathionine
gamma-synthase gene in Arabidopsis. Proc Natl Acad Sci U S A 100:10225–10230
Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Su CL (2006) Regulation of phosphate homeostasis
by MicroRNA in Arabidopsis. Plant Cell 18:412–421
Clarkson DT, Smith FW, Berg PJV (1983) Regulation of sulphate transport in a tropical legume,
Macroptilium atropurpureum, cv. Siratro. J Exp Bot 34:1463–1483
Curien G, Dumas R, Ravanel S, Douce R (1996) Characterization of an Arabidopsis thaliana
cDNA encoding an S-adenosylmethionine-sensitive threonine synthase. Threonine synthase
from higher plants. FEBS Lett 390:85–90
Curtis TY, Powers SJ, Balagiannis D, Elmore JS, Mottram DS, Parry MAJ, Rakszegi M, Bedö Z,
Shewry PR, Halford NG (2010) Free amino acids and sugars in rye grain: implications for
acrylamide formation. J Agric Food Chem 58:1959–1969
Doughty KJ, Kiddle GA, Pye BJ, Wallsgrove RM, Pickett JA (1995) Selective induction of
glucosinolates in oilseed rape leaves by methyl jasmonate. Phytochemistry 38:347–350
Droux M, Ruffet ML, Douce R, Job D (1998) Interactions between serine acetyltransferase and
O-acetylserine (thiol) lyase in higher plants–structural and kinetic properties of the free and
bound enzymes. Eur J Biochem 255:235–245
Eilers T, Schwarz G, Brinkmann H, Witt C, Richter T, Nieder J, Koch B, Hille R, Hansch R,
Mendel RR (2001) Identification and biochemical characterization of Arabidopsis thaliana
sulfite oxidase. A new player in plant sulfur metabolism. J Biol Chem 276:46989–46994
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 83

Essigmann B, Guler S, Narang RA, Linke D, Benning C (1998) Phosphate availability affects the
thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid
biosynthesis in Arabidopsis thaliana. Proc Natl Acad Sci U S A 95:1950–1955
Fahey JW, Zalcmann AT, Talalay P (2001) The chemical diversity and distribution of
glucosinolates and isothiocyanates among plants. Phytochemistry 56:5–51
Fernie AR, Tadmor Y, Zamir D (2006) Natural genetic variation for improving crop quality. Curr
Opin Plant Biol 9:196–202
Fismes J, Vong PC, Guckert A, Frossard E (2000) Influence of sulfur on apparent N-use efficiency,
yield and quality of oilseed rape (Brassica napus L.) grown on a calcareous soil. Eur J Agron
12:127–141
Flæte NES, Hollung K, Ruud L, Sogn T, Færgestad EM, Skarpeid HJ, Magnus EM, Uhlen AK
(2005) Combined nitrogen and sulphur fertilisation and its effect on wheat quality and protein
composition measured by SE-FPLC and proteomics. J Cereal Sci 41:357–369
Francois JA, Kumaran S, Jez JM (2006) Structural basis for interaction of O-acetylserine
sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex.
Plant Cell 18:3647–3655
Friedman M (2003) Chemistry, biochemistry, and safety of acrylamide, a review. J Agric Food
Chem 51:4504–4526
Fujii H, Chiou T-J, Lin S-I, Aung K, Zhu J-K (2005) A miRNA involved in phosphate-starvation
response in Arabidopsis. Curr Biol 15:2038–2043
Garcia I, Castellano JM, Vioque B, Solano R, Gotor C, Romero LC (2010) Mitochondrial beta-
cyanoalanine synthase is essential for root hair formation in Arabidopsis thaliana. Plant Cell
22:3268–3279
Gasber A, Klaumann S, Trentmann O, Trampczynska A, Clemens S, Schneider S, Sauer N,
Feifer I, Bittner F, Mendel RR, Neuhaus HE (2011) Identification of an Arabidopsis solute
carrier critical for intracellular transport and inter-organ allocation of molybdate. Plant Biol
13:710–718
Ghandilyan A, Ilk N, Hanhart C, Mbengue M, Barboza L, Schat H, Koornneef M, El-Lithy M,
Vreugdenhil D, Reymond M, Aarts MG (2009) A strong effect of growth medium and organ
type on the identification of QTLs for phytate and mineral concentrations in three Arabidopsis
thaliana RIL populations. J Exp Bot 60:1409–1425
Gigolashvili T, Berger B, Mock HP, Muller C, Weisshaar B, Flugge UI (2007) The transcription
factor HIG1/MYB51 regulates indolic glucosinolate biosynthesis in Arabidopsis thaliana.
Plant J 50:886–901
Gigolashvili T, Engqvist M, Yatusevich R, Muller C, Flugge UI (2008) HAG2/MYB76 and
HAG3/MYB29 exert a specific and coordinated control on the regulation of aliphatic
glucosinolate biosynthesis in Arabidopsis thaliana. New Phytol 177:627–642
Gilbert SM, Clarkson DT, Cambridge M, Lambers H, Hawkesford MJ (1997) SO42
deprivation
has an early effect on the content of ribulose-1,5-bisphosphate carboxylase/oxygenase and
photosynthesis in young leaves of wheat. Plant Physiol 115:1231–1239
Giovanelli J, Mudd SH, Datko AH (1985) Quantitative analysis of pathways of methionine
metabolism and their regulation in lemna. Plant Physiol 78:555–560
Haas FH, Heeg C, Queiroz R, Bauer A, Wirtz M, Hell R (2008) Mitochondrial serine
acetyltransferase functions as a pacemaker of cysteine synthesis in plant cells. Plant Physiol
148:1055–1067
Hacham Y, Schuster G, Amir R (2006) An in vivo internal deletion in the N-terminus region of
Arabidopsis cystathionine gamma-synthase results in CGS expression that is insensitive to
methionine. Plant J 45:955–967
Hagen G, Guilfoyle T (2002) Auxin-responsive gene expression: genes, promoters and regulatory
factors. Plant Mol Biol 49:373–385
Halford NG, Curtis TY, Muttucumaru N, Postles J, Elmore JS, Mottram DS (2012) The acrylamide
problem: a plant and agronomic science issue. J Exp Bot 63:2841–2851
84 P. Baraniecka and S. Kopriva

Halkier BA, Gershenzon J (2006) Biology and biochemistry of glucosinolates. Annu Rev Plant
Biol 57:303–333
Hänsch R, Lang C, Riebeseel E, Lindigkeit R, Gessler A, Rennenberg H, Mendel RR (2006) Plant
sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent
non-enzymatic reaction step. J Biol Chem 281:6884–6888
Hatzfeld Y, Lee S, Lee M, Leustek T, Saito K (2000a) Functional characterization of a gene
encoding a fourth ATP sulfurylase isoform from Arabidopsis thaliana. Gene 248:51–58
Hatzfeld Y, Maruyama A, Schmidt A, Noji M, Ishizawa K, Saito K (2000b) beta-Cyanoalanine
synthase is a mitochondrial cysteine synthase-like protein in spinach and Arabidopsis. Plant
Physiol 123:1163–1171
Hawkesford MJ (2000) Plant responses to sulphur deficiency and the genetic manipulation of
sulphate transporters to improve S‐utilization efficiency. J Exp Bot 51:131–138
Hawkesford MJ (2003) Transporter gene families in plants: the sulphate transporter gene family –
redundancy or specialization? Physiol Plant 117:155–163
Hawkesford M, Davidian J-C, Grignon C (1993) Sulphate/proton cotransport in plasma-membrane
vesicles isolated from roots of Brassica napus L.: increased transport in membranes isolated
from sulphur-starved plants. Planta 190:297–304
Hell R, Hillebrand H (2001) Plant concepts for mineral acquisition and allocation. Curr Opin
Biotechnol 12:161–168
Hell R, Wirtz M (2008) Metabolism of cysteine in plants and phototrophic bacteria. In: Hell R,
Dahl C, Knaff D, Leustek T (eds) Sulfur metabolism in phototrophic organisms. Springer,
Netherlands, pp 59–91
Hell R, Wirtz M (2011) Molecular biology, biochemistry and cellular physiology of cysteine
metabolism in Arabidopsis thaliana. The Arabidopsis book. Am Soc Plant Biol 9:e0154
Hesse H, Trachsel N, Suter M, Kopriva S, von Ballmoos P, Rennenberg H, Brunold C (2003)
Effect of glucose on assimilatory sulphate reduction in Arabidopsis thaliana roots. J Exp Bot
54:1701–1709
Hesse H, Kreft O, Maimann S, Zeh M, Hoefgen R (2004a) Current understanding of the regulation
of methionine biosynthesis in plants. J Exp Bot 55:1799–1808
Hesse H, Nikiforova V, Gakiere B, Hoefgen R (2004b) Molecular analysis and control of cysteine
biosynthesis: integration of nitrogen and sulphur metabolism. J Exp Bot 55:1283–1292
Hirai MY, Sugiyama K, Sawada Y, Tohge T, Obayashi T, Suzuki A, Araki R, Sakurai N,
Suzuki H, Aoki K, Goda H, Nishizawa OI, Shibata D, Saito K (2007) Omics-based identifi-
cation of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis.
Proc Natl Acad Sci U S A 104:6478–6483
Hopkins L, Parmar S, Blaszczyk A, Hesse H, Hoefgen R, Hawkesford MJ (2005) O-acetylserine
and the regulation of expression of genes encoding components for sulfate uptake and
assimilation in potato. Plant Physiol 138:433–440
Howarth JR, Fourcroy P, Davidian JC, Smith FW, Hawkesford MJ (2003) Cloning of two
contrasting high-affinity sulfate transporters from tomato induced by low sulfate and infection
by the vascular pathogen Verticillium dahliae. Planta 218:58–64
Hsieh LC, Lin SI, Shih AC, Chen JW, Lin WY, Tseng CY, Li WH, Chiou TJ (2009) Uncovering
small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.
Plant Physiol 151:2120–2132
Hubberten HM, Klie S, Caldana C, Degenkolbe T, Willmitzer L, Hoefgen R (2012) Additional
role of O-acetylserine as a sulfur status-independent regulator during plant growth. Plant J
70:666–677
Hussain A, Larsson H, Kuktaite R, Prieto-Linde ML, Johansson E (2012) Towards the under-
standing of bread-making quality in organically grown wheat: dough mixing behaviour,
protein polymerisation and structural properties. J Cereal Sci 56:659–666
Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their
targets, including a stress-induced miRNA. Mol Cell 14:787–799
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 85

Jost R, Altschmied L, Bloem E, Bogs J, Gershenzon J, Hahnel U, Hansch R, Hartmann T,

Kopriva S, Kruse C, Mendel RR, Papenbrock J, Reichelt M, Rennenberg H, Schnug E,
Schmidt A, Textor S, Tokuhisa J, Wachter A, Wirtz M, Rausch T, Hell R (2005) Expression
profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of
primary and secondary sulfur-related pathways in Arabidopsis thaliana. Photosynth Res
86:491–508
Kandlbinder A, Finkemeier I, Wormuth D, Hanitzsch M, Dietz KJ (2004) The antioxidant status of
photosynthesizing leaves under nutrient deficiency: redox regulation, gene expression and
antioxidant activity in Arabidopsis thaliana. Physiol Plant 120:63–73
Karmoker J, Clarkson D, Saker L, Rooney J, Purves J (1991) Sulphate deprivation depresses the
transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare
L.) roots. Planta 185:269–278
Kataoka T, Hayashi N, Yamaya T, Takahashi H (2004a) Root-to-shoot transport of sulfate in
Arabidopsis. Evidence for the role of SULTR3;5 as a component of low-affinity sulfate
transport system in the root vasculature. Plant Physiol 136:4198–4204
Kataoka T, Watanabe-Takahashi A, Hayashi N, Ohnishi M, Mimura T, Buchner P, Hawkesford
MJ, Yamaya T, Takahashi H (2004b) Vacuolar sulfate transporters are essential determinants
controlling internal distribution of sulfate in Arabidopsis. Plant Cell 16:2693–2704
Kawashima CG, Berkowitz O, Hell R, Noji M, Saito K (2005) Characterization and expression
analysis of a serine acetyltransferase gene family involved in a key step of the sulfur assim-
ilation pathway in Arabidopsis. Plant Physiol 137:220–230
Kawashima CG, Yoshimoto N, Maruyama-Nakashita A, Tsuchiya YN, Saito K, Takahashi H,
Dalmay T (2009) Sulphur starvation induces the expression of microRNA-395 and one of its
target genes but in different cell types. Plant J 57:313–321
Kawashima CG, Matthewman CA, Huang S, Lee B-R, Yoshimoto N, Koprivova A, Rubio-
Somoza I, Todesco M, Rathjen T, Saito K, Takahashi H, Dalmay T, Kopriva S (2011) Interplay
of SLIM1 and miR395 in the regulation of sulfate assimilation in Arabidopsis. Plant J
66:863–876
Khan MS, Haas FH, Allboje Samami A, Moghaddas Gholami A, Bauer A, Fellenberg K,
Reichelt M, Hänsch R, Mendel RR, Meyer AJ, Wirtz M, Hell R (2010) Sulfite reductase
defines a newly discovered bottleneck for assimilatory sulfate reduction and is essential for
growth and development in Arabidopsis thaliana. Plant Cell Online 22:1216–1231
Kim H, Hirai MY, Hayashi H, Chino M, Naito S, Fujiwara T (1999) Role of O-acetyl-L-serine in
the coordinated regulation of the expression of a soybean seed storage-protein gene by sulfur
and nitrogen nutrition. Planta 209:282–289
Kobayashi T, Eun C-H, Hanai H, Matsubayashi Y, Sakagami Y, Kamada H (1999)
Phytosulphokine-α, a peptidyl plant growth factor, stimulates somatic embryogenesis in carrot.
J Exp Bot 50:1123–1128
Koornneef M, Alonso-Blanco C, Vreugdenhil D (2004) Naturally occurring genetic variation in
Arabidopsis thaliana. Annu Rev Plant Physiol Plant Mol Biol 55:141–172
Kopriva S (2006) Regulation of sulfate assimilation in Arabidopsis and beyond. Ann Bot
97:479–495
Kopriva S, Koprivova A (2004) Plant adenosine 50 -phosphosulphate reductase: the past, the
present, and the future. J Exp Bot 55:1775–1783
Kopriva S, Rennenberg H (2004) Control of sulphate assimilation and glutathione synthesis:
interaction with N and C metabolism. J Exp Bot 55:1831–1842
Kopriva S, Muheim R, Koprivova A, Trachsel N, Catalano C, Suter M, Brunold C (1999) Light
regulation of assimilatory sulphate reduction in Arabidopsis thaliana. Plant J 20:37–44
Kopriva S, Buchert T, Fritz G, Suter M, Weber M, Benda R, Schaller J, Feller U, Schurmann P,
Schunemann V, Trautwein AX, Kroneck PM, Brunold C (2001) Plant adenosine 5-
0
-phosphosulfate reductase is a novel iron-sulfur protein. J Biol Chem 276:42881–42886
86 P. Baraniecka and S. Kopriva

Kopriva S, Suter M, von Ballmoos P, Hesse H, Krähenbühl U, Rennenberg H, Brunold C (2002)

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor. Plant
Physiol 130:1406–1413
Kopriva S, Fritzemeier K, Wiedemann G, Reski R (2007) The putative moss 3-
0
-phosphoadenosine-50 -phosphosulfate reductase is a novel form of adenosine-5-
0
-phosphosulfate reductase without an iron-sulfur cluster. J Biol Chem 282:22930–22938
Kopriva S, Mugford S, Matthewman C, Koprivova A (2009) Plant sulfate assimilation genes:
redundancy versus specialization. Plant Cell Rep 28:1769–1780
Kopriva S, Mugford SG, Baraniecka P, Lee B-R, Matthewman CA, Koprivova A (2012) Control
of sulfur partitioning between primary and secondary metabolism in Arabidopsis. Front Plant
Sci 3:163. doi:10.3389/fpls.2012.00163
Koprivova A, Suter M, Op den Camp R, Brunold C, Kopriva S (2000) Regulation of sulfate
assimilation by nitrogen in Arabidopsis. Plant Physiol 122:737–746
Koprivova A, North KA, Kopriva S (2008) Complex signaling network in regulation of adenosine
50 -phosphosulfate reductase by salt stress in Arabidopsis roots. Plant Physiol 146:1408–1420
Krueger S, Niehl A, Lopez Martin MC, Steinhauser D, Donath A, Hildebrandt T, Romero LC,
Hoefgen R, Gotor C, Hesse H (2009) Analysis of cytosolic and plastidic serine
acetyltransferase mutants and subcellular metabolite distributions suggests interplay of the
cellular compartments for cysteine biosynthesis in Arabidopsis. Plant Cell Environ 32:349–367
Kuktaite R, Larsson H, Johansson E (2004) Variation in protein composition of wheat flour and its
relationship to dough mixing behaviour. J Cereal Sci 40:31–39
Kutz A, Muller A, Hennig P, Kaiser WM, Piotrowski M, Weiler EW (2002) A role for nitrilase 3 in
the regulation of root morphology in sulphur-starving Arabidopsis thaliana. Plant J 30:95–106
Lambein I, Chiba Y, Onouchi H, Naito S (2003) Decay kinetics of autogenously regulated CGS1
mRNA that codes for cystathionine gamma-synthase in Arabidopsis thaliana. Plant Cell
Physiol 44:893–900
Lass B, Ullrich-Eberius CI (1984) Evidence for proton/sulfate cotransport and its kinetics in
Lemna gibba G1. Planta 161:53–60
Lee B-R, Koprivova A, Kopriva S (2011) The key enzyme of sulfate assimilation, adenosine
50 -phosphosulfate reductase, is regulated by HY5 in Arabidopsis. Plant J 67:1042–1054
Lejay L, Gansel X, Cerezo M, Tillard P, Muller C, Krapp A, von Wiren N, Daniel-Vedele F, Gojon
A (2003) Regulation of root ion transporters by photosynthesis: functional importance and
relation with hexokinase. Plant Cell 15:2218–2232
Leustek T, Martin MN, Bick JA, Davies JP (2000) Pathways and regulation of sulfur metabolism
revealed through molecular and genetic studies. Annu Rev Plant Physiol Plant Mol Biol
51:141–165
Levy M, Wang Q, Kaspi R, Parrella MP, Abel S (2005) Arabidopsis IQD1, a novel calmodulin-
binding nuclear protein, stimulates glucosinolate accumulation and plant defense. Plant J
43:79–96
Liang G, Yang F, Yu D (2010) MicroRNA395 mediates regulation of sulfate accumulation and
allocation in Arabidopsis thaliana. Plant J 62:1046–1057
Lin S-I, Chiang S-F, Lin W-Y, Chen J-W, Tseng C-Y, Wu P-C, Chiou T-J (2008) Regulatory
network of microRNA399 and PHO2 by systemic signaling. Plant Physiol 147:732–746
Logan HM, Cathala N, Grignon C, Davidian JC (1996) Cloning of a cDNA encoded by a member
of the Arabidopsis thaliana ATP sulfurylase multigene family. Expression studies in yeast and
in relation to plant sulfur nutrition. J Biol Chem 271:12227–12233
López-Martı́n MC, Becana M, Romero LC, Gotor C (2008) Knocking out cytosolic cysteine
synthesis compromises the antioxidant capacity of the cytosol to maintain discrete concentra-
tions of hydrogen peroxide in Arabidopsis. Plant Physiol 147:562–572
Loudet O, Saliba-Colombani V, Camilleri C, Calenge F, Gaudon V, Koprivova A, North KA,
Kopriva S, Daniel-Vedele F (2007) Natural variation for sulfate content in Arabidopsis
thaliana is highly controlled by APR2. Nat Genet 39:896–900
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 87

Lunn JE, Droux M, Martin J, Douce R (1990) Localization of ATP sulfurylase and O-Acetylserine
(thiol)lyase in Spinach leaves. Plant Physiol 94:1345–1352
Malhi SS, Gan Y, Raney JP (2007) Yield, seed quality, and sulfur uptake of Brassica oilseed crops
in response to sulfur fertilization. Agron J 99:570–577
Martin MN, Tarczynski MC, Shen B, Leustek T (2005) The role of 50 -adenylylsulfate reductase in
controlling sulfate reduction in plants. Photosynth Res 86:309–323
Maruyama-Nakashita A, Inoue E, Watanabe-Takahashi A, Yamaya T, Takahashi H (2003)
Transcriptome profiling of sulfur-responsive genes in Arabidopsis reveals global effects of
sulfur nutrition on multiple metabolic pathways. Plant Physiol 132:597–605
Maruyama-Nakashita A, Nakamura Y, Watanabe-Takahashi A, Yamaya T, Takahashi H (2004a)
Induction of SULTR1;1 sulfate transporter in Arabidopsis roots involves protein phosphory-
lation/dephosphorylation circuit for transcriptional regulation. Plant Cell Physiol 45:340–345
Maruyama-Nakashita A, Nakamura Y, Yamaya T, Takahashi H (2004b) A novel regulatory
pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4-mediated
cytokinin-dependent regulation. Plant J 38:779–789
Maruyama-Nakashita A, Nakamura Y, Watanabe-Takahashi A, Inoue E, Yamaya T, Takahashi H
(2005) Identification of a novel cis-acting element conferring sulfur deficiency response in
Arabidopsis roots. Plant J 42:305–314
Maruyama-Nakashita A, Nakamura Y, Tohge T, Saito K, Takahashi H (2006) Arabidopsis SLIM1
is a central transcriptional regulator of plant sulfur response and metabolism. Plant Cell
18:3235–3251
Matsubayashi Y, Sakagami Y (1999) Characterization of specific binding sites for a mitogenic
sulfated peptide, phytosulfokine-alpha, in the plasma-membrane fraction derived from Oryza
sativa L. Eur J Biochem 262:666–671
Matsubayashi Y, Sakagami Y (2006) Peptide hormones in plants. Annu Rev Plant Biol
57:649–674
Matsubayashi Y, Ogawa M, Morita A, Sakagami Y (2002) An LRR receptor kinase involved in
perception of a peptide plant hormone, phytosulfokine. Science 296:1470–1472
Matsuzaki Y, Ogawa-Ohnishi M, Mori A, Matsubayashi Y (2010) Secreted peptide signals
required for maintenance of root stem cell niche in Arabidopsis. Science 329:1065–1067
Matthewman CA, Kawashima CG, Huska D, Csorba T, Dalmay T, Kopriva S (2012) miR395 is a
general component of the sulfate assimilation regulatory network in Arabidopsis. FEBS Lett
586:3242–3248
McCouch S (2004) Diversifying selection in plant breeding. PLoS Biol 2:e347
McGrath SP, Zhao FJ (1996) Sulphur uptake, yield responses and the interactions between
nitrogen and sulphur in winter oilseed rape (Brassica napus). J Agric Sci 126:53–62
McGrath SP, Zhao FJ, Withers PJA (1996) Development of sulphur deficiency in crops and its
treatment. Proceedings of the Fertiliser Society
Meng L, Buchanan BB, Feldman LJ, Luan S (2012) CLE-like (CLEL) peptides control the pattern
of root growth and lateral root development in Arabidopsis. Proc Natl Acad Sci U S A
109:1760–1765
Mithen R, Faulkner K, Magrath R, Rose P, Williamson G, Marquez J (2003) Development of
isothiocyanate-enriched broccoli, and its enhanced ability to induce phase 2 detoxification
enzymes in mammalian cells. Theor Appl Genet 106:727–734
Moss H, Wrigley C, MacRichie R, Randall P (1981) Sulfur and nitrogen fertilizer effects on wheat.
II. Influence on grain quality. Aust J Agric Res 32:213–226
Mugford SG, Yoshimoto N, Reichelt M, Wirtz M, Hill L, Mugford ST, Nakazato Y, Noji M,
Takahashi H, Kramell R, Gigolashvili T, Flugge UI, Wasternack C, Gershenzon J, Hell R,
Saito K, Kopriva S (2009) Disruption of adenosine-50 -phosphosulfate kinase in Arabidopsis
reduces levels of sulfated secondary metabolites. Plant Cell 21:910–927
Mugford SG, Matthewman CA, Hill L, Kopriva S (2010) Adenosine-50 -phosphosulfate kinase is
essential for Arabidopsis viability. FEBS Lett 584:119–123
88 P. Baraniecka and S. Kopriva

Murillo M, Leustek T (1995) Adenosine-50 -triphosphate-sulfurylase from Arabidopsis thaliana

and Escherichia coli are functionally equivalent but structurally and kinetically divergent:
nucleotide sequence of two adenosine-50 -triphosphate-sulfurylase cDNAs from Arabidopsis
thaliana and analysis of a recombinant enzyme. Arch Biochem Biophys 323:195–204
Myles S, Peiffer J, Brown PJ, Ersoz ES, Zhang Z, Costich DE, Buckler ES (2009) Association
mapping: critical considerations shift from genotyping to experimental design. Plant Cell
21:2194–2202
Nakayama M, Akashi T, Hase T (2000) Plant sulfite reductase: molecular structure, catalytic
function and interaction with ferredoxin. J Inorg Biochem 82:27–32
Nikiforova V, Freitag J, Kempa S, Adamik M, Hesse H, Hoefgen R (2003) Transcriptome analysis
of sulfur depletion in Arabidopsis thaliana: interlacing of biosynthetic pathways provides
response specificity. Plant J 33:633–650
Noctor G, Gomez L, Vanacker H, Foyer CH (2002) Interactions between biosynthesis, compart-
mentation and transport in the control of glutathione homeostasis and signalling. J Exp Bot
53:1283–1304
Noji M, Inoue K, Kimura N, Gouda A, Saito K (1998) Isoform-dependent differences in feedback
regulation and subcellular localization of serine acetyltransferase involved in cysteine biosyn-
thesis from Arabidopsis thaliana. J Biol Chem 273:32739–32745
Nowak K, Luniak N, Witt C, Wustefeld Y, Wachter A, Mendel RR, Hansch R (2004) Peroxisomal
localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation. Plant Cell
Physiol 45:1889–1894
Ohkama N, Takei K, Sakakibara H, Hayashi H, Yoneyama T, Fujiwara T (2002) Regulation of
sulfur-responsive gene expression by exogenously applied cytokinins in Arabidopsis thaliana.
Plant Cell Physiol 43:1493–1501
Ohkama-Ohtsu N, Oikawa A, Zhao P, Xiang C, Saito K, Oliver DJ (2008) A gamma-glutamyl
transpeptidase-independent pathway of glutathione catabolism to glutamate via 5-oxoproline
in Arabidopsis. Plant Physiol 148:1603–1613
Olsen LR, Huang B, Vetting MW, Roderick SL (2004) Structure of serine acetyltransferase in
complexes with CoA and its cysteine feedback inhibitor. Biochemistry 43:6013–6019
Prosser IM, Purves JV, Saker LR, Clarkson DT (2001) Rapid disruption of nitrogen metabolism
and nitrate transport in spinach plants deprived of sulphate. J Exp Bot 52:113–121
Rae A, Smith F (2002) Localisation of expression of a high-affinity sulfate transporter in barley
roots. Planta 215:565–568
Ravanel S, Gakière B, Job D, Douce R (1998) The specific features of methionine biosynthesis and
metabolism in plants. Proc Natl Acad Sci U S A 95:7805–7812
Ravanel S, Block MA, Rippert P, Jabrin S, Curien G, Rébeillé F, Douce R (2004) Methionine
metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can
import s-adenosylmethionine from the cytosol. J Biol Chem 279:22548–22557
Renosto F, Patel HC, Martin RL, Thomassian C, Zimmerman G, Segel IH (1993) ATP sulfurylase
from higher plants: kinetic and structural characterization of the chloroplast and cytosol
enzymes from spinach leaf. Arch Biochem Biophys 307:272–285
Renwick JA (2001) Variable diets and changing taste in plant-insect relationships. J Chem Ecol
27:1063–1076
Rotte C, Leustek T (2000) Differential subcellular localization and expression of ATP sulfurylase
and 50 -adenylylsulfate reductase during ontogenesis of Arabidopsis leaves indicates that
cytosolic and plastid forms of ATP sulfurylase may have specialized functions. Plant Physiol
124:715–724
Rouached H (2011) Multilevel coordination of phosphate and sulfate homeostasis in plants. Plant
Signal Behav 6:952–955
Rouached H, Berthomieu P, El Kassis E, Cathala N, Catherinot V, Labesse G, Davidian J-C,
Fourcroy P (2005) Structural and functional analysis of the C-terminal STAS (sulfate trans-
porter and anti-sigma antagonist) domain of the Arabidopsis thaliana sulfate transporter
SULTR1.2. J Biol Chem 280:15976–15983
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 89

Rouached H, Wirtz M, Alary R, Hell R, Arpat AB, Davidian J-C, Fourcroy P, Berthomieu P (2008)
Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1
and SULTR1.2, in Arabidopsis. Plant Physiol 147:897–911
Rouached H, Secco D, Arpat B, Poirier Y (2011) The transcription factor PHR1 plays a key role in
the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis. BMC
Plant Biol 11:19
Saito K (2000) Regulation of sulfate transport and synthesis of sulfur-containing amino acids. Curr
Opin Plant Biol 3:188–195
Saito K (2004) Sulfur assimilatory metabolism. The long and smelling road. Plant Physiol
136:2443–2450
Shahsavani S, Gholami A (2008) Effect of sulphur fertilization on breadmaking quality of three
winter wheat varieties. Pak J Biol Sci 11:2134–2138
Shibagaki N, Grossman AR (2004) Probing the function of STAS domains of the Arabidopsis
sulfate transporters. J Biol Chem 279:30791–30799
Shibagaki N, Grossman AR (2010) Binding of cysteine synthase to the STAS domain of sulfate
transporter and its regulatory consequences. J Biol Chem 285:25094–25102
Shibagaki N, Rose A, McDermott JP, Fujiwara T, Hayashi H, Yoneyama T, Davies JP (2002)
Selenate-resistant mutants of Arabidopsis thaliana identify Sultr1;2, a sulfate transporter
required for efficient transport of sulfate into roots. Plant J 29:475–486
Shinmachi F, Buchner P, Stroud JL, Parmar S, Zhao FJ, McGrath SP, Hawkesford MJ (2010)
Influence of sulfur deficiency on the expression of specific sulfate transporters and the
distribution of sulfur, selenium, and molybdenum in wheat. Plant Physiol 153:327–336
Skirycz A, Reichelt M, Burow M, Birkemeyer C, Rolcik J, Kopka J, Zanor MI, Gershenzon J,
Strnad M, Szopa J, Mueller-Roeber B, Witt I (2006) DOF transcription factor AtDof1.1
(OBP2) is part of a regulatory network controlling glucosinolate biosynthesis in Arabidopsis.
Plant J 47:10–24
Smith FW, Ealing PM, Hawkesford MJ, Clarkson DT (1995a) Plant members of a family of sulfate
transporters reveal functional subtypes. Proc Natl Acad Sci U S A 92:9373–9377
Smith FW, Hawkesford MJ, Prosser IM, Clarkson DT (1995b) Isolation of a cDNA from
Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma
membrane. Mol Gen Genet 247:709–715
Smith FW, Hawkesford MJ, Ealing PM, Clarkson DT, Vanden Berg PJ, Belcher AR, Warrilow AG
(1997) Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate
transporter. Plant J 12:875–884
Sønderby IE, Hansen BG, Bjarnholt N, Ticconi C, Halkier BA, Kliebenstein DJ (2007) A systems
biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping
functions in regulation of aliphatic glucosinolates. PLoS One 2:e1322
Suzuki A, Shirata Y, Ishida H, Chiba Y, Onouchi H, Naito S (2001) The first exon coding region of
cystathionine gamma-synthase gene is necessary and sufficient for downregulation of its own
mRNA accumulation in transgenic Arabidopsis thaliana. Plant Cell Physiol 42:1174–1180
Takahashi H, Saito K (1996) Subcellular localization of spinach cysteine synthase isoforms and
regulation of their gene expression by nitrogen and sulfur. Plant Physiol 112:273–280
Takahashi H, Saito K (2008) Molecular biology and functional genomics for identification of
regulatory networks of plant sulfate uptake and assimilatory metabolism. In: Hell R, Dahl C,
Knaff D, Leustek T (eds) Sulfur metabolism in phototrophic organisms. Springer, Netherlands,
pp 149–159
Takahashi H, Watanabe-Takahashi A, Smith FW, Blake-Kalff M, Hawkesford MJ, Saito K (2000)
The roles of three functional sulphate transporters involved in uptake and translocation of
sulphate in Arabidopsis thaliana. Plant J 23:171–182
Takahashi H, Kopriva S, Giordano M, Saito K, Hell R (2011) Sulfur assimilation in photosynthetic
organisms: molecular functions and regulations of transporters and assimilatory enzymes.
Annu Rev Plant Biol 62:157–184
90 P. Baraniecka and S. Kopriva

Tareke E, Rydberg P, Karlsson P, Eriksson S, Tornqvist M (2002) Analysis of acrylamide, a

carcinogen formed in heated foodstuffs. J Agric Food Chem 50:4998–5006
Tomatsu H, Takano J, Takahashi H, Watanabe-Takahashi A, Shibagaki N, Fujiwara T (2007) An
Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of
molybdate from soil. Proc Natl Acad Sci U S A 104:18807–18812
Varadarajan DK, Karthikeyan AS, Matilda PD, Raghothama KG (2002) Phosphite, an analog of
phosphate, suppresses the coordinated expression of genes under phosphate starvation. Plant
Physiol 129:1232–1240
Vauclare P, Kopriva S, Fell D, Suter M, Sticher L, von Ballmoos P, Krahenbuhl U, den Camp RO,
Brunold C (2002) Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine
50 -phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by
thiols. Plant J 31:729–740
Vidmar JJ, Tagmount A, Cathala N, Touraine B, Davidian JE (2000) Cloning and characterization
of a root specific high-affinity sulfate transporter from Arabidopsis thaliana. [Research Sup-
port, Non-U S Gov’t]. FEBS Lett 475:65–69
Wang R, Okamoto M, Xing X, Crawford NM (2003) Microarray analysis of the nitrate response in
Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to
glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol 132:556–567
Watanabe M, Mochida K, Kato T, Tabata S, Yoshimoto N, Noji M, Saito K (2008) Comparative
genomics and reverse genetics analysis reveal indispensable functions of the serine
acetyltransferase gene family in Arabidopsis. Plant Cell 20:2484–2496
White PJ, Brown PH (2010) Plant nutrition for sustainable development and global health. Ann
Bot 105:1073–1080
Wirtz M, Hell R (2006) Functional analysis of the cysteine synthase protein complex from plants:
structural, biochemical and regulatory properties. J Plant Physiol 163:273–286
Wirtz M, Berkowitz O, Droux M, Hell R (2001) The cysteine synthase complex from plants.
Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional
domain for catalysis and protein-protein interaction. Eur J Biochem 268:686–693
Wirtz M, Droux M, Hell R (2004) O-acetylserine (thiol) lyase: an enigmatic enzyme of plant
cysteine biosynthesis revisited in Arabidopsis thaliana. J Exp Bot 55:1785–1798
Xiang C, Oliver DJ (1998) Glutathione metabolic genes coordinately respond to heavy metals and
jasmonic acid in Arabidopsis. Plant Cell Online 10:1539–1550
Yamaguchi Y, Nakamura T, Harada E, Koizumi N, Sano H (1999) Differential accumulation of
transcripts encoding sulfur assimilation enzymes upon sulfur and/or nitrogen deprivation in
Arabidopsis thaliana. Biosci Biotechnol Biochem 63:762–766
Yamakawa S, Sakuta C, Matsubayashi Y, Sakagami Y, Kamada H, Satoh S (1998) The promotive
effects of a peptidyl plant growth factor, phytosulfokine-α, on the formation of adventitious
roots and expression of a gene for a root-specific cystatin in cucumber hypocotyls. J Plant Res
111:453–458
Yang H, Matsubayashi Y, Nakamura K, Sakagami Y (1999) Oryza sativa PSK gene encodes a
precursor of phytosulfokine-alpha, a sulfated peptide growth factor found in plants. Proc Natl
Acad Sci U S A 96:13560–13565
Yang H, Matsubayashi Y, Hanai H, Sakagami Y (2000) Phytosulfokine-alpha, a peptide growth
factor found in higher plants: its structure, functions, precursor and receptors. Plant Cell
Physiol 41:825–830
Yang H, Matsubayashi Y, Nakamura K, Sakagami Y (2001) Diversity of Arabidopsis genes
encoding precursors for phytosulfokine, a peptide growth factor. Plant Physiol 127:842–851
Yatusevich R, Mugford SG, Matthewman C, Gigolashvili T, Frerigmann H, Delaney S,
Koprivova A, Flugge UI, Kopriva S (2010) Genes of primary sulfate assimilation are part of
the glucosinolate biosynthetic network in Arabidopsis thaliana. Plant J 62:1–11
Yoshimoto N, Takahashi H, Smith FW, Yamaya T, Saito K (2002) Two distinct high-affinity
sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots.
Plant J 29:465–473
3 Macronutrient Use Efficiency – Sulfur in Arabidopsis thaliana 91

Yoshimoto N, Inoue E, Saito K, Yamaya T, Takahashi H (2003) Phloem-localizing sulfate

transporter, Sultr1;3, mediates re-distribution of sulfur from source to sink organs in
Arabidopsis. Plant Physiol 131:1511–1517
Yoshimoto N, Inoue E, Watanabe-Takahashi A, Saito K, Takahashi H (2007) Posttranscriptional
regulation of high-affinity sulfate transporters in Arabidopsis by sulfur nutrition. Plant Physiol
145:378–388
Zamir D (2001) Improving plant breeding with exotic genetic libraries. Nat Rev Genet 2:983–989
Zhao F, McGrath SP (1994) Extractable sulphate and organic sulphur in soils and their availability
to plants. Plant Soil 164:243–250
Zhao FJ, Hawkesford MJ, Warrilow AGS, McGrath SP, Clarkson DT (1996) Responses of two
wheat varieties to sulphur addition and diagnosis of sulphur deficiency. Plant Soil 181:317–327
Zhao FJ, Hawkesford MJ, McGrath SP (1999) Sulphur assimilation and effects on yield and
quality of wheat. J Cereal Sci 30:1–17
Chapter 4
Efficient Mineral Nutrition: Genetic
Improvement of Phosphate Uptake and Use
Efficiency in Crops

Astrid Gruen, Martin R. Broadley, Peter Buchner,

and Malcolm J. Hawkesford

Abstract Phosphorus (P) is an essential macronutrient for plants, the lack of which
can be a major constraint for agricultural productivity. Economic, political and
environmental factors have prioritized the need for research on P acquisition
efficiency (PAE), P utilization efficiency (PUE) and P fertiliser uptake efficiency
in crops. P has critical functions in plants and complex interactions in soils.
Appropriate screening approaches and implications of improvement in crop pro-
duction are discussed. P acquisition is mediated by members of phosphate trans-
porter families and the roles of these phosphate transporters as well as enzymes
involved in P partitioning and re-translocation are complex. There is also a critical
importance of regulatory genes including transcription factors, signalling pathways
and apparently other P-responsive genes with unknown function. Furthermore,
morphological and biochemical responses enhance P solubility in the soil and
facilitate uptake and include root plasticity, secretion processes and symbioses.
Exploitation of genetic variation, classical breeding and biotechnological gene
modification of target genes are future routes for crop improvement. There is a
need for selection not just for uptake but also focussing on P storage pools within
cells and tissues, and additionally a consideration of crop P requirements during the
different growth stages of crops. The review concludes with a summary giving an
outlook to future questions related to crop PAE/PUE improvement.

Keywords Phosphorus • Acquisition efficiency • Utilization efficiency • Phosphate

transporters • Mycorrhiza • Signaling • Diversity • Adaptation • QTL

A. Gruen • P. Buchner • M.J. Hawkesford (*)

Rothamsted Research, Harpenden AL5 2JQ, Hertfordshire, UK
e-mail: [email protected]
M.R. Broadley
Plant Science Division, School of Biosciences, University of Nottingham, Loughborough
LE12 5RD, UK

© Springer International Publishing Switzerland 2014 93

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_4
94 A. Gruen et al.

Efficient Mineral Nutrition: Phosphate as Nutritional Trait

of Interest

Implications for Phosphorus Uptake

Phosphorus (P) is an essential macronutrient with multiple functions in plant

macromolecular structures as a component of nucleic acids and phospholipids,
with crucial roles in energy metabolism, participation in signal transduction path-
ways via phosphorylation/dephosphorylation and controlling key enzyme reactions
(Theodorou and Plaxton 1993; Schachtman et al. 1998; Marschner 2012). In many
agricultural systems, P is one of the most limiting nutrients for crop production and
is a major constraint for yield (Vance et al. 2003; Raghothama 2005; Kirkby and
Johnston 2008), with shoot growth, tiller number and tiller weight all influenced by
P availability (Römer and Schilling 1986; Bollons and Barraclough 1997). Critical
P concentrations in wheat range from 0.5 to 0.4 % in dry matter (DM) at tillering
and 0.2–0.3 % in DM at booting (Finck 1991; Marschner 2012). The amount of
readily available P may be raised through P fertilisation and agronomic strategies
such as fertiliser placement or the application of the “critical value” concept (Bahl
and Singh 1986; Strong et al. 1997; Kirkby and Johnston 2008; Syers et al. 2008).
Phosphorus fertiliser derived from rock phosphate is a finite and non-renewable
resource (Cordell et al. 2009). Furthermore, P derived from P fertiliser may cause
environmental problems associated with eutrophication (Gaxiola et al. 2001), espe-
cially as a result of overuse (Fig. 4.1). Hence, a major challenge for future crop
production will be to produce higher yields with fewer inputs such as P fertiliser
(Gregory and George 2011). One strategy would be to develop crop genotypes that
require smaller amounts of fertiliser and therefore using nutrients more efficiently,
bred based on trait-focused screens of germplasm collections (Gregory and George
2011). Nevertheless, little progress has been made in breeding cultivars with high P
utilisation efficiency or P acquisition efficiency (Calderón-Vázquez et al. 2011;
Rose et al. 2011), and the recovery (uptake) of applied P, which ranges between
25 and 60 % depending on the method used, is still modest (Syers et al. 2008).
Nutrient acquisition via the plant root system is a crucial factor for agricultural
productivity and crop yield (Lynch 1995). Consequently, the amount of available P
is not only determined by the ability of the soil to replenish P ions, but is also
influenced by the extent and efficiency of uptake by the plant roots. Thus, cropping
system-specific and plant-specific approaches must be taken into account in order to
raise P acquisition efficiency (PAE) and P use efficiency (PUE) and a consideration
of root traits in genetic selection might result in significant improvements for crops
(Vance et al. 2003).
Soil phosphorus is the most immobile, inaccessible and unavailable of all
macronutrient elements (Holford 1997) and is taken up by plants mainly in its
inorganic form as phosphate (Pi) (George and Richardson 2008).
Edaphic and climate factors and cropping systems have a strong impact on the
proliferation and rate of replenishment of the available Pi pool, which can be
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 95

16000 0.7

14000
0.6
Pasture production (kg dry matter/ha)

12000
0.5

0.01 M CaCl2-P (mg/L)

10000
0.4

8000

0.3
6000

0.2
4000 Control
188 SSP
376 SSP 0.1
2000
0.01M CaCl2-P

0 0.0
0 10 20 30 40
Olsen P (mg/L)
Current Opinion in Biotechnology

Fig. 4.1 The gap between the agronomic optimum in Olsen P for pasture production (green up
arrow) and a potential threshold in Olsen P for P loss in subsurface drainage (as estimated by
0.01 M CaCl2 -P; red down arrow) shows there is little justification in exceeding the agronomic
optimum. Data are from plots receiving different rates of superphosphate (SSP; kg ha 1 year 1) in
a trial in Canterbury, New Zealand (Data from McDowell (2012) and used with permission)

recovered by the plant. Pi transfer from the soil to the root proceeds mainly by
diffusion rather than mass flow, with slow diffusion rates at around 10 15 m s 1 and
a concentration gradient as the driving force towards the roots (Hinsinger 2001;
Rausch and Bucher 2002) resulting in a depletion zone of 1–2 mm around the root
(Jungk 2001). Soil Pi concentrations are extremely low, being generally <10 μM
and typically around 2 μM (Bieleski 1973; Barber 1984; Holford 1997), whereas in
plants, concentrations of over 40 mM can be achieved (Bollons and Barraclough
1997). Hence, plants take up Pi faster than it is supplied by diffusion (Bieleski
1973).
Soil pH, buffer capacity, soil moisture and soil structure affect Pi solubility and
sorptivity (Holford 1997; Syers et al. 2008); Pi can be absorbed on the surface of
clay minerals, Fe- and Al-hydrous oxide surfaces and organic matter complexes or
be fixed in acidic soils as Al-/Fe phosphates or Ca/Mg-phosphates in alkaline soils
(Barber 1984; Bahl and Singh 1986; Holford 1997; Hinsinger 2001).
Significant amounts of soil phosphorus (20–80 %) is bound in organic forms
such as nucleic acids, phospholipids and predominately monophosphate esters, as
phytic acid and derivatives (Richardson 1994). These organic forms have to be
mineralised and/or solubilised into inorganic forms in order to be available for
plants; a process which is either microbiological or plant mediated.
96 A. Gruen et al.

Phosphate Transporters: Uptake and Translocation

Plants acquire P predominately as orthophosphate (H2PO4 /HPO42 ; Pi) from the

soil solution (Bieleski 1973; Holford 1997; Schachtman et al. 1998; Marschner
2012). This process is mediated by plasma membrane-localised phosphate trans-
porters which have been suggested to operate as H+ co-transporters (Daram
et al. 1998; Smith et al. 1999; Mimura 2001; Rae et al. 2003; Raghothama 2005).
Consistent with the physiological pH in many agricultural soils, maximal uptake
rates occur in a pH range 5–6 (Ullrich-Eberius et al. 1981; Furihata et al. 1992; Rae
et al. 2003). A constitutively expressed low-affinity uptake system with a Km of 50–
300 μM and a high-affinity uptake system, which is regulated by Pi availability with
Km of 3–7 μM have been proposed (Ullrich-Eberius et al. 1981; Furihata et al. 1992;
Preuss et al. 2010). Phosphate transporters are classified into distinct families: Pht1,
Pht2, Pht3 and Pht4 (Bucher et al. 2001; Liu et al. 2011).
Pht1 are high-affinity transporters homologous to the yeast PHO84 Pi transporter
and other fungal high-affinity Pi transporters (Pao et al. 1998). However, the
functional characterisation of the Pht1 transporter family in rice and barley (Rae
et al. 2003; Ai et al. 2009) revealed kinetic properties, which are within both high-
and low-affinity ranges. Pht1 transporters belong to the distinct phosphate:H+
symporter (PHS) family which is a member of the major facilitator superfamily
(MFS) of proteins (Pao et al. 1998). All these transporters exhibit high sequence
similarity with each other, being similar in size and having 12 predicted transmem-
brane domains (TMs) with a large hydrophilic loop between TM6 and TM7 that
results in a 6 + 6 configuration (Liu et al. 2011). The N- and C-termini are oriented
towards the inside of the cell and they contain potential sites for phosphorylation
and N-glycosylation (Smith et al. 1999).
Transporters of the Pht2 family have been cloned in Arabidopsis, and have been
suggested to have roles as constitutively expressed low-affinity proton symporters
(H+/Pi cotransporter) for Pi loading in green shoot organs and predominantly leaf
tissues (Daram et al. 1999; Versaw and Harrison 2002). The Pht2 protein is
structurally similar to the Pht1 members, but is more closely related to the putative
Pi transporters from bacteria and mammalian Na+/Pi transporters. Pht2 amino acid
sequences are distinct from Pht1 transporters and have a large hydrophilic loop
between TM8 and TM9 (Daram et al. 1999). In wheat, expression analysis of
TaPht2;1 revealed a predominant expression in a photoperiod-dependent manner
in the leaves, which was significantly enhanced during P starvation (Guo
et al. 2013). GFP fusion studies localised TaPht2;1 to the chloroplast envelope,
suggesting a regulatory role mediating Pi translocation from the cytosol to the
chloroplast as low-affinity transporter with a Km of 225 μM Pi (Guo et al. 2013).
The Pht3 transporters belong to the mitochondrial transporter family (Rausch
and Bucher 2002) and the Pht4 family has been suggested to play a role in Pi
translocation between the cytosol, chloroplast, plastids and the Golgi apparatus
(Guo et al. 2008; Liu et al. 2011).
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 97

Targeting the Pi transporters to the plasma membrane via the secretory traffick-
ing pathway is mediated by PHF genes (phosphate transporter traffic facilitator 1),
which are expressed strongly in root tissues and in leaf mesophyll cells, mimicking
the expression pattern of the Pht1 gene family (González et al. 2005; Chen
et al. 2011). OsPHF1 and OsPHF1L (Chen et al. 2011) and TaPHF1 (Wang
et al. 2013) in rice and wheat, which are homologous genes to AtPHF1 in
Arabidopsis (González et al. 2005; Bayle et al. 2011), are localised specifically in
the endoplasmic reticulum (González et al. 2005; Bayle et al. 2011). AthPHF1
encodes for a plant-specific protein structurally related to SEC12 proteins of the
early secretory pathway (González et al. 2005). The exit of Pht1 transporter from
the endoplasmic reticulum and the targeting through the endosomal compartments
are modulated by phosphorylation (Bayle et al. 2011). The phf1 mutation in
Arabidopsis impairs Pi uptake and Pi transport (González et al. 2005). In rice, it
decreases excessive shoot Pi accumulation and Pi concentrations in leaves and roots
driven by OsPHR2 and OsPHF1 over-expression (Chen et al. 2011). Even under
P-replete experimental conditions, expression of phosphate starvation-induced
genes (PSI) was induced in Osphf1-1 mutants due to impaired plasma membrane
location of the low-affinity P transporter OsPT2 and a high-affinity P transporter
OsPht1;8 (Cheng et al. 2011).
Increasing expression of Pht1 transporters was observed as a response to P
starvation or mycorrhizal infection, exhibiting a large diversity of expression pat-
terns throughout the plant tissues. However, the lack of complete genome sequence
information has hindered detailed investigation in wheat. Pht1 transporters, which
are preferentially or exclusively expressed in roots, were found in barley (Smith
et al. 1999; Rae et al. 2003), wheat (Davies et al. 2002; Teng et al. 2013; Wang
et al. 2013), Arabidopsis (Mudge et al. 2002; Muchhal et al. 1996), rice (Paszkowski
et al. 2002) maize (Nagy et al. 2006) and tomato (Liu et al. 1998; Muchhal and
Raghothama 1999). Furthermore, Pht1 expression in shoot tissues has been reported
e.g. in phloem of older leaves, flag leaves or sheaths of barley (Rae et al. 2003), in the
panicle at the heading stage and flag leaves after heading in rice (Liu et al. 2011), in
mature pollen of Arabidopsis (Mudge et al. 2002) or during mycorrhizal infection in
roots of rice (Paszkowski et al. 2002; Yang et al. 2012), wheat (Glassop et al. 2005),
Brachypodium (Hong et al. 2012) and Medicago (Gaude et al. 2012).
The specificity of Pht1 expression to arbuscular mycorrhiza (AM) colonisation
has been investigated in cereal species and was considered symbiosis specific
(Harrison et al. 2002; Gutjahr et al. 2008; Glassop et al. 2005; Paszkowski
et al. 2002), as for instance for the expression of OsPT1;11 (Gutjahr et al. 2008;
Paszkowski et al. 2002) and OsPT1;13 (Yang et al. 2012; Güimil et al. 2005) in rice.
Homologues of both genes occur in Brachypodium (Hong et al. 2012) and maize
(Nagy et al. 2006), where transcripts also accumulated in non-colonised roots and
leaves, suggesting additional roles during P starvation (Yang et al. 2012).
The Pht1 promoters contain the target elements for transcription factors of the P
signalling network, for instance the P1BS cis-element as a target for PHR1
(Schünmann et al. 2004; Ren et al. 2012a) or the WRKY-binding W-box as a target
98 A. Gruen et al.

for WRKY75 (Devaiah et al. 2007a, b; Miao et al. 2009), suggesting their embed-
ding in the cross-talk of P signalling during P starvation to maintain P homeostasis.
It may be concluded that the regulation of Pi uptake and Pi transport mediated by
phosphate transporters, for which multiple roles in Pi acquisition and Pi
remobilisation have been suggested, are very complex. Roles of transporters in
the genetically diverse trait of tolerance to low P or in the PUE context will be
discussed further but remain elusive.

Morphological and Biochemical Adaptations

of Plants During P Limitation

When sensing nutrient depletion, plants have developed broad morphological and
biochemical strategies to deal with the heterogeneous availability of soil resources.
Root plasticity (morphology, topology and architecture) is a crucial but neglected
factor which is linked with immobile nutrients such as P (Lynch 1995). Plant roots
typically respond to P deficiency through allocation of more carbohydrates towards
the roots, which enhances root growth to maximise the soil volume exploited and
increases root to shoot ratio (Hermans et al. 2006; Hammond and White 2008).
Root hair formation (number, length and surface area) is strongly related to P
depletion (Bates and Lynch 1996; Gahoonia et al. 1997; Jungk 2001; Zhu
et al. 2005a, b) emphasizing a strong role in Pi acquisition from the soil (Gahoonia
and Nielsen 1998; Gahoonia et al. 2001). Topsoil foraging, which is characterised
by enhanced lateral root branching over primary root growth, contributes to effi-
cient P acquisition (Lynch and Brown 2001; Williamson et al. 2001; Pérez-Torres
et al. 2008).
P acquisition is enhanced through symbioses with arbuscular mycorrhizal
(AM) fungi (Barber 1984; Fitter 2006), by substantially increasing the P absorbing
surface for P uptake (Jakobsen et al. 1992) and the ability to access mineralised
organic P sources (Koide and Kabir 2000) and increased expression and secretion
of plant acid phosphatases (Tarafdar and Marschner 1994).
Strigolactones have a role in facilitating symbiosis formation (Gomez-Roldan
et al. 2008), and also stimulate tiller formation (Hong et al. 2012), which is an
important parameter for yield. As pH has a strong influence on the bioavailability of
soil Pi (Barber 1984; Hinsinger 2001) root excreted protons tend to acidify the
rhizosphere and, along with organic acids like malic acid, citric acid or phenolic
compounds that also act as chelators (Raghothama 1999; Vance et al. 2003), will all
help to solubilise Pi in the rhizosphere. Organic acids displace bound Pi from Al3+-,
Fe3+- and Ca2+-phosphates (Dinkelacker et al. 1989; Gerke et al. 1994). In partic-
ular, cluster roots (brush-like root formations) are an adaptation strategy to low soil
Pi availability of many plant species such as white lupin and other members of the
Proteaceae family and induce such chemical changes in the rhizosphere (Neumann
and Martinoia 2002). Plants also respond to P deprivation through the induction of
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 99

various metabolic processes e.g. the induction of a bypass pathway of glycolysis

and mitochondrial electron transport to replace ATP as an energy resource
(Theodorou and Plaxton 1993; Duff et al. 1989). All these metabolic changes
contribute to a better internal P utilisation when Pi is limiting.
The genetic background for these responses and their potential as targets for crop
genetic improvement based on studies with model plants will be described below.

Efficient Phosphate Nutrition in Model Plants

Phosphate acquisition and use efficiency (PAE, PUE) represent capabilities to cope
with either P limiting conditions or growth maintenance under P deficiency stress,
utilising morphological, biochemical and molecular changes without sacrificing
yields (Chiou and Lin 2011). The Pi-starvation adaptation response (PSR)
consisting of the regulation or coordination of the Pi-starvation inducible genes
(PSI) in order to maintain P homeostasis has been investigated predominantly in the
model plant Arabidopsis thaliana, in white lupine, in rice and in maize using
forward (mutants) and reverse genetic approaches (Alexova and Millar 2013;
Calderón-Vázquez et al. 2008; Li et al. 2010; Nilsson et al. 2007, 2010; Bustos
et al. 2010; Oono et al. 2011; Uhde-Stone et al. 2003; Rubio et al. 2001; Aung
et al. 2006; Sánchez-Calderón et al. 2006; Franco-Zorrilla et al. 2007; Miura
et al. 2011; Shin et al. 2006; Duan et al. 2008).
PSI genes and enzymes, which are induced or suppressed at the molecular level,
and commonly used as markers for monitoring PSR, are described below. Bio-
chemical and morphological changes have been elucidated to a much larger extent,
whereas information on phosphate sensing and signal transmission is more limited
(Chiou and Lin 2011). Nonetheless, the lack of studies investigating genotypic
variation in a broad spectrum of cultivars makes it difficult to draw conclusion for
enhancing PUE in crops (Alexova and Millar 2013; Calderón-Vázquez et al. 2011;
Veneklaas et al. 2012).

Root Morphology

Among common responses to P starvation are changes in root morphology such as

increasing root hair density, reduction of primary root growth and promoted lateral
root initiation, which are well described in Arabidopsis (Williamson et al. 2001;
López-Bucio et al. 2000; Sánchez-Calderón et al. 2006) and have been recently
studied in cereals (Hochholdinger and Zimmermann 2008). For instance, a root-
hairless mutant of Arabidopsis (Bates and Lynch 2001) and a barley root-hair-
deficient mutant (Gahoonia et al. 2001) grew poorly under low Pi. The root cap is
the site of sensing local Pi concentrations initiating spatial changes, comprising
inhibition of cell division activity of primary meristematic cells and root cell
100 A. Gruen et al.

elongation (Ticconi et al. 2004; Sánchez-Calderón et al. 2005; Franco-Zorrilla

et al. 2007; Svistoonoff et al. 2007). Phytohormone-related genes have been
reported to mediate root architectural changes under low P growing environments,
particular auxin-responsive genes (Bates and Lynch 1996; Hammond et al. 2004;
Pérez-Torres et al. 2008; Jain et al. 2007; Miura et al. 2011). Expansins are involved
in cell wall extension (Zhao et al. 2012), including root-hair formation
(Yu et al. 2011) and are stimulated by indole-3-acetic acid and abscisic acid
under abiotic stress (Zhao et al. 2012).
For instance, Miura et al. (2011) assumed that genes coding for expansin
17, glycosyl hydrolase 19 and UDP-glycosyltransferase are involved in the regula-
tion of cell wall-loosening and elongation in response to P starvation in
Arabidopsis. Distinct responsiveness to Pi availability among Arabidopsis ecotypes
(Chevalier and Rossignol 2011) and the identification of quantitative trait loci
(QTLs) (Reymond et al. 2006) suggests a genetically determined control of the
root growth response to low Pi. Genetic factors controlling root plasticity have been
investigated using low phosphorus insensitive (lip) mutants (Sánchez-Calderón
et al. 2006). The mutation disrupted not only the root developmental response but
also altered the induction of P deprivation responsive genes, which are relevant for
adaptation to low-Pi, including acid phosphatases (AtPAP1, AtACP5) and phos-
phate transporters (AtPT1, AtPT2) (Sánchez-Calderón et al. 2006). Furthermore,
their findings suggest that the root architectural response is mediated by a specific
nutrient (P) sensing signalling network (Sánchez-Calderón et al. 2006). For
instance, WRKY75 (Devaiah et al. 2007a) and ZAT6 (Devaiah et al. 2007b) are
among several transcription factors, which have a regulatory effect on root archi-
tecture of Arabidopsis and were suggested to have an impact on P starvation
responses. However, genetic selection based on root parameters has been difficult
due to their multigenic nature and the lack of appropriate evaluation methods
(Vance et al. 2003).

P Starvation Signaling

Elicited responses to internal and external nutritional status involve local and
systemic signalling (Chiou and Lin 2011); signalling molecules, their mode-of-
action and interacting pathways are summarised below.
A strong increase of Induced by P starvation gene transcripts (IPS) under Pi
starvation has been reported in Arabidopsis and rice (Rubio et al. 2001; Oono
et al. 2011) and members of the IPS gene family have been widely used as
molecular markers of plant Pi nutritional status (Zhou et al. 2008; Tian
et al. 2012; Wang et al. 2013). IPS genes are involved in the miR399-PHO2
regulatory loop as ribo-regulators (Doerner 2008) and function as miRNA399
antagonists, which negatively alter PHO2 expression at the post-transcriptional
level; a regulatory process which is described as “target mimicry” (Franco-Zorrilla
et al. 2007). It seems likely that they stabilise the initial decrease of PHO2 transcript
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 101

to prevent Pi toxicity via Pi accumulation in the shoots (Bari et al. 2006; Chitwood
and Timmermans 2007). Five IPS genes have been found in the Arabidopsis
genome (Franco-Zorrilla et al. 2007), and two in rice, maize and barley (Hou
et al. 2005). Promoters of the pho4-regulon in yeast have two cis-regulatory
elements, which were also found in Arabidopsis (At4/AtIPS4), tomato (TPSI1),
Medicago truncula (Mt4) and rice (OsPI1) (Hammond et al. 2003). At4 and AtIPS4
in Arabidopsis are involved in Pi allocation between roots and shoot and enhance
lateral root development (Shin et al. 2006; Franco-Zorrilla et al. 2007).
The at4 mutant exhibited P accumulation in shoots (Shin et al. 2006), whereas
over-expression decreased P accumulation (Franco-Zorrilla et al. 2007). AtIPS1
modulates PHR expression, a MYB-CC type transcription factor which is involved
in P starvation responses (Rubio et al. 2001). PHR1 (phosphate starvation respon-
sive 1) plays a pivotal role in sensing P availability (Chiou and Lin 2011) and has
been examined in detail. PHR1 is a member of the MYB-transcription factor family
(15 members) and seems to be a key regulator for downstream P responsive genes
through binding to a P1BS (PHR1 specific binding sequence) cis-element, which is
an imperfect palindromic sequence (GNATATNC) (Rubio et al. 2001; Nilsson
et al. 2007, 2010; Bustos et al. 2010). An important downstream target of
AtPHR1 and possible homologues is miRNA399, which is involved in the PHO2
regulation as previously mentioned (Miura et al. 2005; Schachtman and Shin 2007).
Over-expression of AthPHR1 increased the transcript level of miRNA399 and
decreased expression of PHO2, increased further the Pi content and enhanced root
hair density in rice and Arabidopsis (Nilsson et al. 2007; Zhou et al. 2008; Bustos
et al. 2010). Promoters of several P starvation-induced and repressed genes, includ-
ing IPS and a high-affinity P-transporters, contain the P1BS cis-element (Oono
et al. 2011; Hammond et al. 2003; Rubio et al. 2001; Schünmann et al. 2004; Guo
et al. 2013; Bustos et al. 2010). In wheat, over-expression of TaPHR1 did not
change transcript levels of TaPHF1, TaPHO2 or TaSPX3, whereas TaIPS and
TaPht1;2 exhibited increased expression levels in the transgenic lines suggesting
that transcriptional factors additional to TaPHR1 may be functional in the P
starvation signalling (Wang et al. 2013). However, the Athphr1 mutant impairs a
broad range of P starvation responses and shows impaired root growth and root hair
length (Rubio et al. 2001; Bustos et al. 2010; Nilsson et al. 2007).
In rice, two homologues of AtPHR1, OsPHR1 and OsPHR2, are involved in P
starvation signalling (Zhou et al. 2008; Wang et al. 2009a, b). However only over-
expression of OsPHR2 resulted in increased shoot Pi and altered root morphology
(Zhou et al. 2008; Wu and Wang 2008; Bustos et al. 2010). OsPHR2 positively
regulated the low-affinity phosphate transporter OsPT2 in roots resulting in exces-
sive Pi accumulation in the shoot tissue (Liu et al. 2010). Further, a root-associated
purple acid phosphatase (10a) in rice, OsPAP10a, is controlled and induced by
OsPHR2 (Tian et al. 2012).
SPX proteins (which contain a SPX domain, SYG1, PHO81, XPR1 at the
N-termini) are involved in the downstream responses of PHR1 in Arabidopsis
(Duan et al. 2008) and OsPHR2 and PHO2 in rice (Wang et al. 2009a, b). Members
of the SPX protein family in rice (OsSPX3 and SPX1/2/6) have been shown to be
102 A. Gruen et al.

highly induced (preferentially) in rice roots and shoots where they are involved in
the regulation of PSI and OsIPS1 (Wang et al. 2009a, b; Oono et al. 2011). OsSPX1
over-expression suppressed IPS gene induction, miRNA399 and phosphate trans-
porter Pht1 expression (Wang et al. 2009a, b) and in yeast, an SPX domain limited
the phosphate uptake velocity (Hürlimann et al. 2009). Similar results were
obtained with Arabidopsis mutants for AtSPX1-AtSPX4 affecting the expression
pattern of purple acid phosphatases genes (Duan et al. 2008). Furthermore, OsSPX1
is positively regulated by OsPHR2, involved in the feedback Pi signalling network
in roots by suppressing OsPT2 and other PSI genes in the PHR2/Pho2 background
(Liu et al. 2010), and negatively regulates shoot P accumulation (Wang et al. 2009a,
b). OsSPX1 over-expression counteracted the effect of PHR2 over-expression in
rice, which mimics P starvation and induces PSI gene expression but not the
function of Ospho2 regulating OsPT2 expression (Liu et al. 2010). In summary,
SPX proteins seem to be essential players for maintaining P homeostasis and P
signalling in plants (Rouached et al. 2010; Nilsson et al. 2012; Secco et al. 2012).
The regulatory mechanism of P allocation among different organs during plant
development under P stress remains relatively elusive and investigations have been
mainly focused on screening Arabidopsis mutants with abnormal P distribution.
The Athpho1 mutant showed severe P deficiency in above-ground shoot tissues due
to influencing transfer of Pi to the xylem vessels for subsequent transport to the
shoot and leaves (Poirier et al. 1991; Liu et al. 2012). PHO1 is a membrane-
spanning protein but there is no evidence that it is a transporter itself and it does
not have homology to any other previously known transporter (Hamburger
et al. 2002). Eleven members of the AthPHO1 transporter family are known
which share the same topology (Wang et al. 2004); a SPX tripartite domain in the
N-terminal (SYG1/PHO81/XPR1) and an EXS domain at the C-terminal (ERD1/
XPR1/SYG1). AthPHO1 has been localised to the ER and the Golgi (Liu
et al. 2012). The EXS/SPX domains, and particularly the N-terminal region of
PHO1, have been identified in yeast as being involved in either phosphate transport
or in sorting proteins to endomembranes (Liu et al. 2012; Wang et al. 2004).
AtPHO1 seem to mediate Pi efflux out of root stellar cells along its electrochemical
gradient (Hamburger et al. 2002) and AtPHO1;H1, seems to be regulated by PHR1
(Stefanovic et al. 2007). The roles of the other members, AtPHO1;H2 to AtPHO1;
H9, are unknown (Secco et al. 2010) but show a distinct expression pattern from
that of AtPHO1 and AtPHO1;H1 (Hamburger et al. 2002). The AthPHO1 family
clusters into two clades, which are expressed in a broad range of tissues, including
leaves and predominately in vascular tissues of roots, leaves, stems or flowers. Only
one clade which contains AtPHO1 and AtPHO1;H1 clusters with the three OsPHO1
proteins found in rice (Secco et al. 2010). OsPHO1;2 was mostly expressed in roots
and was relatively lowly and constantly expressed in other tissues. A mutation
affected root-to-shoot Pi transfer (Secco et al. 2012). OsPHO1;1 was predominately
expressed in flowers before and during pollination and OsPHO1;3 was the lowest
expressed, and higher in leaves and flowers (Secco et al. 2010). AthPHO1 is a
downstream component of the AthPHO2 regulatory pathway (Liu et al. 2012).
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 103

PHO1 degradation or down-regulation is not Pi and PHO2 dependent and was

suggested to occur at the post-translational level (Liu et al. 2012; Aung et al. 2006).
The Arabidopsis pho2 mutant exhibits excessively high P concentrations in the
shoots, displaying symptoms of toxicity as a result of enhanced uptake and root-to-
shoot translocation (Delhaize and Randall 1995; Liu et al. 2012). PHO2 is predom-
inantly expressed in the root (Chiou et al. 2006) and its localisation within the cell
has been determined as being with the ER and Golgi (Liu et al. 2012). PHO2 is a
member of the E2 ubiquitin conjugase family (UBC24) (Bari et al. 2006) and is a
target for miRNA399, which suppresses PHO2 expression (Aung et al. 2006; Chiou
et al. 2006; Fujii et al. 2005). Over-expression of miRNA399 or loss of function of
UBC24 resulted both in enhanced Pi accumulation and impairment of Pi
remobilisation from old to young leaves in Arabidopsis (Chiou et al. 2006). Expres-
sion of miRNA399 is an early response to P deficiency and a systemic signal of Pi
deficiency derived from P deplete root and shoot tissues (Lin et al. 2009; Bari
et al. 2006; Aung et al. 2006; Chiou et al. 2006). Its expression is regulated by
PHR1 (Bari et al. 2006; Aung et al. 2006) in a mechanism called “target mimicry”
(Franco-Zorrilla et al. 2007) as previously described. In addition to miRNA399,
various other miRNAs could be identified by deep sequencing in Arabidopsis
(Hsieh et al. 2009; Lundmark et al. 2010) and other plant species (Chiou and Lin
2011), including white lupin (Zhu et al. 2010), soybean (Zeng et al. 2010), tomato
(Gu et al. 2010) and wheat (Zhao et al. 2013). It has been assumed that high-affinity
phosphate transporters, AthPht1;8 and AthPht1 cause the phenotype of the pho2
mutant and miRNA399 over-expressors because they are up-regulated in these
mutants (Aung et al. 2006; Bari et al 2006). In rice shoots, Ospho2 mediated P
accumulation was assumed being the result of induced expression of high- and
low-affinity phosphate transporters OsPht1;2, OsPht1;9 and OsPht1;10 (Liu
et al. 2010). It has been suggested that PHO2 encodes an additional regulator for
the low-affinity Pi translocator protein, AthPT2;1, in green shoot tissues of
Arabidopsis (Daram et al. 1999). Nonetheless, downstream responses of PHO2
are still not completely understood (Liu et al. 2012). PHO1 and PHO2 are both
expressed in vascular root tissues (Hamburger et al. 2002) as is miRNA399 (Aung
et al. 2006).
There is evidence that sucrose is a global regulator of plant P starvation
responses, interacting with P starvation signals (Lloyd and Zakhleniuk 2004;
Karthikeyan et al. 2007; Lei et al. 2011) and root architecture alterations (Niu
et al. 2012). Sucrose phosphate synthase (SPS) has been reported to be more
abundant in P deficiency tolerant cultivars of Brassica napus (Yao et al. 2011).
Exogenously applied sugars or sucrose-enriched growing media could stimulate P
starvation inducible genes such as acid phosphatases (APase), AtIPS1 (Müller
et al. 2005) or an APGase subunit in tobacco (Nielsen et al. 1998).
Phosphate transporters, for instance TaPht1;2 (Miao et al. 2009), seem to belong
to the group of sugar-modulated genes under P-starvation (Jain et al. 2007;
Karthikeyan et al. 2007; Hammond and White 2008).
The Arabidopsis pho3 mutant exhibits a restricted sucrose translocation from
root to shoot caused by a defective sucrose transporter, SUC2, which is involved in
104 A. Gruen et al.

phloem loading (Lloyd and Zakhleniuk 2004). Pho3 shows altered APase induc-
tion/secretion on the root surface, reduced Pi accumulation in both leaves and roots
(Zakhleniuk et al. 2001) and a strongly induced Glc-6-P/phosphate translocator.
This phenomenon is again consistent with the observation that sucrose accumulates
in Pi-starved leaves of various crops (Hammond and White 2008). Microarray
analysis investigating consequences of SUC2 over-expression in the transcriptome
of the hypersensitive to phosphate starvation1 (hps1) Arabidopsis mutant revealed
the induction of Pi starvation induced genes under P replete conditions (Lei
et al. 2011). In conclusion, sugar sensing and signalling is involved in adaptation
responses to P starvation even if the exact mechanism is not understood.
There are other transcription factors induced by P starvation and involved in Pi
availability responses including OsPTF1 (Yi et al. 2005), WRKY75 and ZAT6
(Devaiah et al. 2007a, b), BHLH32 (Chen et al. 2007), WRKY6 (Chen
et al. 2009a, b) and MYB62 (Misson et al. 2005). Two WRKY box (W-box)
elements have been found in the promoter of genes involved in Pi retranslocation
and scavenging, including AtPht1 transporters, AtIPS, acid phosphatase genes
(AtPS2), purple acid phosphatases (PAP11) and PHR1 (Devaiah et al. 2007a, b).
BHLH32 acts a negative regulator for PPCK (phosphoenolpyruvate carboxylase
kinase) expression in P sufficiency, root hair formation and anthocyanin production
(Chen et al. 2007). Plant hormones have been implicated with P signalling mech-
anisms as changes in Pi availability alters the expression of genes involved in the
biosynthesis of phytohormones which in turn may influence PSR (Morcuende
et al. 2007; Chiou and Lin 2011). Pi itself may be important in P starvation
signalling, as is the case for nitrate acting as a signal stimulating root growth
(Zhang and Forde 2000). The Pi flow across the peri-arbuscular cortex membrane
may be among the mechanisms which allow plants to recognise AM fungi from less
beneficial microbes (Yang and Paszkowski 2011). Compelling evidence is provided
by studies using phosphite, a phosphate analogue which is taken up by plants but
cannot be oxidised once inside the cell, mimicking sufficient Pi supply in P starving
plants, which result in an interference and attenuation with PSR at the transcrip-
tional and post-transcriptional level (Chiou and Lin 2011).

Metabolic Changes

Several proteomics studies are contributing to the understanding of the metabolic

responses to P starvation in model plants and crops (Yao et al. 2011; Li et al. 2008a,
b; Chevalier and Rossignol 2011). However, proteins responsible for distinct
PAE/PUE in various species and cultivars have not been identified (Alexova and
Millar 2013).
P limitation alters the TCA cycle metabolism and has been shown in a broad
range of studies. Enzymes involved in the TCA cycle and glycolysis produce
organic acids required for P recycling from phosphorylated glycolytic intermedi-
ates as well as releasing Pi from organic P sources or inorganic bound Pi in the soil
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 105

(Oono et al. 2011). The over-production of citrate in transgenic tobacco (López-

Bucio et al. 2000) as well as mitochondrial citrate synthase in A. thaliana (Koyama
et al. 2000) enhanced Pi uptake. Key enzymes which have been studied in
A. thaliana are citrate synthase, malic enzyme and aconitase, which exhibited
variation in protein abundance between ecotypes during P deficiency (Chevalier
and Rossignol 2011). In other species, the activity of aconitase correlated with
organic acid secretion (Neumann and Römheld 1999) and in alfalfa, the
overexpression of malate dehydrogenase (MDH) resulted in increased P accumu-
lation (Tesfaye et al. 2001). Another approach was the expression of phytase genes
of alfalfa or of a fungal origin in Arabidopsis and tobacco plants, resulting in
improved acquisition of organic P sources (George et al. 2005). However, the
length of P starvation influences the synthesis and degradations of proteins which
are potentially involved in enhancing the plants adaptation to P deficiency. For
instance, genes encoding for isocitrate dehydrogenase were suppressed in rice roots
only after a certain time of exposure to P starvation, resulting in a suppression of
citrate degradation (Oono et al. 2011).
The replacement of phospholipids by galactolipids or sulpholipids is a well-
known adaptation process in plants during P deficiency (Andersson et al. 2003;
Hammond et al. 2003; Byrne et al. 2011), even if phospholipid degradation is
differently mediated in different species (Calderón-Vázquez et al. 2011). For
instance, in potato, an array study identified novel roles for the main storage protein
in potato tubers, the patatin-like proteins, which also have lipase activity and are
potentially involved breakdown of phospholipids for Pi recycling (Hammond
et al. 2011). Numerous studies in model plants or crops reported the induction of
genes related to an altered lipid metabolism, for example UDP-sulfoquinovose
synthase 1 (SQD1) or glycerophosphoryl diester phosphodiesterase (GDPD) or
lipid transfer proteins (Hammond et al. 2011; Oono et al. 2011; Morcuende
et al. 2007; Calderón-Vázquez et al. 2008; Wasaki et al. 2003). Glyceropho-
sphodiester phosphodiesterases (GPX-PDE) catalyse the hydrolysis of phospho-
lipids to glycerol-3-phosphate and the corresponding alcohol. Recently, GPX-PDE
genes were identified which were highly expressed in cluster roots of white lupine
under Pi-deficiency (Cheng et al. 2011; Uhde-Stone et al. 2003).
To date, the knowledge about functional consequences of replacing phospho-
lipids in membranes is very limited (Veneklaas et al. 2012).

Post-translational Modifications

Post-translational modifications are important factors in P signalling and metabolic

pathways involved in PUE (Alexova and Millar 2013; Plaxton and Tran 2011),
highlighted when comparing proteome with transcriptome studies in P-starved
maize (Calderón-Vázquez et al. 2008; Li et al. 2008a, b) and Arabidopsis
(Morcuende et al. 2007). A striking example underpinning the importance of
post-translational modifications within adaptation to low P exposure is the potential
106 A. Gruen et al.

role of a protein kinase, OsPupK46-2, within the Pup1 locus (Phosphorus uptake
1), which is a major QTL for low P tolerance in rice (Gamuyao et al. 2012).
Pht1 phosphate transporter genes are also regulated at the post-transcriptional
level by the recently discovered PHF1 in rice and Arabidopsis (Chen et al. 2011;
Bayle et al. 2011; Chiou and Lin 2011), which is connected to a kinase,
RAPTOR1B. PHR1 is up-regulated at the transcript level and both are
up-regulated at the protein-level in P-starved roots of Arabidopsis (Lan
et al. 2012). The enhanced synthesis of organic acids allows P recycling from
phosphorylated glycolytic intermediates, particularly phosphoenolpyruvate (PEP),
which is mediated via the enzyme phosphoenolpyruvate carboxylase (PEPC).
PEPC is in turn activated by reversible phosphorylation via PPCK (PEPC kinase)
(Gregory et al. 2009). PPCK has been reported as being among up-regulated genes
during P starvation (Morcuende et al. 2007; Müller et al. 2007; Chen et al. 2007).
Therefore, PEP and PEPC are among the metabolites and enzymes, which are more
abundant or active in P starved A. thaliana, B. nigra, O. sativa and T. aestivum
(Morcuende et al. 2007; Johnson et al. 1996; Duff et al. 1989; Oono et al. 2011;
Neumann and Römheld 1999).
In Arabidopsis, PHR1, which initiates P starvation signalling responses, was
hypothesised to be a target for the conjugation of the SUMO superfamily of proteins
via SUMO ligases (sumoylation; Miura et al. 2005; Wang et al. 2013). The
sumoylation is mediated by the small ubiquitin like modifier SUMO E3 ligase,
AtSIZ1, which is more root than shoot abundant and is localised in the nucleus of
the cells (Miura et al. 2005). The homologue, OsSIZ1, have been also detected
among transcripts of P starved rice (Oono et al. 2011). The observation that AtIPS
gene induction was reduced in siz1 mutants, indicating a positive regulation with
AtSIZ1, (Miura et al. 2005), strengthens the hypothesis that post-translational
modifications as essential for the initial stages of P starvation signalling cascades.
Additionally, AtSIZI negatively regulates P starvation-dependent primary root
growth inhibition (increased root hair number and length) through the control of
auxin patterning (Miura et al. 2011), whereas the phr1 mutant does not exhibit
affected root architecture (Rubio et al. 2001).
In conclusion, post-translational modifications may be essential leaders for P
economy in plants (Raven 2008) and potentially for enhancing PUE in crops.

PAE and PUE in Crops

Definitions and Implications

Grain crops like rice, maize, wheat and oilseed rape are essential major staple foods
(FAO 2011) and major contributors to the global phosphorus cycle (Rose and
Wissuwa 2012). Global P flows resulting from, for instance, the rock mineral
fertiliser trade, the global grain trade or manure-delivering livestock production
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 107

Fig. 4.2 Global map of agronomic P imbalances for the year 2000 per unit of cropland area in
each 5  grid cell. The surpluses and deficits are each classified according to quartiles globally (0–
25th, 25–50th, 50–75th, and 75–100th percentiles) (Data taken from MacDonald et al. (2011) and
used with permission)

are unevenly distributed, resulting in massive agronomic imbalances and spatial

surplus or deficit patterns across regions and countries (Tiessen 2008; MacDonald
et al. 2011; Fig. 4.2). Additionally, large amounts of applied P are removed in
harvested products from the fields and across the globe (Lott et al. 2009), and
nutrient recycling, especially organic P sources, from urban areas or returning
biomass is rare (Cordell et al. 2009). Rock P fertilisers are extracted and exported
from only a few countries worldwide, which are geopolitically controversial and
potentially unstable (Cordell et al. 2009; Tiessen 2008; FAO 2011). Demand is still
increasing and estimated to be 1.9 % yearly from 2011 to 2015 (FAO 2011). In
order to meet the requirement for the future demands of an increasing world
population, bearing in mind that P reserves are limited and deposits cannot be
exploited infinitely (Kirkby and Johnston 2008), it is a necessity to enhance P
fertiliser use efficiency (Gregory and George 2011).
There is an essential need for agronomic P efficiency criteria to be defined in
order to be able to exploit and screen genetic variation in grain crops (Rose and
Wissuwa 2012), one of the key strategies for breeding more P efficient crops
(Gregory and George 2011). Phosphorus efficiency is a complex trait and the
genetic determinants, which are involved in enhanced low P tolerance or P effi-
ciency are still not clearly understood. As previously described, the focus of
molecular research lies mainly on P stress responses of individual genotypes or
model plants and it is questionable if these findings are generally applicable for
crops in an agricultural context. Genotypic phosphate efficiency differences that
exist might only rarely be conserved between and within species (Hammond
et al. 2009; Alexova and Millar 2013; Calderón-Vázquez et al. 2008, 2011; Niu
et al. 2012) and need to be identified before they can be exploited for breeding. Due
to the previously described interactions of P accessibility in soils (Holford 1997),
108 A. Gruen et al.

field experiments or experiments in soil might be more suitable for assessing or

identifying traits for P efficiency rather than using solution culture (Hayes
et al. 2004; Gunes et al. 2006; George et al. 2004, 2005).
For agronomic and economic reasons, P efficiency is based on the cropping area
leading to improved P fertiliser recovery and use of soil P (Sattelmacher
et al. 1994). This concept takes into account the low P availability in e.g. tropical
soils (Wissuwa et al. 2009), limited access to P fertilisers in some regions of the
world (Tiessen 2008), costs for fertilisation (FAO 2011) and environmental aspects
including impaired water quality by run-off or drainage due to agricultural inten-
sification (McDowell 2012). This unit is based on the P efficiency properties of the
plant itself, which can be divided into PAE and PUE (Wang et al. 2010a, b). It is of
strong economic interest to not only enhance P acquisition, which might result in
over-mining the soil, but also enhance PUE without increasing P export from the
field via the grain (Batten 1992; Rose and Wissuwa 2012). Both traits are usually
linked, negatively associated with each other and are hard to distinguish
(Su et al. 2006; Su et al. 2009; Rose and Wissuwa 2012), indicating a need to
choose a selection technique achieving an appropriate distinction (Batten 1992) and
a clear positive correlation of biomass ratios with increasing PUE (Rose and
Wissuwa 2012). In contrast to PAE, PUE is much less well understood, lacks
clearly defined and consistent terminologies or screening methodologies across
the literature which is a bottleneck for P efficiency improvement in crops
(Hammond et al. 2009; Wang et al. 2010a, b; Rose and Wissuwa 2012). To achieve
an increase of PUE is especially important in regions of high cropping intensity
facing plateauing yields during the last decade.

Screening Approaches

When P efficiency is to be evaluated across a range of distinct or heterogeneous

genotypes e.g. high-yielding modern varieties and low-yielding land races, a tissue-
specific approach is probably required (Rose and Wissuwa 2012). Using this
approach presupposes knowledge about the different P pools as well as about the
changing P requirements depending on the growth stage of the crop (Veneklaas
et al. 2012).
Phosphate is compartmentalised within plant cells and exists in two main P pools
(Veneklaas et al 2012). The first P pool consists of free inorganic orthophosphate
(Pi), which is either metabolically active in the cytoplasm or stored in the vacuole to
buffer P demands of the cytoplasm (Mimura et al. 1996; Lauer et al. 1989). The
storage Pi can have a diagnostic value (Bollons and Barraclough 1997, 1999),
although shoot growth seems to be reduced before severe Pi depletion of the
vacuolar storage pool occurs (Rouached 2011; Mimura et al. 1996). The second P
pool represents organic forms as P esters, comprising nucleic acids, phospholipids,
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 109

phosphorylated proteins and low relative molecular mass metabolites (Veneklaas

et al. 2012). In the nucleic acid pool, RNA is usually the largest with 40-60 % of this
P pool (Bieleski 1968) with ribosomal RNA (rRNA) having the biggest share,
adjusting with growing pattern (Suzuki et al. 2010; Hensel et al. 1993; Kanda
et al. 1994). Nucleic acid and protein turnover and repair has a large P cost
(Raven 2012). Hence, plants cannot dispense DNA or RNA without affecting
growth (Raven 2008); a target for use efficiency would be the optimising of the
ribosomal pool size, protein biosynthesis and especially protein turnover
(Veneklaas et al. 2012).
Phospholipids in cell membranes fulfil structural roles, serve as substrates for
biochemical signals and are required in abundance by photosynthetic tissues and
cell-expanding/-dividing tissues. The replacement of phospholipids by glycolipids
(galactolipids, sulfolipids) in plastids, as result of P deficiency are known (Wasaki
et al. 2003; Andersson et al. 2003; Essigmann et al. 1998; Oono et al. 2011), in
order to economise the use of P, as the total area of membranes can hardly be
decreased (Veneklaas et al. 2012). In cyanobacteria and algae their replacement can
be complete (Van Mooy et al. 2006, 2009) or partial in rhizobial symbionts (Gaude
et al. 2004), but in plants consequences of such a replacement remain speculative
(Veneklaas et al. 2012).
Phosphate uptake seems to be most critical during vegetative growth whereas
a shift occurs in the late vegetative or early reproductive stage and remobilisation
and optimal allocation becomes another resource of P (Rose et al. 2007; Römer
and Schilling 1986; Veneklaas et al. 2012). There are rankings in the literature
focusing on early growth (Liao et al. 2008) or contrastingly, on grain yield (Jones
et al. 1992). P concentrations in the grain of crops are usually much higher than in
vegetative tissues (Veneklaas et al. 2012), and seed P reserves occur predomi-
nately as phytate (Lott et al. 2009; Raboy 2009; White and Veneklass 2012),
which are the salts of phytic acid with high affinity to Zn and Fe (Michael
et al. 1980). Seed P content as well as phytate content differs between wheat
genotypes (Batten 1992) and declines with decreasing P supply (Mengel and
Kirkby 2001). High P grain content, especially phytate, is not particularly desir-
able, as it acts as an anti-nutrient in animal and human diet which aggravates the
global problem of mineral malnutrition (White et al. 2012) and causes environ-
mental problems in the form of phosphate-rich manure or sewage (Raboy 2009)
once it is removed with the grain as the harvested product from the cropping area.
However, whilst low seed P content or concentration could be appropriate selec-
tion criteria for improved PUE, it is controversial. Grain or seed P reserves
support initial seedling growth until it is supplemented through Pi uptake by the
developing root system (White and Veneklaas 2012) and correlated with the
initial root biomass (Zhu et al. 2005a, b). It is questionable if seed coating or P
fertiliser placement could compensate low grain P (Rebafka et al. 1993), even if
lower root development due to lower seed P reserves can be overcome due to
mycorrhizal infection (Zhu and Smith 2001).
110 A. Gruen et al.

Exploiting Genetic Differences

There are studies showing genotypic differences for P deficiency tolerance

suggesting that P efficiency mechanisms may differ among wheat, rye and triticale
genotypes (Ozturk et al. 2005; Manske et al. 2001; Osborne and Rengel 2002;
Gunes et al. 2006). Hammond et al. (2009) observed a considerable diverse species-
wide variation for shoot P concentrations and several PUE measures in Brassica
oleracea landraces and commercial varieties. Manske et al. (2001) argued that
under P deficient conditions P uptake efficiency plays the key determinant for yield,
whereas P utilisation efficiency was more relevant under well P supplied condi-
tions. There are also several investigations in other Brassica plants aiming to
exploit genetic diversity (Akhtar et al. 2008; Solaiman et al. 2007; Yang
et al. 2010, 2011).
Batten (1992) pointed out that selection of more P efficient wheat occurs
unconsciously during breeding when selecting for higher yields at sub-optimal P
levels. Chin et al. (2010) mentioned a similar observation of an unconscious
selection for a major P deficiency tolerant rice QTL, Pup1, in drought tolerant
varieties developed under unfavourable conditions. Hammond et al. (2009) simi-
larly implicated inadvertent selection in B. oleracea breeding programmes.
The selection process resulting in modern (Brassica) crop varieties with
increased grain yield per mg shoot P (Hammond et al. 2009) might be just a result
of higher HI (Batten and Khan 1987), rather than a selection of physiological traits
(Rose and Wissuwa 2012). Chin et al. (2010) support that hypothesis by observing a
lower frequency of the low P-tolerant QTL Pup1 in modern irrigated rice varieties
compared to traditional varieties. There are further studies showing that enhanced
low P tolerance in wheat was due to enhanced P uptake rather than enhanced P
utilisation (Gahoonia et al. 1996; Gahoonia et al 1999).
A comparison of new CIMMYT wheat varieties with an older Mexican variety
showed that PUE did not differ (Egle et al. 1999). However, P acquisition under low
P conditions was improved mainly due to a better root length density, especially
during the period during anthesis and grain filling (Egle et al. 1999). With P
fertilisation, root length density was not significantly different between the older
and the modern varieties, but higher P uptake rates, especially during grain filling,
of the modern varieties seemed to have contributed to higher P acquisition ability at
appropriate P supply. A higher shoot growth and a subsequently higher sink
capacity of more kernels might have contributed to that effect (Egle et al. 1999).
In maize, several studies have identified distinguishable P uptake amongst geno-
types (Da Silva and Gabelman 1992; Zhu and Lynch 2004; Chen et al. 2009a, b; de
Sousa et al. 2012), without providing large amounts of potential target genes for
exploiting crop biodiversity (Calderón-Vázquez et al. 2008).
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 111

Potential Targets for Genetic Improvement

The design of phenotypic screens for dissecting P acquisition or P utilisation

differences in crops requires an understanding of the underlying molecular mech-
anism of low-P tolerance. This section highlights mechanisms which are known in
crops and that may be potential targets for a breeding approach, integrating
physiological, molecular and genetic strategies. Genes will be listed that come
from model organisms and were subsequently investigated in crop species. A
potential candidate target would be characterised by being a key factor in the
molecular mechanism of the P starvation response, adaptation and genetic diversity
responsible for low P tolerance, keeping in mind that there is a need for different
strategies in low-input and high-input systems, focusing more on PAE or PUE
respectively. Teng et al. (2013) investigated the expression profiling of known P
starvation-induced genes in wheat under different levels of P fertiliser and soil
Olsen P, proving that the turning point for the genetic response was the critical P
level. This observation leads to a more general model (Fig. 4.3) which raises the
question: which strategy would be most suitable to shift the onset of gene expres-
sion for the P starvation signalling response into lower P levels and therefore
decreasing the crop demand for equal yield performance? There were three main
scientific approaches to this problem.
The first approach comprises the majority of studies which are based on com-
parisons of low P-tolerant genotypes with more susceptible genotypes or cultivars
(Pariasca-Tanaka et al. 2009; Li et al. 2008a, b; Hammond et al. 2009; Zhang

Fig. 4.3 Model for of the optimal yield (red up arrow) coinciding with the induction of
P-starvation marker genes (green down arrow) in field-derived wheat roots shifting towards
lower soil Olsen-P levels with improved P efficiency traits (Teng et al. 2013)
112 A. Gruen et al.

et al. 2009; Li et al. 2010; Yao et al. 2011) exposed to a short-term P starvation
period; particularly transcriptome, proteome or metabolite profiling studies (Wang
et al. 2002; Wasaki et al. 2003; Hammond et al. 2004; Calderón-Vázquez
et al. 2008; Huang et al. 2008; Oono et al. 2011; Liang et al. 2010; Oono
et al. 2013). Even if these profiling studies provided a useful tool to study the
response mechanisms with regards to adaptation to nutrient stresses (Hammond
et al. 2004; Nilsson et al. 2010), the majority used hydroponically grown plant
material exposed to short-term P starvation. However, Hammond et al. (2011) used
the transcriptional profiling technology to identify a predictive diagnostic gene set
for detecting the physiological Pi status of potato under field conditions and at a
range of P fertiliser application rates. This group of genes were determined by
investigating the transcriptional P starvation responses in potato leaves grown
hydroponically and were validated for exposure to various nutritional and abiotic
stresses using the Arabidopsis orthologs (Hammond et al. 2011). This aspect is
more focused on increasing the precision of fertiliser application but may also be a
potential tool for genotypic screening PUE and PUE under agronomic conditions in
the future.
The second approach deals with the over-expression of target genes resulting in
partly contradictory observations due to the expression level of their role in the P
sensing and regulating network (Rae et al. 2004; Zhou et al. 2008; Ren et al. 2012a,
b; Tian et al. 2012; Guo et al. 2013; Wang et al. 2013).
The third approach uses quantitative trail loci (QTL) analysis to dissect the
genetic basis of P efficiency and identify superior alleles or loci in different
germplasm (Wissuwa et al. 2005; Zhu et al. 2005a, b; Su et al. 2006; Liang
et al. 2010; Yang et al. 2011; Gamuyao et al. 2012) which should lead, if successful,
to marker-assisted selection (MAS) in breeding for improved nutritional traits. All
three approaches will be described and discussed in more detail within the sections
below.

Root Morphology and Organic Acid Secretion

Due to the low mobility of P in the soil, the root architecture of crops in agricultural
systems is strongly related to P distribution in the soil profile, determined by tillage,
fertiliser and cultivation practices, which influence in turn the chemical dynamics
of soil P and the rhizosphere (Niu et al. 2012). Phenotyping of root (architectural)
parameters are difficult to evaluate as selection criteria as they are both time
consuming and destructive (de Sousa et al. 2012), which makes the in vitro QTL
analysis approach described below very attractive for breeders using MAS (Liang
et al. 2010). Root morphology or primary root growth in maize seems not to be
affected by P availability (Mollier and Pellerin 1999) and the extensive shoot-born
root system and different root types of cereals (Hochholdinger and Zimmermann
2008) emphasises regulatory differences in crops compared to model plants such as
Arabidopsis.
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 113

Nonetheless, genotypic differences in low P tolerance or high yield at low P

availability were often associated with root growth properties or P uptake capability
in crops (Hammond et al. 2009; Pariasca-Tanaka et al. 2009; Yao et al. 2011; Li
et al. 2008a, b; Zhu and Lynch 2004; Zhu et al. 2005a, b; Gahoonia et al. 1996;
1997) showing that there is a large exploitable genetic variation in the root
acquisition trait. There exists considerable genotypic variation in root hairs in
barley and wheat cultivars showing that root hair length was strongly correlated
to the rhizosphere P depletion ability (Gahoonia et al. 1997). A root hairless mutant
in maize conferred significant grain yield loss (Hochholdinger et al. 2008). Similar
observations were made when comparing different maize lines and their root hair
length, plasticity and subsequent performance under low P (Zhu et al. 2010). A
positive and negative regulatory role for transcription factors like PHR1 WRKY75
and BHLH32 has been suggested in Arabidopsis (Chen et al. 2007; Devaiah
et al. 2007a, b; Bustos et al. 2010). Unfortunately, the underlying genetic mecha-
nisms of germplasm variation for the root hair trait are not yet determined. MAS
may facilitate root trait selection for breeding more P efficient cultivars, exempli-
fied by studies showing that root morphology QTLs are likened to P acquisition
efficiency in maize (Zhu et al. 2005a, b), wheat (Ren et al. 2012a, b) or soybean
(Liang et al. 2010). Gene modification is a means of enhancing low P tolerance
(Wang et al. 2013). For example, a ß-expansin gene in soybean, Gm-EXPB2,
enhanced P uptake when it was over-expressed (Guo et al. 2011). Expansins are
involved in cell wall extension (Zhao et al. 2012), including root hair formation
(Yu et al. 2011) and are among up-regulated genes during P starvation (Calderón-
Vázquez et al. 2008), suggesting a remodelling of the cell wall structure and
integrity. Root morphology related genes, Rtcs (rootless concerning crown and
lateral seminal roots; Hochholdinger and Zimmermann 2008), Bk2 (brittle stalk-2;
Brady et al. 2007) and Rth3 (root hairless 3; Hochholdinger et al. 2008), were
determined as being related to differential P responses and PAE capability in the
seedling stage in two contrasting maize lines (de Sousa et al. 2012). However, even
if observable only under low P conditions, root traits exhibited high heritability and
a low coefficient of variation making them exploitable for breeding (de Sousa
et al. 2012). Genes which were investigated belonged to a family of glycosylpho-
phatidylinositol (GPI)-anchored proteins involved in root cell expansion, cell wall
biosynthesis and root hair formation (de Sousa et al. 2012). Comparing the prote-
ome of a low P tolerant and a sensitive oilseed rape genotype revealed that proteins
related to lateral root formation such as auxin-responsive family proteins and
sucrose-phosphate synthase-like proteins were up-regulated in roots and leaves
(Yao et al. 2011) suggesting sources of tolerance lying in the P starvation signalling
mechanism. Furthermore, Li et al. (2008a, b) revealed an increase of low-P
tolerance when the phosphoprotein, phosphatase 2A isoform 4, was increased in
abundance in the more tolerant genotype. This protein family is involved in auxin
transport and reduced activity altered lateral root growth in Arabidopsis (Rashotte
et al. 2001). Significant increases of CDC48 and other regulators of cell division
and cell cycle, including Ran GTPase, MCM6 and importins, were assumed to be
important factors mediating a better root development and accelerated cell prolif-
eration in the meristem under P starvation (Li et al. 2008a, b).
114 A. Gruen et al.

Root system modification and organic acid secretion require a carbon supply
which might be to the detriment of yield or growth but being nevertheless beneficial
under conditions of low phosphorus availability (Zhu and Lynch 2004; Lynch and
Ho 2005; Johnson et al. 1996; Yao et al. 2011). PUE in two different P efficient
maize lines growing in nutrient solution culture was related to proteins that
decreased citrate degradation, increased citrate synthesis and malate dehydrogenase
activity in the roots (Li et al. 2008a). Furthermore, proteins related to carbon and
energy metabolism were expressed to a higher extent in a low P-tolerant Brassica
napus genotype compared to a low P-sensitive one (Yao et al. 2011). It has been
hypothesised that transgenic plants that secret microbial phytases into the rhizo-
sphere have potential for improved acquisition of organic P sources, but when
grown in soil their growing performance matched the control plants (George
et al. 2005; Richardson et al. 2000). Studies on bacterial citrate synthase genes in
tobacco came to similar contradictory results (López-Bucio et al. 2000; Delhaize
et al. 2001) and investigations of genotypic variation in root exuded wheat phos-
phatases activity could not relate their activity to the P content when plants were
grown in soil (George et al. 2008). Nonetheless, Zhang et al. (2009) suggested that
improved acquisition and therefore higher P uptake efficiency of two Brassica
napus genotypes grow in soil was related to the ability to lower the pH or higher
acid phosphatase activity in the rhizosphere. However, higher APase activity was
observed in roots and particularly in shoot tissue in P scarce conditions in rice but
without exhibiting genotypic differences (Yao et al. 2011). Over-expression of a
wheat malate transporter Ta-ALMT1 in barley enhanced P uptake on acid soils in
the short-term but not when the soil was limed (Delhaize et al. 2009). Over-
expression of a root-associated purple phosphatase gene in rice, OsPAP10a, could
promote better growth and a higher tiller number compared to the wild type under P
sufficient conditions (Tian et al. 2012). In conclusion, results from in vivo studies
appear contradictory when tested under soil conditions. However, genotypic vari-
ation of low P tolerance is related to root morphology and secretory traits, which
may be exploitable.

Pi Acquisition via Phosphate Transporters

A strong induction of Pht1 transporters was reported in the majority of transcript

profiling studies where plants were exposed to a short-term P starvation period and
grown mainly in nutrient solution (Wang et al. 2002; Wasaki et al. 2003; Calderón-
Vázquez et al. 2008; Huang et al. 2008; 2011) and in the field (Teng et al. 2013),
which makes them obvious targets for genetic improvement. However, enhanced
induction of TaPht1 transcripts might be either a short-term adaptation to local P
depletion or unevenly distributed patterns of P availability. High persistent induc-
tion during severe long-term scarcity as an adaptation mechanism seems question-
able and should be investigated on field-grown crops as performed by Teng
et al. (2013). Enhancing P acquisition by over-expressing phosphate transporter
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 115

genes has been reported in tobacco cell cultures (Mitsukawa et al. 1997) but could
not be confirmed at the plant level (Rae et al. 2004). Promoter fusion to fluorescent
proteins showed that high expression levels of root expressed phosphate transporter
occur in trichoblast cells (Daram et al. 1998; Mudge et al. 2002; Schünmann
et al. 2004) and remarkably in root tips and root hairs under low P conditions
(Sánchez-Calderón et al. 2006). The increasing rate of Pi uptake during P depriva-
tion via enhanced phosphate transporter expression presumably occurs as a result of
increased Vmax, rather than increased affinity (Km), implying increasing high-
affinity P transporter synthesis with similar kinetic properties (Raghothama
2005). As variability in P depletion profiles in the rhizosphere of wheat genotypes
suggest genetic variability in root hair formation (Gahoonia et al. 1996; 1997),
varietal expression differences with respect to preferentially root expressed Pht1 are
very likely. Until now, there has been no evidence that genotypic variation exists
that could be exploited in breeding (Rose and Wissuwa 2012): this should clearly be
area for future research.

P Partitioning and Re-translocation Within the Crop

Preferential re-translocation of P to the roots was characteristic for more P-efficient

rice genotypes (Wissuwa et al. 2005) or better remobilisation of P from shoot to
root in oilseed rape (Hammond et al. 2009; Akhtar et al. 2008). Two rice genotypes,
distinguishable in their ability to tolerate a P-limited environment, showed that
genes like PEPC (phosphoenolpyruvate carboxylase), which are involved in the
modified glycolysis bypassing ATP-requiring reactions, were up-regulated in the
more tolerant but down-regulated in the more susceptible genotype (Li et al. 2010).
Interestingly, over-expression of the bHLH transcription factor OsPTF1 enhanced
low P tolerance and resulted in increasing expression of Glu-6-P translocator, H+-
ATPase, but also PEP carboxykinase in the shoot (Yi et al. 2005). A proteome study
showed that among the higher over-accumulated proteins in the more P starvation
tolerant genotype was an important enzyme of the pentose phosphate pathway,
6-phosphogluconate dehydrogenase (Li et al. 2008a).
The authors assumed that requirements of the sugar metabolism could be
fulfilled more satisfyingly in the low P-tolerant genotype which was also
represented by the larger proportion of sucrose in the total soluble sugar fraction
(Li et al. 2008a). More abundant pyruvate phosphate dikinase, pyruvate kinase-like
proteins and UDP-glucose pyrophosphorylase, which utilise PPi (pyrophosphate) to
produce ATP or UTP, in the low P-tolerant maize line could be assigned to a higher
in vivo PUE (Li et al. 2008a, b). Another fundamental issue would be assessing the
role or phosphate transporters involved in P loading into the grain (Rose and
Wissuwa 2012).
116 A. Gruen et al.

Genes Involved in P Starvation Signalling Cascades

As discussed above, the transcription factor PHR1 seems to be a key regulator for
downstream P-responsive genes through binding to a PHR1 specific binding
sequence (P1BS) cis-element in model plants (Bustos et al. 2010; Rubio
et al. 2001). In Brassica napus, the homologue BnPHR1 was predominantly
expressed in the roots exposed to P limitation and over-expression enhanced
remarkably the expression of the high-affinity transporter BnPT2 (Ren
et al. 2012a, b). In wheat, three PHR1 homologues genes have been identified
(Wang et al. 2013), which regulate genes such as TaPt2;1 (Tittarelli et al 2007; Guo
et al. 2013) or TaIPS1 (Oono et al. 2013) that have been reported to contain the
P1BS element. TaPht1-A1 transcriptionally activated the expression of the phos-
phate transporter TaPht1;2 in yeast cells (Wang et al. 2013). The promoter of the
high-affinity transporter TaPht1;2 was more abundant in a P-efficient genotype than
in an inefficient genotype (Miao et al. 2009). Furthermore, TaPHR1-A1 over-
expression resulted in an up-regulation of P starvation response genes, stimulated
lateral root branching, enhanced P uptake and P translocation and increased grain
yield but not P distribution from shoot to the grains in pot and field trials under P
deficient conditions (Wang et al. 2013). Pht1 transporter expression of TaPht1;2 in
the roots and TaPht1;6 expression in the shoots increased under high and low P
conditions, whereas other usually P starvation induced genes such as TaIPS1.2,
TaPHO or TaSPX3 did not change their level of expression (Wang et al. 2013).
These results indicated that TaPHR1 is an upstream regulator for Pht1 transporter
but suggested other transcriptional factors being relevant for the induction of other
P starvation induced genes as it is the case for OsPHR2 (Zhou et al. 2008). Oono
et al. (2013) recently published a transcriptome study using de novo transcript
assembly analysis, in order to investigate wheat seedlings, cv. Chinese spring,
exposed to 10 days of P starvation. Genes of the phosphorylation category including
protein kinases were among the up-regulated transcripts (Oono et al. 2013). Fur-
thermore, genes belonging to oxidation-reduction processes, metabolic processes,
carbohydrate metabolism, transcription process, lipid metabolism and transmem-
brane transport were induced, as well as AtWRKY6 and AtPHO1 homologues
(Oono et al. 2013).
Rice-orthologous transcripts of PHR1, PHO2 and SIZ1 were detected but not all
were highly responsive to P starvation (Oono et al. 2011, 2013). Significant was the
induction of TaIPS1 homologous (Oono et al. 2013), suggesting that the
IPS-mediated signalling cascade may also be functional as previously observed in
model species including rice (Oono et al. 2011). This aspect is relevant when taking
in account that genetic variation in PUE of barley exhibited a correlated expression
of the low-affinity phosphate transporters, HvPht1;3 and HvPht1;6, with HvIPS1
expression (Huang et al. 2011). Higher PUE was also a consequence of higher root-
shoot ratios under P limitation indicating an increase in carbohydrate partitioning
(Huang et al. 2011). In model plants, IPS genes have been shown to be a
miRNA399 antagonist and involved in the miR399-PHO2 regulatory loop
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 117

(Franco-Zorrilla et al. 2007; Doerner 2008). In wheat, TaIPS1 transcript levels were
strongly repressed in roots and TaIPS2 transcript levels in shoots of P-deficient
wheat by N deficiency (Li et al. 2008a, b) providing evidence of an influence on the
signalling pathways of P homeostasis by the nitrogen nutritional status. Further-
more, nine wheat miRNAs were identified in addition to miRNA399 as responsive
to P starvation in a variety-dependent manner (Zhao et al. 2013). TamiRNAs
putatively target diverse gene families, which are down-regulated during P defi-
ciency stress including transcriptional regulation, signal transduction, phytohor-
mone and defence responses among several others (Zhao et al. 2013). Transgenic
tomato lines over-expressing miRNA399 from Arabidopsis enhanced the secretion
of acid phosphatases and protons in roots (Gao et al. 2010). A further example is the
over-expression of another P starvation-induced transcription factor, OsPTF1 in
rice, which increased tiller number, shoot biomass, panicle weight and P content
under low Pi conditions (Yi et al. 2005). Genes which are regulated by OsPTF1
contain E-box and G-box elements but do not include high affinity transporters or
acid phosphatases (Yi et al. 2005). In addition, total root length and root surface
area may be increased resulting in higher P uptake rates (Yi et al. 2005). Both
studies provide evidence of a promising method enhancing P uptake in crops
although enhancing low P tolerance via this pathway needs more understanding
of other involved proteins and factors.

Quantitative Trait Loci Identification

Until now, the indirect approach of QTL identification for P deficiency tolerance
has been mostly exploited in rice and Brassica species, and approaches have
focused on P acquisition parameters among different aspects and targets of P
efficiency. To date, Pup1 (Phosphorus uptake 1) is the only major QTL for P
deficiency tolerance in rice coming from a landrace, which could actually be used
by rice breeders for marker-assisted introgression into elite material (Wissuwa and
Ae 2001, Wissuwa et al. 2002; Chin et al. 2010).
There are more known QTLs, often at very early growth stages (Su et al. 2006,
2009; Yang et al. 2010, 2011), related to yield components under low P conditions
(Su et al. 2009; Chin et al. 2010; Ding et al. 2012; Gamuyao et al. 2012; Shi
et al. 2013), to seed P concentrations (Ding et al. 2010; Zhao et al. 2008), to P uptake
capability and morphological adaptation e.g. tiller number (Su et al. 2006; Wissuwa
et al. 1998, 2002; Chin et al. 2010; Gamuyao et al. 2012) or root morphology (Zhu
et al. 2005a, b, 2006; Liang et al. 2010; Yang et al 2011).
In wheat, a large number of QTLs on all chromosomes have been detected in a
double haploid (DH) population derived from a P deficiency tolerant and low
P-sensitive variety implying a polygenetic control of low P sensitivity
(Su et al. 2006, 2009). There were three major loci associated with higher tiller
number, shoot dry weight and shoot phosphate uptake under low P conditions
suggesting that these alleles may be used for MAS (Su et al. 2006). Two of these
118 A. Gruen et al.

loci are liked to genes associated with vernalization requirements including

flowering time and shoot morphology (Su et al. 2006). Hammond et al. (2009)
reported that QTLs associated with PUE measures were related to root traits in
B. oleracea species which puts them back in the focus as potential targets for crop
improvement. QTLs for root hair length and lateral root growth have been identified
in maize under P deficiency, a trait which was observed in previous studies as
associated with improved low P tolerance and may be controlled by many minor
genes or loci with epistatic effects (Zhu et al. 2005a, b). An additional study on
soybean possibly related root traits and P efficiency traits to three QTL clusters
(Liang et al. 2010). As previously mentioned, a major storage form of phosphorus
in grain crops is the non-desirable phytate, which diminishes nutritional quality and
causes environmental problems. Despite being of strong interest for human health,
QTL analysis focusing on seed traits or seed quality has not been performed
extensively. Ding et al. (2012) observed an overlap of a P efficiency QTL with a
QTL for seed P concentrations and in a RIL population (durum wheat x wild emmer
wheat) and eight QTLs found for grain P content co-localised with QTLs for grain
protein content (Peleg et al. 2009) which is correlated with both phytic acid and
total P (Raboy et al. 2009). Zhu et al. (2005a, b) found seed phosphorus reserve-
related QTLs in maize, which was only partly related to seed size. In rice grown
with unlimited nutrients, Stangoulis et al. (2007) found a common QTL for phytate
and P concentrations and interestingly, the phytate QTLs were distinct from those
for micronutrients such as Fe, Zn or Mn.
So far, the underlying genes or their functional mechanisms, which are targeting
a higher low P tolerance, remain still quite elusive (Pariasca-Tanaka et al. 2009;
Chin et al. 2010; Shi et al. 2013). The region of a major QTL for low P tolerance in a
rice cultivar, Pup1 (Phosphate uptake 1), has been mapped and has been predicted
to contains 60 genes. However, their mechanism for low P tolerance as not yet been
identified (Ismail et al. 2007; Wissuwa et al. 2002). In one case, the receptor-like
cytoplasmic Ser/Thr protein kinase PSTOL1 gene, present within the Pup1 QTL
region, seems to be involved in early crown root development and enhances yield
and gene expression of such related to root growth when it was over-expressed
(Gamuyao et al. 2012). Another approach for delivering putative candidate targets
used a comparative mapping technique (in silico mapping) between the model plant
Arabidopsis and the crop Brassica napus (Yang et al. 2010; Ding et al. 2012; Shi
et al. 2013) which revealed yield associated QTLs being linked to genes which are
involved in P homeostasis including P transport, transcriptional control, phospho-
lipid and carbohydrate metabolism (Shi et al. 2013). Among these genes were
glucose-6-phosphate transporters, BnSIZ1-A2, BnPHO1 or BnSQD2 and phos-
phate transporters (Shi et al. 2013) as well as BnIPS2 (Ding et al. 2012), which
therefore confirm their potential roles as targets for crop improvement. Yang
et al. (2010) observed a linkage of two functional gene-based markers (GBMs)
potentially usable for MAS, BnIPS2 and BnGTP1, as well as orthologous genes for
root development, auxin transport with two root morphology related QTLs.
In summary, recent approaches have proven to be a useful tool to dissect the
genetic basis of P efficiency-related traits, and have detected a large number of
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 119

QTLs in several crop species. Nonetheless, its practical and effective application in
MAS of breeding programmes has been rather limited up to now and would require
higher precision, reliability and a proof of consistency across these known QTLs.
Additionally, these findings emphasise the previously mentioned importance of
post-transcriptional modification in the sensing and signalling network of P homeo-
stasis, the low P tolerance, and the association of low P tolerance with root
morphology.

Conclusions
Phosphorus is an essential macronutrient with crucial functions in plant
macromolecular structure, energy metabolism and signal transduction,
which can be a major constraint for high yield when it becomes limiting in
crop production. There are economic, political and environmental reasons
why P efficiency (PAE, PUE) and P fertiliser use in crops should be inves-
tigated for future crop improvement. Therefore, scientific interest has
increased with the aim of finding the underlying molecular mechanisms for
adaptation to low P accessibility and of identifying targets to archive high-
yielding, low P-tolerant crops. Agronomic strategies for raising the amount of
available fertiliser are constantly under assessment but the polygenetic basis
of P deficiency tolerance is not yet understood. Field selection and screening
for PAE/PUE traits, especially in relation to roots, are difficult to realise due
to the complexity of soil P and agronomic practice, which may have signif-
icant impact on P availability and root properties. Another critical point is the
shift from P acquisition to translocation processes when crops become gen-
erative and distribution in grain and seeds becomes predominant. Several
approaches for investigating crops, such as the comparison of individual
genotypes exposed to a short-term P starvation period, the over-expression
of target genes and QTL analysis have resulted in contradictory observations.
Nevertheless many potential target genes, which have been identified previ-
ously in model organisms using forward and reverse genetic approaches, are
also be found in crops and are potentially exploitable. Phosphate transporters,
several transcription factors, genes coding for proteins of the TCA cycle
metabolism, phospholipid degradation, transfer and post-translational modi-
fications are among the candidates who have been detected even if their role
in the genetically diverse low P tolerance or PAE/PUE context seems com-
plex and still rather elusive.

Acknowledgements Work at Rothamsted Research is supported via the 20:20 Wheat®

Programme by the UK Biotechnology and Biological Sciences Research Council. The contribu-
tion of A. Gruen was supported by which received funding from the European Union Seventh
Framework Programme (FP7/2007-2013) under grant agreement n 264296.
120 A. Gruen et al.

References

Ai P, Sun S, Zhao J, Fan X, Xin W, Guo Q, Yu L, Shen Q, Wu P, Miller AJ, Xu A (2009) Two rice
phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties
in uptake and translocation. Plant J 57:798–809
Akhtar MS, Oki Y, Adachi T (2008) Genetic variability in phosphorus acquisition and utilization
efficiency from sparingly soluble P-sources by brassica cultivars under P-stress environment. J
Agron Crop Sci 194:380–392
Alexova R, Millar AH (2013) Proteomics of phosphate use and deprivation in plants. Proteomics
13:609–623
Andersson MX, Stridh MH, Larsson KE, Liljenberg C, Sandelius AS (2003) Phosphate-deficient
oat replaces a major portion of the plasma membrane phospholipids with the galactolipid
digalactosyldiacylglycerol. FEBS Lett 537:128–132
Aung K, Lin SI, Wu CC, Huang YT, Su CL, Chiou TJ (2006) pho2, a phosphate overaccumulator,
is caused by a nonsense mutation in a microRNA399 target gene. Plant Physiol 141:1000–1011
Bahl GS, Singh NT (1986) Phosphorus diffusion in soils in relation to some edaphic factors and its
influence on P uptake by maize and wheat. J Agric Sci 107:335–341
Barber SA (1984) Phosphorus. In: Soil nutrient bioavailability – a mechanistic approach. Wiley,
New York, pp 201–228
Bari R, Pant BD, Stitt M, Scheible WR (2006) PHO2, microRNA399, and PHR1 define a
phosphate-signaling pathway in plants. Plant Physiol 141:988–999
Bates TR, Lynch JP (1996) Stimulation of root hair elongation in Arabidopsis thaliana by low
phosphorus availability. Plant Cell Environ 19:529–538
Bates TR, Lynch JP (2001) Root hairs confer a competitive advantage under low phosphorus
availability. Plant Soil 236:243–250
Batten GD, Khan MA (1987) Uptake and utilization of phosphorus and nitrogen by bread wheats
grown under natural rainfall. Aust J Exp Agr 27:405–410
Batten GD (1992) A review of phosphorus efficiency in wheat. Plant Soil 146:163–168
Bayle V, Arrighi JF, Creff A, Nespoulous C, Vialaret J, Rossignol M, Gonzalez E, Paz-Ares J,
Nussaume L (2011) Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple
levels of posttranslational regulation. Plant Cell 23:1523–1535
Bieleski RL (1968) Effect of phosphorus deficiency on levels of phosphorus compounds in
Spirodela. Plant Physiol 43:1309–1316
Bieleski RL (1973) Phosphate pools, phosphate transport, and phosphate availability. Annu Rev
Plant Physiol 24:225–252
Bollons HM, Barraclough PB (1997) Inorganic orthophosphate for diagnosing the phosphorus
status of wheat plants. J Plant Nutr 20:641–655
Bollons HM, Barraclough PB (1999) Assessing the phosphorus status of winter wheat crops:
inorganic orthophosphate in whole shoots. J Agric Sci 133:285–295
Brady SM, Song S, Dhugga KS, Rafalski JA, Benfey PN (2007) Combining expression and
comparative evolutionary analysis. The COBRA gene family. Plant Physiol 143:172–187
Bucher M, Rausch C, Daram P (2001) Molecular and biochemical mechanisms of phosphate
uptake into plants. J Plant Nutr Soil Sci 164:209–221
Bustos R, Castrillo G, Linhares F, Puga MI, Rubio V, Pérez- Pérez J, Solano R, Leyva A, Paz-Ares
J (2010) Central regulatory system largely controls transcriptional activation and repression
responses to phosphate starvation in Arabidopsis. PLoS Genet 6:e1001102
Byrne SL, Foito A, Hedley PE, Morris JA, Stewart D, Barth S (2011) Early response mechanisms
of perennial ryegrass (Lolium perenne) to phosphorus deficiency. Ann Bot 107:243–254
Calderón-Vázquez C, Ilbarra-Laclette E, Caballero-Perez J, Herrera-Estrella L (2008) Transcript
profiling of Zea mays roots reveals responses to phosphate deficiency and the plant-species-
specific level. J Exp Bot 59:2479–2497
Calderón-Vázquez C, Sawers RJH, Herrera-Estrella L (2011) Phosphate deprivation in maize:
genetics and genomics. Plant Physiol 156:1067–1077
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 121

Chen ZH, Nimmo GA, Jenkins GI, Nimmo HG (2007) BHLH32 modulates several biochemical
and morphological processes that respond to Pi starvation in Arabidopsis. Biochem J 405:191–
198
Chen YF, Li LQ, Xu Q, Kong YH, Wang H, Wu WH (2009a) The WRKY6 transcription factor
modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis. Plant Cell
21:3554–3566
Chen JY, Xu L, Cai YL, Xu J (2009b) Identification of QTLs for phosphorus utilization in maize
(Zea mays L.) across P levels. Euphytica 167:245–252
Chen J, Liu Y, Ni J, Wang Y, Bai Y, Shi J, Gan J, Wu Z, Wu P (2011) Osphf1 regulates the plasma
membrane localization of low- and high-affinity inorganic phosphate transporters and deter-
mines inorganic phosphate uptake and translocation in rice. Plant Physiol 157:269–278
Cheng L, Bucciarelli B, Liu J, Zinn K, Miller S, Patton-Vogt J, Allan D, Shen J, Vance CP (2011)
White lupin cluster root acclimation to phosphorus deficiency and root hair development
involve unique glycerophosphodiester phosphodiesterases. Plant Physiol 156:1131–1148
Chevalier F, Rossignol M (2011) Proteomic analysis of Arabidopsis thaliana ecotypes
with contrasted root architecture in response to phosphate deficiency. J Plant Physiol
168:1185–1890
Chin JH, Lu X, Haefele SM, Gamuyao R, Ismail A, Wissuwa M, Heuer S (2010) Development and
application of gene-based markers for the major rice QTL Phosphorus uptake 1. Theor Appl
Genet 120:1073–1086
Chiou TJ, Lin S (2011) Signaling network in sensing phosphate availability in plants. Annu Rev
Plant Biol 62:185–206
Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Sua CL (2006) Regulation of phosphate
homeostasis by microRNA in Arabidopsis. Plant Cell 18:412–421
Chitwood DH, Timmermans MCP (2007) Target mimics modulate miRNAs. Nat Genet
39:935–936
Cordell D, Drangert J, White S (2009) The story of phosphorus: global food security and food for
thought. Glob Environ Chang 12:292–305
Da Silva AE, Gabelman WH (1992) Screening maize inbred lines for tolerance to low-P stress
conditions. Plant Soil 146:181–187
Daram P, Brunner S, Persson BL, Amrhein N, Bucher M (1998) Functional analysis and cell-
specific expression of a phosphate transporter from tomato. Planta 206:225–233
Daram P, Brunner S, Rausch C, Steiner C, Amrheim N (1999) Pht2;1 encodes a low-affinity
phosphate transporter from Arabidopsis. Plant Cell 11:2153–2166
Davies TGE, Ying J, Li ZS, Li J, Gordon-Weeks R (2002) Expression of analysis of putative high-
affinity transporters in Chinese winter wheats. Plant Cell Environ 25:1325–1339
De Sousa SM, Clark RT, Mendes FF, de Oliveira AC, de Vasconcelos MJV, Parentoni SN,
Kochian LV, Guimarães CT, Magalhães JV (2012) A role for root morphology and related
candidate genes in P acquisition efficiency in maize. Funct Plant Biol 39:925–935
Delhaize E, Randall PJ (1995) Characterization of a phosphate-accumulator mutant of
Arabidopsis-thaliana. Plant Physiol 107:207–213
Delhaize E, Hebb DM, Ryan PR (2001) Expression of a Pseudomonas aeruginosa citrate synthase
gene in tobacco is not associated with either enhanced citrate accumulation or efflux. Plant
Physiol 125:2059–2067
Delhaize E, Taylor P, Hocking PJ, Simpson RJ, Ryan PR, Richardson AE (2009) Transgenic
barley (Hordeum vulgare L.) expressing the wheat aluminium resistance gene (TaALMT1)
shows enhanced phosphorus nutrition and grain production when grown on an acid soil. Plant
Biotech J 7:391–400
Devaiah BN, Karthikeyan AS, Raghothama KG (2007a) WRKY75 transcription factor is a
modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol
143:1789–1801
Devaiah BN, Nagarajan VK, Raghothama KG (2007b) Phosphate homeostasis and root develop-
ment in Arabidopsis are synchronized by the zinc finger transcription factor ZAT6. Plant
Physiol 145:147–159
122 A. Gruen et al.

Dinkelacker B, Römheld V, Marschner H (1989) Citric acid excretion and precipitation of calcium
citrate in the rhizosphere of white lupine. Plant Cell Environ 12:285–292
Ding G, Yang M, Hu Y, Liao Y, Shi L, Xu F, Meng J (2010) Quantitative trait loci affecting seed
mineral concentrations in Brassica napus grown with contrasting phosphorus supplies. Ann
Bot 105:1221–1234
Ding G, Zhao Z, Liao Y, Hu Y, Shi L, Long Y, Xu F (2012) Quantitative trait loci for seed yield
and yield-related traits, and their responses to reduced phosphorus supply in Brassica napus.
Ann Bot 109:747–759
Doerner P (2008) Phosphate starvation signaling: a threesome controls systemic Pi homeostasis.
Curr Opin Plant Biol 11:536–540
Duan K, Yi K, Dang L, Huang H, Wu W, Wu P (2008) Characterization of a sub-family of
Arabidopsis genes with the SPX domain reveals their diverse functions in plant tolerance to
phosphorus starvation. Plant J 54:965–975
Duff SMG, Moorhead GBG, Lefebre DD, Plaxton WC (1989) Phosphate starvation inducible
‘bypasses’ of adenylate and phosphate dependent glycolytic enzymes in Brassica nigra
suspension cells. Plant Physiol 90:1275–1278
Egle K, Manske G, Römer W, Vlek PLG (1999) Improved phosphorus efficiency of three new
wheat genotypes from CIMMYT in comparison with an older Mexican variety. J Plant Nutr
Soil Sci 162:353–358
Essigmann B, Güler S, Narang RA, Linke D, Benning C (1998) Phosphate availability affects the
thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid
biosynthesis in Arabidopsis thaliana. Proc Natl Acad Sci U S A 95:1950–1955
Finck A (1991) Düngermenge nach Pflanzenanalyse. In: Baumeister W (ed) Düngung. Ulmer,
Stuttgart, pp 96–101
Fitter AH (2006) What is the link between carbon and phosphorus fluxes in arbuscular mycorrhi-
zas? A null hypothesis for symbiotic function. New Phytol 172:3–6
Food and Agriculture Organization of The United Nations: current world fertilizer trends and
outlook to 2015. Rome 2011
Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga IM, Rubio-Somoza I, Leyva A,
Weigel D, Garcia JA, Paz-Ares J (2007) Target mimicry provides a new mechanism for
regulation of microRNA activity. Nat Genet 39:1033–1037
Fujii H, Chiou TJ, Lin SI, Aung K, Zhu JK (2005) A miRNA involved in phosphate-starvation
response in Arabidopsis. Curr Biol 15:2038–2043
Furihata T, Suzuki M, Sakurai H (1992) Kinetic characterization of two phosphate uptake systems
with different affinities in suspension-cultured Catharanthus roseus protoplasts. Plant Cell
Physiol 33:1151–1157
Gahoonia TS, Nielsen NE (1998) Direct evidence on participation of root hairs in phosphorus (32P)
uptake from soil. Plant Soil 198:147–152
Gahoonia TS, Care D, Nielsen NE (1996) Variation in acquisition of soil phosphorus among wheat
and barley genotypes. Plant Soil 178:223–230
Gahoonia TS, Care D, Nielsen NE (1997) Root hairs and phosphorus acquisition of wheat and
barley cultivars. Plant Soil 1991:181–188
Gahoonia TS, Nielsen NE, Lyshede OB (1999) Phosphorus (P) acquisition of cereal cultivars in
the field at three levels of P fertilization. Plant Soil 211:269–281
Gahoonia TS, Nielsen NE, Joshi PA, Jahoor A (2001) A root hairless barley mutant for elucidation
genetic of root hairs and phosphorus uptake. Plant Soil 235:211–219
Gamuyao R, Chin JH, Pariasca-Tanaka J, Pesaresi P, Catausan S, Dalid C, Slamet-Loedin I,
Tecson-Mendoza EM, Wissuwa M, Heuer S (2012) The protein kinase Pstol1 from traditional
rice confers tolerance of phosphorus deficiency. Nature 488:535–539
Gao N, Su Y, Mi J, Shen W, Shi W (2010) Transgenic tomato overexpressing ath-miR399d has
enhanced phosphorus accumulation through increased acid phosphatase and proton secretion
as well as phosphate transporters. Plant Soil 334:123–136
Gaude N, Tippmann H, Flemetakis E, Katinakis P, Udvardi M, Dörmann P (2004) The galactolipid
digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing
nodules of soybean and lotus. J Biol Chem 279:34624–34630
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 123

Gaude N, Bortfeld S, Duensing N, Lohse M, Krajinski F (2012) Arbuscule-containing and

non-colonized cortical cells of mycorrhizal roots undergo extensive and specific
reprogramming during arbuscular mycorrhizal development. Plant J 69:510–528
Gaxiola RA, Edwards M, Elser JJ (2001) A transgenic approach to enhance phosphorus use
efficiency in crops as part of a comprehensive strategy for sustainable agriculture.
Chemosphere 84:840–845
George TS, Richardson AE (2008) Potential and limitations to improving crops for enhanced
phosphorus utilization. In: White PJ, Hammond JP (eds) The ecophysiology of plant-
phosphorus interactions, vol 7. Springer, Dordrecht, pp 247–270
George TS, Richardson AE, Hadobas PA, Simpson RJ (2004) Characterization of transgenic
Trifolium subterraneum L. which expresses phyA and releases extracellular phytase: growth
and P nutrition in laboratory media and soil. Plant Cell Environ 27:1351–1361
George TS, Simpson RJ, Hadobas PA, Richardson AE (2005) Expression of a fungal phytase gene
in Nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils. Plant
Biotechnol J 3:129–140
George TS, Gregory PJ, Hocking P, Richardson AE (2008) Variation on root-associated phospha-
tase activities in wheat contributes to the utilization of organic P substrates in vitro, but does
not explain differences in the P-nutrition of plants when grown in soils. Environ Exp Bot
64:2239–2249
Gerke J, Römer W, Jungk A (1994) The excretion of citric and malic-acid by proteoid roots of
lupinus-albus l – effects on soil solution concentrations of phosphate, iron, and aluminum in the
proteoid rhizosphere in samples of an oxisol and a luvisol. Z Pflanz Bodenkunde 157:289–294
Glassop D, Smith SE, Smith FW (2005) Cereal phosphate transporters associated with the
mycorrhizal pathway of phosphate uptake in roots. Planta 222:688–698
Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pagès V, Dun EA, Pillot JP, Letisse F,
Matusova R, Danoun S, Portais JC, Bouwmeester H, Bécard G, Beveridge CA, Rameau C,
Rochange SF (2008) Strigolactone inhibition of shoot branching. Nature 455:189–194
González E, Solano R, Rubio V, Leyva A, Paz-Ares J (2005) Phosphate transporter traffic
facilitator1 is a plant-specific sec12-related protein that enables the endoplasmic reticulum
exit of a high-affinity phosphate transporter in Arabidopsis. Plant Cell 17:3500–3512
Gregory PJ, George TS (2011) Feeding nine billion: the challenge to sustainable crop production. J
Exp Bot 62:5233–5239
Gregory AL, Hurley BA, Tran HT, Valentine AJ, She YM, Knowles VL, Plaxton WC (2009) In
vivo regulatory phosphorylation of the phosphoenolpyruvate carboxylase AtPPC1 in
phosphate-starved Arabidopsis thaliana. Biochem J 420:57–65
Gu M, Xu K, Chen A, Zhu Y, Tang G, Xu G (2010) Expression analysis suggests potential roles of
micro RNAs for phosphate and arbuscular mycorrhizal signaling in Solanum lycopersicum.
Physiol Plant 138:226–237
Gunes A, Inal A, Alpaslan M, Cakmak I (2006) Genotypic variation in phosphorus efficiency
between wheat cultivars grown under greenhouse and field conditions. Soil Sci Plant Nutr
52:470–478
Guo B, Jin Y, Wussler C, Blancaflor EB, Moter CM, Versaw WK (2008) Functional analysis of the
Arabidopsis PHT4 family of intracellular phosphate transporters. New Phytol 177:889–898
Guo W, Zhao J, Li X, Qin L, Yan X, Hong Liao H (2011) A soybean b-expansin gene
GmEXPB2 intrinsically involved in root system architecture responses to abiotic stresses.
Plant J 66:541–552
Guo C, Zhao X, Liu X, Zhang L, Gu J, Li X, Lu W, Xiao K (2013) Function of wheat phosphate
transporter gene TaPHT2;1 in Pi translocation and plant growth regulation under replete and
limited Pi supply conditions. Planta 237:1163–1178
Güimil S, Chang HS, Zhu T, Sesma A, Osbourn A, Roux C, Ioannidis V, Oakeley EJ, Docquier M,
Descombes P, Briggs SP, Paszkowski U (2005) Comparative transcriptomics of rice reveals
an ancient pattern of response to microbial colonization. Proc Natl Acad Sci U S A
102:8066–8070
124 A. Gruen et al.

Gutjahr C, Banba M, Croset V, An K, Miyao A, An G, Hirochika H, Imaizumi-Anraku H,

Paszkowski U (2008) Arbuscular mycorrhiza–specific signaling in rice transcends the common
symbiosis signaling pathway. Plant Cell 20:2989–3005
Hamburger D, Rezzonico E, Petetot JM, Somerville C, Poirier Y (2002) Identification and
characterization of the Arabidopsispho1 gene involved in phosphate loading to the xylem.
Plant Cell 14:889–920
Hammond JP, White PJ (2008) Sucrose transport in the phloem: integrating root responses to
phosphorus starvation. J Exp Bot 59:93–109
Hammond JP, Bennet MJ, Bowen HC, Broadley MR, Eastwood DC, May ST, Rahn CR,
Swarup R, Woolaway KE, White PJ (2003) Changes in gene expression in Arabidopsis shoots
during phosphate starvation and the potential for developing smart plants. Plant Physiol
132:578–596
Hammond JP, Broadley MR, White PJ (2004) Genetic responses to phosphorus deficiency. Ann
Bot 94:323–332
Hammond JP, Broadley MR, White PJ, King GJ, Bowen HC, Hayden R, Meacham MC, Mead A,
Overs T, Spracklen WP, Greenwood DJ (2009) Shoot yield drives phosphorus use efficiency in
Brassica oleracea and correlates with root architecture traits. J Exp Bot 60:1953–1968
Hammond JP, Broadley MR, Bowen HC, Spracklen WP, Hayden RM, White PJ (2011) Gene
expression changes in phosphorus deficient potato (Solanum tuberosum L.) leaves and the
potential for diagnostic gene expression markers. PLoS One 6:e24606
Harrison MJ, Dewbre GR, Liu J (2002) A phosphate transporter from Medicago truncatula
involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi. Plant Cell
14:2413–2429
Hayes JE, Zhu YG, Mimura T, Reid RJ (2004) An assessment of the usefulness of solution culture
in screening for phosphorus efficiency in wheat. Plant Soil 261:91–97
Hermans C, Hammond JP, White PJ, Verbruggen N (2006) How do plants respond to nutrient
shortage by biomass allocation? Trends Plant Sci 11:610–617
Hensel LL, Grbic V, Baumgarten DA, Bleecker AB (1993) Developmental and age-related
processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis.
Plant Cell 5:553–564
Hinsinger P (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-
induced chemical changes: a review. Plant Soil 237:173–195
Hochholdinger F, Zimmermann R (2008) Conserved and diverse mechanisms in root develop-
ment. Curr Opin Plant Biol 11:70–74
Hochholdinger F, Wen TJ, Zimmermann R, Chimot-Marolle P, Silva ODE, Bruce W, Lamkey KR,
Wienand U, Schnable PS (2008) The maize (Zea mays L.) roothairless3 gene encodes a
putative GPI-anchored, monocot specific, COBRA-like protein that significantly affects
grain yield. Plant J 54:888–898
Holford ICR (1997) Soil phosphorus: its measurement, and its uptake by plants. Aust J Soil Res
35:227–239
Hong JJ, Park YS, Bravo A, Bhattarai KK, Daniels DA, Harrison MJ (2012) Diversity of
morphology and function in arbuscular mycorrhizal symbioses in Brachypodium distachyon.
Planta 236:851–865
Hou XL, Wu P, Jiao FC, Jia QJ, Chen HM, Yu J, Song XW, Yi KK (2005) Regulation of the
expression of OsIPS1 and OsIPS2 in rice via systemic and local Pi signalling and hormones.
Plant Cell Environ 28:353–364
Hsieh LC, Lin SI, Shih ACC, Chen JW, Lin WY, Tseng CY, Li WH, Chi TJ (2009) Uncovering
small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.
Plant Physiol 151:2120–2132
Huang CY, Roessner U, Eickmeier I, Gene Y, Callahan DL, Sirley N, Langridge P, Bacie A (2008)
Metabolite profiling reveals distinct changes in carbon and nitrogen metabolism in phosphate-
deficient barley plants (Hordeum vulgare L.). Plant Cell Physiol 49:691–703
Huang CY, Shirley N, Genc Y, Shi B, Langridg P (2011) Phosphate utilization efficiency
correlates with expression of low-affinity phosphate transporters and noncoding RNA, IPS1,
in barley. Plant Physiol 156:1217–1229
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 125

Hürlimann HC, Pinson B, Stadler-Waibel M, Zeeman SC, Freimoser FM (2009) The SPX domain
of the yeast low-affinity phosphate transporter Pho90 regulates transport activity. EMBO Rep
10:1003–1008
Ismail AM, Heuer S, Thomson MJ, Wissuwa M (2007) Genetic and genomic approaches to
develop rice germplasm for problem soils. Plant Mol Biol 65:547–570
Jain A, Poling MD, Karthikeyan AS, Blakeslee JJ, Peer WA, Titapiwatanakun B, Murphy AS,
Raghothama KG (2007) Differential effects of sucrose and auxin on localized phosphate
deficiency-induced modulation of different traits of root system architecture in Arabidopsis.
Plant Physiol 144:232–247
Jakobsen I, Abbott LK, Robson AD (1992) External hyphae of vesicular-arbuscular mycorrhizal
fungi associated with Trifolium subterraneum L. 1. Spread of hyphae and phosphorus inflow
into roots. New Phytol 120:371–380
Jones GPD, Jessop RS, Blair GJ (1992) Alternative methods for the selection of phosphorus
efficiency in wheat. Field Crops Res 30:29–40
Johnson JF, Vance CP, Allan DL (1996) Phosphorus deficiency in Lupinus albus. Plant Physiol
112:31–41
Jungk A (2001) Root hairs and the acquisition of plant nutrients from soil. J Plant Nutr Soil Sci
164:121–129
Kanda H, Kasukabe Y, Fujita H, Washino T, Tachibana S (1994) Effect of low root temperature on
ribonucleic-acid concentrations in figleaf gourd and cucumber roots differing in tolerance to
chilling temperature. J Jpn Soc Hortic Sci 63:611–618
Karthikeyan AS, Varadarajan DK, Jain A, Held MA, Carpita NC, Raghothama KG (2007)
Phosphate starvation responses are mediated by sugar signaling in Arabidopsis. Planta
225:907–918
Kirkby EA, Johnston AEJ (2008) Soil and fertilizer phosphorus in relation to crop nutrition. In:
White PJ, Hammond JP (eds) The ecophysiology of plant-phosphorus interactions, vol
7. Springer Science+Business Media B.V., Dordrecht, pp 177–223
Koide RT, Kabir Z (2000) Extraradical hyphae of the mycorrhizal fungus Glomus intraradices can
hydrolyse organic phosphate. New Phytol 148:511–517
Koyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata D (2000) Overexpression of
mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-
limited soil. Plant Cell Physiol 41:1030–1037
Lan P, Li W, Schmidt W (2012) Complementary proteome and transcriptome profiling in
phosphate-deficient Arabidopsis roots reveals multiple levels of gene regulation. Mol Cell
Proteomics 11:1156–1166
Lauer MJ, Blevins DG, Sierzputowska-Gracz H (1989) 31P-nuclear magnetic resonance determi-
nation of phosphate compartmentation in leaves of reproductive soybeans (Glycine max L.) as
affected by phosphate nutrition. Plant Physiol 89:1331–1336
Lei M, Liu Y, Zhang B, Zhao Y, Wang X, Zhou Y, Raghothama KG, Liu D (2011) Genetic and
genomic evidence that sucrose is a global regulator of plant responses to phosphate starvation
in Arabidopsis. Plant Physiol 156:1116–1130
Li K, Xu C, Li Z, Zhang K, Yang A, Zhang J (2008a) Comparative proteome analyses of
phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant
reveal root characteristics associated with phosphorus efficiency. Plant J 55:927–939
Li Y, Tong Y, Bin L, Zhao H, Zhang X, Li Z (2008b) Expression of TaIPS genes in
wheat seedlings with nitrogen and phosphorous starvation. Acta Bot Boreal Occident Sin
27:1303–1307
Li L, Liu C, Lian X (2010) Gene expression profiles in rice roots under low phosphorus stress.
Plant Mol Biol 72:423–432
Liang Q, Cheng X, Mei M, Yan X, Liao H (2010) QTL analysis of root traits as related to
phosphorus efficiency in soybean. Ann Bot 106:223–234
Liao M, Hocking PJ, Dong B, Delhaize E, Richardson AE, Ryan PR (2008) Variation in early
phosphorus-uptake efficiency among wheat genotypes grown on two contrasting Australian
soils. Aust J Agric Res 59:157–166
126 A. Gruen et al.

Lin WY, Lin SI, Chiou TJ (2009) Molecular regulators of phosphate homeostasis in plants. J Exp
Bot 60:1427–1438
Liu C, Muchal US, Uthappa M, Kononowicz AK, Raghotama KG (1998) Tomato phosphate
transporter genes are differentially regulated in plant tissues by phosphorus. Plant Physiol
116:91–99
Liu F, Wang Z, Ren H, Shen C, Li Y, Ling HQ, Wu C, Lian X, Wu P (2010) OsSPX1 suppresses
the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in
shoots of rice. Plant J 62:508–517
Liu F, Chang X-J, Ye Y, Xie W-B, Wu P, Lian X-M (2011) Comprehensive sequence and
whole-life-cycle expression profile analysis of the phosphate transporter gene family in rice.
Mol Plant 4:1105–1122
Liu TY, Huang TK, Tseng CY, Lai YS, Lin SI, Lin WY, Chen JW, Chiou T (2012) PHO2-
dependent degradation of PHO1 modulates phosphate homeostasis in Arabidopsis. Plant Cell
24:2168–2183
Lloyd JC, Zakhleniuk OV (2004) Responses of primary and secondary metabolism to sugar
accumulation revealed by microarray expression analysis of the Arabidopsis mutant, pho3.
J Exp Bot 55:1221–1230
López-Bucio J, de la Vega OM, Guevara-Garcı́a A, Herrera-Estrella L (2000) Enhanced
phosphorus uptake in transgenic tobacco plants that overproduce citrate. Nat Biotechnol
18:450–453
Lott JNA, Bojarski M, Kolasa J, Batten GD, Campbell LC (2009) A review of the phosphorus
content of dry cereal and legume crops of the word. Int J Agric Resour Govern Ecol 8:351–370
Lundmark M, Kørner CJ, Nielsen TH (2010) Global analysis of microRNA in Arabidopsis in
response to phosphate starvation as studied by locked nucleic acid-based microarrays. Physiol
Plant 140:57–68
Lynch J (1995) Root architecture and plant productivity. Plant Physiol 109:7–13
Lynch J, Brown KM (2001) Topsoil foraging – an architectural adaptation of plants to low
phosphorus availability. Plant Soil 237:225–237
Lynch JP, Ho MD (2005) Rhizoeconomics: carbon costs of phosphorus acquisition. Plant Soil
269:45–56
MacDonald GK, Bennet EM, Potter PA, Ramakutty N (2011) Agronomic phosphorus imbalances
across the word’s croplands. Proc Natl Acad Sci U S A 108:3086–3091
Manske GGB, Ortiz-Monasterio JI, van Ginkel M, González RM, Fischer RA, Rajaram S, Vlek
PLG (2001) Importance of P uptake efficiency versus P utilization for wheat yield in acid and
calcareous soils in Mexico. Eur J Agron 14:261–274
Marschner P (2012) Phosphorus. In: Marschner P (ed) Marschner’s mineral nutrition of higher
plant, 3rd edn. Academic Press/Elsevier Ltd, London
McDowell RW (2012) Minimising phosphorus losses from the soil matrix. Curr Opin Biotechnol
23:860–865
Mengel K, Kirkby EA (2001) Principles of plant nutrition, 5th edn. Kluwer Academic Publishers,
Dordrecht, p 849
Miao J, Sun J, Liu D, Li B, Zhang A, Li Z (2009) Characterization of the promoter of phosphate
transporter TaPHT1.2 differentially expressed in wheat varieties. J Genet Genomics
36:455–466
Michael B, Zink F, Lantzsch HJ (1980) Effect of phosphate application of phytin-P and other
phosphate fractions in developing wheat grains. Z Planzenernähr Bodenk 143:369–376
Mimura T (2001) Physiological control of phosphate uptake and phosphate homeostasis in plant
cells. Aust J Plant Physiol 28:653–658
Mimura T, Sakano K, Shimmen T (1996) Studies on the distribution, re-translocation and
homeostasis of inorganic phosphate in barley leaves. Plant Cell Environ 19:311–320
Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, Bligny R, Ortet P, Creff A, Somerville S,
Rolland N, Doumas P, Nacry P, Herrerra-Estrella L, Nussaume L, Thibaud MC (2005) A
genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips deter-
mined plant responses to phosphate deprivation. Proc Natl Acad Sci U S A 102:11934–11939
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 127

Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T, Harashima S, Shibata D (1997)

Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco
cultured cells enhances cell growth under phosphate-limited conditions. Proc Natl Acad Sci U
S A 94:7098–7102
Miura K, Rus A, Sharkhuu A, Yokoi S, Karthikeyan AS, Raghothama KG, Baek D, Koo YD, Jin
JB, Bressan RA, Yun DJ, Hasegawa PM (2005) The Arabidopsis SUMO E3 ligase SIZ1
controls phosphate deficiency responses. Proc Natl Acad Sci U S A 102:7760–7765
Miura M, Lee J, Gong Q, Ma S, Jin JB, Yoo CY, Miura T, Sato A, Bohnert HJ, Hasegawa PM
(2011) SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves
the control of auxin accumulation. Plant Physiol 155:1000–1012
Mollier A, Pellerin S (1999) Maize root system growth and development as influenced by
phosphorus deficiency. J Exp Bot 50:487–497
Morcuende R, Bari R, Gibon Y, Zhen W, Pant BD, Bläsing O, Usadel B, Czechowski T, Udvardi
MK, Stitt M, Scheible W-R (2007) Genome-wide reprogramming of metabolism and regula-
tory networks of Arabidopsis in response to phosphorus. Plant Cell Environ 30:85–112
Muchhal US, Pardo JM, Raghothama KG (1996) Phosphate transporters from the higher plant
Arabidopsis thaliana. Proc Natl Acad Sci U S A 93:10519–10523
Muchhal US, Raghothama KG (1999) Transcriptional regulation of plant phosphate transporters.
Proc Natl Acad Sci U S A 96:5868–5872
Mudge SR, Rae AL, Diatloff E, Smith FW (2002) Expression analysis suggests novel roles for
members of the Pht1 family of phosphate transporters in Arabidopsis. Plant J 31:341–353
Müller R, Nilsson L, Krintel C, Nielsen TH (2004) Gene expression during recovery from
phosphate starvation in roots and shoots of Arabidopsis thaliana. Physiol Plant 122:233–243
Müller R, Nilsson L, Nielsen LK, Nielsen TH (2005) Interaction between phosphate starvation
signalling and hexokinase-independent sugar sensing in Arabidopsis leaves. Physiol Plant
124:81–90
Nagy R, Vasconcelos MJV, Zhao S, McElver J, Bruce W, Amrhein N, Raghothama KG, Bucher M
(2006) Differential regulation of five Pht1 phosphate transporters from maize (Zea mays L.).
Plant Biol 8:186–197
Neumann G, Martinoia E (2002) Cluster roots – an underground adaptation for survival in extreme
environments. TRENDS Plant Sci 7:162–167
Neumann G, Römheld V (1999) Root excretion of carboxylic acids and protons in phosphorus-
deficient plants. Plant Soil 211:121–130
Nielsen TH, Krapp A, Röper-Schwarz U, Stitt M (1998) The sugar-mediated regulation of genes
encoding the small subunit of Rubisco and the regulatory subunit of ADP glucose
pyrophosphorylase is modified by phosphate and nitrogen. Plant Cell Environ 12:443–454
Nilsson L, Müller R, Nielsen TH (2007) Increased expression of the MYB-related transcription
factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant Cell Environ
30:1499–1512
Nilsson L, Müller R, Nielsen TH (2010) Dissecting the plant trascriptome and the regulatory
response to tomato deprivation. Physiol Plant 139:129–143
Nilsson L, Lundmark M, Jensen PE, Nielsen TH (2012) The Arabidopsis transcription factor
PHR1 is essential for adaptation to high light and retaining functional photosynthesis during
phosphate starvation. Physiol Plant 144:35–47
Niu YF, Chai RS, Jin GL, Wang H, Tang CX, Zhang YS (2012) Responses of root architecture
development to low phosphorus availability: a review. Ann Bot 112:391–408
Oono Y, Kawahara Y, Kanamori H, Mizuno H, Yamagata H, Yamamoto M, Hosokawa S,
Ikawa H, Akahane I, Zhu Z, Wu J, Itoh T, Matsumoto T (2011) mRNA-seq reveals a
comprehensive transcriptome profile of rice under phosphate stress. Rice 4:50–65
Oono Y, Kobayashi F, Kawahara Y, Yazawa T, Handa H, Itoh T, Matsumoto T (2013) Charac-
terisation of the wheat (Triticum aestivum L.) transcriptome by de novo assembly for the
discovery of phosphate starvation-responsive genes: gene expression in Pi-stressed wheat.
BMC Genomics 14:77
Osborne LD, Rengel Z (2002) Screening cereals for genotypic variation in efficiency of phospho-
rus uptake and utilisation. Aust J Agric Res 53:295–303
128 A. Gruen et al.

Ozturk L, Eker S, Torun B, Cakmak I (2005) Variation in phosphorus efficiency among 73 bread
and durum wheat genotypes grown in a phosphorus-deficient calcareous soil. Plant Soil
269:69–80
Pao SS, Paulsen IT, Saier MH (1998) Major facilitator superfamily. Microbiol Mol Biol Rev
63:1–134
Pariasca-Tanaka J, Satoh K, Rose T, Mauleon R, Wissuwa M (2009) Stress response versus stress
tolerance: a transcriptome analysis of two rice lines contrasting in tolerance to phosphorus
deficiency. Rice 2:167–185
Paszkowski U, Kroken S, Roux C, Briggs SP (2002) Rice phosphate transporters include an
evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis.
Proc Natl Acad Sci U S A 99:13324–13329
Peleg Z, Cakmak I, Ozturk L, Yazici A, Jun Y, Budak H, Korol AB, Fahim TA, Saranga Y (2009)
Quantitative trait loci conferring grain mineral nutrient concentrations in durum wheat x wild
emmer wheat RIL population. Theor Appl Genet 119:353–369
Pérez-Torres CA, López-Bucio J, Cruz-Ramı́rez A, Ibarra-Laclette E, Dharmasiri S, Estelle M,
Herrera-Estrella L (2008) Phosphate availability alters lateral root development in Arabidopsis
by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor. Plant Cell
20:3258–3272
Plaxton W, Tran HT (2011) Metabolic adaptation of phosphate-starved plants. Plant Physiol
156:1006–1015
Poirier Y, Thoma S, Somerville C, Schiefelbein J (1991) A mutant of Arabidopsis deficient in
xylem loading of phosphate. Plant Physiol 97:1087–1093
Preuss C, Huang CY, Gilliham M, Tyerman SD (2010) Channel-like characteristics of the
low-affinity barley phosphate transporter Pht1;6 when expressed in xenopus oocytes. Plant
Physiol 152:1431–1441
Raboy V (2009) Approaches and challenges to engineering seed phytate and total phosphorus.
Plant Sci 177:281–296
Rae AL, Cybinski DH, Jarmey JM, Smith FW (2003) Characterization of two phosphate trans-
porters from barley; evidence for diverse function and kinetic properties among members of
the Pht1 family. Plant Mol Biol 53:27–36
Rae AL, Jarmey JM, Mudge SR, Smith FW (2004) Over-expression of a high-affinity
transporter in transgenic barley plants does not enhance phosphate uptake rates. Funct Plant
Biol 31:141–148
Raghothama KG (1999) Phosphate acquisition. Annu Rev Plant Physiol Plant Mol Biol
50:665–693
Raghothama KG (2005) Phosphorous. In: Broadley MR, White PJ (eds) Plant nutritional geno-
mics. Blackwell Publishing Ltd., Oxford, pp 112–126
Rashotte AM, DeLong A, Muday GK (2001) Genetic and chemical reductions in protein phos-
phatase activity alter auxin transport, gravity response, and lateral root growth. Plant Cell
13:1683–1697
Rausch C, Bucher M (2002) Molecular mechanisms of phosphate transport in plants. Planta
216:23–37
Raven JA (2008) Phosphorus and the future. In: White PJ, Hammond JP (eds) The ecophysiology
of plant-phosphorus interactions. Springer, Dordrecht, pp 271–283
Raven JA (2012) Protein turnover and plant RNA and phosphorus requirements in relation to
nitrogen fixation. Plant Sci 188:25–35
Rebafka FP, Bationo A, Marschner H (1993) Phosphorus seed coating increases phosphorus
uptake, early growth and yield of pearl-millet (pennisetum-glaucum (l) r br) grown on an
acid sandy soil in niger, west-africa. Fertil Res 35:151–160
Ren F, Guo QQ, Chang LL, Chen L, Zhao CZ, Zhong H, Li XB (2012a) Brassica napus PHR1
gene encoding a myb-like protein functions in response to phosphate starvation. PLoS One
7:e44005
Ren Y, He X, Liu D, Li J, Zhao X, Li B, Tong Y, Zhang A, Li Z (2012b) Major quantitative trait
loci for seminal root morphology of wheat seedlings. Mol Breed 30:139–148
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 129

Reymond M, Svistoonof FS, Loudet O, Nussaume L, Desnos T (2006) Identification of QTL

controlling root growth response to phosphate starvation in Arabidopsis thaliana. Plant Cell
Environ 29:115–125
Richardson AE, Hadobas PA, Hayes JE (2000) Acid phosphomonoesterase and phytase activities
of wheat (Triticum aestivum L.) roots and utilization of organic phosphorus substrates by
seedings grown in sterile culture. Plant Cell Environ 23:397–405
Richardson AE (1994) Soil microorganisms and phosphorus availability. In: Pankhurst CE, Doube
BM, Gupta VVS, Grace PR (eds) Soil biota: management in sustainable farming systems.
CSIRO Australia, Melbourne, pp 50–62
Römer R, Schilling G (1986) Phosphorus requirements of the wheat plant in various stages of its
life cycle. Plant Soil 91:221–229
Rose TJ, Wissuwa M (2012) Rethinking internal phosphorus utilization efficiency: a new approach
is needed to improve PUE in grain crops. In: Sparks DL (ed) Advances in agronomy. Elsevier,
London, pp 185–218
Rose TJ, Rengel Z, Ma Q, Bowden JW (2007) Differential accumulation patterns of phosphorus
and potassium by canola cultivars compared to wheat. J Plant Nutr Soil Sci 170:404–411
Rose TJ, Rose MT, Pariasca-Tanaka J, Heuer S, Wissuwa M (2011) The frustration with utiliza-
tion: why have improvements in internal phosphorus utilization efficiency in crops remained so
elusive? Front Plant Sci 2:73. doi:10.3389/fpls.2011.00073
Rouached H, Arpat AB, Poirier Y (2010) Regulation of phosphate starvation responses in plants:
signaling players and cross-talks. Mol Plant 3:288–299
Rouached H (2011) Multilevel coordination of phosphate and sulfate homeostasis in plants. Plant
Signal Behav 6:952–955
Rubio V, Linhares F, Solano R, Martı́n AC, Iglesias J, Leyva A, Paz-Ares J (2001) A conserved
MYB transcription factor involved in phosphate starvation signaling both in vascular plants
and in unicellular algae. Gene Dev 15:2122–2133
Sánchez-Calderón L, López-Bucio J, Chacón-López A, Cruz-Ramı́rez A, Nieto-Jacobo F,
Dubrovsky JG, Herrera-Estrella L (2005) Phosphate starvation induces a determinate devel-
opmental program in the roots of Arabidopsis thaliana. Plant Cell Physiol 46:174–184
Sánchez-Calderón L, López-Bucio J, Chacón-López A, Gutiérrez-Ortega A, Hernández-Abreu E,
Herrera-Estrella L (2006) Characterization of low phosphorus insensitive mutants reveals a
crosstalk between low phosphorus-induced determinate root development and the activation of
genes involved in the adaptation of Arabidopsis to phosphorus deficiency. Plant Pysiol
140:879–889
Sattelmacher B, Horst WJ, Becker HC (1994) Factors that contribute to genetic variation for
nutrient efficiency of crop plants. Z Pflanzen Bodenk 157:215–224
Schachtman DP, Shin R (2007) Nutrient sensing and signaling: NPKS. Annu Rev Plant Biol
58:47–69
Schachtman DP, Reid RJ, Ayling SM (1998) Phosphorus uptake by plants: from soil to cell. Plant
Physiol 116:447–453
Schünmann PHD, Richardson AE, Smith FW, Delhaize E (2004) Characterization of promoter
expression patterns derived from the Pht1 phosphate transporter genes of barley (Hordeum
vulgare L.). J Exp Bot 55:855–865
Secco D, Baumann A, Poirier Y (2010) Characterization of the rice PHO1 gene family reveals a
key role for OSPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in
dicotyledons. Plant Physiol 152:1693–1704
Secco D, Wang C, Arpat BA, Wang Z, Poirier Y, Tyerman SD, Wu P, Shou H, Whelan J (2012)
The emerging importance of the SPX domain-containing proteins in phosphate homeostasis.
New Phytol 193:842–851
Shi T, Li R, Zhao Z, Ding G, Long Y, Meng J, Xu F, Shi L (2013) QTL for yield traits and their
association with functional genes in response to phosphorus deficiency in Brassica napus.
PLoS One 8:e54559
Shin H, Shin HS, Chen R, Harrison MJ (2006) Loss of At4 function impacts phosphate distribution
between the roots and the shoots during phosphate starvation. Plant J 45:712–726
130 A. Gruen et al.

Smith FW, Cybinski DH, Rae AL (1999) Regulation of expression of genes encoding phosphate
transporters in barley roots. In: Gissel-Nielsen G, Jensen A (eds) Plant nutrition-molecular
biology and genetics. Kluwer Academic Publisher, Dordrecht, pp 145–150
Solaiman Z, Marschner P, Wang D, Rengel Z (2007) Growth, P uptake and rhizosphere properties
of wheat and canola genotypes in an alkaline soil with low P availability. Biol Fertil Soils
44:143–153
Stangoulis JCR, Huynh BL, Welch RM, Choi EY, Graham RD (2007) Quantitative trait loci for
phytate in rice grain and their relationship with grain micronutrient content. Euphytica
154:289–294
Stefanovic A, Ribot C, Rouached H, Wang Y, Chong J, Belbahri L, Delessert S, Poirier Y (2007)
Members of the PHO1 gene family show limited functional redundancy in phosphate
transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways. Plant J
50:982–994
Strong WM, Best EK, Cooper JE (1997) Phosphate fertilizer residues in wheat-growing soils of the
western downs. Qld Aust J Soil Res 35:341–354
Su J, Xiao Y, Li M, Liu Q, Li B, Tong Y, Jia J, Li Z (2006) Mapping QTLs for phosphorus-
deficiency tolerance at wheat seedling stage. Plant Soil 281:25–36
Su J, Zheng Q, Li HW, Li B, Jing RL, Tong YP, Li ZS (2009) Detection of QTLs for phosphorus
use efficiency in relation to agronomic performance of wheat grown under phosphorus
sufficient and limited conditions. Plant Sci 176:824–836
Suzuki Y, Kihara-Doi T, Kawazu T, Miyake C, Makino A (2010) Differences in rubisco content
and its synthesis in leaves at different positions in Eucalyptus globulus seedlings. Plant Cell
Environ 33:1314–1323
Svistoonoff S, Creff A, Reymond M, Sigoillot-Claude C, Ricaud L, Blanchet A, Nussaume L,
Desnos T (2007) Root tip contact with low-phosphate media reprograms plant root architec-
ture. Nat Genet 39:792–796
Syers JK, Johnston AE, Curtin D (2008) Efficiency of soil and fertilizer phosphorus use –
reconciling changing concepts of soil phosphorus behaviour with agronomic information.
FAO Fertil Plant Nutr Bull 18:27–44
Tarafdar JC, Marschner H (1994) Phosphatase activity in the rhizosphere and hydrosphere of a
mycorrhizal wheat supplied with inorganic and organic phosphorus. Soil Biol Biochem
26:387–395
Teng W, Deng Y, Chen YP, Xu XF, Chen RY, Lv Y, Zhao YY, Zhao XQ, He X, Li B, Tong YP,
Zhang FS, Li ZS (2013) Characterization of root response to phosphorus supply from mor-
phology to gene analysis in field-grown wheat. J Exp Bot 64:1403–1411
Theodorou ME, Plaxton WC (1993) Metabolic adaptations of plant respiration to nutritional
phosphate deprivation. Plant Physiol 101:339–344
Tesfaye M, Temple SJ, Allan DL, Vance CP, Samac DA (2001) Overexpression of malate
dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to
aluminum. Plant Physiol 127:1836–1844
Tian J, Wang C, Zhang Q, He X, Whelan J, Shou H (2012) Overexpression of OsPAP10a, a root-
associated acid phosphatase, increased extracellular organic phosphorus utilization in rice. J
Integr Plant Biol 54:631–639
Ticconi CA, Delatorre CA, Lahner B, Salt DE, Abel S (2004) Arabidopsis pdr2 reveals a
phosphate-sensitive checkpoint in root development. Plant J 37:801–814
Tiessen H (2008) Phosphorus in a global environment. In: White PJ, Hammond JP (eds) The
ecophysiology of plant-phosphorus interactions, vol 7. Springer, Dordrecht, pp 1–7
Tittarelli A, Milla L, Vargas F, Morales A, Neupert C, Meisel LA, Salvo-G H, Peñaloza E,
Muñoz G, Corcuera LJ, Silva H (2007) Isolation and comparative analysis of the wheat
TaPT2 promoter: identification in silico of new putative regulatory motifs conserved between
monocots and dicots. J Exp Bot 58:2573–2582
Uhde-Stone C, Zinn KE, Ramirez-Yanez M, Li A, Vance CP (2003) Nylon filter arrays reveal
differential gene expression in proteoid roots of white lupin in response to phosphorus
deficiency. Plant Physiol 131:1064–1079
4 Efficient Mineral Nutrition: Genetic Improvement of Phosphate Uptake and Use. . . 131

Ullrich-Eberius CI, Novacky A, Fischer E, Lüttge U (1981) Relationship between

energy-dependent phosphate uptake and the electrical membrane potential in Lemna gibba
G1. Plant Physiol 67:797–801
Van Mooy BAS, Rocap G, Fredricks HF, Evans CT, Devol AH (2006) Sulfolipids dramatically
decrease phosphorus demand by picocyanobacteria in oligotrophic marine environments. Proc
Natl Acad Sci U S A 103:8607–8612
Van Mooy BAS, Fredricks HF, Pedler BE, Dyhrman ST, Karl DM, Koblizek M, Lomas MW,
Mincer TJ, Moore LR, Moutin T, Rappe MS, Webb EA (2009) Phytoplankton in the ocean use
non-phosphorus lipids in response to phosphorus scarcity. Nature 458:69–72
Vance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by
plants for securing a non-renewable resource. New Phytol 157:423–447
Veneklaas EJ, Lambers H, Bragg J, Finnegan PM, Lovelock CE, Plaxton WC, Price CA, Scheible
WR, Shane MW, White PJ, Raven JA (2012) Opportunities for improving phosphorus-use
efficiency in crop plants. New Phytol 195:306–320
Versaw WK, Harrison MJ (2002) A chloroplast phosphate transporter PHT2;1 influences
allocation of phosphate within the plant and phosphate-starvation responses. Plant Cell
14:1751–1766
Wang YH, Garvin DF, Kochian LV (2002) Rapid induction of regulatory and transporter genes in
response to phosphorus, potassium, and iron deficiencies in tomato roots. Evidence for cross
talk and root/rhizosphere-mediated signals. Plant Physiol 130:1361–1370
Wang Y, Ribot C, Rezzonico E, Poirier Y (2004) Structure and expression profile of the
Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis.
Plant Physiol 135:400–411
Wang C, Ying S, Huang H, Li K, Wu P, Shou H (2009a) Involvement of OsSPX1 in phosphate
homeostasis in rice. Plant J 57:895–904
Wang Z, Hu H, Huang H, Duan K, Wu ZA, Wu P (2009b) Regulation of OsSPX1 and OsSPX3 on
expression of OsSPX domain genes and Pi-starvation signaling in rice. J Integr Plant Biol
51:663–674
Wang X, Shen J, Liao H (2010a) Acquisition or utilization, which is more critical for enhancing
phosphorus efficiency in modern crops? Plant Sci 179:302–306
Wang L, Chen F, Zhang F, Mi G (2010b) Two strategies for achieving higher yield under
phosphorus deficiency in winter wheat grown in field conditions. Field Crops Res 118:36–42
Wang J, Sun J, Miao J, Guo J, Shi Z, He M, Chen Y, Zhao X, Li B, Han FP, Tong Y, Li Z (2013) A
wheat phosphate starvation response regulator Ta-PHR1 is involved in phosphate signalling
and increases grain yield in wheat. Ann Bot. doi:10.1093/aob/mct080
Wasaki J, Yonetani R, Kuroda S, Shinano T, Yazaki J, Fujii F, Shimbo K, Yamamoto K, Sakata K,
Sasaki T, Kishimtot N, Kikuchi S, Yamagishi MY, Osaki M (2003) Transcriptomic analysis of
metabolic changes by phosphorus stress in rice roots. Plant Cell Environ 26:1515–1523
White PJ, Veneklaas EJ (2012) Nature and nurture: the importance of seed phosphorus content.
Plant Soil 357:1–8
White PJ, Broadley MR, Gregory PJ (2012) Managing the nutrition of plants and people. Appl
Environ Soil Sci 2012: Article ID 104826
Williamson LC, Ribrioux SPCP, Fitter AH, Leyser HMO (2001) Phosphate availability regulates
root system architecture in Arabidopsis. Plant Physiol 126:875–882
Wissuwa M, Yano M, Ae N (1998) Mapping of QTLs for phosphorus-deficiency tolerance in rice
(Oryza sativa L.). Theor Appl Genet 97:777–783
Wissuwa M, Ae N (2001) Further characterization of two QTLs that increase phosphorus uptake of
rice (Oryza sativa L.) under phosphorus deficiency. Plant Soil 237:275–286
Wissuwa M, Wegner J, Ae N, Yano M (2002) Substitution mapping of Pup1: a major QTL
increasing phosphorus uptake of rice from a phosphorus-deficient soil. Theor Appl Genet
105:890–897
Wissuwa M, Gamat G, Ismail AM (2005) Is root growth under phosphorus deficiency affected by
source or sink limitations? J Exp Bot 56:1943–1950
Wissuwa M, Mazzola M, Picard C (2009) Novel approaches in plant breeding for rhizosphere-
related traits. Plant Soil 321:409–430
132 A. Gruen et al.

Wu P, Wang XM (2008) Role of OsPHR2 on phosphorus homeostasis and root hairs development
in rice (Oryza sativa L.). Plant Signal Behav 3:674–675
Yang YS, Paszkowski U (2011) Phosphate import at the arbuscule: just a nutrient? MPMI
24:1296–1299
Yang M, Ding G, Shi L, Feng J, Xu F, Meng J (2010) Quantitative trait loci for root morphology in
response to low phosphorus stress in Brassica napus. Theor Appl Genet 121:181–193
Yang M, Ding G, Shi L, Xu F, Meng J (2011) Detection of QTL for phosphorus efficiency at
vegetative stage in Brassica napus. Plant Soil 339:97–111
Yang SY, Grønlund M, Jakobsen I, Suter-Grotemeyer M, Rentsch D, Miyao A, Hirochika H,
Kumar CS, Sundaresan V, Salamin N, Catausan S, Mattes N, Heuer S, Paszkowskia U (2012)
Nonredundant regulation of rice arbuscular mycorrhizal symbiosis by two members of the
phosphate transporter1 gene family. Plant Cell 24:4236–4251
Yao Y, Sun H, Xu F, Zhang X, Liu S (2011) Comparative proteome analysis of metabolic changes
by low phosphorus stress in two Brassica napus genotypes. Planta 233:523–537
Yi K, Wu Z, Zhou J, Du L, Guo L, Wu Y, Wu P (2005) OsPTF1, a novel transcription factor
involved in tolerance to phosphate starvation in rice. Plant Physiol 138:2087–2096
Yu Z, Kang B, He X, Lv S, Bai Y, Ding W, Chen M, Cho H, Wu P (2011) Root hair-specific
expansins modulate root hair elongation in rice. Plant J 66:725–734
Zakhleniuk OV, Raines CA, Lloyd JC (2001) pho3: a phosphorus-deficient mutant pf Arabidopsis
thaliana (L.) Heynh. Planta 212:529–534
Zeng HQ, Zhu YY, Huang SQ, Yang ZM (2010) Analysis of phosphorus-deficient responsive
miRNAs and cis-elements from soybean (Glycine max L.). J Plant Physiol 167:1289–1297
Zhang H, Forde BG (2000) Regulation of Arabidopsis root development by nitrate availability.
J Exp Bot 51:51–59
Zhang H, Huang Y, Ye X, Shi L, Xu F (2009) Genotypic differences in phosphorus acquisition and
the rhizosphere properties of Brassica napus in response to low phosphorus stress. Plant Soil
320:91–102
Zhao J, Jamar DCL, Lou P, Wang Y, Wu J, Wang X, Bonnema G, Koornneef M, Vreugdenhil D
(2008) Quantitative trait loci analysis of phytate and phosphate concentrations in seeds and
leaves of Brassica rapa. Plant Cell Environ 31:887–900
Zhao MR, Han YY, Feng YN, Li F, Wang W (2012) Expansins are involved in cell growth
mediated by abscisic acid and indole-3-acetic acid under drought stress in wheat. Plant Cell
Rep 31:671–685
Zhao X, Liu X, Guo C, Gu J, Xiao K (2013) Identification and characterization of microRNAs
from wheat (Triticum aestivum L.) under phosphorus deprivation. J Plant Biochem Biotechnol
22:113–123
Zhou J, Jiao FC, Wu Z, Li Y, Wang X, He X, Zhong W, Wu P (2008) OsPHR2 is involved in
phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants. Plant
Physiol 146:1673–1686
Zhu J, Lynch JP (2004) The contribution of lateral rooting to phosphorus acquisition efficiency in
maize (Zea mays) seedlings. Funct Plant Biol 31:949–958
Zhu YG, Smith SE (2001) Seed phosphorus (P) content affects growth, and P uptake of wheat
plants and their association with arbuscular mycorrhizal (AM) fungi. Plant Soil 231:105–112
Zhu J, Shawn M, Kaepple SM, Lynch JP (2005a) Mapping of QTLs for lateral root branching and
length in maize (Zea mays L.) under differential phosphorus supply. Theor Appl Genet
111:688–695
Zhu J, Shawn M, Kaepple SM, Lynch JP (2005b) Mapping of QTL controlling root hair length in
maize (Zea mays L.) under phosphorus deficiency. Plant Soil 270:299–310
Zhu J, Mickelson SM, Kaeppler SM, Lynch JP (2006) Detection of quantitative trait loci for
seminal root traits in maize (Zea mays L.) seedlings grown under differential phosphorus
levels. Theor Appl Genet 113:1–10
Zhu J, Zhang C, Lynch JP (2010) The utility of phenotypic plasticity of root hair length for
phosphorus acquisition. Funct Plant Biol 37:313–322
Chapter 5
Micronutrient Use Efficiency – Cell Biology
of Iron and Its Metabolic Interactions
in Plants

Ilaria Forieri and Ruediger Hell

Abstract Iron (Fe) is an intriguing nutrient due to its dual nature. Its redox
properties make it essential for different vital processes in plant cells. But an excess
of Fe can be toxic as it catalyses the formation of reactive oxygen species.
Therefore Fe homeostasis must be tightly regulated. Different mechanisms con-
tribute to the regulation, including the control of uptake, the intracellular chelation
by different molecules and the partitioning into the organelles and storage loca-
tions. Despite its high abundance in soil, Fe solubility is extremely low. Fe
availability represents a significant constraint to plant growth and plants have
developed distinct strategies to ensure Fe solubilisation and uptake. The Fe-S
clusters in the electron transport chain of mitochondria and chloroplasts represent
an important sink of Fe. Recent observations suggest that a co-regulation exists
between Fe and sulfur metabolism. This is most likely the outcome of the high
demand for Fe and S required for the biosynthesis of Fe-S clusters. In the following
chapter the uptake strategies and their regulation mechanisms will be introduced.
Moreover, different aspects of the regulation of Fe homeostasis in the cell will be
presented, including the partitioning in the organelles. In the last section different
evidences towards the interaction between Fe and S metabolism will be discussed.

Keywords Iron • Micronutrients • Homeostasis • Regulation • Iron transporters

• Deficiency • Partitioning

Introduction: Fe Importance for Plant Nutrition

Iron (Fe) is a chemical element with atomic number 26, which belongs, together
with manganese, cobalt, nickel, copper and zinc, to the metals of the first transition
series in the periodic table. Fe is the most abundant element found on planet Earth,
as it constitutes a significant part of the inner and outer core. It is the fourth most

I. Forieri • R. Hell (*)

Department for Plant Molecular Biology, Centre for Organismal Studies (COS),
University of Heidelberg, Heidelberg, Germany
e-mail: [email protected]

© Springer International Publishing Switzerland 2014 133

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_5
134 I. Forieri and R. Hell

common element in the crust, after oxygen, silicon and aluminium. As a transition
metal, Fe can easily accept and donate electrons. Thus its oxidation state can vary in
a broad range between
2 and +6, but the most common under current atmospheric
conditions are +2 (ferrous iron) and +3 (ferric iron). This redox property of Fe and
its capability to form complexes with different ligands make this element indis-
pensable for different biological processes in all the living organisms. Indeed
several proteins (Fe-proteins) found in different organisms rely on Fe as a cofactor
for proper functioning. Fe is also essential for plant metabolism, where it partici-
pates in vital cellular functions such as photosynthesis, respiration and chlorophyll
biosynthesis.
Despite its essential role for life, an excess of free Fe can be detrimental to the
cell because it can react with oxygen catalysing the formation of reactive oxygen
species (ROS) such as superoxide (O2∙
) and hydroxyl radical (OH∙) via the Fenton
reaction (reviewed by Hell and Stephan 2003):

Fe3þ þ O2 
! Fe2þ þ O2
Fe2þ þ H2 O2 ! Fe3þ þ OH
þ OH
Resulting in : O2 
þ H2 O2 ! O2 þ OH
þ OH

These radicals constitute a severe danger for the cell particularly the hydroxyl
radical, which is very reactive and can indiscriminately oxidise DNA, polyunsatu-
rated fatty acids in lipids (lipid peroxidation), amino acids in proteins and sugars.
To avoid potentially toxic reactions, protein-bound Fe is found incorporated into
structures such as heme or coordinated with sulfur (S) to form Fe-S cluster. Heme
contains a Fe atom in the centre of a large heterocyclic organic ring, the porphyrin,
made of four pyrrolic groups joined by methine bridges. Among heme proteins, a
fundamental role is played by hemoglobin and myoglobin in vertebrates as they
contain a Fe atom which binds to oxygen. The most common heme proteins in
plants are cytochromes, which participate in the electron transport process of
mitochondria and chloroplasts. Other heme proteins are catalase and peroxidases
that are involved in scavenging of ROS.
Fe-S clusters are versatile and ubiquitous cofactors of many different enzymes
that participate in respiration, photosynthesis, DNA repair and replication, sulfur
and nitrogen assimilation and ribosome biosynthesis (Balk and Pilon 2011). They
are formed from Fe atoms and sulfur in the form of acid-labile sulfide and are bound
to proteins via the sulfhydryl groups of cysteine residues. Different forms are found
in plants, the most common are the 2Fe-2S and 4Fe-4S bound to four cysteine (Cys)
residues. Other types include the 2Fe-2S Rieske-type cluster coordinated by 2 Cys
and 2 His residues and 3Fe-4S liganded by 3 Cys (reviewed by Couturier
et al. 2013).
Toxicity of free Fe ions may be avoided by chelation by different compounds
such as the non-proteinogenic amino acid nicotianamine, citrate and the storage via
ferritin proteins.
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 135

Ferrous Fe is relatively soluble but it is easily oxidised to ferric Fe by atmo-

spheric oxygen. The solubility of Fe3+ is highly influenced by the soil pH. Indeed in
alkaline conditions Fe3+ hydrolyses water producing Fe(OH)3 that polymerises and
precipitates together with inorganic anions. Free Fe3+ is soluble up to 10
6 M at pH
3.3, but in aerated soil at neutral-basic pH, the concentration of free Fe3+ and Fe2+ is
estimated to be less than 10
15 M (Marschner 1995). This is much lower than the
optimal concentration needed by plants that require between 10
4 and 10
8 M Fe3+.
This solubility problem strongly impacts Fe availability that represents a severe
constraint for plant development and yield. In particular, Fe is considered the third
most limiting plant nutrient after nitrogen and phosphorus. Hence, even though Fe
is quite abundant in soil, Fe deficiency represents a major problem for worldwide
agriculture in calcareous-alkaline soil, which comprises 30 % of all arable land.
Upon Fe deficiency, plants display typical symptoms such as leaf chlorosis and
reduced growth. Therefore the quest for Fe use efficient crop plants is a goal of plant
breeding and biotechnology.
Plants are always dealing with the dual nature of Fe and have developed
sophisticated mechanisms to ensure adequate Fe acquisition from the soil and at
the same time to make it available for biological processes in the cell avoiding toxic
reactions.
Fe content in crop plants, which constitute a widely utilised staple food, signif-
icantly influences Fe assimilation by the human population. Fe deficiency is one of
the most diffuse nutritional problems in the world, with around 30 % of the world
population affected according to World Health Organization (http://www.who.int/
nutrition/topics/ida/en/). Therefore, understanding the mechanisms used by plants
to cope with changing Fe availability is a prerequisite not only for improving crop
yield but also for a positive impact on human nutrition.
In this chapter, the strategies adopted by plants for Fe uptake will be reviewed,
with focus on mechanisms of regulation. The partitioning of Fe in the cell and the
interaction with other nutrients such as sulfur will also be presented.

Fe Deficiency and Plant Responses

Higher plants use distinct strategies to ensure Fe solubilisation and uptake. In the
1980s, Römheld and Marschner (1986) divided plants into two groups according to
their Fe uptake mechanisms. Dicotyledonous and non-graminaceous monocotyle-
donous plants belong to the Strategy I or reduction strategy group, whereas Poaceae
to the Strategy II or chelation strategy group.
Strategy I is based on (1) soil acidification to increase Fe solubility, (2) reduction
of Fe3+ to Fe2+ in the rhizosphere and (3) uptake of Fe2+ across the root plasma
membrane (see Fig. 5.1). This strategy was first characterised in Lycopersicum
esculentum (tomato) and Pisum sativum (pea) as model crop plants. Recently, most
of the studies in this respect have focused on Arabidopsis thaliana, which repre-
sents a powerful tool for cell biology investigations. The genes responsible for the
136 I. Forieri and R. Hell

Fig. 5.1 Fe deficiency responses in plants. Strategy I (non-graminaceous plants) and Strategy II
(graminaceous plants) are presented. In the rectangles the key enzymes of the two strategies are
shown. Abbreviations: AHA2 Arabidopsis H+-ATPase, DMAS deoxymugineic acid synthase,
FRO2 ferric chelate reductase 2, IRT1 iron regulated transporter 1, MAs mugineic acids, NAAT
nicotianamine aminotransferase, NAS nicotianamine synthase, TOM transporter of mugineic acids,
YS yellow stripe, YSL yellow stripe like. YSL refers to orthologs of YS in plants other than maize

different steps of the strategy I have been identified and cloned and Fe deficiency
results in an up-regulation of their expression. Firstly, the H+-ATPase family
(HA) excretes protons into the rhizosphere to increase Fe solubility (Palmgren
2001). In Arabidopsis the HA2 gene particularly is induced in Fe deficiency
(Santi and Schmidt 2009). The reduction of Fe3+ is catalysed by the ferric-chelate
reductase oxidase 2 (FRO2) in Arabidopsis (Robinson et al. 1999) and by FRO1 in
pea (Waters et al. 2002). FRO proteins are integral membrane proteins that belong
to a superfamily of flavocytochromes and can transfer electrons from cytosolic
NADPH to FAD across the plasma membrane (Robinson et al. 1999). FRO2 was
isolated as allelic to the frd1 mutants in Arabidopsis (Yi and Guerinot 1996). These
mutants are not able to induce the Fe chelate reductase activity, although they are
still able to acidify the rhizosphere upon Fe deficiency. Moreover, these mutants
cannot translocate radiolabeled Fe from root to the shoot when Fe is provided as
chelated Fe3+. Altogether these results shown that FRO activity is uncoupled from
the HA activity and that Fe3+ reduction to Fe2+ is a prerequisite for the transport.
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 137

After reduction, the uptake of Fe2+ in the root epidermal cells is performed by
the iron-regulated transporter 1 (IRT1) in Arabidopsis (Vert et al. 2003). Orthologs
of IRT1 were cloned in both pea and tomato (Cohen et al. 1998; Eckhardt
et al. 2001). The Arabidopsis knock-out mutant irt1 is lethal unless plants are
watered with an excess of Fe (Vert et al. 2002). Another transporter, IRT2, is also
induced in roots when exposed to Fe shortage (Vert et al. 2001) but its knock-out
mutant is not affected under normal Fe conditions. The attempt to complement the
irt1 mutant with IRT2 driven by a constitutive promoter did not rescue the pheno-
type, showing that the two transporters have different roles in Fe uptake (Varotto
et al. 2002). Both transporters belong to the zinc-regulated transporter iron-
regulated transporter like protein family (ZIP) that takes its name from the first
transporter that was identified, the zinc regulated transporter ZRT. Indeed IRT1 and
IRT2 are not highly specific for Fe and can mediate the import of a broad spectrum
of metal species including zinc Zn2+, manganese Mn2+ and cadmium Cd2+. The
Arabidopsis thaliana genome encodes for 16 ZIP proteins (Mäser et al. 2001) and
they function in the uptake of different bivalent metal ions.
IRT1 is regulated at different levels. Its expression promptly responded after the
plants were transferred to Fe deficiency (Connolly et al. 2002). In particular, the
mRNA accumulated after 24 h and the protein level peaked after 72 h. After Fe
resupply the IRT1 protein was already almost undetectable after 12 h, indicating
that its expression is tightly regulated. An over-expressing line of IRT1 showed
accumulation of the protein only under Fe deficiency, indicating a fine regulation of
Fe homeostasis at the uptake level. Indeed IRT1 protein is rapidly degraded in
response to changing Fe conditions and this degradation is mediated by
ubiquitination (Kerkeb et al. 2008; Barberon et al. 2011). Recently the specific
E3 ubiquitin ligase IRT1 DEGRADATION FACTOR 1 (IDF1) was identified in a
screen of insertional mutants (Shin et al. 2013).
The main feature of Strategy II is the excretion of chelating compounds such as
mugineic acids (MAs) in the root rhizosphere that chelate Fe3+, to enhance solu-
bility and allow for mobilisation of Fe3+ (Fig. 5.1). The name mugineic acid is
derived from the Japanese word komugi for wheat from which these compounds
had first been isolated. The MAs biosynthetic pathway is conserved among the
Poaceae family, which comprises many of the most important food plants: rice,
wheat and maize, and starts from S-adenosyl-L-methionine (SAM). Three mole-
cules of SAM are converted into nicotianamine (NA) in one reaction by
nicotianamine synthase (NAS). NA is a precursor for MAs in strategy II plants
but additionally functions as Fe chelator in different plant organs, and seems to be
essential not only for the uptake of Fe from the soil, but also for the cell-to-cell and
long distance transport within the plant (Schuler et al. 2012). Studies on NA started
with the analysis of the tomato mutant chloronerva. This mutant is NA-free and
shows retarded growth and intercostal chlorosis of young leaves. Map-based clon-
ing revealed that chloronerva is a single copy gene in tomato and encodes for NAS
(Ling et al. 1999). The NAS genes have been cloned from other plant species such
as barley, rice and Arabidopsis, showing that NA carries out functions in strategy I
and II species (reviewed by Hell and Stephan 2003; Klatte et al. 2009). The
138 I. Forieri and R. Hell

expression of most NAS genes is strongly induced upon Fe deficiency. NA is then

further processed by NA aminotransferase (NAAT) and deoxymugineic acid
synthase (DMAS) to form 20 -deoxymugineic acid (DMA). DMA is the starting
point for the synthesis of all the other chemical forms of MAs (Nakanishi
et al. 2000). The secretion of MAs is diurnally regulated, with a peak in the morning
(Cakmak et al. 1998). The transporter of mugineic acid family phytosiderophores
1 (TOM) has been identified in rice and barley as responsible for the secretion of
MAs (Nozoye et al. 2011). After secretion, MAs can bind to Fe3+ and the MA-Fe3+
complexes are taken up by the root YELLOW STRIPE 1 (YS1) and YELLOW
STRIPE 1-like transporters (YSL1) (Curie et al. 2009; Inoue et al. 2009). The study
of these transporters started with the analysis of the maize mutant yellow stripe 1.
This mutant shows leaf chlorosis and fails to take up phytosiderophores from the
soil (Von Wiren et al. 1994). The YELLOW STRIPE 1 gene was then mapped and
cloned (Curie et al. 2001) and found to encode for a membrane transporter that
mediates the uptake of phytosiderophores bound to Fe3+. The biochemical function
is directly shown by the ability of YS1 to restore the growth of a yeast strain
deficient in Fe uptake, when phytosiderophores are present in the media. Its mRNA
accumulates significantly under Fe deficiency in both root and shoot. The latter was
surprising as phytosiderophores were not expected to be transported in green tissues
as they are only present in the root (Curie et al. 2001). Indeed, although the release
of phytosiderophores is a prerogative of graminaceous plants, YSL transporters are
found also in non-graminaceous taxa (Chu et al. 2010, 2013). In particular,
Arabidopsis thaliana has eight YSL genes (Mäser et al. 2001). In addition, YSL
transporters have been found to transport NA-Fe3+ complexes (reviewed by Chu
et al. 2013), adding to the fundamental role of NA as Fe chelator in both strategy I
and strategy II plants.
Interestingly, rice additionally possesses an iron transporter, OsIRT1, an
ortholog of AtIRT1. However, rice roots show a very low ferric-chelate reductase
activity, suggesting a role of OsIRT1 in the direct uptake of Fe2+ in anaerobic
growth conditions that is typical for this crop (Ishimaru et al. 2006).
Both graminaceous and non-graminaceous plants possess other divalent metal
transporters, which can facilitate Fe assimilation. The NRAMP (natural resistance-
associated macrophage protein) family transporters have been first found in mam-
mals and have then been cloned from Arabidopsis thaliana (Curie et al. 2000) There
are six genes encoding for NRAMP proteins in Arabidopsis (Mäser et al. 2001).
They can mediate the uptake of several divalent metals, including Fe2+, zinc Zn2+,
manganese Mn2+, nickel Ni2+ and cadmium Cd2+. The expression of three of these
transporters, NRAMP1, NRAMP2 and NRAMP4, is induced by Fe deficiency in
roots and leaves. In particular, NRAMP1 is thought to mediate the uptake of Fe and
other essential nutrients, such as manganese from the soil (Curie et al. 2000;
Cailliatte et al. 2010). The other two transporters NRAMP3 and NRAMP4 are
involved in the Fe distribution to developing seeds in low Fe conditions (Lanquar
et al. 2005).
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 139

Regulation of the Strategies

The regulation of Fe deficiency responses is very complex and requires the coor-
dination of several regulatory elements. The presence of different pathway and
feedback signals constitute an important aspect in the regulation.
Several transcription factors that are involved in the regulation of the Fe uptake
machinery have been already identified in different Strategy I plants (Fig. 5.2a).
The key element/regulator in this respect was first identified in the tomato fer
mutant. Map-based cloning revealed that FER encodes for a basic helix-loop-
helix (bHLH) transcription factor (Ling et al. 2002). Arabidopsis possesses an
ortholog of FER, which has been named FIT (FER-like iron deficiency-induced
transcription factor, also named before FIT1/FRU/AtbHLH029; Bauer et al. 2007).
FIT expression is repressed upon full Fe supply, whereas the expression is highly
induced in Fe deficiency. FIT positively regulates the expression of different
Fe-responsive genes, including IRT1 and FRO2. The fit mutant shows leaf chlorosis
and decreased Fe content and fails to induce the typical Strategy I responses
(Colangelo and Guerinot 2004). Moreover, the mutant dies at the seedling stage
unless watered with additional Fe. FRO2 mRNA level is severely downregulated in
the mutant and the FRO activity cannot be detected. The transcript level of IRT1 is
decreased but still detectable in fit plants whereas the protein IRT1 is not present.
These results suggest that FIT can control the Strategy I responses at different level,
regulating the gene expression but also the turnover of IRT1 protein.
FIT can also interact directly with other bHLH factors, such as bHLH038 and
bHLH039. This interaction is thought to serve in modulating the plant response to
Fe deficiency (Yuan et al. 2008). These two factors belong together with bHLH100
and bHLH101 to a specific sub-group of bHLH and their expression is strongly
induced upon Fe deficiency (Wang et al. 2007). They are also functioning inde-
pendently from FIT to mediate Fe deficiency responses (Sivitz et al. 2012).
FIT can also directly interact with ETHYLENE INSENSITIVE 3 and ETHYL-
ENE INSENSITIVE 3–LIKE1 (Lingam et al. 2011). This interaction provides the
molecular link between ethylene and the responses to Fe starvation, which was
elusive before. Ethylene is known to be a positive regulator of the induction of
different Fe responsive genes. The ethylene downstream transcription factors EIN3
and EIL1 are required for FIT accumulation and therefore thought to inhibit its
proteasomal degradation, thus enhancing the plant responses to Fe deficiency
(Lingam et al. 2011).
Microarray analysis aimed at finding new regulatory candidates identified the
bHLH transcription factor POPEYE (PYE) (Long et al. 2010). PYE is upregulated
specifically in the cells of the root perycicle upon Fe deficiency. The mutant pye
displays severely impaired growth under – Fe condition; therefore PYE seems to
play a fundamental role in the roots of plants exposed to Fe deficiency. Moreover,
PYE is proposed to negatively regulate a cluster of Fe-responsive genes, amongst
these NAS4 and FRO3. PYE can directly interact with PYE homologues, such as
IAA-Leu Resistant3 (ILR3) and bHLH115. ILR3 in turn interacts with another
140 I. Forieri and R. Hell

Fig. 5.2 Regulation of Fe deficiency responses in (a) Strategy I and (b) Strategy II plants.
Rectangles indicate important regulatory transcription factors of the two Strategies and down-
stream Fe responsive genes. Arrows indicate positive or negative regulation. Abbreviations: bHLH
basic helix-loop-helix, FIT FER-like iron deficiency induced, EIN3/EIL1 ETHYLENE INSENSI-
TIVE 3/ETHYLENE INSENSITIVE 3–LIKE, PYE POPEYE, ILR3, BTS BRUTUS, IDEF iron
deficiency responsive element-binding factor, IRO iron-related transcription factor

regulatory protein named BRUTUS (BTS). BTS possesses three different domains,
one with putative E3 ligase activity, one for transcriptional regulation and one for
Fe binding. Unlike pye, bts mutants appear more resistant to Fe deprivation and
show a better growth in – Fe conditions, with longer roots and greener shoots. A
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 141

direct interaction between PYE and BTS has not been reported, but interestingly
BTS interacts with the PYE interactors ILR3 and bHLH115. It is therefore specu-
lated that this interaction participates in the regulation of Fe deficiency responses in
the root. The induction of PYE under Fe limiting conditions might serve to regulate
Fe homeostasis in the plant. Additionally BTS, the antagonist of PYE, might help in
this regulation controlling PYE activity (Long et al. 2010).
Other regulatory elements have been identified in Strategy II plants (Fig. 5.2b).
The analysis was based on stepwise promoter analysis of the barley IDS2 gene in
tobacco and allowed the identification of two key regulators of Fe deficiency
responses, the cis-acting iron deficiency responsive element 1 (IDE1) and IDE2
(Kobayashi et al. 2003). IDE1 and IDE2 were the first discovered cis-acting
elements related to nutrient deficiency. From sequence alignment of the promoters
of several Fe responsive genes it emerged that these cis-elements are quite con-
served among different plant species. Indeed they have been found in several genes,
e.g. HvNAAT, HvNAS, OsNAS2, OsNAS3, OsIRT1, AtIRT1 and AtFRO2. IDE1 and
IDE2 can interact with two rice transcription factors IDE-binding factor 1 (IDEF1)
and IDEF2 (Kobayashi et al. 2007; Ogo et al. 2008). These two factors are members
of the ABI3/VP1 (ABSCISIC ACID INSENSITIVE 3/VIVIPAROUS 1) family and
NAC (NO APICAL MERISTEM, Arabidopsis transcription activation factor and
CUP SHAPED COTYLEDON) family, respectively. IDEF1 and IDEF2 are con-
stitutively expressed in vegetative tissues and can regulate two different sets of
genes (Kobayashi et al. 2009). IDE1 regulates most of the Fe related genes in
normal Fe conditions and during the early responses to Fe deficiency. Interestingly,
IDEF1 can switch its target genes in the late stages of Fe deficiency. IDEF2 instead
maintains the same target genes during the responses to Fe deficiency and it is
known to positively regulate the expression of OsYSL2 (Kobayashi et al. 2010).
Therefore, IDEF2 is also involved in the correct partitioning of Fe between roots
and shoot.
Many regulators from graminaceous plants have been identified by a microarray
analysis approach. The most extensively studied candidate is OsIRO2, which
encodes a bHLH transcription factor (Ogo et al. 2011). Its expression is positively
regulated by IDEF1 in Fe deficiency. OsIRO2 can in turn positively regulate
different Strategy II genes, such as OsNAS1, OsNAS2, OsNAAT1, TOM1 and
OsYSL15. Another bHLH transcription factor, OsIRO3, is present in rice and its
expression is induced by Fe deficiency (Zheng et al. 2010). It seems to be a negative
regulator of several genes related to Fe deficiency responses.
Intriguingly, sequence comparison with Arabidopsis transcription factors has
shown that OsIRO2 is similar to AtbHLH038, 039, 100 and 101, whereas IRO3 is
similar to PYE (Ogo et al. 2006). Thus far, no correspondent of FIT has been found
in graminaceous plants and no orthologue of IDEF1 and IDEF2 has been found in
non-graminaceous plants. Therefore, it seems that the regulatory mechanisms are
only partially conserved between Strategy I and Strategy II plants.
Apart from the positive regulator ethylene, other signaling molecules and plant
hormones participate in the regulation of Fe deficiency responses. Among these
nitric oxide (NO), carbon dioxide and auxin also contribute to the induction of
142 I. Forieri and R. Hell

several Fe-responsive genes. In contrast, cytokinin and jasmonic acid can nega-
tively regulate the expression of different Fe genes such as IRT1, FRO2 and FIT
(reviewed by Kobayashi and Nishizawa 2012).
While the regulation of the Fe deficiency responses has been elucidated, the Fe
sensing mechanism in the root remains unidentified. Recently the IDEF1 transcrip-
tion factor has been found to bind directly to Fe and other divalent metals via its
proline-rich domains and histidine-asparagine residues (Kobayashi et al. 2012).
Thus, this transcription factor might be one factor for the sensing of the actual Fe
situation in the cell and thus the Fe availability.

Fe Homeostasis in the Cell

After uptake in the root, further steps are required in order to allocate Fe in the rest
of the plant. Fe must first be transported from the root epidermis through the root
tissues to be loaded into the xylem. Due to its low solubility, a symplastic transport
is assumed, but little is known about the mechanism and possible carrier (Morrissey
and Guerinot 2009). Once it reaches shoot tissues, other mechanisms must be
involved for the unloading and the transport into the different cellular
compartments.
The solubility problem requires that Fe must always be in a chelated form during
transport within the plant. Another important reason for the chelation is the poten-
tial toxicity of free ionic Fe that can catalyse the formation of reactive oxygen
species (ROS) causing cell damage. Citrate, NA and MAs are the known predom-
inant Fe chelators. In particular, citrate plays a fundamental role in chelating Fe in
the xylem. Indeed, citrate-Fe(III) complexes have been found in the xylem sap of
tomato plants (Rellan-Alvarez et al. 2010). FERRIC REDUCTASE DEFECTIVE
3 (FRD3) is an Arabidopsis multidrug and toxin efflux (MATE) transporter that
plays a fundamental role in balancing Fe homeostasis. Its orthologue, OsFRDL1,
has been found in rice. Both transporters mediate the efflux of citrate into the
xylem. The frd3 mutant displays chlorotic and dwarf phenotype, constitutive
up-regulation of the Fe deficiency genes and of FRO activity. A high level of Fe
is found in the roots of the mutant, due to inefficient Fe translocation to the shoot,
emphasising the importance of citrate for Fe transport from root to shoot (Durrett
et al. 2007; Rogers and Guerinot 2002). As FRD3 and FRDL1 efflux citrate in the
Fe-free form, other transporters must be involved in the transport of Fe into the
xylem. The Arabidopsis ferroportin 1/iron regulated 1 (AtFPN/AtREG1) could be
involved in this process. Although direct evidence is still lacking, the localisation,
the promoter activity and the mutant phenotype make this transporter a promising
candidate (Morrissey et al. 2009).
Other transporters are involved in unloading the xylem into phloem. Members of
the YSL family are widely expressed in different tissues also of non-graminaceous
plants, suggesting a role in Fe translocation from xylem to phloem besides the
uptake of MA-Fe complexes from the soil. Indeed YSL transporters have been
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 143

found to mediate the transport of NA-Fe complexes (Curie et al. 2009). The
Arabidopsis YSL1 and YSL2 proteins were found to localise to the plasma mem-
brane and to function in yeast complementation assay (Chu et al. 2010). They are
active in leaves and in flowers and are therefore required for the fertility and the
development of seeds and for the distribution of Fe to the seeds. The two trans-
porters and AtYSL3 are quite closely related but they have distinct functions in the
plant, as neither YSL1 nor YSL2 under control of YSL3 promoter could complement
the double mutant ysl1ysl3. YSL4 and YSL6 were found to localise to the chloro-
plast (Divol et al. 2013), to the tonoplast and to internal membranes (Chu
et al. 2013). They are thought to mediate the release of Fe from the chloroplast in
case of Fe overload, thus controlling Fe homeostasis.
NA certainly represents the principal Fe chelator in the cell for different reasons
(reviewed by Hell and Stephan 2003). It can form complexes with both Fe3+ and
Fe2+ at neutral and basic pH, NA-Fe complexes are unlikely to react with oxygen in
the Fenton reaction, NA is found in all plant tissues and also all plant species its
concentration positively correlates with the root areas of Fe uptake. NA is also
involved in loading the seeds with Fe (Klatte et al. 2009). It is, however, also able to
bind and transport other transition metals such as zinc (Haydon et al. 2012).

Partitioning of Fe in the Organelles

The partitioning of Fe to the organelles must be tightly regulated, due to the high
requirement for the biosynthesis of Fe-S clusters in both chloroplasts and mito-
chondria. In addition, synthesis of cytosolic Fe-S depends on provision of a
precursor from the mitochondria (Balk and Pilon 2011). The import of Fe into the
chloroplast also represents an important strategy to store Fe in a non-toxic and
available form. Indeed the largest amount of Fe in plant cells is found in the
chloroplast, where 80–90 % of Fe is accumulated (Marschner 1995). The members
of the ferritin family (FER) play a fundamental role to prevent oxidative damage in
case of Fe overload. Ferritins are spherical protein complexes formed by 24 sub-
units. They can internalise Fe atoms in their central cavity and can release them
when needed (Briat et al. 2010). Animal ferritins are regulated mostly at the
translational level, while phytoferritins are mainly subjected to transcriptional
regulation.
In Arabidopsis, there are four ferritin isoforms (FER1, FER2, FER3 and FER4).
A loss-of-function approach was used to investigate the role of this protein in
different plant tissues (Ravet et al. 2009). The analysis showed that plants lacking
ferritins were more sensitive to excess of Fe, with reduced growth and defects in
flower development. Moreover, loss-of-function mutant plants presented differen-
tial regulation of genes related to Fe uptake and higher level of ROS and conse-
quently higher activity of detoxifying enzymes. Electron microscopy has shown
that plant ferritins localise to the plastids, mainly to non-photosynthetic ones such
as proplastids, etioplasts and amyloplasts (Seckback 1982). The loss-of-function
144 I. Forieri and R. Hell

approach provided more evidence that ferritins are not actually required for the
proper formation of the photosynthetic chloroplast or for the functioning of the
photosynthetic apparatus. Indeed, ferritins seem to play a fundamental role in the
protection against oxidative stress (Ravet et al. 2009).
Other studies have attempted to further elucidate the localisation of ferritins in
plant cells.
According to Zancani et al. (2004) ferritins can also localise to the mitochondria.
Indeed, according to bioinformatics analyses, the Arabidopsis AtFER4 is the
isoform most likely to be targeted to the mitochondria. A study was conducted
based on the knock-out mutant atfer4. An antibody against FER was applied to
protein fractions of isolated mitochondria and a ferritin signal was found in
mitochondria isolated from wild type plants subjected to high Fe supply. The signal
was not present in the fraction isolated from the mutant plants. This mitochondrial
isoform seems to be of great importance for balancing Fe homeostasis in hetero-
trophic tissues, as shown by work on suspension cell cultures (Tarantino
et al. 2010). Petit et al. (2001) identified a cis-element in the region of maize ferritin
gene ZmFER1 and in its orthologue from Arabidopsis AtFER1. This regulator
named iron-dependent regulatory sequence (IDRS) is able to repress the transcrip-
tion of the gene under low Fe conditions. IDRS also has additional functions in
Arabidopsis, where it triggers the expression of AtFER1 under dark-induced senes-
cence but not in age-dependent senescence and in seedlings (Tarantino et al. 2003).
This suggests that more regulatory elements must be involved in the regulation of
AtFER1 expression under such conditions.
Time for coffee (TIC) has been found in a luciferase-based genetic screen of the
AtFER1 promoter (Duc et al. 2009). TIC has been previously described as a nuclear
component of the circadian clock. Mutants of TIC are chlorotic unless supplied
with exogenous iron and are hypersensitive to iron during the early stages of
development. Thus TIC is a central regulator of AtFER1 as it represses its expres-
sion in low Fe conditions, in a way that is independent from IDRS. The tic mutants
also fail to repress other genes induced by Fe overload under low Fe, pointing out
that TIC-dependent pathways are fundamental for the response to Fe overload.
Another Fe binding protein, which has been reported to be involved in the
protection against photo-oxidative damage is frataxin. This protein has been
hypothesised to participate in the mitochondrial biosynthesis of Fe-S cluster acting
as Fe donor. Its importance for plant cell has been demonstrated by analysis of
T-DNA insertion mutants in Arabidopsis. Indeed frataxin knock-out mutants are
lethal, while the knockdown ones are viable but accumulate high levels of ROS and
induce the expression of genes encoding for ROS scavenging proteins (Busi
et al. 2006).
The import of Fe into the chloroplast is linked to ferritin function. The permease
in chloroplast 1 (PIC1) was first identified as member of the chloroplast inner
membrane translocon complex TIC (Teng et al. 2006). This transporter emerged
as a possible candidate in a bioinformatic screening for the Fe importer in the
chloroplast proteome, due to its biochemical characteristics such as hydrophobicity,
basic isoelectric point and predicted transmembrane domains, and was therefore
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 145

renamed permease in chloroplast 1 PIC1 (Duy et al. 2007). The loss-of-function

mutants of PIC1 display chlorotic phenotype and severely impaired growth. More-
over, the mutation causes severe problems in chloroplast development and leads to
disturbed metal homeostasis in leaves.
FRO7, one of the members of the Arabidopsis FRO family, localises to the inner
chloroplast membrane and it is thought to be essential for seedling development.
The growth of the fro7 mutant is significantly impaired in alkaline conditions and in
media lacking sugar. Moreover, chloroplasts isolated from the mutant exhibit
significantly lower FRO activity and Fe content compared to the wild type (Jeong
et al. 2008). These results suggested an important role of FRO7 as a chloroplast Fe
transporter during photosynthesis and development.
In addition, the mitochondria contribute to cellular Fe homeostasis. Indeed a
mutation in the STARIK/ATM3 gene that encodes for a mitochondria ABC trans-
porter leads to chlorosis and reduced growth. The mitochondria of the starik mutant
accumulate more non-heme and non-protein bound Fe, as the biosynthesis of Fe-S
clusters in the mitochondria is linked to the intracellular Fe by this transporter
(Kushnir et al. 2001; Bernard et al. 2009). A mitochondria iron transporter (MIT)
has been identified in a screening of T-DNA (transfer DNA) of rice in Fe deficiency
conditions (Bashir et al. 2011). The homozygous knock-out mutant mit is lethal,
highlighting the importance of this transporter for plant growth. In contrast, the
heterozygous mutant is viable but severely impaired in growth, exhibiting an
accumulation of Fe in the shoot with less Fe in the mitochondria.
The vacuole also functions to store Fe and avoid toxicity. The vacuolar iron
transporter (VIT) mediates the transport of Fe from the cytosol into the vacuole and
plays a fundamental role in seed and seedling development, as shown by the
analysis of the vit1-1 mutant (Kim et al. 2006). In contrast, NRAMP3 and
NRAMP4 are influx transporters that function to export Fe from the vacuole into
the cytosol during seed germination (Lanquar et al. 2005). Their contribution is
essential to provide Fe during development until the seedlings can start to take up
Fe from the environment.

Interaction of Fe and S Metabolism (Uptake, Fe-S Clusters)

Uptake and homeostasis of Fe is known to be linked to other metals such as zinc

(Lin et al. 2009; Deinlein et al. 2012). More recently the connection between Fe and
sulfur (S) metabolism is being recognised, mainly because Fe is required together
with S for the biosynthesis of the Fe-S clusters.
The thylakoids in chloroplasts harbour ferredoxin, photosystem I (PSI) and
cytochrome b6f complex, which belong to the photosynthetic electron transport
chain. In the stroma, other Fe-S proteins are found; among these there are nitrite
reductase and two key enzymes for sulfur metabolism, sulfite reductase and APR.
In mitochondria, major Fe-S proteins are Complex I, II and III of the respiratory
146 I. Forieri and R. Hell

chain and aconitase. Other Fe-S proteins are found in the nucleus and function in
DNA replication (Balk and Pilon 2011) and damage repair (Liu et al. 2003).
The coordination of the plant’s demand for Fe-S clusters supports the hypothesis
of a co-evolution between the Fe and the S metabolisms and the development of
interaction traits between them.
Chelated Fe and reduced S in form of cysteine represent the substrates of the
biosynthetic pathway. The Fe donor molecule is not known yet, whereas it is known
that sulfur is mobilised from cysteine by a cysteine desulfurase. The protein frataxin
has received attention for its putative role as Fe donor in the mitochondria assembly
pathway (Busi et al. 2006).
As sulfur is present as acid-labile sulfide (S
2) in the Fe-S cluster, two additional
electrons are needed to reduce elemental sulfur S0 to S
2 (Lill 2009). In the first step
of the pathway, the Fe-S cluster is assembled on scaffold proteins. In the second
step, the cluster is transferred to the specific apoprotein, which provides free amino
acidic residues to bind it. Additional carrier proteins are involved in this step. The
assembly machineries have been characterised in plants: chloroplasts contain the
ISC (iron-sulfur cluster) biosynthetic pathway, whilst mitochondria contain the
SUF-like (sulfur mobilization) pathway and in the cytoplasm the CIA (cytosolic
iron-sulfur cluster assembly) pathway (reviewed by Balk and Pilon 2011; Couturier
et al. 2013). The CIA is dependent on the mitochondria SUF assembly machinery,
which provided the sulfide-containing compound that is used for the biosynthesis of
the cluster. The mitochondrial ABC transporter, STARIK/ATM3, is thought to be
involved in this process. Indeed the mutant atm3 shows severely impaired activity
of cytosolic aconitase while the mitochondria and plastidic isoforms are unaffected.
Furthermore, atm3 does not accumulate Fe in the mitochondria and the general Fe
homeostasis is not affected (Bernard et al. 2009).
Upon nutrient deficiency, a complex reprogramming of cell metabolism occurs,
in order to maintain viability. The strong requirement of Fe and S for the biosyn-
thesis of Fe-S clusters in the organelles might constitute a feedback signal for the
co-regulation of the assimilation pathways. Indeed the existence of such signals has
been recently proposed for Fe and S metabolism (Vigani et al. 2013; Chan
et al. 2013). The plant responses triggered by Fe or S deficiency have been well
characterised. The consequences of the combined shortage of these nutrients
however have only seldom been investigated, and might impact particularly on
Fe-S cluster assembly. Significant interactions between Fe uptake mechanisms and
external sulfate supply have been reported. It has been shown that S deficiency
limits the capacity to cope with Fe shortage in tomato plants, preventing the
expression of Fe chelate reductase FRO1 and reducing the activity of Fe2+ trans-
porter (Zuchi et al. 2009). Other studies using barley plants reported a positive
correlation between S supply in the growth media and the plant capability of coping
with Fe deficiency. Indeed phytosiderophores represent another important junction
between Fe and S metabolism, as they are derived from the S-containing amino acid
methionine. The release rate of phytosiderophores was diminished in barley plants
upon sulfate deficiency, due to a decrease of the methionine level. After sulfate
resupply, plants increased the release of phytosiderophores when exposed to Fe
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 147

shortage (Astolfi et al. 2010, 2012). Recently the effect of Fe deficiency on sulfur
metabolism has been analyzed in durum wheat (Ciaffi et al. 2013). Wheat plants
grown under sufficient S supply showed an up-regulation of certain S deficiency
responses when exposed to Fe deficiency. The expression of the high affinity sulfate
transporters was increased in the root, as well as of several genes of the S metabolic
pathway.
Recently we found that also in Arabidopsis thaliana the expression of the two
key genes for the uptake of Fe (IRT1) and of S (SULTR1;1) correlates with the
supply of both Fe and S in the growth media. The expression is differentially
regulated in case of double nutrient shortage (Forieri et al. 2013). We suggested
that Fe-S cluster availability might function in sensing and signalling of combined
Fe and S deficiencies. Altogether, these analyses strongly support the existence of a
co-regulation between the metabolic pathways, as the limitation of one nutrient
influences the uptake of the other one. Such a co-regulation is very likely to be the
outcome of a complex remodelling of the whole plant metabolism upon nutrient
limitation as known for the prolonged deficiency of the single nutrients (Schuler
et al. 2011; Nikiforova et al. 2003). Hence, we propose different signals that might
contribute to this co-regulation, such as the sensing of the Fe and S concentrations
in the root rhizosphere or within root cells, ROS, metabolism intermediates, Fe-S
cluster assembly machineries or Fe-S proteins.

Conclusions
Fe is one of the most fascinating elements for life functions due to its redox
properties and is of great importance for human nutrition. Crop plants are the
direct or indirect source of Fe in our food, and research on the dicot model
plant Arabidopsis thaliana and also on rice has provided tremendous
advances in our understanding of plant Fe homeostasis in the past years. In
particular, the primary uptake processes into the root are now based on
molecular evidence for the genes involved in Strategy I (reduction based)
and Strategy II (chelation based). The allocation of Fe from the rhizodermis
to xylem and phloem for supply of young and growing tissues and
recirculation to roots is, however, much less well understood. Finally, trans-
port processes inside cells are beginning to be unraveled, explaining how iron
homeostasis is mediated between cytosol, plastids, mitochondria and the
vacuole. The key genes involved in these processes also represent possible
candidates in the search for Fe use efficient plants.
A crucial process in homeostatic control is the chelation of the almost
insoluble and redox active free Fe ions. Fe chelated by nicotianamine is
carried across plasmalemma and endomembranes and also for long distance
transport. Future work needs to address the mechanisms of donation of Fe
from chelators to acceptor molecules such as heme, Fe-S clusters and pro-
teins. In addition, the regulatory networks of transcription factors that

(continued)
148 I. Forieri and R. Hell

function in the sensing of Fe deficiency, adaptation of root morphology and

coordination with uptake of other nutrients are only beginning to be discov-
ered. Recent observations indicate a co-regulation of Fe homeostasis with the
uptake and metabolism of S, most likely triggered by the demand for Fe-S
clusters in the electron transport chains of plastids and mitochondria. Detailed
understanding of Fe homeostasis is prerequisite for the generation of Fe
efficient plants and enhanced Fe contents in food.

References

Astolfi S, Zuchi S, Hubberten H-M, Pinton R, Hoefgen R (2010) Supply of sulphur to S-deficient
young barley seedlings restores their capability to cope with iron shortage. J Exp Bot
61:799–806
Astolfi S, Zuchi S, Neumann G, Cesco S, Di Toppi LS, Pinton R (2012) Response of barley plants
to Fe deficiency and Cd contamination as affected by S starvation. J Exp Bot 63:1241–1250
Balk J, Pilon M (2011) Ancient and essential: the assembly of iron–sulfur clusters in plants. Trends
Plant Sci 16:218–226
Barberon M, Zelazny E, Robert S, Conejero G, Curie C, Friml J, Vert G (2011) Monoubiquitin-
dependent endocytosis of the iron-regulated transporter 1 (IRT1) transporter controls iron
uptake in plants. Proc Natl Acad Sci U S A 108:E450–E458
Bashir K, Ishimaru Y, Shimo H, Nagasaka S, Fujimoto M, Takanashi H, Tsutsumi N, An G,
Nakanishi H, Nishizawa NK (2011) The rice mitochondrial iron transporter is essential for
plant growth. Nat Commun 2:322
Bauer P, Ling HQ, Guerinot ML (2007) FIT, the FER-like iron deficiency induced transcription
factor in Arabidopsis. Plant Physiol Biochem 45:260–261
Bernard DG, Cheng Y, Zhao Y, Balk J (2009) An allelic mutant series of ATM3 reveals its key
role in the biogenesis of cytosolic iron-sulfur proteins in Arabidopsis. Plant Physiol
151:590–602
Briat JF, Duc C, Ravet K, Gaymard F (2010) Ferritins and iron storage in plants. Biochim Biophys
Acta 1800:806–814
Busi MV, Maliandi MV, Valdez H, Clemente M, Zabaleta EJ, Araya A, Gomez-Casati DF (2006)
Deficiency of Arabidopsis thaliana frataxin alters activity of mitochondrial Fe-S proteins and
induces oxidative stress. Plant J 48:873–882
Cailliatte R, Schikora A, Briat JF, Mari S, Curie C (2010) High-affinity manganese uptake by the
metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions.
Plant Cell 22:904–917
Cakmak I, Erenoglu B, Gülüt K, Derici R, Römheld V (1998) Light-mediated release of
phytosiderophores in wheat and barley under iron or zinc deficiency. Plant Soil 202:309–315
Chan KX, Wirtz M, Phua SY, Estavillo GM, Pogson BJ (2013) Balancing metabolites in drought:
the sulfur assimilation conundrum. Trends Plant Sci 18:18–29
Chu HH, Chiecko J, Punshon T, Lanzirotti A, Lahner B, Salt DE, Walker EL (2010) Successful
reproduction requires the function of Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-
Like3 metal-nicotianamine transporters in both vegetative and reproductive structures. Plant
Physiol 154:197–210
Chu H-H, Conte SS, Chan Rodriguez D, Vasques K, Punshon T, Salt DE, Walker EL (2013)
Arabidopsis thaliana Yellow Stripe1-Like4 and Yellow Stripe1-Like6 localize to internal
cellular membranes and are involved in metal ion homeostasis. Front Plant Sci 4. doi:10.
3389/fpls.2013.00283
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 149

Ciaffi M, Paolacci AR, Celletti S, Catarcione G, Kopriva S, Astolfi S (2013) Transcriptional and
physiological changes in the S assimilation pathway due to single or combined S and Fe
deprivation in durum wheat (Triticum durum L.) seedlings. J Exp Bot 64:1663–1675
Cohen CK, Fox TC, Garvin DF, Kochian LV (1998) The role of iron-deficiency stress responses in
stimulating heavy-metal transport in plants. Plant Physiol 116:1063–1072
Colangelo EP, Guerinot ML (2004) The essential basic helix-loop-helix protein FIT1 is required
for the iron deficiency response. Plant Cell 16:3400–3412
Connolly EL, Fett JP, Guerinot ML (2002) Expression of the IRT1 metal transporter is controlled
by metals at the levels of transcript and protein accumulation. Plant Cell 14:1347–1357
Couturier J, Touraine B, Briat J-F, Gaymard F, Rouhier N (2013) The iron-sulfur cluster assembly
machineries in plants: current knowledge and open questions. Front Plant Sci 4. doi:10.3389/
fpls.2013.00259
Curie C, Alonso JM, Le Jean M, Ecker JR, Briat JF (2000) Involvement of NRAMP1 from
Arabidopsis thaliana in iron transport. Biochem J 347(Pt 3):749–755
Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF, Walker EL (2001) Maize yellow
stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409:346–349
Curie C, Cassin G, Couch D, Divol F, Higuchi K, Le Jean M, Misson J, Schikora A, Czernic P,
Mari S (2009) Metal movement within the plant: contribution of nicotianamine and yellow
stripe 1-like transporters. Ann Bot 103:1–11
Deinlein U, Weber M, Schmidt H, Rensch S, Trampczynska A, Hansen TH, Husted S, Schjoerring
JK, Talke IN, Kramer U, Clemens S (2012) Elevated nicotianamine levels in Arabidopsis
halleri roots play a key role in zinc hyperaccumulation. Plant Cell 24:708–723
Divol F, Couch D, Conejero G, Roschzttardtz H, Mari S, Curie C (2013) The Arabidopsis Yellow
Stripe LIKE4 and 6 transporters control iron release from the chloroplast. Plant Cell
25:1040–1055
Duc C, Cellier F, Lobreaux S, Briat JF, Gaymard F (2009) Regulation of iron homeostasis in
Arabidopsis thaliana by the clock regulator time for coffee. J Biol Chem 284:36271–36281
Durrett TP, Gassmann W, Rogers EE (2007) The FRD3-mediated efflux of citrate into the root
vasculature is necessary for efficient iron translocation. Plant Physiol 144:197–205
Duy D, Wanner G, Meda AR, Von Wiren N, Soll J, Philippar K (2007) PIC1, an ancient permease
in Arabidopsis chloroplasts, mediates iron transport. Plant Cell 19:986–1006
Eckhardt U, Mas Marques A, Buckhout TJ (2001) Two iron-regulated cation transporters from
tomato complement metal uptake-deficient yeast mutants. Plant Mol Biol 45:437–448
Forieri I, Wirtz M, Hell R (2013) Towards new perspectives on the interaction of iron and sulfur
metabolism in plants. Front Plant Sci 4. doi:10.3389/fpls.2013.00357
Haydon MJ, Kawachi M, Wirtz M, Hillmer S, Hell R, Kramer U (2012) Vacuolar nicotianamine
has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis.
Plant Cell 24:724–737
Hell R, Stephan U (2003) Iron uptake, trafficking and homeostasis in plants. Planta 216:541–551
Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M, Nakanishi H,
Mori S, Nishizawa NK (2009) Rice OsYSL15 is an iron-regulated iron (III)-deoxymugineic
acid transporter expressed in the roots and is essential for iron uptake in early growth of the
seedlings. J Biol Chem 284:3470–3479
Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S,
Matsuhashi S, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2006) Rice plants take up
iron as an Fe3+-phytosiderophore and as Fe2+. Plant J 45:335–346
Jeong J, Cohu C, Kerkeb L, Pilon M, Connolly EL, Guerinot ML (2008) Chloroplast Fe(III)
chelate reductase activity is essential for seedling viability under iron limiting conditions. Proc
Natl Acad Sci U S A 105:10619–10624
Kerkeb L, Mukherjee I, Chatterjee I, Lahner B, Salt DE, Connolly EL (2008) Iron-induced
turnover of the Arabidopsis IRON-REGULATED TRANSPORTER1 metal transporter
requires lysine residues. Plant Physiol 146:1964–1973
150 I. Forieri and R. Hell

Kim SA, Punshon T, Lanzirotti A, Li L, Alonso JM, Ecker JR, Kaplan J, Guerinot ML (2006)
Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1.
Science 314:1295–1298
Klatte M, Schuler M, Wirtz M, Fink-Straube C, Hell R, Bauer P (2009) The analysis of
Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron
loading and iron deficiency responses. Plant Physiol 150:257–271
Kobayashi T, Nishizawa NK (2012) Iron uptake, translocation and regulation in higher plants.
Annu Rev Plant Biol 63:131–152
Kobayashi T, Nakayama Y, Itai RN, Nakanishi H, Yoshihara T, Mori S, Nishizawa NK (2003)
Identification of novel cis-acting elements, IDE1 and IDE2, of the barley IDS2 gene promoter
conferring iron-deficiency-inducible, root-specific expression in heterogeneous tobacco plants.
Plant J 36:780–793
Kobayashi T, Ogo Y, Itai RN, Nakanishi H, Takahashi M, Mori S, Nishizawa NK (2007) The
transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants.
Proc Natl Acad Sci U S A 104:19150–19155
Kobayashi T, Itai RN, Ogo Y, Kakei Y, Nakanishi H, Takahashi M, Nishizawa NK (2009) The rice
transcription factor IDEF1 is essential for the early response to iron deficiency and induces
vegetative expression of late embryogenesis abundant genes. Plant J 60:948–961
Kobayashi T, Ogo Y, Aung MS, Nozoye T, Itai RN, Nakanishi H, Yamakawa T, Nishizawa NK
(2010) The spatial expression and regulation of transcription factors IDEF1 and IDEF2. Ann
Bot 105:1109–1117
Kobayashi T, Itai RN, Aung MS, Senoura T, Nakanishi H, Nishizawa NK (2012) The rice
transcription factor IDEF1 directly binds to iron and other divalent metals for sensing cellular
iron status. Plant J 69:81–91
Kushnir S, Babiychuk E, Storozhenko S, Davey MW, Papenbrock J, De Rycke R, Engler G,
Stephan UW, Lange H, Kispal G, Lill R, Van Montagu M (2001) A mutation of the mitochon-
drial ABC transporter Sta1 leads to dwarfism and chlorosis in the Arabidopsis mutant starik.
Plant Cell 13:89–100
Lanquar V, Lelievre F, Bolte S, Hames C, Alcon C, Neumann D, Vansuyt G, Curie C, Schroder A,
Kramer U, Barbier-Brygoo H, Thomine S (2005) Mobilization of vacuolar iron by AtNRAMP3
and AtNRAMP4 is essential for seed germination on low iron. EMBO J 24:4041–4051
Lill R (2009) Function and biogenesis of iron-sulphur proteins. Nature 460:831–838
Lin YF, Liang HM, Yang SY, Boch A, Clemens S, Chen CC, Wu JF, Huang JL, Yeh KC (2009)
Arabidopsis IRT3 is a zinc-regulated and plasma membrane localized zinc/iron transporter.
New Phytol 182:392–404
Ling HQ, Koch G, Baumlein H, Ganal MW (1999) Map-based cloning of chloronerva, a gene
involved in iron uptake of higher plants encoding nicotianamine synthase. Proc Natl Acad Sci
U S A 96:7098–7103
Ling HQ, Bauer P, Bereczky Z, Keller B, Ganal M (2002) The tomato fer gene encoding a bHLH
protein controls iron-uptake responses in roots. Proc Natl Acad Sci U S A 99:13938–13943
Lingam S, Mohrbacher J, Brumbarova T, Potuschak T, Fink-Straube C, Blondet E, Genschik P,
Bauer P (2011) Interaction between the bHLH transcription factor FIT and ETHYLENE
INSENSITIVE3/ETHYLENE INSENSITIVE3-LIKE1 reveals molecular linkage between
the regulation of iron acquisition and ethylene signaling in Arabidopsis. Plant Cell
23:1815–1829
Liu Z, Hong SW, Escobar M, Vierling E, Mitchell DL, Mount DW, Hall JD (2003) Arabidopsis
UVH6, a homolog of human XPD and yeast RAD3 DNA repair genes, functions in DNA repair
and is essential for plant growth. Plant Physiol 132:1405–1414
Long TA, Tsukagoshi H, Busch W, Lahner B, Salt DE, Benfey PN (2010) The bHLH transcription
factor POPEYE regulates response to iron deficiency in Arabidopsis roots. Plant Cell
22:2219–2236
Marschner H (1995) Mineral nutrition of higher plants. Academic/Harcourt Brace & Company,
London, pp 313–324
5 Micronutrient Use Efficiency – Cell Biology of Iron and Its Metabolic. . . 151

Mäser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN, Amtmann A, Maathuis
FJ, Sanders D, Harper JF, Tchieu J, Gribskov M, Persans MW, Salt DE, Kim SA, Guerinot ML
(2001) Phylogenetic relationships within cation transporter families of Arabidopsis. Plant
Physiol 126:1646–1667
Morrissey J, Guerinot ML (2009) Iron uptake and transport in plants: the good, the bad, and the
ionome. Chem Rev 109:4553–4567
Morrissey J, Baxter IR, Lee J, Li L, Lahner B, Grotz N, Kaplan J, Salt DE, Guerinot ML (2009)
The ferroportin metal efflux proteins function in iron and cobalt homeostasis in Arabidopsis.
Plant Cell 21:3326–3338
Nakanishi H, Yamaguchi H, Sasakuma T, Nishizawa NK, Mori S (2000) Two dioxygenase genes,
Ids3 and Ids2, from Hordeum vulgare are involved in the biosynthesis of mugineic acid family
phytosiderophores. Plant Mol Biol 44:199–207
Nikiforova V, Freitag J, Kempa S, Adamik M, Hesse H, Hoefgen R (2003) Transcriptome analysis
of sulfur depletion in Arabidopsis thaliana: interlacing of biosynthetic pathways provides
response specificity. Plant J 33:633–650
Nozoye T, Nagasaka S, Kobayashi T, Takahashi M, Sato Y, Sato Y, Uozumi N, Nakanishi H,
Nishizawa NK (2011) Phytosiderophore efflux transporters are crucial for iron acquisition in
graminaceous plants. J Biol Chem 286:5446–5454
Ogo Y, Itai RN, Nakanishi H, Inoue H, Kobayashi T, Suzuki M, Takahashi M, Mori S, Nishizawa
NK (2006) Isolation and characterization of IRO2, a novel iron-regulated bHLH transcription
factor in graminaceous plants. J Exp Bot 57:2867–2878
Ogo Y, Kobayashi T, Nakanishi Itai R, Nakanishi H, Kakei Y, Takahashi M, Toki S, Mori S,
Nishizawa NK (2008) A novel NAC transcription factor, IDEF2, that recognizes the iron
deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants. J
Biol Chem 283:13407–13417
Ogo Y, Itai RN, Kobayashi T, Aung MS, Nakanishi H, Nishizawa NK (2011) OsIRO2 is
responsible for iron utilization in rice and improves growth and yield in calcareous soil.
Plant Mol Biol 75:593–605
Palmgren MG (2001) Plant plasma membrane H+-ATPases: powerhouses for nutrient uptake.
Annu Rev Plant Physiol Plant Mol Biol 52:817–845
Petit JM, Van Wuytswinkel O, Briat JF, Lobreaux S (2001) Characterization of an iron-dependent
regulatory sequence involved in the transcriptional control of AtFer1 and ZmFer1 plant ferritin
genes by iron. J Biol Chem 276:5584–5590
Ravet K, Touraine B, Boucherez J, Briat JF, Gaymard F, Cellier F (2009) Ferritins control
interaction between iron homeostasis and oxidative stress in Arabidopsis. Plant J 57:400–412
Rellan-Alvarez R, Giner-Martinez-Sierra J, Orduna J, Orera I, Rodriguez-Castrillon JA, Garcia-
Alonso JI, Abadia J, Alvarez-Fernandez A (2010) Identification of a tri-iron (III), tri-citrate
complex in the xylem sap of iron-deficient tomato resupplied with iron: new insights into plant
iron long-distance transport. Plant Cell Physiol 51:91–102
Robinson NJ, Procter CM, Connolly EL, Guerinot ML (1999) A ferric-chelate reductase for iron
uptake from soils. Nature 397:694–697
Rogers EE, Guerinot ML (2002) FRD3, a member of the multidrug and toxin efflux family,
controls iron deficiency responses in Arabidopsis. Plant Cell 14:1787–1799
Romheld V, Marschner H (1986) Evidence for a specific uptake system for iron phytosiderophores
in roots of grasses. Plant Physiol 80:175–180
Santi S, Schmidt W (2009) Dissecting iron deficiency-induced proton extrusion in Arabidopsis
roots. New Phytol 183:1072–1084
Schuler M, Keller A, Backes C, Philippar K, Lenhof H-P, Bauer P (2011) Transcriptome analysis
by GeneTrail revealed regulation of functional categories in response to alterations of iron
homeostasis in Arabidopsis thaliana. BMC Plant Biol 11:87
Schuler M, Rellan-Alvarez R, Fink-Straube C, Abadia J, Bauer P (2012) Nicotianamine functions
in the phloem-based transport of iron to sink organs, in pollen development and pollen tube
growth in Arabidopsis. Plant Cell 24:2380–2400
152 I. Forieri and R. Hell

Seckback J (1982) Ferreting out the secrets of plant ferritin
a review. J Plant Nutr 5:369–394
Shin LJ, Lo JC, Chen GH, Callis J, Fu H, Yeh KC (2013) IRT1 DEGRADATION FACTOR1, a
RING E3 ubiquitin ligase, regulates the degradation of IRON-REGULATED TRANS-
PORTER1 in Arabidopsis. Plant Cell 25:3039–3051
Sivitz AB, Hermand V, Curie C, Vert G (2012) Arabidopsis bHLH100 and bHLH101 control iron
homeostasis via a FIT-independent pathway. PLoS One 7:e44843
Tarantino D, Petit JM, Lobreaux S, Briat JF, Soave C, Murgia I (2003) Differential involvement of
the IDRS cis-element in the developmental and environmental regulation of the AtFer1 ferritin
gene from Arabidopsis. Planta 217:709–716
Tarantino D, Santo N, Morandini P, Casagrande F, Braun H-P, Heinemeyer J, Vigani G, Soave C,
Murgia I (2010) AtFer4 ferritin is a determinant of iron homeostasis in Arabidopsis thaliana
heterotrophic cells. J Plant Physiol 167:1598–1605
Teng YS, Su YS, Chen LJ, Lee YJ, Hwang I, Li HM (2006) Tic21 is an essential translocon
component for protein translocation across the chloroplast inner envelope membrane. Plant
Cell 18:2247–2257
Varotto C, Maiwald D, Pesaresi P, Jahns P, Salamini F, Leister D (2002) The metal ion transporter
IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana.
Plant J 31:589–599
Vert G, Briat JF, Curie C (2001) Arabidopsis IRT2 gene encodes a root-periphery iron transporter.
Plant J 26:181–189
Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML, Briat JF, Curie C (2002) IRT1, an
Arabidopsis transporter essential for iron uptake from the soil and for plant growth. Plant Cell
14:1223–1233
Vert GA, Briat JF, Curie C (2003) Dual regulation of the Arabidopsis high-affinity root iron uptake
system by local and long-distance signals. Plant Physiol 132:796–804
Vigani G, Zocchi G, Bashir K, Philippar K, Briat JF (2013) Signals from chloroplasts and
mitochondria for iron homeostasis regulation. Trends Plant Sci 18:305–311
Von Wiren N, Mori S, Marschner H, Romheld V (1994) Iron inefficiency in maize mutant ys1 (Zea
mays L. cv Yellow-Stripe) is caused by a defect in uptake of iron phytosiderophores. Plant
Physiol 106:71–77
Wang HY, Klatte M, Jakoby M, Baumlein H, Weisshaar B, Bauer P (2007) Iron deficiency-
mediated stress regulation of four subgroup Ib BHLH genes in Arabidopsis thaliana. Planta
226:897–908
Waters BM, Blevins DG, Eide DJ (2002) Characterization of FRO1, a pea ferric-chelate reductase
involved in root iron acquisition. Plant Physiol 129:85–94
Yi Y, Guerinot ML (1996) Genetic evidence that induction of root Fe(III) chelate reductase
activity is necessary for iron uptake under iron deficiency. Plant J 10:835–844
Yuan Y, Wu H, Wang N, Li J, Zhao W, Du J, Wang D, Ling HQ (2008) FIT interacts with
AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in
Arabidopsis. Cell Res 18:385–397
Zancani M, Peresson C, Biroccio A, Federici G, Urbani A, Murgia I, Soave C, Micali F,
Vianello A, Macrı̀ F (2004) Evidence for the presence of ferritin in plant mitochondria. Eur J
Biochem 271:3657–3664
Zheng L, Ying Y, Wang L, Wang F, Whelan J, Shou H (2010) Identification of a novel iron
regulated basic helix-loop-helix protein involved in Fe homeostasis in Oryza sativa. BMC
Plant Biol 10:166
Zuchi S, Cesco S, Varanini Z, Pinton R, Astolfi S (2009) Sulphur deprivation limits Fe-deficiency
responses in tomato plants. Planta 230:85–94
Chapter 6
Boron: A Promising Nutrient for Increasing
Growth and Yield of Plants

Himanshu Bariya, Snehal Bagtharia, and Ashish Patel

Abstract Boron (B) is a vital nutrient for plant growth and metabolism. Lack of B
in plant tissues causes reductions in crop yields, whilst an excess supply of B may
also seriously damage plant tissues and sometimes leads to plant death. Appropriate
amounts of B in plants are crucial for normal growth and it significantly increases
seed germination and seedling growth. Moreover B has positive effects on the
uptake and utilization of other nutrients at the whole plant level and it may improve
nutrient use efficiency (NUE) and nutrient demand and supply (NDS). NUE mainly
reflects efficiency of extraction of mineral nutrients from soil along with their
integration and recycling, whereas NDS nutrient shows how efficiently plant can
fulfil the demand and supply rate of required nutrient at different stages and
conditions of plant life cycle. Despite a substantial existing literature, the under-
standing of B interactions with other nutrients remains unclear.

Keywords Boron • NUE (nitrogen use efficiency) • Soil interactions • Deficiency

• Reproductive growth • Photosynthesis • Nutrient interactions

Introduction: Boron in the Soil and in the Plant

Boron belongs to the metalloid elements having properties of both metals and
non-metals (Marschner 1995). There is very low abundance of B in nature (Kot
2009) but it is broadly distributed in all the layers of the soil. B abundance range in
rocks averages about 10–20 mg B kg
1. In sea water it can range from 1 to 10 mg B
kg
1 and as far as river is concern the B concentration is about 1/350 that of sea
water (Power and Woods 1997).
Warington (1923) established the requirement of B for plant growth and func-
tioning, and many recent reports suggest an essentiality of B for all vascular plants.

H. Bariya • A. Patel (*)

Department of Biotechnology, Hemchandra North Gujarat University,
Patan 384265, Gujarat, India
e-mail: [email protected]
S. Bagtharia
Gujarat State Biotechnology Mission (GSBTM), DST – Government of Gujarat,
Gandhinagar 382017, Gujarat, India

© Springer International Publishing Switzerland 2014 153

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_6
154 H. Bariya et al.

B deficiency or toxicity affects various metabolic and physiological processes

(Blevins and Lukaszewski 1998; Bolaños et al. 2004). Less than 10 mg kg
1 B in
the soil is considered to be a B deficient soil (Woods 1994). Moreover, most of this
B is in a bound form in rocks and is not readily available to plants. Boric acid is the
most common form of B liberated during weathering of rocks (Nable et al. 1997)
and is easily absorbed by plant roots, but this represents only 10 % of the total B in
the soil (Power and Woods 1997).
Soil pH, texture, temperature, and organic matter affect soil B availability;
among these the soil pH is one of the most significant characteristics for B
availability (Goldberg 1997). Boric acid is a very weak acid and when the pH is
below 7, it appears in its undissociated form; at alkaline pH, boric acid dissociates
to form the borate anion:

ðpKa 9:25Þ

Therefore, at common soil pH values (5.5–7.5), B exists mainly as soluble

uncharged boric acid (B(OH)3), and in this form B is absorbed by plant roots
(Hu and Brown 1997; Power and Woods 1997; Sathya et al. 2013). In flooded
conditions, B is easily solubilised and leached resulting in a deficiency of B for
growing plants.
A plant requirement for B varies between species and is dependent on the
conditions in which they grow. The B requirement for one plant species may be
toxic or deficient for other species (Blevins and Lukaszewski 1998). There are three
main groups of plants based on B requirements, one consists of graminaceous
species, a second are monocotyledons and a third are dicotyledons and lactifers,
having minimum, moderate and high B demands, respectively (Blevins and
Lukaszewski 1998; Goldbach et al. 2001). It is necessary to study mechanisms of
B uptake and transport, as well as translocation in plant systems, to optimise
agricultural production. For example, B deficiency results in major disorders,
which may decrease plant growth in soil with excess water. Molecular mechanisms
involved in B transport from the soil to root cells and xylem have two different
routes. Transport of B is dependent mainly on its availability and it is mostly
transported into the plant from the soil by passive diffusion, which is possible
when adequate B is available. When B availability is low or under deficiency
conditions, uptake is facilitated by active transport, which requires energy.
B absorbed by roots must be delivered to the xylem for further transport around
the plant. When there is sufficient B availability, absorbed B moves by passive
diffusion involving MIPs channels (Dannel et al. 2002; Miwa and Fujiwara 2010).
In limited or deficient B conditions, transport of B towards the xylem is facilitated
via a specific B transporter (BOR), which is an energy dependent transport process
(Fig. 6.1). Takano et al. (2002) had identified such a transporter of B under limiting
conditions, known as BOR1 in Arabidopsis. Subsequently, BOR1-like genes have
been reported in Eucalyptus (Domingues et al. 2005) and in rice (Nakagawa
et al. 2007). Expression of two genes, NIP5;1 and BOR1, are decreased by
transcriptional and post-translational regulation respectively, under sufficient B
supply (Miwa and Fujiwara 2010; Yang et al. 2013).
6 Boron: A Promising Nutrient for Increasing Growth and Yield of Plants 155

Fig. 6.1 Schematic diagram of boron transport from soil to xylem via root tissue. In sufficient B
condition NIP5;1 imports B from the soil to epidermal, cortex and endodermal cells, and BOR
1 exports B from stelar cell (xylem loading)

After loading in the xylem, B is transported to the shoot through the transpiration
stream (Wimmer and Eichert 2013; Bogiani et al. 2014). Transport of B through
phloem was also reported, and such transport differs between species (Brown and
Hu 1996; Brown and Shelp 1997; Ganie et al. 2013). In certain plants, B is
transported and translocated to reproductive and vegetative tissue via the phloem
(Matoh and Ochiai 2005). It is also suggested that there is a formation of a boron-
diol complex involving a sugar alcohol, which acts as the transport molecule
(Brown and Hu 1996; Hu et al. 1997). Transgenic tobacco and rice with enhanced
sorbitol production had a higher ability to transport B through the phloem towards
the plant shoot (Brown et al. 1999; Bellaloui et al. 2003). Plants that produce sugar
alcohols like sorbitol and trehalose, have the ability of B transport via the phloem,
whereas plants without any production of sugar alcohols have no such transport
system (Stangoulis et al. 2001; Takano et al. 2001; Matoh and Ochiai 2005).

Role of Boron in Plant Functioning

Since the beginning of twentieth century, B has been considered to be an important

micronutrient for plant growth, but there are very few records regarding its actual
biochemical role. Deficiency of B is common all over the world, which has an
156 H. Bariya et al.

important agronomic impact (Gupta 1979). In soils with high percolatory water,
B is easily leached downward in the soil and hence it is not readily available for
plants (Blevins and Lukaszewski 1998). Adequate B nutrition is required for high
production as well as for quality of crops. B deficiency results in biochemical,
metabolic and physiological abnormalities, and causes diverse disease symptoms in
plants and hence adequate supply is a critical challenge for plant nutrition.
In the last 10 years, several roles have been demonstrated for B in plant function,
including as a cell wall component, an involvement in membrane structure and
integrity, and involvement in metabolic process (Bolaños et al. 2004). To date, one
of the most accepted roles of B in plant physiological function is the formation of an
ester with one apiose residue of rhamnogalacturonan ІІ (RG II) in the cell wall
(Kobayashi et al. 1996), which is essential for maintaining cell wall permeability
and also for rigidity (Fleischer et al. 1999; Ryden et al. 2003). Moreover, B
deficiency led to a decrease of gene transcription of various hydrolytic enzymes
such as xyloglucan endotransglucosylase/hydrolases (XTHs), expansins, pectin
methylesterases, and pectin lyases in Arabidopsis roots (Camacho-Cristóbal
et al. 2002). These enzymes play key roles in cell wall loosening, necessary for
cell elongation (Cosgrove 1999). Camacho-Cristobal et al. (2011) suggested an
influence of B in transcriptional level regulation of genes, which are responsible for
cell wall synthesis and its modification.
Many research reports suggested roles of B in plasma membrane transport
processes, and in membrane integrity by cross-linking the membrane molecules
containing hydroxlated ligands such as glycoproteins and glycolipids (Goldbach
et al. 2001; Wimmer et al. 2009). The membrane potential in Daucus carota is
changed under limited B (Blaser-Grill et al. 1989) and activity of the proton-
pumping ATPase was reduced in Helianthus annuus roots (Ferrol and Donaire
1992). Similarly, it has been reported that B deficiency alters plasma membrane
permeability for ions and other solutes (Wang et al. 1999; Carmen Rodriguez
Hernandez et al. 2013). The impact of B on ion fluxes can be mediated by direct
or indirect effects of B on plasma membrane-bound proton-pumping ATPase
(Cara et al. 2002). The activity of the K+ stimulated ATPase in B-deficient maize
roots was considerably lower than in control plants (Pollard et al. 1977). These
results indicated that the action of B could be associated with membrane com-
ponents. It is still unclear whether B directly interacts with membrane proteins or
indirectly modifies membrane properties with subsequent changes in enzymatic
activities.
The literature indicates possible roles of B in several other metabolic func-
tions. For instance, it has been shown that B deficiency causes qualitative and
quantitative changes in phenol metabolism (Pandey and Archana 2012; Hajiboland
et al. 2013). Additionally B deficiency affects nitrogen metabolism (Bolaños
et al. 1994). B-deficient plants showed lower nitrate reductase activity and enhanced
amounts of nitrate; these observations strongly suggest a role of B in the de
novo synthesis of the nitrate reductase protein or facilitation of nitrate absorption
(Ruiz et al. 1998).
6 Boron: A Promising Nutrient for Increasing Growth and Yield of Plants 157

Effect of B on Photosynthesis

Photosynthesis is a complex series of reactions that culminate in the reduction of

carbon dioxide. The effect of B nutrition on photosynthetic processes has been
rarely studied. Decreases in B supply reduce soluble proteins and chlorophyll in
leaves, which are important constituents for Hill reactions and photosynthesis
(Mukhopdhyaya et al. 2013).
There was no direct involvement of B on rate of photosynthesis, but from recent
research it is evident that B has a positive effect on photosynthesis under normal
(optimum) level. Moreover, there are reports of increases in chlorophyll pigment
and carotenoids by foliar spraying of B leading to increases in photosynthetic rate
(Thurzo et al. 2010). Ganie et al. (2013) also showed that net photosynthetic rate
was increased due to increases in plant light harvesting pigments such as chloro-
phyll and carotene in the leaves.
B deficiency negatively affects photosynthesis by decreasing photosynthetic
oxygen evolution rates and hence the efficiency of photosystem II (Kastori
et al. 1995; El-Shintinawy 1999). Photosynthetic rate (Pn) drastically decreased in
cotton plants when grown in B deficient soil (Zhao and Oosterhuis 2002). It was
reported that inhibition of photosynthesis was a result of reduced Hill reactions and
low intercellular CO2 concentrations (Sharma and Ramchandra 1990). Some exper-
imental evidence indicated close relationships between gas exchange parameters
and B deficiency, implying that these parameters were possibly affected by external
B supply and in turn prejudiced growth. Previous studies, under B deficiency,
supported a change in photosynthetic enzyme activities that were undoubtedly
involved in a decrease in Pn (Sharma and Ramchandra 1990). B deficiency was
followed by reduction in the leaf stomatal conductance (gs) and rate of photosyn-
thesis (Huang et al. 2005; Han et al. 2008). Related declines in plant gs at B
deficiency were initiated by high oxidative damage in leaves (Huang et al. 2005).
Moreover, low gs also decreases E (Han et al. 2008). It was advocated that the
presence of free hexoses can elicit regulation of the Calvin cycle and, hence, can
obstruct Pn (Han et al. 2008). A diminution in pigments under B deficiency was
shown for citrus (Han et al. 2008). The synthesis of unnecessary starch possibly
disrupts chloroplast structure, leading to poorer CO2 assimilation and decreased
chlorophyll content (Han et al. 2008). It is likely that B deficiency caused changes
in chloroplast structure, which eventually affected pigment content (Pandey and
Pandey 2008). B deficiency may also affect photosynthetic responses by the
modifications in the structure and function of chloroplast thylakoids.

Effect on Reproductive Growth

According to Loomis and Durst (1992) B is essential for generative growth. Boron
is involved in metabolism of carbohydrates and phenolic acids, which are crucial
158 H. Bariya et al.

for growth of pollen tubes. Decreases in crop propagative yield or seed/fruit quality
in low B soils can be due to diminished reproductive development early or late in
the flowering/fruiting cycle. It has often been seen that reproductive growth, mainly
flowering, fruit and seed set and seed yield, is particularly sensitive to B deficiency
compared to asexual growth (e.g. Woodbridge et al. 1971; Dear and Lipsett 1987;
Noppakoonwong et al. 1997). Likewise, substantial yield decreases can arise
without expression of indications of deficiency during prior somatic growth. B
also plays an important role in synthesis and metabolism of nucleic acids (Hundt
et al. 1970).
As described above, B is responsible for enhancing chlorophyll content and rate
of photosynthesis (Pn), as well as inducing dry matter production in plants, and
therefore may result both in enhancing flowering and also the transport of photo-
synthetic products to reproductive stages, ultimately leading to yield improvement
(Du Ying Qiong et al. 1999).
Factors influencing the impact of a low external B supply on sexual reproduc-
tion in flowering plants are likely to include: the capacity of roots to obtain B
from soil (Hu and Brown 1997); the mobility of B in the phloem (Brown and
Shelp 1997); the relative sink size in floral parts for photosynthate; the capacity to
redistribute B from vegetative tissues to reproductive organs; the rate of transpi-
ration by floral organs; the functional necessity for B in reproductive tissues;
and the distribution and richness of B-binding compounds in the apoplastic
pathway between the vein endings and the most distal floral tissue. The B
requirement for flowering is indicated by the sensitivity of pollen development
to low B and the generally high concentrations of B that occur in reproductive
parts of the flower. Under conditions of low external B supply, levels in
the anthers and pistils do not decline to the low levels measured in leaves.
B concentrations are higher in the stamen than in the pistil. The physiological
roles for B in sexual reproduction have yet to be fully defined and there is a need
for experimentation in this area.
Many of the studies that have been undertaken do not give definitive informa-
tion as to whether plants were critically deficient in B at the time of flowering, or
the B status of floral tissues was not determined at the time of impairment in
cellular development/function, or cell structure and metabolism were examined
long after the primary effect of B took place. An example is the observation that B
deficiency results in male sterility, a condition that can be induced by deficiencies
in other nutrients (e.g. Mn, Cu) or unfavourable environmental conditions
(e.g. water deficit, high/low temperature). These observations reveal nothing
about the processes that are being affected by low B supply and do not enable
us to conclude whether the effect of low B supply on male sterility is a direct or
indirect event. As root function is greatly impaired in severely B-deficient plants
in containers and this can impact on whole plant physiology, the requirement of
pollen development for B should be studied under conditions of controlled
external supply.
6 Boron: A Promising Nutrient for Increasing Growth and Yield of Plants 159

Flowering Response

There is no evidence that B deficiency prevents initiation of or delays floral develop-

ment. When plants are grown without B or are transferred into B-free nutrient solutions
in pot experiments, the apical meristems may abort and therefore flowers do not
develop, as shown for peach (Prunus persica; Kamali and Childers 1970). However,
under field conditions, where B deficiency stresses occur more gradually, plants may
have time to adapt to deficiency. For example, in peanut (Arachis hypogaea), the
flowering period was extended in B-deficient plants (Harris and Brolman 1966),
resulting in low-B plants producing as many flowers as B-adequate plants. In species
where the flowers occur in compact inflorescences, and these are terminal on the stem
(e.g. sunflower, wheat), low B has a greater impact on reproduction because the plant
has less ability to modulate reproductive growth than species with axillary inflores-
cences and indeterminate growth. The first group of plants is more prone to pollen
sterility under low B than the latter group of plants. Experiments on wheat by Li
et al. (1978), suggested that absorbed B was transported from soil to floret and spikelet
organs, which resulted in accumulation of B in seeds. Finally, low B can result in plants
being functionally male sterile (e.g. wheat, Li et al. 1978; rice, Garg et al. 1979;
barley, Simojoki 1972), although cases of female sterility have been reported
(e.g. maize, Vaughan 1977; avocado, Coetzer and Robbertse 1987). The external B
supply does not appear to alter the frequency of unisexual flowers, although some
workers have attempted unsuccessfully to do this by applying B sprays (Singh 1994).

Effect of B on Nutrient Use Efficiency, Demand and Supply

Nutrient use efficiency (NUE) may be expressed as productivity of the plant per
content of applied nutrients. Enhancement of NUE is vital for improvement of crop
production in marginal lands with poor nutrient availability. NUE for plants is
reliant on the ability to efficiently take up nutrients from the soil, but also on
translocation, storage and usage within the plant, and on the environment (North
et al. 2009). NUE is largely dependent on nutrient availability in the soil or applied
medium. Nutrient demand and supply (NDS) is how efficiently the plant can fulfil
the demand and supply rate for required nutrients at different stages and conditions
of plant growth and development. Under limiting conditions of nutrients, plants
showed decreases in nutrient uptake compared to sufficient nutrient conditions
(preserving the nutrition for future demand).
Large variations in defining nutrient efficient plants and methods used in calcu-
lating nutrient use efficiency, makes it difficult to compare results of different
studies. The effort to measure yield response to an applied nutrient is further
confounded by other factors, such as variable soil fertility levels, climatic condi-
tions, crop rotations, and changes in production practices that affect nutrient use
efficiency (Stewart et al. 2005). In simple terms, efficiency is the ratio of output
160 H. Bariya et al.

Fig. 6.2 Mulder’s chart shows some of the interactions between plant nutrients. Interaction: A
decrease in availability to the plant of a nutrient by the action of another nutrient (see direction of
arrow). Stimulation: An increase in the need for a nutrient by the plant because of the increase in
the level of another nutrient (dotted line)

(economic yield) to input (fertilisers) for a process or complex system (Crop

Science Society of America 1992).
Plant nutrients rarely work in isolation. Interactions among nutrients are impor-
tant because a deficiency of one restricts the uptake and use of another. Figure 6.2
shows interactions of major plant nutrients with each other (Khan Towhid Osman
2013). Numerous studies have demonstrated that interactions between B and other
nutrients, primarily N, P and K, impact crop yields and nutrient efficiency.
For example, for experiments carried out in our research group on groundnut it was
evident that uptake of almost all macro and micronutrients by straw and seeds showed
a linear relationship ( p < 0.05) among the different nutrients, which were absorbed by
roots. B at a level of 1 kg ha
1 resulted in significant uptake of macro and
micronutrients from soil to seed and straw. Data strongly support improvement of
6 Boron: A Promising Nutrient for Increasing Growth and Yield of Plants 161

NUE as well as NDS in groundnut at 1.0 kg ha
1 B supply. Other levels of B treatment
did not significantly increase the uptake of nutrients in groundnut. The same trend of
nutrient uptake by plants at different levels of applied B has also been reported by
Nadia et al. (2006) and 200 ppm B sprayed on groundnut plant showed an increase in
uptake of N, P, K and Fe, Mn and Zn. The highest value of yield and yield components
were received from the plant treated with 200 ppm B (Nadia et al. 2006). Leaf
venation, xylem stream, and transpiration are the primarily factors involved in the
accumulation of B in leaves (Oertli and Richardson 1970; Shelp and Shattuck 1987).
These results are similar to the observations of McIlrath et al. (1960). It has been
suggested that selected nutrients in the soil have antagonist and/or synergistic effect
on the uptake of other nutrients by roots of developing plants (Malvi 2011). More-
over, B interactions, either synergistic and/or antagonistic, may affect plant nutrition
under both deficiencies as well as in toxic conditions (Tariq and Mott 2007).
Deficiency, sufficiency and toxicity of B may exert an effect on mineral nutrient
content of plants but such an interaction has not been well studied or reported. The
results of many reports in this direction are conflicting, which may be due to
different experimental systems with different crop plants and varieties (Lombin
and Bates 1982; Mozafar 1989). B is directly or indirectly involved in many
physiological and biochemical processes and may affect other plant nutrients
(Bolaños et al. 2004). Therefore, one might expect relationships between B and
other nutrient utilization to be very complex. Examples of effects of B on avail-
ability and uptake of plant nutrients other than B are described below.
Parks et al. (1944) were the first researchers who reported that with graded B
levels, the concentrations of NH4-N, NO3-N, Org-N, P, K, Ca, Mg, Na, Zn, Cu, Fe,
Mn, Mo and B were altered in the tomato leaflets as much as several-fold. In
addition, they stated that B supply had specific effects, and the trends found were
completely dissimilar with respect to different elements. In the absence of B, the
concentrations of N, K, Ca, Mg, Na, Cu and Mn in tobacco leaves were increased
and the concentrations of P, Fe and Al were decreased as compared to plants fed
with a B adequate nutrient solution (Steinburg et al. 1955).
Baker and Cook (1959) reported that P, K and Mg were higher and Ca was lower
in severely B deficient alfalfa plants compared to healthy plants, perhaps due to the
dilution effect which occurred in the healthy plants. In increasing B conditions, the
concentration of Cu, Fe, Mn, Mo, and B were increasing in perennial fodder grass,
but the reverse trend occurred in the case of uptake and ash content for
micronutrients accept B (Mcllarth et al. 1960). Cu and K content in grass showed
highly significant positive correlations, while Ca and Mg contents showed negative
correlations with B contents for 98 grasses at the flowering stage when grown in
increasing B nutrition (Tolgyesi and Kozma 1974). Touchton and Boswell (1975)
observed that P, K, Ca, Mg, Na, Zn, Cu, Fe, Mn, Mo and Al concentrations varied
slightly with location, but were not affected by the method or rate of B application.
Only the B concentration in tissues was significantly increased with regard to
method rate and location. Increasing B nutrition enhanced phytotoxicity and
some interactions among nutrients due to increased concentrations of Zn, Cu, Fe
and Mn in the leaves, stem and roots of bush bean plants (Wallace et al. 1977). But
162 H. Bariya et al.

contradictory results were reported by Leece (1978), who observed that with high
levels of applied B, the concentrations of N, P, K, Ca, Zn, Cu, Fe and Mn (not Mg)
in maize crop were depressed. The reverse results were obtained when no B was
applied. Increasing B supply in soil resulted in the decrease of leaf N and P in
tomato, suggesting B antagonism. The contrary was the case with a B effect on
leaf K, Ca, Mg and Na (Aduayi 1978). Yadav and Manchanda (1979) noted that
with an increase in the B content of soil, Ca and Mg concentration in wheat and
Gramineae crops significantly decreased, whereas N, P and K contents were
significantly increased.
Moreover, with differential supply of B in nutrient solution, the concentration of
Fe, Mn, P and Ca in the shoots and roots of tomato increased, and B reduced the
translocation of Mn, P and Fe whilst Ca remained unchanged (Alvarez-Tinaut
et al. 1980). Addition of B in the nutrient solution decreased the absorption of N,
P Ca, Mg and B, induced K accumulation, while Na remained unaffected in lamina
stem and roots of Cabernet sauvignon wine plants (Downton and Hawker 1980).
Gomez-Rodriguez et al. (1981) found a highly significant inverse correlation
between B and Mn concentrations in leaves of sunflower, while Cu, Fe and Zn
concentrations were not changed by different B levels in the nutrient solution.
Marked reductions in Fe and Mn adsorption, but an increase in Zn uptake were
recorded in bean plants grown in B deficient medium. The transport of Fe, Mn and
Zn was increased in the trifoliate leaves, while that in shoots was reduced. It appears
that, B is involved in the physiological processes controlling uptake and transport of
nutrients like Mn, Fe and Zn (Dave and Kannan 1981).
Lombin and Bates (1982) found that with increasing B levels, the uptake of K,
Mn, Zn, Cu, Mo and B was increased in alfalfa, peanut and soybean crops, but had
no apparent effect on that uptake of Ca ad Mg in all crops. Similar detrimental
effects of B on the uptake of Ca and Mg were reported by Singh and Singh (1983),
who observed varying B level significantly increased the concentration of N, P, K,
Na and B and decreased Ca and Mg concentrations in lentil plants. Applied B
increased the N, P, K, Na and B content but decrease Ca and Mg contents of barley
crops, whilst uptake of N, P, Na and B in grain and straw significantly increased,
and K uptake remained unaffected (Singh and Singh 1983). Francois (1986)
reported that with increasing B in the soil solution the concentration of B, P, K
and Mg tended to increase in tomato leaves, whilst Ca and Na showed inconsistence
trends. Studies on the chemical composition of radish, using sand culture tech-
niques, indicated that Ca and P concentrations decreased significantly and K, Mg
and Na remained unchanged with the increasing B levels (Francois 1986). Morsey
and Taha (1986) reported that applied soil B and foliar application increased the
concentration and uptake of N, P, K, Mn and B in both shoots and roots of sugar
plants. Patel and Golakia (1986) demonstrated the effect of soil B on the uptake
of N, P, K, Ca, Zn, Cu, Fe and Mn by a groundnut crop. Interestingly they outlined
the mechanisms of action for some nutrients in relation to the B effect: for example,
B increased an uptake and could be responsible for a favourable effect on nodula-
tion. A positive effect of B and P uptake, which altered the permeability of plasma
lemma at the root surface, resulted in increased P absorption. Uptake of K increased
6 Boron: A Promising Nutrient for Increasing Growth and Yield of Plants 163

because of their mutual synergistic relationship, but Ca decreased due to antago-

nistic effect. Uptake of Fe and Cu were positively correlated, while Mn and Zn
negatively correlated with applied B. The deficient state of B resulted in decreased
the leaf N, P, Ca, Mg, Fe, Cu, Zn and B in tomato. On the other hand, excess B
increased the concentration of nutrients with greater significance for K, Mg and Fe
followed by Ca and Mn and in smaller quantity Cu and Zn (Carpena-Artes and
Carpena-Ruiz 1987).
B toxicity had no consistent effects on the tissue concentration of P, K, Ca, Mg,
Zn, Cu, Fe and Mn for five barley and six wheat cultivars grown in nutrient solution
and no interactions were found among B nutrients and cultivars (Nable 1989).
Higher levels of applied B significantly depressed the N and enhanced P and K
contents in three cuttings of Trifolium alexandrinum (berseem; Pal et al. 1989).
Singh et al. (1990) reported that the concentration of P, Mg and Zn in wheat
increased and Ca, K, Cu, Fe and Mn decreased with increasing B in soil. On the
other hand, an increasing supply of B significantly decreases the uptake of P, K, Ca,
Mg and Mn, while that of Zn, Cu and Fe increased. They concluded that high levels
of applied B had an antagonistic effect on the uptake of nutrients and this could be
due to the toxic effect of B on root cells, resulting in impaired nutrient absorption
processes. Alvarez-Tinaut (1990) found positive correlations between B and Fe and
Cu contents of sunflower, suggesting that B could indirectly affect catalase activity
via Fe and Cu. Positive correlations between Zn and B also indicated that B could
indirectly affect the enzyme through modification of the Zn content. Concentra-
tions, total uptake and ratios of certain nutrients in radish top and root change with
differential B supply to nutrient solution (Tariq 1997). However, this study also
suggested that changes occurring were mainly due to the B effect and partially due
to antagonism between Ca and B. It is clear from the reported literature, that B
interactions, either synergism or/and antagonism, can affect plant nutrition under
both deficiency and toxicity conditions.
With increasing B supply in nutrient medium, leaf content of P became high
(usually younger leaves shows higher P concentration) and there was a minor
decrease in K, Ca and Mg compared to the average concentration for leaves (Furlani
et al. 2003).
Yang and Gu (2004) demonstrated the effect of B on Al toxicity for soybean
seedlings. They showed that high supply of B was found to induce Al toxicity by
significantly increasing growth parameters including root length at 2 mM, and fresh
weight at 5 mM Al for two different cultivars. Similar results have been described
by Hossain and Hossain (2004), who confirmed the relationship of B with Al. The
ratio between Ca and B in the plant is sometimes used to identify B deficiency. In
one study, the supply of Ca and B to four maize cultivars considerably improved
shoot dry matter production (Kanwal et al. 2008).
B is responsible for changes in other nutrients in soil-plant interactions (Tariq
and Mott 2006). They also showed optimum productivity of radish plants at 0.5 mg
l
1 B supply. Toxic effects confirmed by significant productivity decreases were
found at increased levels of B supply. The amount of B, Zn and Cu in plants was
increased and amount of Fe, Mn and Mo were reduced. Except B, the net uptake of
164 H. Bariya et al.

all microelements decreased with increasing levels of B in the nutrient supply, and
exerted close connection to the growth response of radish plants. Moreover, Zn/Cu
ratio increased and ratio of Mn/Fe and Mn/Zn decreased, while Fe/Cu exerted
unpredictable trend with increasing B levels. Inoculation with biofertilisers (Rhi-
zobium strains) alone or combined with different levels of B increased significantly
the uptake of N, P, K, Fe, Mn, Zn and B by shoot and seeds of peanut in both
seasons as compared with the corresponding treatments without biofertilisers. The
highest values of N, P, K, Fe and Mn uptake by straw and seeds of peanut plants
were obtained by using (200 ppm of B + Rhizobium spp.) in two successive seasons,
while the highest values of Zn and B uptake by straw and seeds in both seasons were
obtained by using (300 ppm of B + inoculation with Rhizobium; Nadia et al. 2006).
B interactions (synergism and/or antagonism) can affect plant nutrition under both
deficient and toxic levels (Tariq and Mott 2007).
There is no significant effect on the residual Fe in the soil when using B fertiliser.
Results suggest that Zn and B fertilisers had no role in the changes of residual Fe
and Mn in the soil relative to the normal levels, and other factors are operative on
the accumulation of residual Fe and Mn in the soil relative to its normal levels. The
effect of Zn and B interaction on the residual Fe and Mn in the soil was insignificant
(Aref 2010).
Our findings showed that different level of B applied to the groundnut plant
affect uptake of nutrients in an irregular fashion. Interactions between nutrients and
applied B indicted uptake of N, P, K, Mg, Mn, Zn and Fe are indirectly dependent
on increasing supply of B resulting in increasing NUE and NDS, but at a certain
level. Our recent observations suggested that at 1 kg ha
1 B level, NUE of
groundnut plant was higher in terms of absorption of mineral nutrients compared
to the 0.5 kg ha
1 B level. At the 2.0 kg ha
1 B level, groundnut varieties showed
decreases in nutrients uptake capacity, resulting in decreases in NUE. Increases in
supply of nutrients may affect nutrient uptake capacity of plants, which was
confirmed when levels of absorbed macro- and micronutrients in groundnut plants
at 2.0 kg ha
1 B supply were determined. At 0.5 and 1.0 kg ha
1 B, NDS in
groundnut plants was higher. NDS was improved in both varieties of groundnut at
1.0 kg ha
1 B supply. Higher levels of B might be exerting antagonist effects on
uptake of nutrients, which might be affecting NUE and NDS to groundnut plants.
Observations of nutrient uptake capacity of groundnut plants in all three conditions
(limiting, sufficient and toxic) strongly suggested that 1.0 kg ha
1 B improved NUE
and NDS in groundnut plants. Despite substantial literature, the mechanism of B
interaction with other nutrients still remains unclear and needs more investigation
in terms of improvement in NUE and NDS. NUE is usually studied for only one
nutrient and improving NUE for any one nutrient may affect the NUE of other
nutrients; this is still a question of interest.
6 Boron: A Promising Nutrient for Increasing Growth and Yield of Plants 165

Concluding Remarks
Boron is a crucial element for normal development and growth of plants. This
chapter highlights the positive effects of boron on crops and also describes the
interaction of B with the uptake of other nutrients. It further highlights the
importance of understanding the mechanisms of B action in plants and
determining the molecular mechanisms of plant responses to toxicity and
deficiency of B, to allow improvement of crops for tolerance to both condi-
tions. Deficient or toxic amounts of B may both have adverse effect on plants
and alter the uptake of other nutrients by direct or indirect interactions.
Increasing supply of B may result in increasing nutrient use efficiency
(NUE) and nutrient demand and supply (NDS). Alternatively higher levels
of B may exert antagonist effects on uptake of nutrients, negatively affecting
NUE and NDS. The mechanism underlying the B interaction with other
nutrients still remains unclear and requires further investigation. NUE is
studied typically for only one nutrient at a time and improving NUE for any
one nutrient almost certainly affects the NUE of other nutrients. However, the
interactions of B with other plant elements are complex and may exert
antagonistic or synergistic effects, which may be specific for species, growth
medium and different environments.

References

Aduayi EA (1978) Role of boron on growth components and elemental composition of Ife plum
tomato. Commun Soil Sci Plant Anal 9:1–11
Alvarez-Tinaut MC, Leal A, Recalde-Martinez L (1980) Iron-manganese interaction and its
relation to boron levels in tomato plants. Plant Soil 55:377–388
Alvarez-Tonaut MC (1990) Correlation between boron and other micronutrients. In: Behaviour,
function and significance of boron in agriculture. Report on an International Workshop at
St. John’s College, Oxford, England. 23–25 July, 1990. Borax Consolidated Limited, London,
SW 1P 1HT
Aref F (2010) Influence of zinc and boron interaction on residual available iron and manganese in
the soil after corn harvest. Am Eur J Agric Environ Sci 8:767–772
Baker AS, Cook RL (1959) Green house studies on alfalfa with soil type, soil reaction and borax
fertilization as variables. Agron J 51:1–4
Bellaloui N, Yadavc RC, Chern MS, Hu H, Gillen AM, Greve C, Dandekar AM, Ronald PC,
Brown PH (2003) Transgenically enhanced sorbitol synthesis facilitates phloemboron mobility
in rice. Physiol Plant 117:79–84
Blaser-Grill J, Knoppik D, Amberger A, Goldbach H (1989) Influence of boron on the membrane
potential in Elodea densa and Helianthus annuus roots and H+ extrusion of suspension cultured
Daucus carota cells. Plant Physiol 90:280–284
Blevins DG, Lukaszewski KM (1998) Boron in plant structure and function. Annu Rev Plant
Physiol Plant Mol Biol 49:481–500
Bogiani JC, Sampaio TF, Abreu-Junior C, Rosolen CA (2014) Boron uptake and translocation in
some cotton cultivars. Plant Soil 375:241–253
166 H. Bariya et al.

Bolaños L, Esteban E, de Lorenzo C, Fernández-Pascual M, de Felipe MR, Garate A, Bonilla I

(1994) Essentiality of boron for symbiotic dinitrogen fixation in pea (Pisum sativum) rhizo-
bium nodules. Plant Physiol 104:85–90
Bolaños L, Lukaszewski K, Bonilla I, Blevins D (2004) Why boron? Plant Physiol Biochem
42:907–912
Brown PH, Hu H (1996) Phloem mobility of boron is species dependent: evidence of phloem
mobility in sorbitol-rich species. Ann Bot 77:497–505
Brown PH, Shelp BJ (1997) Boron mobility in plants. Plant Soil 193:85–101
Brown PH, Bellaloui N, Hu H, Dandekar A (1999) Transgenically enhanced sorbitol synthesis
facilitates phloem boron transport and increases tolerance of tobacco to boron deficiency. Plant
Physiol 119:17–20
Camacho-Cristóbal JJ, Anzellotti D, González-Fontes A (2002) Changes in phenolic metabolism
of tobacco plants during short-term boron deficiency. Plant Physiol Biochem 40:997–1002
Camacho-Cristóbal JJ, Rexach J, Herrera-Rodrı́guez MB, Navarro-Gochicoa MT, González-
Fontes A (2011) Boron deficiency and transcript level changes. Plant Sci 181:85–89
Cara FA, Sánchez E, Ruiz JM, Romero L (2002) Is phenol oxidation responsible for the short-term
effects of boron deficiency on plasma-membrane permeability and function in squash roots?
Plant Physiol Biochem 40:853–858
Carmen Rodriguez- Hernandez M, Moreno DA, Carvajal M, Carmen–Martinez- Ballesta M
(2013) Interactive effects of boron and NaCl stress on water and nutrient transport in two
broccoli cultivars. Funct Plant Biol 40(7):739–748
Carpena-Artes O, Carpena-Ruiz RO (1987) Effect of boron in tomato plant. Leaf evaluations.
Agrochimica 31:391–400
Coetzer LA, Robbertse PJ (1987) Pollination biology of Persea Americana Fuerte. S Afr Avocado
Growers’ Assoc Yearb 10:43–45
Cosgrove DJ (1999) Enzymes and other agents that enhance cell wall extensibility. Annu Rev
Plant Physiol Plant Mol Biol 50:391–417
Crop Science Society of America (CSSA) (1992) Glossary of crop science terms. CSSA,
Madison
Dannel F, Pfeffer H, Römheld V (2002) Update on boron in higher plant. Uptake, primary
translocation and compartmentation. Plant Biol 4:193–204
Dave IC, Kannan S (1981) Influence of boron deficiency on micronutrients absorption by
Phaseoulus vulgaris and protein contents in cotyledons. Acta Physiol Plant 3:27–32
Dear B, Lipsett J (1987) The effect of boron supply on the growth and seed production of
subterranean clover (Trifloium subtereneum L). Aust J Agric Res 38:537–546
Domingues DS, Leite SMM, Farro APC, Coscrato VE, Mori ES, Furtado EL, Wilcken CF,
Velini ED, Guerrini IA, Maia IG, Marino CL (2005) Boron transport in Eucalyptus. 2. Identi-
fication in silico of a putative boron transporter for xylem loading in eucalypt. Genet Mol Biol
28:625–629
Downton WJS, Hawkar JS (1980) Interaction of boron and chloride on growth and mineral
composition of cabernet sauvignon vines. Am J Enol Vitic 31:277–282
Du Ying Q, Rong LX, Hua HJ, Zhiyao H (1999) Influence of B and/or Mo application on the
growth and yield of peanut. Chin J Oil Crop Sci 21:61–66
El-Shintinawy F (1999) Structural and functional damage caused by boron deficiency in sunflower
leaves. Photosynthetica 36:565–573
Ferrol N, Donaire JP (1992) Effect of boron on plasma membrane proton extrusion and redox
activity in sunflower cells. Plant Sci 86:41–47
Fleischer A, O’Neill MA, Ehwald R (1999) The pore size of non-graminaceous plant cell walls is
rapidly decreased by borate ester cross-linking of the pectic polysaccharide rhamnoga-
lacturonan II. Plant Physiol 121:829–838
Francosis LE (1986) Effect of excess boron on broccoli, cauliflower and radish. Am Soc Hortic Sci
111:494–498
6 Boron: A Promising Nutrient for Increasing Growth and Yield of Plants 167

Furlani ÂMC, Carvalho CP, de Freitas JG, Verdial MF (2003) Wheat cultivar tolerance to boron
deficiency and toxicity in nutrient solution. Sci Agric 60:359–370
Ganie MZ, Akhter F, Bhat MA, Malik AR, Mihd Junaid J, Shah MA, Bhat AH, Bhat TA (2013)
Boron- a critical nutrient element for plant growth and productivity with reference to temperate
fruits. Curr Sci 104:76–85
Garg OK, Sharma AN, Kona GRSS (1979) Effect of boron on the pollen vitality and yield of rice
plants (Oryza sativa L. var. Jaya). Plant Soil 52:575–578
Goldbach HE, Yu Q, Wingender R, Schulz M, Wimmer M, Findeklee P, Baluska F (2001) Rapid
response reactions of roots to boron deprivation. J Plant Nutr Soil Sci 164:173–181
Goldberg S (1997) Reactions of boron with soil. Plant Soil 193:35–48
Gomez-Rodriguez MV, Gomez-Ortega M, Alvarez-Tinaut MC (1981) Boron, copper, iron, man-
ganese and zinc content in leaves of flowering sunflower plants (Helianthus annuus L.) grown
with different boron supplies. Plant Soil 62:461–464
Gupta U (1979) Boron nutrition of crops. Adv Agron 31:273–307
Hajiboland R, Rad B, Bastani S (2013) Phenolic metabolism in boron deficient tea (Camellia
sinensis L. O. Kuntze) plants. Acta Biol Hung 64:196–206
Han S, Chen L, Jiang H, Smith BR, Yang L, Xie C (2008) Boron deficiency decreases growth and
photosynthesis, and increases starch and hexoses in leaves of citrus seedlings. J Plant Physiol
165:1331–1341
Harris HC, Brolmann JB (1966) Comparison of calcium and boron deficiencies of the peanut
II. seed quality in relation to histology and viability. Agron J 58:578–582
Hossain AKMZ, Hossain MA (2004) Effects of aluminum and boron supply on growth of
seedlings among 15 cultivars of wheat (Triticum aestivum L.) grown in Bangladesh. Soil Sci
Plant Nutr 50:189–195
Hu HN, Brown PH (1997) Absorption of boron by plant roots. Plant Soil 193:49–58
Hu H, Penn SG, Lebrilla CB, Brown PH (1997) Isolation and characterization of soluble B
complexes in higher plants. The mechanism of phloem mobility of boron. Plant Physiol
113:649–655
Huang L, Ye Z, Bell WR, Dell B (2005) Boron nutrition and chilling tolerance of warm climate
crop species. Ann Bot 96:755–767
Hundt I, Schilling G, Fisher F, Bergmann W (1970) Investigations on the influence of the
micronutrient boron on nucleic acid metabolism. Albrecht-Thaer-Arch 14:825–837
Kamali AR, Childers NF (1970) Growth and fruiting of peach in sand culture as affected by boron
and fritted form of trace elements. J Am Soc Hortic Sci 95:652–656
Kanwal S, Rahmatullah Aziz T, Maqsood MA, Abbas N (2008) Critical ratio of calcium and boron
in maize shoot for optimum growth. J Plant Nutr 31:1535–1542
Kastori R, Plesnicar M, Pankovic D, Sakac Z (1995) Photosynthesis, chlorophyll fluorescence and
soluble carbohydrates in sunflower leaves as affected by boron deficiency. J Plant Nutr
18:1751–1763
Khan Towhid Osman (2013) Plant nutrients and soil fertility management. In: Soils: principles,
properties and management. Springer, Dordrecht/Heidelberg/New York, pp 129–159
Kobayashi M, Matoh T, Azuma J (1996) Two chains of rhamnogalacturonan II are crosslinked by
borate-diol ester bonds in higher plant cell walls. Plant Physiol 110:1017–1020
Kot FS (2009) Boron sources, speciation and its potential impact on health. Rev Environ Sci
Biotechnol 8:3–28
Leece DR (1978) Effects of boron on the physiological activity of zinc in maize. Aust J Agric Res
29:739–749
Li B, Li H, Kui WH, Chao MC, Jern WS, Li HP, Chu WJ, Wang L (1978) Studies on cause of
sterility of wheat. J Northeast Agric Coll 3:1–19
Lombin GL, Bates TE (1982) Comparative responses of peanut, alfalfa and soybeans to varying
rates of boron and manganese on two calcareous Ontario soils. Can J Soil Sci 62:1–9
Loomis WD, Durst RW (1992) Chemistry and biology of boron. Biofactors 3:229–239
168 H. Bariya et al.

Malvi UR (2011) Interaction of micronutrients with major nutrients with special reference to
potassium. Karnataka J Agric Sci 24:106–109
Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic, San Diego
Matoh T, Ochiai K (2005) Distribution and partitioning of newly taken-up boron in sunflower.
Plant Soil 278:351–360
McIlrath WJ, Debruyn JA, Skok J (1960) Influence of boron supply on the micronutrients content
of setaria shoots. Soil Sci 89:117–121
Miwa K, Fujiwara T (2010) Boron transport in plants: co-ordinated regulation of transporters. Ann
Bot 105:1103–1108
Morsey MA, Taha EM (1986) Effect of boron, manganese and their combination on sugar beet
under El-Minia conditions. 2: concentration and uptake of N, P, K, B and Mn. Ann Agric Sci
Ain Shams Univ Cairo 31:1241–1259
Mozafar A (1989) Boron effect on mineral nutrients of maize. Agron J 81:285–290
Mukhopadhyaya M, Ghosh PD, Mondal TK (2013) Effect of boron deficiency on photosynthesis
and antioxidant responses of young tea plantlets. Russ J Plant Physiol 60:633–639
Nable RO (1989) Effect of boron toxicity upon the mineral nutrient composition of barley and
wheat cultivars. Div Rep Div Soils, CSIRO No. 104: 1–10
Nable RO, Bañuelos GS, Paull JG (1997) Boron toxicity. Plant Soil 193:181–198
Nadia MA, Badran M, Abd El-Hamide AF (2006) Response of peanut to foliar spray with boron
and/or rhizobium inoculum. J Appl Sci Res 2:1330–1337
Nakagawa Y, Hanaoka H, Kobayashi M, Miyoshi K, Miwa K, Fujiwara T (2007) Cell-type
specificity of the expression of OsBOR1, a rice efflux boron transporter gene, is regulated in
response to boron availability for efficient boron uptake and xylem loading. Plant Cell
19:2624–2635
Noppakoonwong RN, Rerkasem B, Bell RW, Dell B, Loner Gan JF (1997) Prognosis and
diagnosis of boron deficiency in blackgram (Vigna mungo L. Hepper) in the field by using
plant analysis. In: Bell RW, Rerksaem B (eds) Proceedings of boron in soil and plants. Kluwer
Academic Publishers, Dordrecht, pp 89–93
North KA, Ehlting B, Kopriva A, Rennenberg H, Kopriva S (2009) Natural variation in
Arabidopsis adaptation to growth at low nitrogen conditions. Plant Physiol Biochem
47:912–918
Oertli JJ, Richardson WF (1970) The mechanism of boron immobility in plants. Plant Physiol
23:108–116
Pal B, Jadaun SPS, Raghav CS (1989) Effect of phosphorous and boron application on dry matter
yield and nutrient contents in berseem. J Indian Soc Soil Sci 37:579–581
Pandey N, Archana (2012) Effect of boron on seed germination and biochemical changes in
linseed at seeling stage. Indian J Agric Biochem 25:167–170
Pandey DK, Pandey N (2008) Screening of wheat genotypes for their susceptibility to boron
deficiency. Res Environ Life Sci 1:37–42
Parks RQ, Lyon CB, Hood SL (1944) Some effect of boron supply on the chemical composition of
tomato leaflets. Plant Physiol 19:404–419
Patel MS, Golakia BA (1986) Effect of calcium carbonate and boron application on yield and
nutrient uptake by groundnut. J Indian Soc Soil Sci 34:815–820
Pollard AS, Parr AJ, Loughman BC (1977) Boron in relation to membrane function in higher
plants. J Exp Bot 28:831–834
Power PP, Woods WG (1997) The chemistry of boron and its speciation in plants. Plant Soil
193:1–13
Ruiz JM, Baghour M, Bretones G, Belakbir A, Romero L (1998) Nitrogen metabolism in tobacco
plants (Nicotiana tabacum L.): role of boron as a possible regulatory factor. Int J Plant Sci
159:121–126
Ryden P, Sugimoto-Shirasu K, Smith AC, Findlay K, Reiter WD, McCann MC (2003) Tensile
properties of arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar
network and rhamnogalacturonan II-borate complexes. Plant Physiol 132:1033–1040
6 Boron: A Promising Nutrient for Increasing Growth and Yield of Plants 169

Sathya S, Mahendran PP, Arulmozhiselven K (2013) Influence of soil and foliar application of
borax on fractions of boron under tomato cultivation in boron deficient soil of typic Haplustalf.
Afr J Agric Res 8:2567–2571
Sharma PN, Ramchandra T (1990) Water relations and photosynthesis in mustard plants subjected
to boron deficiency. Indian J Plant Physiol 33:150–154
Shelp BJ, Shattuck VI (1987) Boron nutrition and mobility, and its relation to hollow stem and the
elemental composition of greenhouse grown cauliflower. J Plant Nutr 10:143–162
Simojoki P (1972) Boron deficiency, pollen sterility and ergot disease of barley. Ann Agric Fenn
11:333–341
Singh AL (1994) Micronutrient nutrition and crop productivity in groundnut. In: Singh K, Purohit
SS (eds) Plant productivity under environmental stress. Agro Botanical Publishers, Bikaner, pp
67–72
Singh V, Singh SP (1983) Effect of applied boron on the chemical composition of lentil plants. J
Indian Soc Soil Sci 31:169–170
Singh JP, Dahia DJ, Narwal RP (1990) Boron uptake and toxicity in wheat in relation to zinc
supply. Fert Res 24:105–110
Stangoulis JCR, Brown PH, Bellaloui N, Reid RJ, Graham RD (2001) The efficiency of boron
utilization in canola. Aust J Plant Physiol 28:1109–1114
Steinburg RA, Specht AW, Roller EM (1955) Effect of micronutrient deficiency on mineral
composition, nitrogen fraction, ascorbic acid and burn of tobacco grown flowering in water
culture. Plant Physiol 30:123–129
Stewart WM, Dibb DW, Johnston AE, Smyth TJ (2005) The contribution of commercial fertilizer
nutrients to food production. Agron J 97:1–6
Takano J, Yamagami M, Noguchi K, Hayashi H, Fujiwara T (2001) Preferential translocation of
boron to young leaves in Arabidopsis thaliana regulated by the BOR1 gene. Soil Sci Plant Nutr
47:345–357
Takano J, Noguchi K, Yasumori M, Kobayashi M, Gajdos Z, Miwa K, Hayashi H, Yoneyama T,
Fujiwara T (2002) Arabidopsis boron transporter for xylem loading. Nature 420:337–339
Tariq M (1997) Effect of boron supply on the availability of nutrients in soil and uptake by radish
(Raphanus sativas L.) Ph.D. thesis, University of Reading, England
Tariq M, Mott CJB (2006) Effect of applied boron on the accumulation of cations and their ratios
to boron in radish (Raphanus sativus L.). Soil Environ 25:40–47
Tariq M, Mott CJB (2007) Effect of boron on behavior of nutrients in soil-plant systems – a
review. Asian J Plant Sci 6:195–202
Thurzo S, Szabo Z, Nyeki J, Silva AP, Nagy PT, Goncalves B (2010) Effect of boron and calcium
sprays on photosynthetic pigments, total phenols and flavonoid content of sweet cherry
(Prunus avium L.). Acta Hortic 868:457–461
Tolgyesi G, Kozma A (1974) Investigation on factors affecting boron uptake by grasses.
Agrokemia es Talajatan 23:83–98
Touchton JT, Boswell FC (1975) Effects of boron on soyabean yield, chemical composition and
related characteristics. Agron J 67:417–420
Vaughan AKF (1977) The relation between the concentration of boron in the reproductive and
vegetative organ of maize plants and their development. Rhod J Agric Res 15:163–170
Wallace A, Romney EM, Alexander GV, Kinnear J (1977) Phytotoxic and some interactions of the
essential metals iron, manganese, molybdenum, zinc, copper and boron. Commun Soil Sci
Plant Anal 8:741–750
Wang ZY, Tang YL, Zhang FS, Wang H (1999) Effect of boron and low temperature on membrane
integrity of cucumber leaves. J Plant Nutr 22:543–550
Warington K (1923) The effect of boric acid and borax on the broad bean and certain other plants.
Ann Bot 37:629–672
Wimmer MA, Eichert T (2013) Review: mechanism for boron deficiency mediated changes in
plant water relations. Plant Sci 203–204:25–32
170 H. Bariya et al.

Wimmer MA, Lochnit G, Bassil E, Mühling KH, Goldbach HE (2009) Membrane associated,
boron-interacting proteins isolated by boronate affinity chromatography. Plant Cell Physiol
50:1292–1304
Woodbridge CG, Venegas A, Carndall PC (1971) The boron content of developing pear, apple and
cherry flower buds. J Am Soc Hortic Sci 96:613–615
Woods WG (1994) An introduction to boron: history, sources, uses, and chemistry. Environ Health
Perspect 102:5–11
Yadav OP, Manchanda HR (1979) Boron tolerance studies in gram and wheat grown on a sierozem
sandy soil. J Indian Soc Soil Sci 27:174–180
Yang YH, Gu HJ (2004) Effects of boron on aluminum toxicity on seedlings of two soybean
cultivars. Water Air Soil Pollut 154:239–248
Yang L, Zhang Q, Dou J, Li L, Guo L, Shi L, Xu F (2013) Characteristics of root boron nutrition
confer high boron efficiency in Brassica napus cultivars. Plant Soil 371:95–104
Zhao D, Oosterhuis DM (2002) Cotton carbon exchange, non-structural carbohydrates, and boron
distribution in tissues during development of boron deficiency. Field Crop Res 78:75–87
Chapter 7
Role of Autophagy in Plant Nutrient
Deficiency

Milagros Collados Rodrı́guez, Katarzyna Zientara-Rytter,

and Agnieszka Sirko

Abstract One of the environmental stresses frequently encountered by plants is

nutrient deficiency. Therefore, reuse of valuable cellular nutrients is an important
trait in nutrient use efficiency (NUE). High NUE is a desired trait in plants at all
developmental steps to reach maximum potentials with minimum inputs. Two
highly conserved evolutionary mechanisms are responsible for protein turnover at
the cellular level, the ubiquitin-proteasome system (UPS) and the autophagy path-
way. Generally, UPS recycles short-lived regulatory proteins while autophagy
recycles long-lived proteins, protein aggregates or organelles. The proteins,
which are destined for degradation, are marked by a special polypeptide tag,
ubiquitin. The features of this tag, as well as activity of ubiquitinating and deubiqui-
tinating enzymes, are determinants that allocate the protein into one or the other
degradation systems. Apart from the common subset of over 30 proteins required
for the “core autophagy”, there exist selective autophagy cargo receptors. These
proteins perform the quality control function by recognizing ubiquitinated cargoes
(ready for degradation) and linking them to the autophagy machinery. Adequate
knowledge of the processes of selective autophagy will be beneficial for agricul-
tural production and the environment by delivering the methods and means for
obtaining crops with improved NUE, higher yield and better stress tolerance.

Keywords NUE (nutrient use efficiency) • Autophagy • UPS (ubiquitin

proteasome system) • Nutrient deficiency • Protein turnover • Protein homeostasis
• PCD (programmed cell death)

M. Collados Rodrı́guez • K. Zientara-Rytter • A. Sirko (*)

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul, Pawinskiego 5A,
02-106 Warsaw, Poland
e-mail: [email protected]

© Springer International Publishing Switzerland 2014 171

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_7
172 M. Collados Rodrı́guez et al.

Introduction

Response to Nutrient Deficiency in Plants

Plants convert CO2, light and water into biomass, however they also require
essential nutrients to complete their lifecycle. The sessile nature of plants makes
them vulnerable to the environmental fluctuations that cause nutrient limitation
(among other stresses). In plants, response to nutrient deficit is complex and
depends on the kind of limiting nutrient (macro- or micronutrient) and the extent
of the shortage. Nutrient deficits result in phenotypic adaptation, such as root length
and branching, shoot biomass reduction and faster transfer from vegetative stage of
growth into the generative one (Amtmann and Armengaud 2009; Gruber
et al. 2013). Plant responses to nutrient deficit may also be monitored at various
molecular levels, such as gene expression, posttranslational modification of various
proteins, qualitative and quantitative changes of proteins and metabolites.
Researchers from many laboratories have revealed that transcriptome, metabolome
and proteome profiles are reprogrammed in plants which encounter nutrient deficit
(for example, Hoefgen and Nikiforova 2008; Howarth et al. 2008; Liang
et al. 2013). Limitation of the particular nutrient (and/or an increased internal
requirement for that nutrient) is sensed by plants and this information is transferred
to the appropriate effectors, which modify the pathway responsible for assimilation
and metabolism of the nutrient. The sensing and regulatory mechanisms are not
completely characterised in most nutrients. In addition, the need to coordinate
assimilation and metabolism of various nutrients makes this regulation even more
complicated. The growing body of evidence suggests that protein degradation
processes not only adjust plant metabolism to long lasting starvation periods, but
they are also important in early responses to short-term nutrient deficit.

Ubiquitin and Protein Turnover Processes

Ubiquitin (Ub) is a 76 amino acid polypeptide that folds up into a compact globular
structure and is heat-stable. It mediates selective proteolysis after enzymatic con-
jugation to the target proteins that can be either monoubiquitinated, when a single
Ub unit is attached through its C-terminal glycine (G-76) to one lysine (K) residue
of the substrate, or multi-monoubiquitinated, when single Ub units are attached
through G-76 to several K residues of the substrate or polyubiquitinated when
multiple Ub units are attached to the single K residue (Schreiber and Peter 2013;
Vierstra 2012). Ub is evolutionary conserved and contains seven highly conserved
lysines that can be used for poly(Ub) chain formation in vivo. Some of these K
residues are traditionally associated with particular degradation systems, for exam-
ple, K-48-linked (poly)Ub appears to be linked rather to proteasomal degradation
while K-63–linked (poly)Ub appears to be more linked to autophagic degradation.
In addition, the role of unconventional ubiquitination in targeting proteins for
degradation indicates the complexity of the process (Xu et al. 2009).
7 Role of Autophagy in Plant Nutrient Deficiency 173

Ubiquitins are encoded by a small-to-medium-sized multigene family compris-

ing two gene classes, monomeric and polymeric. Monomeric ubiquitin genes
consist of 228 nucleotides (76 codons) with an additional C-terminal sequence
that encodes a ribosomal protein. By contrast, polymeric genes known as
polyubiquitins are composed of tandem repeats of a 228-bp gene with no spacer
sequence between them (Nei et al. 2000). In Arabdopsis, the Ub gene family
consists of at least 14 members (Callis et al. 1995).
Three enzymes (E1-E3), working in a hierarchical cascade, attach a Ub tag to the
target proteins (Sadanandom et al. 2012). E1 (ubiquitin-activating enzyme) hydro-
lyses ATP to adenylate the C-terminal carboxyl of Ub and form a thiolester bond
with E1 cysteinyl sulphydyl residue (E1-Ub intermediate). After completion of this
activation, E2 (ubiquitin-conjugating enzyme) accepts Ub on its cysteinyl
sulphydryl group and a thiolester bond is formed again (E2-Ub intermediate).
When conjugation is completed, E2 can either bind to E3 (ubiquitin ligase) to
transfer Ub to the protein substrate or transfer Ub to E3, which subsequently
transfers Ub to the target. Usually a Lys residue of the substrate accepts the
C-terminal Gly-76 of Ub. The E3 Ub ligases recognise and bind specific degrada-
tion signals in substrate proteins and thus confer specificity to the ubiquitination-
mediated degradation.
This ubiquitination cycle can be repeated multiple times. To add more complex-
ity, the protein family of deubiquitinating enzymes form an additional regulatory
step. A comprehensive repository of ubiquitinating and deubiquitinating enzymes
from 50 distinct genomes belonging to four of the five major phylogenetic super-
groups of eukaryotes was recently completed (Hutchins et al. 2013) and it is
publicly available at the Database of Ubiquitinating and Deubiquitinating Enzymes
[http://www.DUDE-db.org].
Proteins targeted for recycling by linkage of the Ub tag are degraded in two
evolutionary conserved degradation systems. The ubiquitin-proteasome system
(UPS) takes over the short-lived regulatory proteins (Dantuma et al. 2000; Vierstra
2009), while the autophagy pathway sequesters within double membrane structures
the longer-lived and bigger proteinaceous material, such as protein aggregates
(Yang and Klionsky 2010). Autophagy and the UPS are critical in the maintenance
of cellular homeostasis, thus their activities need to be carefully orchestrated
(Korolchuk et al. 2010). The limited degradative capacity of UPS is complemented
with autophagy and crosstalk mechanisms between both systems exist (Schreiber
and Peter 2013). Moreover, both recycling mechanisms are in crosstalk with
senescence, programmed cell death (PCD) and prematurate aging (Madeo
et al. 2010). The literature shows that when some autophagy related genes (ATG)
are impaired, accelerated senescence overtakes the plant destiny (Hanaoka
et al. 2002; Thompson and Vierstra 2005) presumably by impairment of autophagic
recycling that contributes to plant energy availability (Izumi et al. 2013).
An overview of the ubiquitination cascade and UPS is shown in Fig. 7.1. The
26S proteasome structures in mammals, yeast and plants indicate a similar overall
design (Sadanandom et al. 2012). However, UPS-dependent regulation of signaling
and metabolic pathways appears to be more complex and prevalent in plants than in
yeast and animals.
174 M. Collados Rodrı́guez et al.

Fig. 7.1 Ubiquitin/26S proteasome system (UPS) overview (a) and schematic structure of 26S
proteasome (b). (a) Ubiquitin (Ub) is in an ATP-dependent manner activated by E1 enzyme,
transferred to an E2 enzyme and delivered to the target protein due to E3 attachment. Rounds of
ubiquitin conjugation cascades polyubiquitinates the substrate to be recognised by 26S proteasome
complexes, which hydrolyse peptide bonds and releases oligopeptides (consisting of 2–10 resi-
dues) to the cytoplasm; the Ub moieties are reused. Su substrate protein, G Gly-76 C-terminal
residue of Ub, K Lys residue of the target protein, S thiolester bond, DUBs deubiquitinating
enzymes. (b) One or two 19S regulatory complexes (RP) composed of base and lid subcomplexes
cap the 20S core complex (CP) formed by two external and two internal rings of seven α-subunits
and seven β-subunits respectively

The proteasome exists in multiple forms, and contains two major assemblies, the
28-subunit core particle (CP, also known as the 20S core) and the regulatory subunit
(RP, also known as the 19S complex and PA700) consisting of 19 subunits in yeast,
and probably the same number in other eukaryotes (Finley 2009). The CP is
composed of four rings: two external rings made of seven α-subunits (α 1-7)
flanking two rings of β-subunits (β 1-7 (A-G in plants). The CP functions as a
non-specific ATP and Ub-independent protease with the capacity to cleave most
peptide bonds due to peptidylglutamyl, trypsin-like and chymotrypsin-like activi-
ties. One or two RP complexes, each consisting of two parts base and lid, can be
attached to the CP. The base consists of a ring of six related AAA+ATPase subunits
(RPT1-6) and three non-ATPase subunits (RPN1, 2 and 10) and is attached directly
to the CP. The lid contains non-ATPase subunits (RPN3, 5–9 an 11–12). RPN11 is a
metaloprotease, which allows recycling of the substrate recognition tag, ubiquitin.
The RP is responsible for recognition of K48 linked poly(Ub) chains, removal of
7 Role of Autophagy in Plant Nutrient Deficiency 175

Ub moieties, unfolding and pore gating the substrate. The proteasome has the
ability to bind different lids or lid-like structures depending on the cellular needs.
A growing body of evidence indicates that monoubiquitinated proteins can be also
targeted to proteasomes indicating that monoubiquitination is sufficient for degra-
dation of small proteins (shorter than 150 aa) containing the unstructured region
(Shabek et al. 2012). For more information about 26S proteasomes in plants, see
reviews and references within (Sullivan et al. 2003; Vierstra 2003, 2009;
Sadanandom et al. 2012).

What Is Autophagy?

Autophagy is a an important cellular process, and one of the two major degradation
pathways along with UPS, which are involved in maintaining homeostasis during
normal development and response to environmental stresses. Different kinds of
autophagy have been reported, including microautophagy, macroautophagy and
chaperone-mediated autophagy. Here (as in majority of other publications) the term
“autophagy” refers to the best-characterised type, macroautophagy. The general
scheme of the process is common in all eukaryotic organisms (Fig. 7.2). The
process starts by the formation of a cup-like structure that engulfs selected cyto-
plasmic content (cargo) and progresses into a double-membrane autophagosome.
The outer membrane of the autophagosome then fuses with the tonoplast (in yeast
or plant cells) or lysosomal membrane (animal cells), while the inner membrane,
together with the cargo composes the autophagic body (or autolysosome in animal
cells). When such an organelle is present in the vacuolar lumen, its contents are
attacked by vacuolar proteases and then released through vacuolar permeases to the
cytosol for reuse (Klionsky 2007).
Over 30 evolutionary conserved autophagy-related (ATG) proteins play a role in
the “core” autophagy pathway in yeast in mammals. Due to the high conservation of
autophagy among eukaryotes, mechanistic explanations for plants are often taken
from yeast or mammalian models (Thompson and Vierstra 2005; Diaz-Troya
et al. 2008; Nakatogawa et al. 2009; Klionsky et al. 2011). However, some ATG
proteins (e.g. ATG29 and ATG31) are not found in plants. The ATG11 protein was
also considered nonexistent in plants (Suzuki et al. 2007) but a current TAIR search
shows its potential existence and hybrid similarity to ATG17; both proteins have
scaffolding properties in yeast.
Autophagy has an important role in regulating PCD. Both, excessive autophagy
as well as inefficient or defective autophagy may lead to cell death either by self-
eating (excessive autophagy) or by accumulation of damaged proteins and organ-
elles (defective autophagy) (Guiboileau et al. 2010). For clarification of the nomen-
clature relating to different types of PCD in animals, the reader is referred to the
published guidance (Kroemer et al. 2009) and for classification of plant PCD and
their merging with categories of PCD recognised in animals, to the review by van
Doorn (2011).
176 M. Collados Rodrı́guez et al.

Fig. 7.2 Macroautophagy steps. The cup shaped double membrane (phagophore or isolation
membrane) forms and expands trapping portions of the cell material. When the phagophore
seals and produces the (double membrane) autophagosome, this encloses the cargo, which is
transported along with the inner membrane of autophagosome into the vacuole (yeast or plants) or
into the lysosome (animals), where it is recycled

TOR Signaling Pathway and Autophagosome Biogenesis

In yeasts, animals and plants, induction of autophagy is possible after inactivating

the Target of Rapamycin (TOR) Ser/Thr kinase belonging to the family of
phosphatidynositol kinase-related kinases (Noda and Ohsumi 1998; Pattingre
et al. 2008; Liu and Bassham 2010). The TOR’s name results from sensitivity to
macrocyclic bacterial lactone rapamycin. TOR is involved in switching cellular
responses depending on the nutritional status (John et al. 2011). During nutrient-
rich conditions an active TOR (the TORC1 complex) consists of TOR, LST8-1,
LST8-2 and RAPTOR (Regulatory Associated Protein of TOR) (John et al. 2011).
TORC1 together with other kinases promote anabolic processes and cell growth
while keeping autophagy switched off. Arabidopsis contains two RAPTOR pro-
teins, RAPTOR1A and RAPTOR1B, whose function is to recruit TOR substrate
proteins. The complex maintains the ATG1 (Autophagy Related Gene1) and
ATG13 kinases, the latter in complex with ATG17 and ATG101 accessory proteins,
dissociated through hypophosphorylation of ATG1 (one phosphorylated site) and
hyperphosporylation of ATG13 (three phosphorylated sites). In nutrient deficient
7 Role of Autophagy in Plant Nutrient Deficiency 177

Fig. 7.3 Initiation of autophagy and phagophore formation. When nutrients are available, TOR is
active in the complex TORC1 (see text for details) and keeps ATG1 dissociated from ATG13 (and
associated proteins) trough hypo- and hyper-phosphorylation, respectively. During nutrient star-
vation the TORC2 complex is formed and the phosphorylation status reverses, allowing the
complex ATG1-ATG13-ATG17-ATG101 to promote the autophagosome nucleation to the PAS
by C-I and ATG8-PE docking to the phagopore inner surface. The C-I is composed of ATG6,
hypothetically ATG14 and their kinase subunits VPS15 and VPS34. The Atg17 scaffold protein is
a recruiting point for further ATG proteins as ATG9 (in complex with ATG2-ATG18-ATG27),
which mediates membrane delivery contributing to the phagophore elongation. Once the
autophagosome closes it can fuse to the plant vacuole for cargo degradation. Dashed arrows
passing through proteins indicate the proteins, which are governed by the protein present in the tail
of the arrow. TOR Target of rapamycin, PAS phagophore assembly site, C-I phosphatidylinositol
3-kinase complex I, Vps vacuolar protein sorting

conditions, an inactive TOR (the TORC2 complex) includes RICTOR (rapamycin-

insensitive companion of TOR) instead of RAPTORs allowing it to swap the ATG1
and ATG13 phosphorylation status to the active one (autophosphorylation of ATG1
and dephosphorylation of ATG13). This, in turn, promotes the nucleation of the
autophagosome by involvement of a downstream hypothetical (not yet identified)
phosphorylation target. Arabidopsis contains four ATG1 proteins (ATG1a-c and a
truncated ATG1t, present only in plants) and two ATG13 proteins (ATG13a and
ATG13b). The phosphorylation-dependent regulatory mechanism has been con-
firmed for ATG1a and ATG13a (Suttangkakul et al. 2011; Li and Vierstra 2012b).
The activated ATG1-ATG13 kinase complex relocates to the phagophore assembly
site (PAS) and promotes ATG9-mediated membrane delivery to the expanding
phagophore (Fig. 7.3). The ATG1-ATG13 kinase complex also docks to ATG8–
PE bound to the inner surface of the phagophore, and is captured and delivered to
178 M. Collados Rodrı́guez et al.

the vacuole for breakdown (Suttangkakul et al. 2011). In addition, it has been
reported that induction of autophagy upon nutrient starvation and salt stress
depends on the activity of a plasma membrane NADPH oxidase, while induction
of autophagy upon osmotic stress (by mannitol) is NADPH oxidase-independent
(Liu et al. 2009).
At the cellular level, the phagophore assembly site (PAS) initiates the
pre-autophagosomal nucleation and formation of the autophagy unit, the
autophagosome. The PAS is molecularly defined by having ATG1-ATG13-
ATG17-ATG101 and class III phosphatidylinositol 3-kinase (PIK3) complexes
bound by lipidated ATG8 (ATG8-PE). In yeast and animals the PIK3 complex is
composed of ATG6, ATG14 and kinase subunits, VPS15 and VPS34 (Rabinowitz
and White 2010; Johansen and Lamark 2011). In yeast, ATG14 targets the kinase
complex to the probable site of autophagosome formation, while the scaffold
protein, ATG17, organises the PAS and creates a recruiting point for further ATG
proteins, which contribute to the elongation step (Suzuki et al. 2007). A membrane
delivery protein, ATG9, contributes to the phagophore expansion (Li and Vierstra
2012a; Orsi et al. 2012) and works in conjunction with integral membrane protein,
ATG27, and with proteins ATG2 and ATG18. ATG18 attaches phosphatidy-
linositol 3-phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)
P2) and together with ATG2 is involved in retrograde transport of ATG9 (Klionsky
et al. 2011). ATG2 and ATG18 are functionally related, independently localise to
the PAS by means of ATG1, and can interact and form a complex but do not recruit
other proteins to the PAS; ATG1 is drawn by ATG13 to the PAS. The plant
orthologues of ATG14, ATG17, ATG22 and ATG27 remain unidentified (see
also Table 7.1).
The steps described above, namely autophagy initiation and nucleation
(phagophore assembly) are followed by a step called vesicle elongation, where
two pathways utilising UPS-like E1-E3 enzymatic cascades are involved in activa-
tion of two ubiquitin-like proteins, ATG8 and ATG12. In the first cascade the
Cys-protease, ATG4, exposes the C-terminal Gly of ATG8. It allows ATG7 (E1) to
bind to the C-terminus of ATG8 that, in turn, permits ATG3 (E2) to lipidate the
C-terminus of ATG8 with phosphatidylethanolamine (PE). The PE tag enables
ATG8 anchoring into the autophagosomal membrane. In the second cascade,
ATG7 (E1) also participates by activating the ubiquitin like protein ATG12,
enabling its conjugation to ATG10 (E2) which in turn, conjugates ATG12 to
ATG5 (Noda et al. 2013). The ATG12-ATG5 platform oligomerizes with ATG16
and forms the E3-like ATG12-ATG5-ATG16 ligase complex. This complex further
promotes ATG8 lipidation and phagophore growth (Fig. 7.4; Maiuri et al. 2007;
Johansen and Lamark 2011). On the other hand, ATG7 activates also the C-terminal
residue of ATG3 (E2) enabling its conjugation to ATG12. In addition, ATG4
protease acts as deubiquitinating (DUB) enzyme and recycles ATG8 through PE
deconjugation from the outer autophagosomal membrane in a reactive oxygen
species (ROS) regulated way (Rabinowitz and White 2010; Johansen and Lamark
2011).
Table 7.1 Components of the core and selective autophagy in Saccharomyces cerevisiae, Homo
sapiens and Arabidopsis thaliana and the examples of loss of function phenotypes of autophagy
mutants in plants
Saccharomyces Selected
cerevisiae Homo sapiens Arabidopsis Loss of function references
(number of (number of thaliana (number phenotype in related to plant
residues) residues) of residues, gene) plants phenotypes
Initiation (ATG1 kinase complex)a
TOR1 (2470); FRAP1/mTOR TOR (2841, Cell, organ, Leiber
TOR2 (2474) (2549) At1g50030) seed production et al. (2010),
and resistance to Johansen and
osmotic stress Lamark (2011),
decrease. Post- and Deprost
germinative et al. (2007)
arrest in growth
and develop-
ment. Early
senescence
ATG1 (897) ULK1 (1050), ATG1a Accelerated Suttangkakul
ULK2 (1036) (626, At3g61960); senescence, et al. (2011)
ATG1b both fixed C and
(711, At3g53930); N starvation
ATG1c sensitivity
(733, At2g37840);
ATGt
(408, At1g49180)
ATG13 (738) HARBI1/ ATG13a Accelerated Suttangkakul
mATG13 (349) (618, At3g49590); senescence, et al. (2011)
ATG13b both fixed C and and Kim
(625, At3g18770) N starvation et al. (2012)
sensitivity
ATG17 (417) FIP200/RBCC1 ATG17 (three No phenotype –
(1594) potential isoforms: data. Exact
1022, At2g37420; yeast homo-
376, At1g59580 logue still
[weak interaction unclearb
with ATG9]; 1516,
At4g28710)) –see
next column
ATG29 (213) Not found Not found – –
ATG31 (196) Not found Not found – –
Not found ATG101 (218) ATG101 No phenotype –
(215, At5g66930) data. Exact
–see next column yeast homo-
logue still
unclearb
KOG1/RAP- RAPTOR RAPTOR1/1b Post-embryonic Anderson
TOR (1557) (1335) (1344, meristem- et al. (2005)
At3g08850); RAP- driven growth and Deprost
TOR2/1a (1336, affected in dou- et al. (2005)
At5g01770) ble mutants.
Single mutant
phenotypes
differ
(continued)
180 M. Collados Rodrı́guez et al.

Table 7.1 (continued)

Saccharomyces Selected
cerevisiae Homo sapiens Arabidopsis Loss of function references
(number of (number of thaliana (number phenotype in related to plant
residues) residues) of residues, gene) plants phenotypes
LST8 (303) mLST8 (260) LST8-1 Altered apical Moreau
(305, At3g18140); dominance, et al. (2012)
LST8-2 (313; meristem cell
At2g22040) proliferation
and leaf devel-
opment (bushy)
Accumulation
of amino acid
and sensitivity
to osmotic
stress
ATG9 complexc
ATG2 (1592) mATG2a ATG2 (1839; Accelerated Yoshimoto
(1938); mAtg2b At3g19190) senescence. et al. (2009),
(2078) Unrestricted Wang
PCD et al. (2011a,
b), Kim
et al. (2012),
and Liu and
Bassham
(2012)
ATG9 (997) mATG9a (839); ATG9 (866; Unrestricted Hofius
mATG9b (924) At2g31260) PCD et al. (2009),
Hanaoka
et al. (2002),
and Kim
et al. (2012)
ATG18 (500) WIPI1 (previ- ATG18a Accelerated Xiong
ously WIPI49) (425, At3g62770); senescence, et al. (2005),
(446) ATG18b both fixed C and (2007), Liu
(312, At4g30510); N starvation et al. (2009),
ATG18c sensitivity; Wang
(393, At2g40810); enhanced sensi- et al. (2011b),
ATG18d tivity to and Liu and
(391, At3g56440); osmotic, oxida- Bassham
ATG18e tive, drought (2012)
(374, At5g05150); and salt stresses.
ATG18f Unrestricted
(763, At5g54730); PCD
ATG18g
(959, At1g03380);
ATG18h
(927, At1g54710)
ATG21 (496) WIPI2/Atg21 a- Not found – –
f (454- 310)
ATG23 (453) Not found Not found – –
(continued)
Table 7.1 (continued)
Saccharomyces Selected
cerevisiae Homo sapiens Arabidopsis Loss of function references
(number of (number of thaliana (number phenotype in related to plant
residues) residues) of residues, gene) plants phenotypes
Nucleation (class III phosphatidylinositol 3-phosphate kinase complex)d
ATG6/VPS30 BECN1 (450) ATG6 Male sterility Harrison-Lowe
(557) (517, At3g61710) and Olsen
(2008) and
Kim
et al. (2012)
ATG14 (344) ATG14L/ ATG14 No phenotype –
BARKOR (492) (506, At4g01270) data. Exact
homologue still
unclear
VPS15 (1454) PIK3R4/VPS15 VPS15 (1494, Defective pol- Wang
BCL2 (1358) At4g29380) len germination et al. (2012)
VPS34 (224) PIK3c3/VPS34 VPS34a Inhibited Welters
(887) (814, At1g60490)
growth and et al. (1994)
development
ATG20 (640) Not found ATG20 (402; No phenotype
At5g06140) data
ATG27 (271) ATG27 ATG27 (276; Not studied in –
At2g40316) plants
Expansion and autophagosome formation (ATG8 and ATG12 conjugation systems)e
ATG3 (310) ATG3 (314) ATG3 No phenotype –
(313, At5g61500) data
Atg4 (507) ATG4a-d/ ATG4a Accelerated Yoshimoto
autophagin1-4 (467, At2g44140); senescence, oxi- et al. (2004),
(398; 392; 458; ATG4b dative stress, Scherz-
474 respectively) (477, At3g59950) fixed C and N Shouval
starvation et al. (2007),
sensitivity Chung
et al. (2010),
Kim
et al. (2012),
and Tsai
et al. (2012)
ATG5 (294) ATG5 (275) ATG5 Accelerated Thompson
(337, At5g17290) senescence, et al. (2005),
both fixed C and Phillips
N starvation et al. (2008),
sensitivity; Yoshimoto
enhanced sensi- et al. (2009),
tivity to heat, Chung
oxidative, et al. (2010),
drought and salt Kwon
stresses. et al. (2010),
Delayed differ- Suttangkakul
entiation of tra- et al. (2011),
cheary ele- Kim
ments. et al. (2012),
Unrestricted Liu and
PCD Bassham
(2012),
(continued)
182 M. Collados Rodrı́guez et al.

Table 7.1 (continued)

Saccharomyces Selected
cerevisiae Homo sapiens Arabidopsis Loss of function references
(number of (number of thaliana (number phenotype in related to plant
residues) residues) of residues, gene) plants phenotypes
Tsai
et al. (2013),
Minina
et al. (2013),
and Zhou
et al. (2013)
ATG7 (630) ATG7 (703; 676; ATG7 Accelerated Doelling
623) (697, At5g45900) senescence, et al. (2002),
both fixed C and Thompson
N starvation et al. (2005),
sensitivity; Phillips
enhanced sensi- et al. (2008),
tivity to heat, Chung
oxidative, et al. (2010),
drought and salt Lenz
stresses. et al. (2011),
Unrestricted Suttangkakul
PCD et al. (2011),
Enhanced ROS Kim
production et al. (2012), Li
and Vierstra
(2012b), Liu
and Bassham
(2012), Minina
et al. (2013),
Zhou
et al. (2013),
and Hofius
et al. (2009)
ATG8 (117) MAPLC3A ATG8a Accelerated Thompson and
(125) (137, At4g21980); senescence, Vierstra (2005)
ATG8b both fixed C and and Liu and
(122, At4g04620); N starvation Bassham
ATG8c sensitivity (2012)
MAPLC3B (133, At1g62040); No phenotype
(125) ATG8d data. All family
MAPLC3C (120, At2g05630); mutant
(147) ATG8e necessary
(122, At2g45170);
GABARAP
ATG8f
(117)
(121, At4g16520);
GABARAP- ATG8g
L1/GEC-1/ (121, At3g60640);
ATG8L (117) ATG8h
GABAPAP-L2/ (120, At3g06420);
GATE-16/ ATG8i
GEF2 (117) (116, At3g15580)
GABARAP-L3
(117)
(continued)
Table 7.1 (continued)
Saccharomyces Selected
cerevisiae Homo sapiens Arabidopsis Loss of function references
(number of (number of thaliana (number phenotype in related to plant
residues) residues) of residues, gene) plants phenotypes
ATG10 (167) ATG10 (220) ATG10 Accelerated Tsai
(226, At3g07525) senescence, et al. (2012),
both fixed C and Phillips
N starvation et al. (2008),
sensitivity. Chung
Unrestricted et al. (2010),
PCD Kim
et al. (2012),
and Liu and
Bassham
(2012)
ATG12 (186) ATG12 (140; ATG12a Accelerated Thompson
74) (96, At1g54210); senescence, et al. (2005),
ATG12b both fixed C and Chung
(994, At3g13970) N starvation et al. (2010),
sensitivity and Kim
et al. (2012)
ATG16 (150) ATG16 1 a-f ATG16 No phenotype –
(607-284); (509, At5g50230) data
ATG16 2 a-f
(619-131)
Autophagy receptors and cargo ligandsf
ATG11 (1178) ATG11 (1591) ATG11 (1148, No phenotype
At4g30790) data
Exact yeast
homologue still
unclearb
ATG19 (415) Not found Not found –
ATG26 (1198) Not found Not found –
ATG30 (384) Not found Not found –
ATG32 (529) NIX/BNIP3L/ ATG32 (953; No phenotype
ATG32 (219) At4g29060) data
Exact homo-
logue still
unclear
ATG34 (412) Not found ATG34 (614; No phenotype
AT1G20950) data
Exact homo-
logue still
unclear
Not found p62/SQSTM NBR1 (704; Increased sensi- Zhou
(440) At4g24690) tivity to some et al. (2013)
Not found NBR1 (966) abiotic stresses
Not found (only NDP52 (446) Not found –
NDP52 gives optineurin (577)
some low hits) TBK (729)
UBQL4 (601)
(continued)
184 M. Collados Rodrı́guez et al.

Table 7.1 (continued)

Saccharomyces Selected
cerevisiae Homo sapiens Arabidopsis Loss of function references
(number of (number of thaliana (number phenotype in related to plant
residues) residues) of residues, gene) plants phenotypes
Bph1p (2167) ALFY (3601) At4g02660; Not studied in
At1g03060 plants. No phe-
notype data.
Exact homo-
logue still
unclear
Some of the proteins cited in this table are represented by more than one alternative spliced
isoform; only the longest one is indicated in brackets. For the function description of most of the
proteins shown here see Klionsky et al. (2011)
a
FRAP1 [FKBP12-rapamycin complex-associated protein (FK506-binding protein 12-rapamycin
complex-associated protein 1)], mTOR (mammalian target of rapamycin), ATG (autophagy-related
protein), ULK1 (Unc-51-like kinase 1), ATG1s ATG1t is represented longer than in Suttangkakul et
al. (2011) because manual removing of the introns in this locus reveals cDNA predicted to encode
408 aa protein (instead of 267 aa). After ELM (EukarioticLinear Motif) domain search a conflict
with Li and Vierstra 2012 appears because the regulatory domain exists although the protein is
shorten in the middle (mostly disordered region), C (carbon), N (nitrogen), LST8 (Lethal with
SEC13 protein 8), RAPTOR (regulatory-associated protein of TOR), FIP200 (FAK family kinase-
interacting protein of 200 kDa), RBCC1 (RB1-inducible coiled-coil protein 1)
b
Suttangkakul et al. (2011) comment to have found possible ATG17 and ATG101 orthologues but
do not state exactly which one. Similarly we failed to identify the ATG29 and ATG31 plant
orthologues but we propose homologues of AtATG101, AtATG11, AtATG17, and ATG32
(At4g29060 has mitochondrial and chloroplastic location based on BLAST, MOTIFSCAN,
TMPRED and ELM search/predictions). Souval et al. (2007) showed oxidative stress sensitivity
in atg4 mutants (a and b) of mammal cells
c
ATG9 in plant ATG9 no transmembrane region is predicted by ELM (weakly by TMPRED),
hypothetically plant ATG9 may attach to the membrane by means of ATG27, WIPI1 (WD repeat
domain, phosphoinositide interacting 1)
d
BECN1 (BECLIN 1), BARKOR (Beclin 1-associated autophagy-related key regulator), PIK3R4
(phosphoinositide 3-kinase regulatory subunit 4), VPS15 (vacuolar protein sorting-associated
protein 15), ATG27 according to Yen et al. (2007) the N terminus is in the vesicular lumen while
that C terminus is in the cytosolic part in yeast; TM and ELM predicts that and plant Nt also ends into
the membrane and has a Ct TM. PIK3c3 (phosphatidylinositol 3-kinase catalytic/class subunit type 3)
e
LC3 (light chain 3), GABARAP (gamma-aminobutyrate receptor associated protein), GATE16
(golgi-associated ATPase enhancer of 16 kDa), GEF2 (ganglioside expression factor 2)
f
BNIP3L (BCL2/NIP3-like), SQSTM (Sequestrosome), NBR1 [next to BRCA1 (breast cancer)
gene 1], TBK (tank binding kinase), UBQL4 (Ubiquilin4), Bph1p (beige protein homologue 1),
ALFY (autophagy linked FYVE protein)

How Many Autophagy Forms Exist in Plants?

The process of autophagy in plants was initially considered as non-selective bulk

degradation of cellular contents, however now it is clear that the process is selective
(Floyd et al. 2012; Li and Vierstra 2012a). For example, the following specific
forms of autophagy were reported each involved in degradation of a specific target,
7 Role of Autophagy in Plant Nutrient Deficiency 185

Fig. 7.4 Recruitments of ATG8 and ATG12 proteins to the autophagosome. The ubiquitin-like
proteins, ATG12 and ATG8, are processed in a UPS-like E1-E3 manner through two separate
conjugation cascades with some overlapping elements. In the first, ATG12 covalently linked to
ATG5 (by subsequent action of E1-like ATG7 and E2-like ATG10) promotes formation of
ATG12-ATG5-ATG16 complex needed for creation of pre-autophagosomal structure. In the
second system, cleavage of ATG8 by ATG4 enables attachment of phosphatidylethanolamine
(PE) to the C-terminus of ATG8 by subsequent action of E1-like ATG7 and E2-like ATG3.
Activated ATG8 (ATG8-PE) associates with the expanding autophagosome membranes and
remains through autophagosome maturation until fusion with the vacuole. This property is
commonly exploited in many labs for reliable autophagy monitoring (Figure based on Suzuki
et al. (2007) and Li and Vierstra (2012a))

such as mitochondria (mitophagy), peroxisomes (pexophagy), ribosomes

(ribophagy), ER (reticulophagy or ERphagy) lipid droplets (lipophagy), pathogens
(xenophagy), protein aggregates (aggrephagy) and choroplast material
(chlorophagy). In addition, in yeast a mechanism also exists called CVT (Cyto-
plasm to Vacuole Targeting; (Bassham et al. 2006), which is only hypothesised in
plants (Nakatogawa et al. 2009; Li and Vierstra 2012a), leaving open the question
of how functional vacuolar proteins reach the plant vacuole.
Work on mitophagy in yeast indicated that the ATG32 mitochondrial outer
membrane protein is the main factor involved in selective degradation in fungal
mitochondria. Following the induction of mitophagy, Atg32 binds Atg11, an
adaptor protein for selective types of autophagy, then it is recruited to mitochondria
and imported into the vacuole along with mitochondria. Therefore, in yeast Atg32
confers selectivity for mitochondrial sequestration as a cargo and is necessary
for recruitment of this organelle by the autophagy machinery for mitophagy
186 M. Collados Rodrı́guez et al.

(Kanki et al. 2009). In plants, much less work on mitophagy has been performed.
Electron microscopy pictures of mitochondria being fused to acidic compartments
(presumably autophagosomes) suggest that mitophagy also takes place in plant
cells (Toyooka et al. 2006). However, autophagosomes colocalise preferentially
with protein aggregates rather than with mitochondria. The autophagy postulated
by Toyooka et al. (2006) seems to be independent of starvation. Although the role
of mitochondria in oxidative stress-induced autophagy and the process of
mitophagy in plants have been recently reviewed (Minibayeva et al. 2012), further
studies on causes of mitophagy in higher plants are desirable.
Literature related to plant pexophagy (selective degradation of peroxisomes) is
limited. It has been shown that the process of pexophagy contributes to virulence of
fungal pathogen, Colletotrichum orbiculare, since the atg26 mutant of this patho-
gen, specifically affected in peroxisomes failed to penetrate the host cells (Asakura
et al. 2009).
Autophagic degradation of chloroplastic material (chlorophagy) via Rubisco-
containing bodies (RCBs) is specifically linked to carbon deficiency (caused by
darkness) but not to nitrogen deficiency. This process supports energy availability
at night for normal growth by providing amino acids (Izumi et al. 2013). On the
other hand, autophagy also contributes to leaf starch degradation at night (Wang
et al. 2013). This process takes place in vacuoles and is independent of the classic
chloroplast pathway of starch degradation. No proof for degradation of entire
chloroplasts is available. On the other hand, vacuolar localisation of the chloro-
plasts in protoplasts from the mesophyll leaves of the senescing wheat leaves seem
to support such possibility (Wittenbach et al. 1982). Plastid degradation has mostly
been studied in the conditions of starvation or in leaves undergoing senescence. It
has been suggested that remobilisation of Rubisco in RCBs represents the first step
of chloroplasts degradation i.e. chloroplast downsizing due to RCB formation and
subsequent deposition into the vacuole (Wada et al. 2009). Rubisco is the most
abundant plant protein and catalyses carboxylation and oxidation competing reac-
tions of photosynthesis. It is a substantial nitrogen source stored in chloroplasts that
the plant may use in case of N deficiency. Plants rely on chloroplasts to survive;
therefore they undergo senescence only when the day becomes short and the
respiratory period surpasses the photosynthetic period. During stress and senes-
cence a compromise is made between maintaining functional chloroplasts and
reusing the nutrients accumulated in the form of Rubisco (Wada et al. 2009). The
extraplastidic degradation processes, namely chlorophagy and RCB rely on
autophagy, demonstrated as some autophagy-deficient mutants (atg5) do not
show RBCs (Ishida et al. 2008). Moreover, it was recently shown that degradation
of stromal proteins takes place within chloroplasts largely via the autophagy-
independent process. However, autophagy is required for the complete degradation
of these proteins (Lee et al. 2013).
The existence in plants of the ribophagy-like mechanism, targeting ribosomes
for recycling under normal growth conditions and similar to ribophagy observed in
yeast under nutrients deficiency, has been suggested (Hillwig et al. 2011). The
7 Role of Autophagy in Plant Nutrient Deficiency 187

authors suggest that this mechanism is not only active during nutritional stress, but
also has a housekeeping role. In addition, autophagy was demonstrated to be
responsible for delivering endoplasmic reticulum (ER) to the vacuole during ER
stress (Liu et al. 2012).
Some differences in autophagy seem to depend on the plant species (Bassham
et al. 2006; Toyooka et al. 2006). These observations point to the possible existence
of lysosome/endosome-like organelles in plants such as tobacco (which also fuse
with the central vacuole) but not in most species such as barley or Arabidopsis.
Toyooka et al. (2006) observed two transport pathways in BY-2 tobacco cells:
direct delivery of autophagosomes to the central vacuole (in the absence of E-64
autophagy inhibitor) versus engulfment of autophagosomes by small vacuoles after
E-64 treatment. This second pathway may be a consequence of incomplete
autophagy inhibition, which could be solved with 3MA (3-methyl-adenine) but
further research on this possibility is required. It is possible that the time of
observation of E-64 treated plants has to be varied (to extract the same conclusions)
depending on the generation time of the plant. In other words, 24 h of E-64
treatment for Arabidopsis (a plant with a 3 month generation time) may have
proportionally stronger effect than such treatment for tobacco (a plants with
6 month generation time). Another reason for discrepancies may be the comparison
of protoplast or BY-2 cells obtained results with those derived from plant organs.

Genes for Plant Autophagy and Typical Phenotypes of atg

Mutants

Nomenclature of autophagy related genes (ATG, some were previously called

APG) originates from yeast heterologous comparisons (Bassham et al. 2006), occa-
sionally leading to confusion between unique yeast and metazoan proteins with
plant ones. A common subset of over 30 proteins is required for all forms of
autophagy (Table 7.1). The significant subset of these proteins represent the com-
ponents of the so called “core autophagy” machinery required for both selective and
non-selective autophagy (Lynch-Day and Klionsky 2010). These proteins belong to
five large complexes responsible for the subsequent steps of autophagosome for-
mation, namely induction, nucleation and extension. In addition to this “core”
subset more proteins are required for certain pathways. Some proteins are present
only in one group of organisms and it is difficult to identify their orthologues
because of low sequence conservation. These proteins are involved in, so called,
selective autophagy and perform a quality control function by distinguishing
between cargoes ready for degradation and their functional counterparts (see
review: Schreiber and Peter 2013). Mutations in ATG proteins impair autophagy
in different ways. The effects depend to which complex the impaired protein
belongs. For example, atg5 or atg7 mutations cause the accumulation of
188 M. Collados Rodrı́guez et al.

downstream pathway proteins and speed up the plant life span (Minina et al. 2013).
Some typical phenotypes of Arabidopsis autophagy mutants are shown in Table 7.1.

Role of Autophagy in Plant Response to Nutrient Deficiency

Plants deficient in autophagy (atg mutants) do not show evident phenotypic differ-
ences from wild type during normal non-stress conditions (see Table 7.1). How-
ever, some atg mutants show accelerated life cycle and early senescence when the
light intensity is low and during short day photoperiods (Thompson et al. 2005).
This suggests that a basal or constitutive autophagy is important for normal plant
growth (Minina et al. 2013). The senescence features, such as leaf yellowing in a
consequence of Rubisco breakdown and chlorophyll dismantling, are more evident
in atg mutants than in the wild type grown under nitrogen (N) or carbon
(C) deficiency (Doelling et al. 2002; Hanaoka et al. 2002; Thompson et al. 2005;
Phillips et al. 2008). Apparently, autophagy is important for recycling and deliver-
ing nutrients to the reproductive organs during senescence and as such it can be
considered as a nutrient redistribution process. In addition, autophagy plays an
important role in N management at the whole-plant level through the control of
remobilisation, under both limiting and ample nitrate conditions (Guiboileau
et al. 2012). For example, heterologous expression of soybean ATG8c in
Arabidopsis led to better performance of the transgenic lines under both starvation
and normal conditions (Xia et al. 2012). Although the protein and soluble sugar
concentrations were similar in the wild type and transgenic line, the fresh weight of
the transgenic lines was significantly larger in both, N sufficient and N deficient
conditions. The transgenic plants survived the period of carbon-limiting conditions
induced by extended darkness much better than the wild type and promptly
recovered and resumed growth when moved back to normal conditions. Compar-
ison of the growth parameters in soil under a long-day photoperiod indicated that
the transgenic plants grew faster than the wild type plants, reached a larger size
before bolting, entered the reproductive stage slightly earlier, and produced more
flowers, siliques and seeds. The authors concluded that over-expression of soybean
ATG8 in Arabidopsis promoted the vegetative growth and facilitated the transition
into reproductive stage.
Similarly, Arabidopsis plants overproducing ATG8 protein fused on its N
terminus to green fluorescent protein (GFP) showed enhanced growth and leaf
size, accelerated flowering under nutrient-limiting and short-day growth, however
they were also slightly more sensitive to mild salt and osmotic stress (Slavikova
et al. 2008). This study also suggested that ATG8 participates in cytokinin-
mediated root-shoot communication possibly by sequestration of proteins involved
in cytokinin transport or signaling.
It has been shown that ATG18a protein from Arabidopsis is required for the
formation of autophagosomes under nitrogen deficiency and sucrose starvation
(Xiong et al. 2005). Interestingly, the mutants with silenced expression of
7 Role of Autophagy in Plant Nutrient Deficiency 189

ATG18a are more sensitive to high salt and drought than the wild-type plants,
demonstrating a role for autophagy in the response to these stresses (Liu
et al. 2009). The same authors also reported that induction of autophagy upon
nutrient starvation and salt stress depends on the activity of plasma membrane
NADPH oxidase and that induction of autophagy upon osmotic stress (caused by
mannitol) is NADPH oxidase-independent.
Nutrient deficit up-regulates autophagy. For example, induced transcription of
some ATG genes (e.g. ATG4, ATG8a-i, ATG3, ATG7) was observed upon C
starvation in Arabidopsis (Rose et al. 2006), and of ATG8 upon N and sulfur
starvation in tobacco (Zientara-Rytter et al. 2011). Additionally, some atg mutants
(e.g. atg7, atg5, atg10) are hypersensitive to nutrient deficit (Li and Vierstra
2012a).
Autophagy participation in plant responses to other nutrient stresses such as
phosphorus or iron starvation has been discussed only in the context of participating
Ub-ligases and the UPS (Lyzenga and Stone 2012; Rojas-Triana et al. 2013).
However, no attempts to test the role of autophagy in this process were undertaken.
Interestingly, it has been recently reported that sulfide, which is generated from
cysteine by cysteine desulfhydrase in the cytosol of Arabidopsis plants, acts as a
negative regulator of autophagy and that this effect is independent from the sulfur
status of the plant (Alvarez et al. 2012a, b). Such a conclusion came from the
observation that plants defective in the activity of cysteine desulfhydrase (des1
mutants) show premature leave senescence and increased expression of senescence
associated genes. In addition, externally added hydrogen sulfide was able to alle-
viate some of the observed phenotypes. The mechanism of action of sulfide is
completely unknown. However, the authors suggested that some enzymes neces-
sary for autophagy process might be possible targets of such regulation
(e.g. through reversible S-thiolation), for example E1 and E2 enzymes involved
in ubiquitination or ATG4 cysteine protease. It is worthwhile mentioning that
hydrogen sulfide is already recognised as an important signaling molecule in
mammalian systems (Lowicka and Beltowski 2007; Szabo 2007; Gadalla and
Snyder 2010).
In conclusion, autophagy is an important process involved in plant response to
nutritional stresses. Emerging data suggest that the controlled enhancement of
autophagy can be exploited for improvement of crop performance in normal and
nutrient-limiting conditions.

Selective Autophagy

The process of selective autophagy was first described in yeast (see above, CVT).
Paradoxically, this process is a part of biosynthetic pathway and it is involved in a
selective delivery of two lysosomal enzymes to the vacuole described neither for
animals nor plants (Lynch-Day and Klionsky 2010). As mentioned before, a
number of organelles, storage structures, pathogens or protein aggregates are
190 M. Collados Rodrı́guez et al.

known to be specifically targeted by autophagy (Weidberg et al. 2011). Each

specific cargo was used as a base of providing the names for different autophagy
types: mitophagy, pexophagy, ribophagy, xenophagy, aggrephagy etc. (see also
above). In most cases the molecular machinery involved in specificity of cargo
recognition is not characterised. However, it is evident that the various cargos in
animal cells are specifically recognised and targeted for lysosomal degradation by
the action of specific cargo-recognising receptors, called selective autophagy
receptors (Lynch-Day and Klionsky 2010; Johansen and Lamark 2011; Behrends
and Fulda 2012; Isakson et al. 2013). The main feature of these proteins is their
ability to interact directly with both the ATG8 proteins through the LIR (LC3-
interating region) motif and the cargo. The selective cargo receptors are degraded
along with the cargo in the autophagosomes (Komatsu and Ichimura 2010). Rec-
ognition of the cargo designed for degradation from the one needed in the cell
requires Ub as a specific ligand. The ubiquitinated cargo is specifically recognised
by cargo receptors, such as p62, NBR1, NDP52, possess the Ub-binding domain
(Johansen and Lamark 2011). Yet, some cargoes can be targeted for lysosomal
degradation in a Ub-independent mechanism and some selective autophagy recep-
tors, which have a Ub-binding domain, directly recognise cargo in a
Ub-independent mechanism (Komatsu et al. 2010). In addition to cargo receptors
able to bind to both the cargo and ATG8 proteins, another category of proteins,
autophagy adaptors (defined as scaffold proteins capable of binding both the cargo-
receptor complex and the core components of autophagic machinery) have been
characterised. The best described examples of such proteins are the mammalian
ALFY (autophagy-linked FYVE protein) and ATG11 from yeast (Lynch-Day and
Klionsky 2010; Isakson et al. 2013).
Figure 7.5 illustrates selective autophagy types reported for plants. Some have
already been discussed above. There is no indication that in every case a specific
autophagy receptor exists. For example, although importance of lipophagy in
animals is well documented (Christian et al. 2013), few data for plants are available
and the presence of this process in plants can only be inferred by accumulation of
lipid peroxides in atg mutants (Xiong et al. 2007). The involvement of mammalian
p62 and NBR1 proteins in degradation of protein aggregates is well known and
therefore it seems reasonable that the plant NBR1-like proteins work as selective
receptors for protein aggregates and possibly also for single Ub-tagged proteins.
The involvement of autophagy in response to biotic stresses has been well studied
in plants but the authors of this review are not aware of any report about direct
targeting of viral or bacterial pathogens by selective cargo receptors. Xenophagy
involving specific cargo receptors is reported for animal pathogens only (Mostowy
et al. 2011). An interesting example are anthocyanins, plant pigments of various
functions maintained in the vacuole: a significant change in anthocyanin profiles
(overall reduction) was observed in atg mutants (Pourcel et al. 2010). Although no
direct evidence exists for the specific authophagy-dependent vacuolar transport of
anthocyanins, it is tempting to speculate that it might be an example of autophagy
involvement in a plant anabolic process (compare to CVT in yeast as an example of
such a process).
7 Role of Autophagy in Plant Nutrient Deficiency 191

Fig. 7.5 Examples of selective autophagy in plants

The last example of selective autophagy shown in Fig. 7.5 deals with haem and
porphyrins degradation (Vanhee et al. 2011). The authors indicated that the
membrane-located tryptophan-rich sensory protein (TPSO), which is induced by
various stresses, is in fact involved in haeme scavenging and targeting for degra-
dation via autophagy. TSPO has the LIR motif and co-localizes with ATG8 in plant
cells. In addition, haem-binding and functional LIR motif are necessary for
autophagy-dependent degradation of TSPO in vitro. It is probable that the TSPO
protein is also involved in binding and degradation of porphyrins, because over-
expression of TSPO in plants alleviated porphyrin-induced cytotoxity in plant cells.
Although the study still leaves many unanswered questions, such as how the plastid
– synthesised haem reaches the cytosol, it revealed the novel autophagy-based
regulatory mechanisms involved in switching off the response to stress when no
longer needed. The increased requirement for ROS scavengers (appearing as a
result of stress) leads to the increased requirements for porphyrin cofactors and
up-regulated tetrapyrrole biosynthesis. These compounds are cytotoxic and their
level needs to be carefully regulated. TSPO binds porphyrins and is along with this
cargo, degraded via autophagy.
The plant-specific ATI1 and ATI2 proteins (autophagy interacting proteins 1 and
2), identified in young seedlings upon carbon starvation were not included in
Fig. 7.5, however they both should be considered a part of selective autophagy
192 M. Collados Rodrı́guez et al.

and both bind ATG8 (Honig et al. 2012a, b). Although in the case of these proteins
the cargo is unknown, and it was speculated that ATI1 and ATI2 are involved either
in removal of germination inhibiting compounds and their deposition into the
vacuole or in transport of germination-promoting compounds to the vacuole.
They probably transport membrane proteins or non-protein compounds into the
vacuole, especially after C starvation.
Additionally, the tomato AGC protein kinase Adi3, known to function as a
suppressor of programmed cell death, was shown to bind to ATG8 (Devarenne
2011). Although the interaction was not further investigated, the phenotypes of the
double atg (atg3, atg6 and atg7) and adi3 mutants suggest that Adi3 might work in
coordination with autophagy to control cell death. However, it is unclear if it can be
considered a selective autophagy cargo receptor.
The evolutionary conserved NBR1-like proteins, which were described also in
plants as selective autophagy cargo receptors, will be discussed below.
An interesting example of the specific target of autophagic degradation in plants
is ARGONAUTE1 (AGO1), a key component of RNA-induced silencing complex
(RISC) (Derrien et al. 2012). Autophagy is involved in the turnover of this protein.
Thus, it is appealing to hypothesize that autophagy is involved in degradation of
proteins from this family during cellular stress, namely when the fast
reprogramming of the RISC is needed following rapid changes of the population
of miRNA and siRNA.
Knowledge of the selective autophagy cargo receptors in plants is very limited
and extensive work is needed to fill the multiple gaps in this field.

NBR1-Like Proteins in Plants

The plant NBR1-like selective cargo receptors, AtNBR1 and Joka2 were identified
independently by two research groups in Arabidopsis (Svenning et al. 2011) and
tobacco (Zientara-Rytter et al. 2011), respectively. Plant NBR1-like proteins seem
to be good autophagy markers (Svenning et al. 2011; Zientara-Rytter et al. 2011;
Minina et al. 2013). Moreover, it has been shown that AtNBR1, similarly to the
mammalian p62 and NBR1, is degraded by the autophagy pathway (Minina
et al. 2013). With respect to the phenotype, the enhanced sensitivity of nbr1 mutants
to heat, oxidative, drought and salt stress was reported (Zhou et al. 2013).
NBR1-like proteins identified in plants are functional hybrids of the mammalian
NBR1 (next to breast cancer 1 gene 1) and p62/SQSTM1 (Sequestosome 1) and
have a modular composition (Fig. 7.6). They have four main domains: PB1 (Phox
and Bem1), ZZ (ZZ-type Zinc finger domain), NBR1/FW and double UBA
(ubiquitin-associated domain), UBA1 and UBA2. The roles of individual domains
and the protein partners specifically binding to these domains are characterised in
mammalian cargo receptors (Kirkin et al. 2009; Mardakheh et al. 2010; Salminen
et al. 2012).
7 Role of Autophagy in Plant Nutrient Deficiency 193

Fig. 7.6 The proteins are drawn to scale. The PB1, ZZ, NBR1, UBA1 and UBA2 domains are
marked. The experimentally verified nuclear localization signals (NLS) and mapped LIR motifs
are shown

PB1 Domain

The PB1 domain is a well-known interaction module, highly conserved among

animals, plants, fungi and amoebae (Sumimoto et al. 2007) that can interact with
various proteins by creating salt bridges between basic and acidic residues located
on the PB1 domain to modify their functions. The PB1 domains are present in
nearly 200 proteins participating in diverse biological processes in all eukaryotes
(Letunic et al. 2002). The PB1 domains could be classified into three groups based
on structure. Type-A is represented by PB1 domains possessing acidic OPCA
motif. Type-B includes PB1 domains with lysine residue/s on the first beta strand
carrying basic charge. The third group contains PB1 domains with both acidic and
basic clusters (Type-A+B).
The PB1 domain of human NBR1 has been classified as Type-A (Muller
et al. 2006). No homodimers can be formed by this domain, while the PB1 domain
of human p62 is classified as Type-A+B, having both acidic and basic clusters, and
is involved in the formation of p62-p62 homodimers and in p62-NBR1
heterodimers. Similarly to p62, AtNBR1 (NBR1 from Arabidopsis) and Joka2
(a tobacco homologue) have been shown to form homodimers through the PB1
domain (Svenning et al. 2011; Zientara-Rytter et al. 2011).
Oligomerisation of the selective autophagy cargo receptors is crucial for degra-
dation process since mutants of p62 and AtNBR1, with substituted amino acids that
are particularly important for polymerisation, lost the ability to aggregate and are
not degraded by autophagy. Consequently, the human NBR1, possessing only an
OPCA motif is not able to self-interact through PB1 and an additional CC (coiled-
coil) region is required for self-oligomerisation.
194 M. Collados Rodrı́guez et al.

ZZ Domain

The Zinc finger (ZZ) domain is located after the N-terminal PB1 domain. The role
of the ZZ domain in the autophagy pathway is unknown, since the deletion of ZZ
region from the p62 protein did not disturb aggregation of p62 in HeLa cells and
only the PB1 and UBA domains are needed for p62 to form cytoplasmic bodies
required for autophagy clearance (Bjorkoy et al. 2005). Nevertheless, in mammals,
the ZZ domain of p62 interacts with RIP1 (receptor-interacting protein) and TRAF6
(TNF receptor associated factor 6) to modulate the NFκB pathway (Sanz
et al. 2000). In plants, the ZZ domain of Joka2 has been shown to interact with
the tobacco UP9C, a homologue of LSU1, which is implicated in the response to
sulfur deficiency (Wawrzynska et al. 2005; Zientara-Rytter et al. 2011). Joka2 was
identified in yeast two hybrid search with UP9C (up-regulated by sulfur deficiency)
as a bait using the cDNA library from tobacco (Nicotiana plumbaginifolia) seed-
lings grown in nutrient sufficient conditions (Lewandowska et al. 2010). Interac-
tions between proteins from both families have been confirmed in planta using
N. tobaccum and Arabidopsis orthologues by bimolecular fluorescence comple-
mentation (BiFC) (K. Zientara-Rytter, A. Sirko – unpublished). Due to the
unknown molecular function of UP9/LSU the role of such binding has yet to be
discovered.

NBR1/FW Domain

The NBR1/FW domain, (consisting of two sets of three β-strands separated by an

unstructural linker) is located between the ZZ and UBA domains and contains four
highly conserved tryptophan (W) residues. The NBR1/FW domain is present in all
NBR1-like proteins throughout the eukaryotic kingdom (Svenning et al. 2011). The
function of the NBR1/FW domain is unknown. However, it has been recently
shown that the mammalian selective autophagy receptor, NBR1, binds to the
light chain of the microtubule-associated protein MAP1B through the NBR1/FW
domain (Marchbank et al. 2012).

UBA Domains

The ubiquitin-binding domain (UBA) is short, containing only about 45 residues. It

is a commonly present in many proteins, which are involved in degradation
pathways requiring Ub tagged to the substrate as a signal for its degradation. In
the selective autophagy receptors the UBA domain is located at the C-terminus as a
single domain as in p62 or it could be duplicated as in AtNBR1, Joka2 and other
plant homologues. The UBA domain present in selective autophagy cargo receptors
7 Role of Autophagy in Plant Nutrient Deficiency 195

prefers (poly)Ub tails bound by lysine in position 63 (K-63) and has a moderate
affinity to the Ub bound to the lysine residue in position 48 (Johansen and Lamark
2011). The UBA domains serve to ensure transport of cargo to the appropriate
process (autophagy or UPS) (Raasi et al. 2005). Deletion of UBA domains
completely inhibit the function of selective autophagy receptors since they are
unable to recognise and bind substrates for autophagy clearance (Seibenhener
et al. 2004).

LIR Motif

The LIR (LC3-interacting region) motif is a short (eight amino acids) sequence
responsible for directly binding of the selective autophagy receptors to ATG8
proteins attached to autophagosomal membranes. Interaction between the LIR
motif of the receptor and the ATG8 proteins is crucial for targeting cargo for
degradation. In a typical LIR motif (X 1X 2X 3W/F/YX1X2L/I/VX3) the hydro-
phobic amino acids (W, F or Y and L, I or V) are essential for binding and occupy
the W-site and L-site created on the surface of ATG8 proteins. Amino acids in
positions X 1, X 2, X 3, X1, X2, X3 are not crucial for interaction but they can
enhance the strength of binding, particularly if acidic residues (D or E) are located
at positions X 1, X 2 and X 3 or X1 (Noda et al. 2008, 2010; Yamaguchi
et al. 2010; Johansen and Lamark 2011). For the AtNBR1 protein, an established
LIR motif (VSEWDPIL) was identified in a strongly conserved region between the
UBA1 and UBA2 domains.
A high conservation of the interaction between autophagy receptors and ATG8
proteins has been revealed. In HeLa cells, AtNBR1 is sequestered into
autophagosomes when human GABARAP subfamily members are also over-
expressed, demonstrating high evolutionary conservation of the selective
autophagy pathway and high structural similarity of ATG8 proteins among differ-
ent kingdoms (Svenning et al. 2011).

Nuclear Localisation Signals

Selective autophagy cargo receptors can be involved in several different pathways

in addition to autophagy. Human p62, for example, also transports substrates
designated for degradation to the proteasome and continuously shuttles between
cytoplasm and nucleus. Proteins larger than 40 kDa, such as p62, are actively
transported between these two cellular compartments and need nuclear import
and export signals. Both importins and exportins transfer cargo protein into the
nucleus or cytoplasm in cooperation with the nuclear pore complex. Nuclear
localization signals (NLS) and nuclear export signal (NES) are short well-described
sequences recognised by nuclear-cytosolic transport receptors. Two functional
196 M. Collados Rodrı́guez et al.

basic monopartite NLS signals (NLS1, NLS2) and one NES motif are present in
p62 (Pankiv et al. 2010), however the role of p62 in the nucleus is unclear. It is
speculated that, since in the nucleus in contrast to the cytoplasm, only proteasomal
degradation system takes place, p62 may act as a polyubiquitin targeting factor
carrying substrates to a nuclear pool of proteasomes. It could be also involved in the
transport of other proteins or it could act as a sensor of nuclear and cytosolic
proteotoxic stress. The role of NLS and NES motifs and the significance of nuclear
localization have not yet been investigated in NBR1-like proteins.

Conclusions and Future Perspectives

Within this chapter multiple reasons to continue studies on autophagy in
plants have been highlighted. Autophagy plays a protective role during
nutrient starvation (and other environmental stresses) by enhancing the
recycling of unwanted cellular materials. The process is evolutionarily con-
served. In animals, induction of autophagy results in extension of the life span
(Madeo et al. 2010; Rubinsztein et al. 2011), while defective autophagy has
been related to cancer, various neurodegenerative and immune-related dis-
eases (Todde et al. 2009). Various genetic, pharmacological or nutritional
approaches designed to improve autophagy in human may be a future strategy
of choice to avoid or delay aging-associated pathologies. Such potential
therapies still need further extensive investigation. Considering the strong
conservation of the process and the participating proteins, studies on
autophagy in plants can give clues to this area of medical research by
providing inexpensive testing models for novel drugs and treatments. Addi-
tionally, in plants, autophagy is involved in regulation of the lifespan (Minina
et al. 2013).
Sufficient knowledge of these mechanisms may allow for controlled
genetic or molecular manipulations of plant metabolism. All modifications
leading to moderate up-regulation of autophagy may have a positive impact
on plant biomass and crop yield. Studies on regulation of the specificity of
autophagy and its role in maintaining proteostasis (protein homeostasis), as
well as overall homeostasis, have become very active areas of research.
Nevertheless many important questions remain unanswered. The problems
still to be elucidated include the regulation of the specificity of the autophagy
process and its role in maintaining proteostasis (protein homeostasis), as well
as overall homeostasis in plants and other living organisms, including
humans.

Acknowledgements M.C.-R. was supported by the Marie Curie Initial Training Network
BIONUT (project No. 264296). K. Z-R is supported by National Science Centre, Poland (project
No. 201/05/N/NZ1/00699). Research in A.S. lab is also supported by the Polish Ministry of
Science and Higher Education (project No. W16/7.PR/2011).
7 Role of Autophagy in Plant Nutrient Deficiency 197

References

Alvarez C, Garcia I, Romero LC, Gotor C (2012a) Mitochondrial sulfide detoxification requires a
functional isoform O-acetylserine(thiol)lyase C in Arabidopsis thaliana. Mol Plant 5:1217–
1226
Alvarez C, Garcia I, Moreno I, Perez-Perez ME, Crespo JL, Romero LC, Gotor C (2012b)
Cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the
transcriptional profile in Arabidopsis. Plant Cell 24:4621–4634
Amtmann A, Armengaud P (2009) Effects of N, PK and S on metabolism: new knowledge gained
from multi-level analysis. Curr Opin Plant Biol 12:275–283
Anderson GH, Veit B, Hanson MR (2005) The Arabidopsis AtRaptor genes are essential for post-
embryonic plant growth. BMC Biol 3:12
Asakura M, Ninomiya S, Sugimoto M, Oku M, Yamashita S, Okuno T, Sakai Y, Takano Y (2009)
Atg26-mediated pexophagy is required for host invasion by the plant pathogenic fungus
Colletotrichum orbiculare. Plant Cell 21:1291–1304
Bassham DC, Laporte M, Marty F, Moriyasu Y, Ohsumi Y, Olsen LJ, Yoshimoto K (2006)
Autophagy in development and stress responses of plants. Autophagy 2:2–11
Behrends C, Fulda S (2012) Receptor proteins in selective autophagy. Int J Cell Biol 2012: 673290
Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H, Johansen T
(2005) p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective
effect on huntingtin-induced cell death. J Cell Biol 171:603–614
Callis J, Carpenter T, Sun CW, Vierstra RD (1995) Structure and evolution of genes encoding
polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia. Genetics
139:921–939
Christian P, Sacco J, Adeli K (2013) Autophagy: emerging roles in lipid homeostasis and
metabolic control. Biochim Biophys Acta 1831:819–824
Chung T, Phillips AR, Vierstra RD (2010) ATG8 lipidation and ATG8-mediated autophagy in
Arabidopsis require ATG12 expressed from the differentially controlled ATG12A and
ATG12B loci. Plant J 62:483–493
Dantuma NP, Lindsten K, Glas R, Jellne M, Masucci MG (2000) Short-lived green fluorescent
proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat
Biotechnol 18:538–543
Deprost D, Truong HN, Robaglia C, Meyer C (2005) An Arabidopsis homolog of RAPTOR/
KOG1 is essential for early embryo development. Biochem Biophys Res Commun 326:844–
850
Deprost D, Yao L, Sormani R, Moreau M, Leterreux G, Nicolai M, Bedu M, Robaglia C, Meyer C
(2007) The Arabidopsis TOR kinase links plant growth, yield, stress resistance and mRNA
translation. EMBO Rep 8:864–870
Derrien B, Baumberger N, Schepetilnikov M, Viotti C, De Cillia J, Ziegler-Graff V, Isono E,
Schumacher K, Genschik P (2012) Degradation of the antiviral component ARGONAUTE1 by
the autophagy pathway. Proc Natl Acad Sci U S A 109:15942–15946
Devarenne TP (2011) The plant cell death suppressor Adi3 interacts with the autophagic protein
Atg8h. Biochem Biophys Res Commun 412:699–703
Diaz-Troya S, Perez-Perez ME, Florencio FJ, Crespo JL (2008) The role of TOR in autophagy
regulation from yeast to plants and mammals. Autophagy 4:851–865
Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra RD (2002) The APG8/12-
activating enzyme APG7 is required for proper nutrient recycling and senescence in
Arabidopsis thaliana. J Biol Chem 277:33105–33114
Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome.
Annu Rev Biochem 78:477–513
Floyd BE, Morriss SC, Macintosh GC, Bassham DC (2012) What to eat: evidence for selective
autophagy in plants. J Integr Plant Biol 54:907–920
Gadalla MM, Snyder SH (2010) Hydrogen sulfide as a gasotransmitter. J Neurochem 113:14–26
198 M. Collados Rodrı́guez et al.

Gruber BD, Giehl RF, Friedel S, von Wiren N (2013) Plasticity of the Arabidopsis root system
under nutrient deficiencies. Plant Physiol 163:161–179
Guiboileau A, Sormani R, Meyer C, Masclaux-Daubresse C (2010) Senescence and death of plant
organs: nutrient recycling and developmental regulation. C R Biol 333:382–391
Guiboileau A, Yoshimoto K, Soulay F, Bataille MP, Avice JC, Masclaux-Daubresse C (2012)
Autophagy machinery controls nitrogen remobilization at the whole-plant level under both
limiting and ample nitrate conditions in Arabidopsis. New Phytol 194:732–740
Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata D, Tabata S, Ohsumi Y (2002) Leaf
senescence and starvation-induced chlorosis are accelerated by the disruption of an
Arabidopsis autophagy gene. Plant Physiol 129:1181–1193
Harrison-Lowe NJ, Olsen LJ (2008) Autophagy protein 6 (ATG6) is required for pollen germina-
tion in Arabidopsis thaliana. Autophagy 4:339–348
Hillwig MS, Contento AL, Meyer A, Ebany D, Bassham DC, Macintosh GC (2011) RNS2, a
conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants.
Proc Natl Acad Sci U S A 108:1093–1098
Hoefgen R, Nikiforova VJ (2008) Metabolomics integrated with transcriptomics: assessing sys-
tems response to sulfur-deficiency stress. Physiol Plant 132:190–198
Hofius D, Schultz-Larsen T, Joensen J, Tsitsigiannis DI, Petersen NH, Mattsson O, Jorgensen LB,
Jones JD, Mundy J, Petersen M (2009) Autophagic components contribute to hypersensitive
cell death in Arabidopsis. Cell 137:773–783
Honig A, Avin-Wittenberg T, Galili G (2012a) Selective autophagy in the aid of plant germination
and response to nutrient starvation. Autophagy 8:838–839
Honig A, Avin-Wittenberg T, Ufaz S, Galili G (2012b) A new type of compartment, defined by
plant-specific Atg8-interacting proteins, is induced upon exposure of Arabidopsis plants to
carbon starvation. Plant Cell 24:288–303
Howarth JR, Parmar S, Jones J, Shepherd CE, Corol DI, Galster AM, Hawkins ND, Miller SJ,
Baker JM, Verrier PJ, Ward JL, Beale MH, Barraclough PB, Hawkesford MJ (2008)
Co-ordinated expression of amino acid metabolism in response to N and S deficiency during
wheat grain filling. J Exp Bot 59:3675–3689
Hutchins AP, Liu S, Diez D, Miranda-Saavedra D (2013) The repertoires of ubiquitinating and
deubiquitinating enzymes in eukaryotic genomes. Mol Biol Evol 30:1172–1187
Isakson P, Holland P, Simonsen A (2013) The role of ALFY in selective autophagy. Cell Death
Differ 20:12–20
Ishida H, Yoshimoto K, Izumi M, Reisen D, Yano Y, Makino A, Ohsumi Y, Hanson MR, Mae T
(2008) Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the
vacuole by an ATG gene-dependent autophagic process. Plant Physiol 148:142–155
Izumi M, Hidema J, Makino A, Ishida H (2013) Autophagy contributes to nighttime energy
availability for growth in Arabidopsis. Plant Physiol 161:1682–1693
Johansen T, Lamark T (2011) Selective autophagy mediated by autophagic adapter proteins.
Autophagy 7:279–296
John F, Roffler S, Wicker T, Ringli C (2011) Plant TOR signaling components. Plant Signal Behav
6:1700–1705
Kanki T, Wang K, Cao Y, Baba M, Klionsky DJ (2009) Atg32 is a mitochondrial protein that
confers selectivity during mitophagy. Dev Cell 17:98–109
Kim SH, Kwon C, Lee JH, Chung T (2012) Genes for plant autophagy: functions and interactions.
Mol Cells 34:413–423
Kirkin V, Lamark T, Sou YS, Bjorkoy G, Nunn JL, Bruun JA, Shvets E, McEwan DG, Clausen
TH, Wild P, Bilusic I, Theurillat JP, Overvatn A, Ishii T, Elazar Z, Komatsu M, Dikic I,
Johansen T (2009) A role for NBR1 in autophagosomal degradation of ubiquitinated sub-
strates. Mol Cell 33:505–516
Klionsky DJ. (2007) Autophagy: from phenomenology to molecular understanding in less than a
decade. Nat Rev Mol Cell Biol 8:931–937
7 Role of Autophagy in Plant Nutrient Deficiency 199

Klionsky DJ, Baehrecke EH, Brumell JH, Chu CT, Codogno P, Cuervo AM, Debnath J, Deretic V,
Elazar Z, Eskelinen EL, Finkbeiner S, Fueyo-Margareto J, Gewirtz D, Jaattela M, Kroemer G,
Levine B, Melia TJ, Mizushima N, Rubinsztein DC, Simonsen A, Thorburn A, Thumm M,
Tooze SA (2011) A comprehensive glossary of autophagy-related molecules and processes
(2nd edition). Autophagy 7:1273–1294
Komatsu M, Ichimura Y (2010) Physiological significance of selective degradation of p62 by
autophagy. FEBS Lett 584:1374–1378
Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, Sou YS, Ueno I,
Sakamoto A, Tong KI, Kim M, Nishito Y, Iemura S, Natsume T, Ueno T, Kominami E,
Motohashi H, Tanaka K, Yamamoto M (2010) The selective autophagy substrate p62 activates
the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nat Cell Biol
12:213–223
Korolchuk VI, Menzies FM, Rubinsztein DC (2010) Mechanisms of cross-talk between the
ubiquitin-proteasome and autophagy-lysosome systems. FEBS Lett 584:1393–1398
Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny
MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA,
Malorni W, Nunez G, Peter ME, Tschopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G,
Nomenclature Committee on Cell, D (2009) Classification of cell death: recommendations of
the Nomenclature Committee on Cell Death 2009. Cell Death Differ 16:3–11
Kwon SI, Cho HJ, Jung JH, Yoshimoto K, Shirasu K, Park OK (2010) The Rab GTPase RabG3b
functions in autophagy and contributes to tracheary element differentiation in Arabidopsis.
Plant J 64:151–164
Lee TA, Vande Wetering SW, Brusslan JA (2013) Stromal protein degradation is incomplete in
Arabidopsis thaliana autophagy mutants undergoing natural senescence. BMC Res Notes 6:17
Leiber RM, John F, Verhertbruggen Y, Diet A, Knox JP, Ringli C (2010) The TOR pathway
modulates the structure of cell walls in Arabidopsis. Plant Cell 22:1898–1908
Lenz HD, Vierstra RD, Nurnberger T, Gust AA (2011) ATG7 contributes to plant basal immunity
towards fungal infection. Plant Signal Behav 6:1040–1042
Letunic I, Goodstadt L, Dickens NJ, Doerks T, Schultz J, Mott R, Ciccarelli F, Copley RR, Ponting
CP, Bork P (2002) Recent improvements to the SMART domain-based sequence annotation
resource. Nucleic Acids Res 30:242–244
Lewandowska M, Wawrzynska A, Moniuszko G, Lukomska J, Zientara K, Piecho M, Hodurek P,
Zhukov I, Liszewska F, Nikiforova V, Sirko A (2010) A contribution to identification of novel
regulators of plant response to sulfur deficiency: characteristics of a tobacco gene UP9C, its
protein product and the effects of UP9C silencing. Mol Plant 3:347–360
Li F, Vierstra RD (2012a) Autophagy: a multifaceted intracellular system for bulk and selective
recycling. Trends Plant Sci 17:526–537
Li F, Vierstra RD (2012b) Regulator and substrate: dual roles for the ATG1-ATG13 kinase
complex during autophagic recycling in Arabidopsis. Autophagy 8:982–984
Liang C, Tian J, Liao H (2013) Proteomics dissection of plant responses to mineral nutrient
deficiency. Proteomics 13:624–636
Liu Y, Bassham DC (2010) TOR is a negative regulator of autophagy in Arabidopsis thaliana.
PLoS One 5:e11883
Liu Y, Bassham DC (2012) Autophagy: pathways for self-eating in plant cells. Annu Rev Plant
Biol 63:215–237
Liu Y, Xiong Y, Bassham DC (2009) Autophagy is required for tolerance of drought and salt stress
in plants. Autophagy 5:954–963
Liu Y, Burgos JS, Deng Y, Srivastava R, Howell SH, Bassham DC (2012) Degradation of the
endoplasmic reticulum by autophagy during endoplasmic reticulum stress in Arabidopsis.
Plant Cell 24:4635–4651
Lowicka E, Beltowski J (2007) Hydrogen sulfide (H2S) – the third gas of interest for pharmacol-
ogists. Pharmacol Rep 59:4–24
200 M. Collados Rodrı́guez et al.

Lynch-Day MA, Klionsky DJ (2010) The Cvt pathway as a model for selective autophagy. FEBS
Lett 584:1359–1366
Lyzenga WJ, Stone SL (2012) Abiotic stress tolerance mediated by protein ubiquitination. J Exp
Bot 63:599–616
Madeo F, Tavernarakis N, Kroemer G (2010) Can autophagy promote longevity? Nat Cell Biol
12:842–846
Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007) Self-eating and self-killing: crosstalk
between autophagy and apoptosis. Nat Rev Mol Cell Biol 8:741–752
Marchbank K, Waters S, Roberts RG, Solomon E, Whitehouse CA (2012) MAP1B interaction
with the FW domain of the autophagic receptor Nbr1 facilitates its association to the micro-
tubule network. Int J Cell Biol 2012:208014
Mardakheh FK, Auciello G, Dafforn TR, Rappoport JZ, Heath JK (2010) Nbr1 is a novel inhibitor
of ligand-mediated receptor tyrosine kinase degradation. Mol Cell Biol 30:5672–5685
Minibayeva F, Dmitrieva S, Ponomareva A, Ryabovol V (2012) Oxidative stress-induced
autophagy in plants: the role of mitochondria. Plant Physiol Biochem 59:11–19
Minina EA, Sanchez-Vera V, Moschou PN, Suarez MF, Sundberg E, Weih M, Bozhkov PV (2013)
Autophagy mediates caloric restriction-induced lifespan extension in Arabidopsis. Aging Cell
12:327–329
Moreau M, Azzopardi M, Clement G, Dobrenel T, Marchive C, Renne C, Martin-Magniette ML,
Taconnat L, Renou JP, Robaglia C, Meyer C (2012) Mutations in the Arabidopsis homolog of
LST8/GbetaL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and
metabolic adaptation to long days. Plant Cell 24:463–481
Mostowy S, Sancho-Shimizu V, Hamon MA, Simeone R, Brosch R, Johansen T, Cossart P (2011)
p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy
pathways. J Biol Chem 286:26987–26995
Muller S, Kursula I, Zou P, Wilmanns M (2006) Crystal structure of the PB1 domain of NBR1.
FEBS Lett 580:341–344
Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y (2009) Dynamics and diversity in autophagy
mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 10:458–467
Nei M, Rogozin IB, Piontkivska H (2000) Purifying selection and birth-and-death evolution in the
ubiquitin gene family. Proc Natl Acad Sci U S A 97:10866–10871
Noda T, Ohsumi Y (1998) Tor, a phosphatidylinositol kinase homologue, controls autophagy in
yeast. J Biol Chem 273:3963–3966
Noda NN, Kumeta H, Nakatogawa H, Satoo K, Adachi W, Ishii J, Fujioka Y, Ohsumi Y, Inagaki F
(2008) Structural basis of target recognition by Atg8/LC3 during selective autophagy. Genes
Cells 13:1211–1218
Noda NN, Ohsumi Y, Inagaki F (2010) Atg8-family interacting motif crucial for selective
autophagy. FEBS Lett 584:1379–1385
Noda NN, Fujioka Y, Hanada T, Ohsumi Y, Inagaki F (2013) Structure of the Atg12-Atg5
conjugate reveals a platform for stimulating Atg8-PE conjugation. EMBO Rep 14:206–211
Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, Tooze SA (2012) Dynamic
and transient interactions of Atg9 with autophagosomes, but not membrane integration, are
required for autophagy. Mol Biol Cell 23:1860–1873
Pankiv S, Lamark T, Bruun JA, Overvatn A, Bjorkoy G, Johansen T (2010) Nucleocytoplasmic
shuttling of p62/SQSTM1 and its role in recruitment of nuclear polyubiquitinated proteins to
promyelocytic leukemia bodies. J Biol Chem 285:5941–5953
Pattingre S, Espert L, Biard-Piechaczyk M, Codogno P (2008) Regulation of macroautophagy by
mTOR and Beclin 1 complexes. Biochimie 90:313–323
Phillips AR, Suttangkakul A, Vierstra RD (2008) The ATG12-conjugating enzyme ATG10 is
essential for autophagic vesicle formation in Arabidopsis thaliana. Genetics 178:1339–1353
Pourcel L, Irani NG, Lu Y, Riedl K, Schwartz S, Grotewold E (2010) The formation of
anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration
of anthocyanin pigments. Mol Plant 3:78–90
7 Role of Autophagy in Plant Nutrient Deficiency 201

Raasi S, Varadan R, Fushman D, Pickart CM (2005) Diverse polyubiquitin interaction properties

of ubiquitin-associated domains. Nat Struct Mol Biol 12:708–714
Rabinowitz JD, White E (2010) Autophagy and metabolism. Science 330:1344–1348
Rojas-Triana M, Bustos R, Espinosa-Ruiz A, Prat S, Paz-Ares J, Rubio V (2013) Roles of
ubiquitination in the control of phosphate starvation responses in plants(f). J Integr Plant
Biol 55:40–53
Rose TL, Bonneau L, Der C, Marty-Mazars D, Marty F (2006) Starvation-induced expression of
autophagy-related genes in Arabidopsis. Biol Cell 98:53–67
Rubinsztein DC, Marino G, Kroemer G (2011) Autophagy and aging. Cell 146:682–695
Sadanandom A, Bailey M, Ewan R, Lee J, Nelis S (2012) The ubiquitin-proteasome system:
central modifier of plant signalling. New Phytol 196:13–28
Salminen A, Kaarniranta K, Haapasalo A, Hiltunen M, Soininen H, Alafuzoff I (2012) Emerging
role of p62/sequestosome-1 in the pathogenesis of Alzheimer’s disease. Prog Neurobiol
96:87–95
Sanz L, Diaz-Meco MT, Nakano H, Moscat J (2000) The atypical PKC-interacting protein p62
channels NF-kappaB activation by the IL-1-TRAF6 pathway. EMBO J 19:1576–1586
Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z (2007) Reactive oxygen species are
essential for autophagy and specifically regulate the activity of Atg4. EMBO J 26:1749–1760
Schreiber A, Peter M (2013) Substrate recognition in selective autophagy and the ubiquitin-
proteasome system. Biochim Biophys Acta. doi:10.1016/j.bbamcr.2013.03.019 [Epub ahead
of print]
Seibenhener ML, Babu JR, Geetha T, Wong HC, Krishna NR, Wooten MW (2004) Sequestosome
1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation.
Mol Cell Biol 24:8055–8068
Shabek N, Herman-Bachinsky Y, Buchsbaum S, Lewinson O, Haj-Yahya M, Hejjaoui M, Lashuel
HA, Sommer T, Brik A, Ciechanover A (2012) The size of the proteasomal substrate deter-
mines whether its degradation will be mediated by mono- or polyubiquitylation. Mol Cell
48:87–97
Slavikova S, Ufaz S, Avin-Wittenberg T, Levanony H, Galili G (2008) An autophagy-associated
Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and
abiotic stresses. J Exp Bot 59:4029–4043
Sullivan JA, Shirasu K, Deng XW (2003) The diverse roles of ubiquitin and the 26S proteasome in
the life of plants. Nat Rev Genet 4:948–958
Sumimoto H, Kamakura S, Ito T (2007) Structure and function of the PB1 domain, a protein
interaction module conserved in animals, fungi, amoebas, and plants. Sci STKE 2007:re6
Suttangkakul A, Li F, Chung T, Vierstra RD (2011) The ATG1/ATG13 protein kinase complex is
both a regulator and a target of autophagic recycling in Arabidopsis. Plant Cell 23:3761–3779
Suzuki K, Kubota Y, Sekito T, Ohsumi Y (2007) Hierarchy of Atg proteins in pre-autophagosomal
structure organization. Genes Cells 12:209–218
Svenning S, Lamark T, Krause K, Johansen T (2011) Plant NBR1 is a selective autophagy
substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and
p62/SQSTM1. Autophagy 7:993–1010
Szabo C (2007) Hydrogen sulphide and its therapeutic potential. Nat Rev Drug Discov 6:917–935
Thompson AR, Vierstra RD (2005) Autophagic recycling: lessons from yeast help define the
process in plants. Curr Opin Plant Biol 8:165–173
Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD (2005) Autophagic nutrient recycling in
Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol 138:2097–
2110
Todde V, Veenhuis M, van der Klei IJ (2009) Autophagy: principles and significance in health and
disease. Biochim Biophys Acta 1792:3–13
Toyooka K, Moriyasu Y, Goto Y, Takeuchi M, Fukuda H, Matsuoka K (2006) Protein aggregates
are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells.
Autophagy 2:96–106
202 M. Collados Rodrı́guez et al.

Tsai YC, Koo Y, Delk NA, Gehl B, Braam J (2012) Calmodulin-related CML24 interacts with
ATG4b and affects autophagy progression in Arabidopsis. Plant J. doi:10.1111/tpj.12043
[Epub ahead of print]
Tsai IT, Chen YH, Chen YH, Wang YH (2013) Amikacin-induced fin reduction is mediated by
autophagy. J Toxicol Pathol 26:79–82
van Doorn WG (2011) Classes of programmed cell death in plants, compared to those in animals. J
Exp Bot 62:4749–4761
Vanhee C, Zapotoczny G, Masquelier D, Ghislain M, Batoko H (2011) The Arabidopsis
multistress regulator TSPO is a heme binding membrane protein and a potential scavenger of
porphyrins via an autophagy-dependent degradation mechanism. Plant Cell 23:785–805
Vierstra RD (2003) The ubiquitin/26S proteasome pathway, the complex last chapter in the life of
many plant proteins. Trends Plant Sci 8:135–142
Vierstra RD (2009) The ubiquitin-26S proteasome system at the nexus of plant biology. Nat Rev
Mol Cell Biol 10:385–397
Vierstra RD (2012) The expanding universe of ubiquitin and ubiquitin-like modifiers. Plant
Physiol 160:2–14
Wada S, Ishida H, Izumi M, Yoshimoto K, Ohsumi Y, Mae T, Makino A (2009) Autophagy plays a
role in chloroplast degradation during senescence in individually darkened leaves. Plant
Physiol 149:885–893
Wang Y, Wu Y, Tang D (2011a) The autophagy gene, ATG18a, plays a negative role in powdery
mildew resistance and mildew-induced cell death in Arabidopsis. Plant Signal Behav 6:1408–
1410
Wang Y, Nishimura MT, Zhao T, Tang D (2011b) ATG2, an autophagy-related protein, negatively
affects powdery mildew resistance and mildew-induced cell death in Arabidopsis. Plant J
68:74–87
Wang WY, Zhang L, Xing S, Ma Z, Liu J, Gu H, Qin G, Qu LJ (2012) Arabidopsis AtVPS15 plays
essential roles in pollen germination possibly by interacting with AtVPS34. J Genet Genomics
39:81–92
Wang Y, Yu B, Zhao J, Guo J, Li Y, Han S, Huang L, Du Y, Hong Y, Tang D, Liu Y (2013)
Autophagy contributes to leaf starch degradation. Plant Cell 25:1383–1399
Wawrzynska A, Lewandowska M, Hawkesford MJ, Sirko A (2005) Using a suppression subtrac-
tive library-based approach to identify tobacco genes regulated in response to short-term
sulphur deficit. J Exp Bot 56:1575–1590
Weidberg H, Shvets E, Elazar Z (2011) Biogenesis and cargo selectivity of autophagosomes. Annu
Rev Biochem 80:125–156
Welters P, Takegawa K, Emr SD, Chrispeels MJ (1994) AtVPS34, a phosphatidylinositol 3-kinase
of Arabidopsis thaliana, is an essential protein with homology to a calcium-dependent lipid
binding domain. Proc Natl Acad Sci U S A 91:11398–11402
Wittenbach VA, Lin W, Hebert RR (1982) Vacuolar localization of proteases and degradation of
chloroplasts in mesophyll protoplasts from senescing primary wheat leaves. Plant Physiol
69:98–102
Xia T, Xiao D, Liu D, Chai W, Gong Q, Wang NN (2012) Heterologous expression of ATG8c
from soybean confers tolerance to nitrogen deficiency and increases yield in Arabidopsis. PLoS
One 7:e37217
Xiong Y, Contento AL, Bassham DC (2005) AtATG18a is required for the formation of
autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J
42:535–546
Xiong Y, Contento AL, Bassham DC (2007) Disruption of autophagy results in constitutive
oxidative stress in Arabidopsis. Autophagy 3:257–258
Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, Robert J, Rush J, Hochstrasser M, Finley D, Peng
J (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in
proteasomal degradation. Cell 137:133–145
7 Role of Autophagy in Plant Nutrient Deficiency 203

Yamaguchi M, Noda NN, Nakatogawa H, Kumeta H, Ohsumi Y, Inagaki F (2010) Autophagy-

related protein (Atg) 8-family interacting motif in Atg3 mediates the Atg3-Atg8 interaction
and is crucial for the cytoplasm-to-vacuole targeting pathway. J Biol Chem 285:29599–29607
Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12:814–822
Yen WL, Legakis JE, Nair U, Klionsky DJ (2007) Atg27 is required for autophagy-dependent
cycling of Atg9. Mol Biol Cell 18: 581–593
Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y (2004) Processing of
ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant
autophagy. Plant Cell 16:2967–2983
Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consonni C, Panstruga R, Ohsumi Y, Shirasu K
(2009) Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic
acid signaling during senescence and the innate immune response in Arabidopsis. Plant Cell
21:2914–2927
Zhou J, Wang J, Cheng Y, Chi YJ, Fan B, Yu JQ, Chen Z (2013) NBR1-mediated selective
autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. PLoS
Genet 9:e1003196
Zientara-Rytter K, Lukomska J, Moniuszko G, Gwozdecki R, Surowiecki P, Lewandowska M,
Liszewska F, Wawrzynska A, Sirko A (2011) Identification and functional analysis of Joka2, a
tobacco member of the family of selective autophagy cargo receptors. Autophagy 7:1145–1158
Chapter 8
Mineral Nutrient Depletion Affects Plant
Development and Crop Yield

Sarah J. Whitcomb, Elmien Heyneke, Fayezeh Aarabi, Mutsumi Watanabe,

and Rainer Hoefgen

Abstract Optimal plant development depends on the availability of light, water,

favourable temperatures and mineral nutrients. Insufficient availability of plant
mineral nutrients leads to growth impairments and yield depressions. In natural
environments as well as in agricultural systems, mineral nutrient availability is
changing in space and time over the growth season of a plant. Therefore, plants
have developed adaptation strategies to cope with nutrient deficiencies. Fully
understanding these mechanisms at the molecular level is a necessity for breeding
nutrient use efficient crops. Plant systems biology approaches contribute to this
endeavour as agriculture and plant breeding face-increasing challenges to achieve
sustainable and effective agricultural production.

Keywords Nutrient deficiency • Development • Senescence • Omics • Systems

biology • Nitrogen • Phosphorus • Sulfur

Introduction – The Importance of Mineral Nutrient Use

Efficiency for a Sustainable Agriculture

Sustainable crop production will be one of the most critical challenges in the next
50 years due to predicted worldwide population growth and increasing meat
consumption, especially in the developing world (Dyson 1999; Long and Ort
2010; Tester and Langridge 2010; Mueller et al. 2012). Conventional breeding
and improved agronomical practices are currently producing only incremental and
insufficient increases in crop yield potential, and factors such as global climate
change will increasingly compromise the ability to reach this theoretical yield
potential (Jaggard et al. 2010). With respect to climate change, increasing night
temperatures and altered water availability patterns are particularly problematic
(Semenov and Halford 2009; Long and Ort 2010; Dolferus et al. 2011;

S.J. Whitcomb • E. Heyneke • F. Aarabi • M. Watanabe • R. Hoefgen (*)

Max Planck Institute of Molecular Plant Physiology, Science Park Potsdam-Golm,
14424 Potsdam, Germany
e-mail: [email protected]

© Springer International Publishing Switzerland 2014 205

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_8
206 S.J. Whitcomb et al.

Li et al. 2011). Additionally, the necessary shift from a fossil fuel to a biofuel based
economy and from petrochemicals to biochemicals increases the pressure on crop
productivity and will demand development of crop cultivars with a wider range of
agronomic features. The biofuel and biochemical industries require regenerative
plant commodities with high vegetative biomass or high seed content of specific
compounds such as particular modified starches, while food and feed production
rely mostly upon annual crops bred for high seed yield and complex quality traits.
For food and feed production, annual, hapaxanthic crop plants dominate agri-
culture, top among them the cereals wheat, rice and maize (Dyson 1999). As they
complete their life cycle from seed to seed in one vegetative period, crop produc-
tivity and quality is tightly coupled to senescence. During the growth season, plants
follow a genetically controlled developmental programme leading to organ and
plant senescence, including regulated allocation of nutrients from vegetative tissues
to the seeds, and finally to plant death (Himelblau and Amasino 2001; Lim
et al. 2007; Wu et al. 2012; Watanabe et al. 2013). Importantly, developmental
senescence programmes are responsive to environmental conditions such as light,
temperature, water and nutrient availability (Watanabe et al. 2012). Adverse envi-
ronmental conditions outside of the plant’s ecologically optimal niche usually result
in stress for the plant and trigger developmental acceleration, premature senes-
cence, and eventually result in reduced yields. This chapter discusses plant
responses to mineral nutrient deficiencies and whether the ability of crop plants
to utilise such nutrients can be improved to meet the global need for more nutrient
use efficient cultivars.
Agroecosystems are open systems where nutrient ions are mobilised from the
soil into crop biomass and removed with the harvest. Crop productivity depends on
mineral nutrient ions in the root environment, the availability of which is deter-
mined by soil parameters such as water content and pH as well as microbial
degradation of biomass such as straw, humus or manure, generally termed
mineralisation (von Liebig 1840; Marschner 2012; Buchanan et al. 2007; Haensch
and Mendel 2009; Amtmann and Blatt 2009; Amtmann and Armengaud 2009).
Mineral fertilisers are applied to compensate for nutrient losses in conventional
agriculture. Usually the provision of macronutrients (N, P, S, Mg, K, Ca) is
sufficient, while micronutrients or trace elements (Fe, Cu, Mn, Ni, Zn, Cl, B, Mo)
are supplemented depending upon the needs of specific soil types or crops
(Watanabe et al. 2012). Nitrogen and phosphate have the largest effect on produc-
tion costs due to the quantities applied and their cost. Optimal crop production
requires a balanced supply of nutrients, as a deficiency of one nutrient cannot be
compensated for by others according to Liebig’s law of the minimum (von Liebig
1840). Furthermore, both deficiency and over-accumulation can lead to negative
effects on plants due to the interaction and competition between minerals at the
level of uptake and assimilation (Hoefgen and Hesse 2008). For example iron
uptake in barley and tomato has been shown to be dependent on sulfate availability.
Under sulfate depletion iron transporters and iron reductase are downregulated,
preventing uptake even in the presence of iron. Further, phytosiderphore biosyn-
thesis and internal iron transport are impaired as nicotianamine synthase is essen-
tially switched off (Astolfi et al. 2010; Zuchi et al. 2009; Cassin et al. 2009; Klatte
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 207

et al. 2009). Especially among the micronutrients, including REDOX-active

transition metals such as copper or zinc, the concentration range between deficiency
and toxicity is often narrow, both having negative effects on plant growth
(He et al. 2005).
Plants are able to respond flexibly to varied environmental conditions. In
addition to light, water and temperature, mineral nutrient availability determines
plant growth and propagation, or, in an agricultural context, crop yield. The
ecological niche of a plant is determined by the specific range of conditions a
certain plant species can adapt to. Crop cultivars are usually the result of adapted
breeding for agricultural conditions and, as a result of this selection process, they
are disposed to narrow genome variability (Tanksley and McCouch 1997). There
are different avenues to re-introduce increased genetic variability, but all
approaches have to take into account the need to retain the very high yield potential
of modern crop cultivars, otherwise the necessary goal of sustainably and afford-
ably providing more food for a growing population on less land will not be met.
Mineral nutrient deficiencies typically result in yield depression and quality
impairment in crop plants. Here we compare what is known about the effects of
mineral depletion on plant development and metabolic composition to natural
developmental senescence of plants. The model plant Arabidopsis thaliana pro-
vides all the necessary tools to perform such studies at a systems biology level. The
conclusions from such investigations will be helpful for developing concepts for
breeding of nutrient use efficient crops and will provide directives for more
sustainable agricultural practice.

Senescence – A Developmentally Controlled Process Critical

for Seed Development

Leaf and whole plant senescence is a genetically programmed and highly regulated
process termed developmental senescence (Wu et al. 2012; Thomas 2013;
Watanabe et al. 2013). This is essentially a self-destructive programme, which
directs breakdown of cellular structures and components in source tissues for export
to sink tissues such as young leaves, roots, flowers, tubers or seeds.
The developmental cycle of an annual plant (Fig. 8.1) starts with germination
and a growth phase leading to leaf expansion, during which young leaves act as a
sink for carbohydrates and root-derived mineral ions. This is followed by a phase of
maturity, in which fully expanded leaves are photosynthetically and biochemically
most active and provide metabolites to developing leaves or other sink tissues such
as roots and developing seeds. Older leaves successively senesce, but after anthesis
and fertilisation, developing seeds act as the major sink and the plant is primed for
general senescence. Upon the switch from vegetative to generative growth, no new
leaves develop and existing leaves enter senescence to provide nutrients to the
seeds. During this phase the plant has to find a compromise between degradation of
macromolecules to remobilise nutrients and leaf functional integrity for photosyn-
thesis and carbohydrate production. For example in rice, wheat and barley, up to
208 S.J. Whitcomb et al.

Fig. 8.1 Schematic life cycle of an annual plant. Light and temperature determine the potential
growth season. The life cycle of an annual plant starts with germination. Seedling leaf develop-
ment initially depends on resources from the seed, and young leaves act as a sink organs until they
develop sufficient photosynthetic activity to function as source tissue by exporting sugars and
nutrients to other parts of the plant. Here the chlorophyll content of leaves is graphed as a proxy for
photosynthetic capacity in leaves. Individual leaves eventually enter a senescence phase resulting
essentially in a programmed self-destruction. During this phase leaf constituents such as proteins,
lipids and mineral nutrients are degraded and exported to sinks. Each subsequent leaf growing
from the shoot apical meristem follows this developmental progression. During the vegetative
phase, the plant accumulates biomass, establishes a canopy and grows. Changes in light or other
environmental conditions trigger flower formation from the shoot apical meristem, constituting a
switch from a vegetative to generative growth phase and eventually results in seed production and
seed maturation. During the seed developmental phase, leaves undergo senescence and remobilize
necessary nutrients to the seeds. Mature seeds then enter a dormancy phase until germination in the
next growth season

90 % of nitrogen in the seed is remobilised from vegetative tissues (Hirel

et al. 2007; Masclaux-Daubresse et al. 2010; Masclaux-Daubresse and Chardon
2011). As a consequence, in these crops grain yield and quality depend primarily on
pre-anthesis uptake of nitrogen. In contrast, 35–55 % of seed nitrogen in maize is
taken up from the soil during grain filling (Hirel et al. 2007; Gregersen et al. 2008).
Starch accumulation is the major factor determining seed yield, but other grain
parameters are equally important for farmers and consumers. For example, nutri-
tional quality is primarily a function of seed protein content and amino acid
balance. Therefore the supply and remobilisation of nitrogen, and to a lesser extent
sulfur for the sulfur-containing amino acids cysteine and methionine, represent
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 209

critical control points for crop quality (Hoefgen and Galili 2002; Hoefgen
et al. 2004). Chloroplasts contain approximately 80 % of total leaf nitrogen, mostly
in the form of Rubisco, and so not surprisingly, the major nitrogen resources for
export to the developing seeds are derived from chloroplast degradation in
senescing leaves (Feller et al. 2008; Kato und Sakamoto 2010). Leaf senescence
is further characterised by the formation of senescence-associated vacuoles,
involvement of the autophagy machinery (see chapter of Collados-Rodriguez
et al. in this book), and activation of proteolytic and other degradative enzymes
(Ishida et al. 2008; Wada et al. 2009; Izumi et al. 2010; Hörtensteiner 2012;
Yamada et al. 2001; Martinez et al. 2008; Watanabe et al. 2010). As a result of
these degradation processes, cellular integrity is compromised and reactive oxygen
species (ROS) accumulate, which may regulate senescence associated gene (SAG)
expression and additionally induce stress mitigation responses to maintain cellular
function as long as possible (Navabpour et al. 2003; Guo and Crawford 2005).
For breeders focused on increasing crop yields, prolongation of leaf integrity has
long been a trait of interest. Some cultivars with extremely late onset of senescence,
so called “stay-green” cultivars, do indeed show higher starch filling and yields
(Richards 2000). However, as robust senescence is required for maximal
remobilisation of nutrients, seed quality might be impaired in late-senescing vari-
eties (Bogard et al. 2011; Gong et al. 2005; Hirel et al. 2007; Masclaux-Daubresse
and Chardon 2011; Gregersen et al. 2008). Further, the effects of late onset
senescence have been found to vary depending on the species, genotype, and
environmental conditions. In wet and cool conditions wheat yield was negatively
correlated with the stay-green characteristic (Naruoka et al. 2012), while on the
other hand, the stay-green trait is often correlated with increased drought tolerance
or accentuated by low nitrogen supply (Yan et al. 2004; Robson et al. 2004; Jordan
et al. 2012). Thus, the relation of gross physiological characteristics such as stay-
green to final biomass and seed yield is still not well understood. We are still far
from being able to provide general rules, particularly as the stay-green phenotype is
caused by different mutations, some of which do not affect senescence parameters
only chlorophyll content (for summary see: Gregersen et al. 2013).
The case of stay-green cultivars highlights the need to consistently describe plant
development and senescence processes at the systems level. This kind of integrative
knowledge would greatly facilitate breeding schemes as it may identify those
processes most relevant for crop breeding.

Transcriptomic and Metabolomic Consequences of Plant

Maturation and Senescence

Arabidopsis thaliana provides a tractable and sufficiently valid model for systems
level analysis of senescence processes. Several detailed transcriptomics and
metabolomic studies (Guo et al. 2004; Buchanon-Wollaston et al. 2005; Breeze
210 S.J. Whitcomb et al.

et al. 2011; Guo and Gan 2012; Masclaux-Daubresse et al. 2005; Watanabe
et al. 2013), and numerous targeted investigations have greatly expanded our
understanding of the shift from anabolic to catabolic metabolism and senescence-
associated gene (SAG and SDG) expression changes.
Arabidopsis thaliana is a long-day plant with a basal rosette and a life cycle of
about 2 months belonging to the brassicaceae family (Fig. 8.2a). Older leaves
successively senesce during development in a highly ordered physiological process
with new appearing leaves acting as sinks. Upon inflorescence formation, a set of
upper rosette leaves as well as small leaves at the flower stem are generated. As
seeds ripen in siliques, the leaves further senesce and finally die off. The senescence
process at the level of genes or metabolites is the same in all leaves and appears to
follow a time shift according to the sequence of emergence (Breeze et al. 2011;
Watanabe et al. 2013). Even within a single leaf, a trajectory of senescence is
established along the basipetal axis with the (older) tip senescing earlier than the
base.
The characteristic shift from anabolic to catabolic processes is readily apparent
when analysing the metabolic composition of senescing plants or tissues over a time
course (Fig. 8.2c; Watanabe et al. 2013). Over the course of senescence, chloro-
phyll contents and chloroplast lipids such as mono- and di galactosyldiglycerides
(MGDGs and DGDGs) decrease, which point to the long-described senescence-
associated chloroplast degradation and decrease in photosynthetic capacity. Despite
this fact, the major sugars remain constant (sucrose) or increase (glucose and
fructose) in senescing leaves, and storage lipids such as triacylglycerides (TAGs)
increase. Protein contents are reduced due to proteolysis. Free branched-chain
amino acids, aromatic amino acids, and stress-related amino acids such as proline,
beta-alanine, and gamma-aminobutyric acid (GABA) accumulate. Conversely, the
major N-containing amino acids glutamine, arginine and their precursors glutamate
and aspartate decrease, probably due to export to sinks.
Furthermore, when looking at nutrient ions, nitrate and phosphate are efficiently
exported from the senescing leaf. In addition to inorganic phosphate, nucleic acids
are a likely source of phosphate for export, as indicated by the induction of RNases
(Himelblau and Amasino 2001; Morcuende et al. 2007; Lers et al. 2006, Watanabe
et al. 2013) and the concomitant induction of phosphate transporters (Chapin and
Jones 2009). The regulatory response network of Arabidopsis to phosphate
ä
Fig. 8.2 (continued) are presented as fold-change from stage 1 in log2 scale. No change to stage
1 is indicated by the dashed line, decrease by negative and increase by positive values. Typically
chlorophyll contents are reduced as well as chloroplast associated lipids as mono- and di
galactosyldiglycerides (MGDS, DGDS) and storage lipids as triacylglycerides (TAGs) accumu-
late. Protein degradation is accompanied by typical accumulation patterns of amino acids, most
importantly the phloem-transported amino acids (asp, glu, gln, and arg) are reduced, while
branched chain amino acids rather accumulate. Sulfur-rich glucosinolates are reduced and reduced
sulfur probably mobilized to the seeds and the nutrient ions phosphate and nitrate are mobilised to
the seeds, while sulfate is not mobilised. Sugars and anthocyanins as well as stress related amino
acids accumulate, these latter processes being linked to senescence induced reactive oxygen
species (ROS) accumulation. The slight increase of most metabolites at the end of the senescence
phase is due to a decrease of fresh weight due to water loss (wilting)
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 211

a Development senescence of Arabidopsis thaliana b Transcript changes during leaf senescence

SAG12 SDGs
12.0 8.0
6.0 CAB1
8.0

Relative change
RBCS1A
4.0
4.0
2.0
0.0 0.0
-2.0
-4.0
-4.0
-8.0
-6.0
-12.0 -8.0
1 2 3 4 1 2 3 4
Stage1 Stage2 Stage3 Stage4 stage stage

c Metabolite changes during leaf senescence

Chlorophyll MGDGs (12 species) DGDGs (14 species) TAGs (38 species)
3.0 6.0 4.0 6.0
3.0
Relative change

2.0 4.0 4.0

2.0
1.0 2.0 2.0
1.0
0.0 0.0 0.0 0.0
-1.0
-1.0 -2.0 -2.0
-2.0
-2.0 -4.0 -4.0
-3.0
-3.0 -6.0 -4.0 -6.0
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

Protein AAAs and BCAAs Asp, Asn, Glu, Gln, Arg Stress AAs
2.0 5.0 2.0 4.0
Asp
1.5 4.0 1.5 3.0
Relative change

Asn
3.0 Glu
1.0 1.0 Gln 2.0
2.0 Arg
0.5 1.0 0.5 1.0
0.0 0.0 0.0 0.0
Leu
-0.5 -1.0 Val -0.5 -1.0
Pro
-2.0 Ile
-1.0 Trp -1.0 -2.0 beta-Alanine
-3.0
Tyr GABA
-1.5 -4.0 -1.5 -3.0
Phe
-2.0 -5.0 -2.0 -4.0
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Sugars Nutrient ions Met-glucosinolates Anthocyanin
6.0 3.0 4.0 3.0
Sulfate 4MSOB
3.0 5MSOP
4.0 2.0 2.0
Relative change

Nitrate 6MSOH
Phosphate 2.0 7MSOH
2.0 1.0 8MSOO 1.0
1.0 4MTB

0.0 0.0 0.0 0.0

-1.0
-2.0 Sucrose -1.0 -1.0
Glucose -2.0
-4.0 Fructose -2.0 -2.0
-3.0
-6.0 -3.0 -4.0 -3.0
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
stage stage stage stage

Fig. 8.2 Developmental senescence of Arabidopsis thaliana. (a) The senescence process in
Arabidopsis is schematically depicted to illustrate the changes accompanied with progressing
senescence during the life cycle for a whole plant. A commonly visible phenotype is the
development of leaf chlorosis due to degradation of chlorophyll in the leaves. The Fig. is modified
from Watanabe et al. 2013 (www.plantphysiol.org.; Copyright American Society of Plant Biolo-
gists). (b) Genes associated with senescence belong to various functional classes. Typical senes-
cence associated genes (SAGs, induced upon senescence) and genes down regulated during
senescence (SDGs) are used as markers for senescence in plants. Expression changes of SAG12
and two SDGs, CAB and RBCS1A, are presented here for an individual life cycle of Arabidopsis
thaliana as fold change to stage 1 in log2 scale. No change to stage 1 is indicated by the dashed
line, decrease by negative and increase by positive values. (c) Metabolite changes during leaf
senescence are depicted. Metabolite contents in rosette leaves during developmental senescence
212 S.J. Whitcomb et al.

starvation is already highly resolved (Scheible et al. 2004; Morcuende et al. 2007;
Doerner 2008). In contrast, sulfate is often poorly mobilised from the vacuole,
probably because soil sulfate uptake is sufficient for seed filling and vacuolar
sulfate export transporters, which are known to be responsive to sulfate starvation,
are not always induced (Davidian and Kopriva 2010). When different regimes of
sulfate were applied to soil grown wheat, persistent sulfate deficiency led to yield
reductions and poor protein quality of the seeds. Application of sulfate, even at the
seed filling stage, was able to revert the phenotype with respect to improved starch
deposition and seed protein quality, especially of sulfur-rich seed proteins though it
could not compensate for previous losses in biomass accumulation (Zörb
et al. 2012; Steinfurth et al. 2012). Interestingly, the sulfur-rich defense compounds
of brassicaceae, the glucosinolates, are actively degraded in leaves during senes-
cence and might provide a source of reduced sulfur for export in contrast to
vacuolar sulfate (Yoshimoto and Saito 2012; Watanabe et al. 2013). However,
various sulfated compounds are not mobilised. Therefore sulfate and sulfurous
organic compounds are a substantial part of the leaf litter and contribute to the
fact that in humus rich soils the majority of nutrient sulfate is released by bacteria
from organic sources (Kertesz 1999; Kertesz and Mirleau 2004; Schmalenberger
et al. 2009).
Senescence processes are clearly extremely complex, and the functions of
individual genes, not to mention the regulatory networks, are still not fully under-
stood. However, transcriptomic and metabolomic studies on developmental senes-
cence in Arabidopsis thaliana (Fig. 8.2) are building a framework at the molecular
level for gluing together more detailed and targeted observations from Arabidopsis
and other species including crops.

Inadequate Availability of Mineral Nutrients Leads

to Nutrient Depletion Induced Senescence

Crops in an agricultural environment are exposed to varying and often non-optimal

environmental conditions, which usually negatively affect carbohydrate synthesis
and nutrient uptake. Moreover, stresses typically occur in combinations: drought
stress is usually accompanied by high temperatures and high light (Long and Ort
2010; Krasensky and Jonak 2012), while unavailability of water in the root zone
hinders nutrient ion uptake and stomatal closure inhibits transpiration and in planta
transport of nutrients. Deficiencies of mineral nutrients leads to nutrient depletion
induced senescence (NuDIS) in plants (Watanabe et al. 2010). NuDIS is accompa-
nied by a mixture of transcriptomic and metabolomic responses depending on
which nutrient ion or combination of ions is depleted. These responses are to
some extent specific for particular nutrients, e.g. the induction of high affinity
transport systems (Hawkesford 2000, 2003; Gojon et al. 2009; Davidian and
Kopriva 2010; Stitt et al. 2002; Xu et al. 2012; Bouguyon et al. 2012). Interestingly,
overlapping molecular responses occur under various nutrient depletions
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 213

(Fig. 8.5a). In particular, later responses all channel into a general senescence
programme comparable to developmental senescence (Fig. 8.5b; Watanabe
et al. 2010, 2012, 2013).
Nutrient ion availability in the field depends on physical parameters such as soil
type and soil chemistry, weather conditions, leaching and run-off and on agricul-
tural parameters such as the amount of minerals removed from the field with the
harvest and fertilisation to replenish resources (Hawkesford 2012). Fertilisation is
usually employed to counteract deficits, though this becomes increasingly costly
due to rising energy costs, e.g. for nitrogen production, and the fact that some
minerals, such as phosphate, might become limiting in the future which would
likely increase prices (Gamuyao et al. 2012). Furthermore, fertilisation carries the
risk of polluting the environment, and it is therefore necessary to improve applica-
tion procedures and to breed mineral nutrient-efficient crop cultivars (McAllister
et al. 2012). In order to strategically support breeding efforts, it is necessary to
understand the processes and identify the genes affecting nutrient use efficiency.
However, the situation in a field can be very complex as nutrients can be available
in varying amounts and combinations. Deficiency might exist from germination on
poor soils or might become established during growth due to active depletion of soil
minerals by the plants or by environmental conditions such as heavy rainfalls.
Sufficient minerals might even be present in a given soil but not accessible to the
plant due to unfavourable soil pH, structure or drought. The effect of distinct
mineral nutrient stresses on plants is well documented from agricultural practice
and from long-term, systematic field experiments, such as the Broadbalk field trial
at Rothamsted Research, UK (Lu et al. 2005; Shinmachi et al. 2010). Plants react to
mineral nutrient depletion with gross phenotypic responses (Fig. 8.3a), such as
reduction of biomass and seed yield, alteration of leaf pigment contents and
distribution, and alteration of root morphology (Lopez-Bucio et al. 2006; Gruber
et al. 2013, Nikiforova et al. 2004; Walch-Liu et al. 2006; Marschner 2012). It is
suggested that the plant response to nutrient deprivation can be divided into two
major phases (Fig. 8.3b).
During the initial rescue phase, the plant tries to counteract mineral deficien-
cies by inducing reversible adaptation mechanisms including increased uptake in
the root zone, mobilisation of internal resources, and by preventing biosynthetic
investments dependent on the depleted nutrient. High-affinity transporters get
induced in roots, which allow the plant to accumulate ions against steep con-
centration gradients present between soil with very low ion concentrations and
root tissue. Lateral root development is decreased in favour of exploratory growth
of the main roots (Gruber et al. 2013; Hubberten et al. 2009, 2012b; Drew and
Saker 1975; Walch-Liu et al. 2006), assimilatory enzymes are activated and the
respective genes are induced (Hoefgen and Hesse 2008; Davidian and Kopriv
2010). Additionally, previously deposited internal stores of limiting elements
such as those found in the vacuole are utilised by inducing vacuolar transporters
in the tonoplast. In the face of continued deficiency, these primary responses are
supported by strategies promoting degradation processes or by delaying anabolic
processes. Even mild nitrogen starvation quickly affects polysome loading and
214 S.J. Whitcomb et al.

Fig. 8.3 Generalised response scheme of annual plants to mineral nutrient availability. Plants
have evolved complex and flexible response mechanisms to cope with variable mineral nutrient
availability, from optimal supply to complete depletion of one or several nutrients. (a) With
decreasing nutrient availability, the relative growth rate, and hence biomass and eventually yield is
reduced. As the plant at the same time “invests” in exploratory root growth, a relative increase of
the shoot to root ratio can be observed. Severe starvation leads to alterations in the plant life cycle
including earlier senescence. (b) When nutrient depletion occurs during the growth phase, the
plant seeks to maintain homeostasis by activating rescue programmes to adapt its metabolism
(points a–d). If nutrient starvation persists, a point of no return is reached when metabolism is
irreversibly shifted to emergency programmes including senescence and early flower formation
and seed production while cannibalizing internal resources (points e.g.). The “goal” is to produce
as many seeds as possible even under continuing mineral nutrient starvation

hence reduces protein synthesis rates (Tschoep et al. 2009) and in the case of
sulfur starvation in Arabidopsis, degradation of glucosinolates (sulfur-rich
secondary metabolites) increases and their synthesis rates decrease (Yoshimoto
and Saito 2012). Additionally, any macronutrient starvation immediately negatively
affects photosynthesis (Fig. 8.6), thereby decreasing carbon supply, essentially
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 215

stalling growth (Wulff-Zottele et al. 2010). Mineral nutrient deficiencies also result in
ROS accumulation, which triggers stress and senescence responses such as the
accumulation of anthocyanins. During this initial response phase, resupply or acqui-
sition of nutrients can restore normal cellular functions and rescue the plant, though
yield reductions are likely as biomass accumulation was reduced during the time of
nutrient deprivation.
Continued starvation of one or more mineral nutrients leads to an emergency
response phase. The plant irreversibly switches its developmental programme to
maturation and senescence (NuDIS; Watanabe et al. 2010). This transition marks a
point of no return. Resupply of nutrients in this phase does not reverse the
maturation programme leading to flower formation, seed ripening and terminal
leaf senescence. The plant apparently invests all remaining resources in producing
as many seeds as possible. The programme actually resembles that of developmen-
tal senescence (Fig. 8.1), which also irreversibly propagates from the vegetative to
the generative phase when the decision for flower formation has been made.
However, mineral nutrient starvation shifts the timing for senescence to an earlier
time point, thus shortening the individual life span.
The effect of NuDIS on yield can be dramatic in crop plants. This has been
demonstrated for example for potassium deficiency (Armengaud et al. 2004;
Amtmann and Armengaud 2009), phosphate deficiency (Batten and Wardlaw
1987) or nitrogen depletion (Parrot et al. 2010). For example, potassium exerts
multiple functions in metabolism and is the second most abundant mineral in plants
after nitrogen. Deficiency leads to multiple negative effects on osmoregulation,
photosynthesis, protein synthesis, enzyme activities and results in light-dependent
chlorosis and necrosis of leaf tissues, all of which impair growth and yield. Wheat
grown on insufficient phosphate senesce far more rapidly and have drastically
reduced seed yield (Batten and Wardlaw 1987). The flag leaf, which provides 51–
89 % of grain phosphorus (Batten et al. 1986; Fig. 8.4), starts to rapidly senesce about
20 days post- anthesis (DPA). This early and steep decay in chlorophyll content is
associated with slower grain filling, probably due to inadequate carbon supply from
reduced photosynthetic capacity. About 40 DPA, flag leaves of phosphate-depleted
plants are essentially devoid of chlorophyll and unable to perform photosynthesis for
carbon supply to the grain. Grain filling plateaus at about 60 % of the potential grain
dry weight result in a yield penalty of about 40 %. Under sufficiently high phosphate
supply, chlorophyll decay also starts about 20 DPA, although at a much slower rate
and accelerates only at about 40 DPA when grains have already reached about 80 %
of their final dry weight. Under phosphate-depleted conditions seed filling stops at
about 35 DPA while under phosphate-sufficient conditions grain filling continues for
an additional 20 days. Thus, early senescence in vegetative tissues truncates seed
maturation and reduces yield (Batten and Wardlaw 1987; Lynch and White 1992;
Lauer et al. 1989; Fig. 8.4).
Notably, under optimal phosphate supply conditions, approximately 30 % of
wheat flag leaf chlorophyll is intact when grain filling is complete, and therefore a
substantial amount of leaf nitrogen remains immobilised in the leaf (Fig. 8.4; Batten
and Wardlaw 1987). Similarly, it has been shown in Arabidopsis that N and P are
substantially but incompletely mobilised during leaf senescence, while significant
216 S.J. Whitcomb et al.

Fig. 8.4 Effect of phosphate starvation on plant senescence and yield (Adapted from Batten and
Wardlaw 1987). Under ample phosphate supply (solid line) chlorophyll content, which is a proxy
for photosynthetic activity, decreases slowly for the first 40 days post anthesis (DPA). Grain dry
weight (dashed line) increases during this period and reaches a plateau at about 50 DPA. Only in
the latest stages of grain filling does chlorophyll breakdown accelerate, even though complete
degradation is not reached. Under phosphate deficient conditions (dotted line) chlorophyll content
declines steeply starting at about 20 DPA and is completely degraded by 30–40 DPA, indicating
that the flag leaf is fully senesced. Due to this early and enhanced senescence, the flag leaf is
unable to provide photosynthates to the developing grain. Grain filling (dash – dotted line) is
truncated by about 20 days, and final grain dry weight reaches only about 60 % of the weight under
full phosphate supply

quantities of sulfate actually remain in the leaf, though organic sulfur compounds
appear to be exported from the senescing leaf (Watanabe et al. 2013). Breeding
efforts to improve nutrient use efficiency need to take into account the inevitable
compromise between nutrient mobilisation and maintenance of leaf functionality,
especially photosynthesis, as already discussed for the stay-green phenotype.
Rather than gross changes, fine-tuning of senescence processes to the actual growth
condition and for each crop are more likely to produce reliable increases in crop
nutrient use efficiency.

Plant Systems Biology Approaches to Nutrient Depletion

Responses

In order to understand and eventually to manipulate nutrient use efficiency in crops

it is necessary to understand the interplay of nutrient availability, nutrient depletion
induced senescence and nutrient use efficiency. As these are not simple causal
relationships but rather dynamic networks of multiple factors which cross-influence
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 217

each other, systems biology approaches will be necessary to unravel these

relationships. Complicating this analysis, the biochemistry of a plant under nutrient
deprivation typically shows pleiotropic effects, which are difficult to directly
connect to the initial nutrient stress (Nikiforova et al. 2004, 2005). Metabolites
related to a distinct nutrient are often involved in diverse biochemical processes
different from the immediate assimilatory pathway. For example, sulfate starvation
results in reduced S-adenosylmethionine (SAM) levels in plants. As SAM is one of
the main methyl group donors for biochemical reactions, pleiotropic effects of
sulfate starvation are inevitable (Nikiforova et al. 2005; Morcuende et al. 2007).
Therefore, it is necessary to differentiate between early response processes, such
as the induction of high affinity transporters or assimilatory genes, later processes
triggered by already downstream effects (Fig. 8.3b) and general processes such
as the NuDIS induced induction of SAGs (Figs. 8.2b, 8.3b, and 8.5b).
Arabidopsis thaliana, due to its amenability to systems biology approaches, has
been widely used to investigate the effects of single nutrient starvations on the
transcriptome (Wang et al. 2003; Amtmann and Armengaud 2009; Watanabe
et al. 2010, 2013) and the metabolome (Nikiforova et al. 2005; Morcuende
et al. 2007). Taken together, these studies reveal that responses to distinct nutrient
stresses overlap to a certain extent (Figs. 8.5a and 8.6), even when focusing on the
subfraction of the SAGs (Fig. 8.5b). Some trends are obvious and corroborate
previous findings. During senescence protein and chlorophyll levels are reduced
(Fig. 8.2c). Anthocyanin accumulation, a typical feature of stressed plants, is shared
across N, P, and S starvation due to the induction of the senescence-associated
transcription factor PAP1 (MYB75; Fig. 8.5a, b; Tohge et al. 2005; Watanabe
et al. 2010, 2012). Also, one of the most responsive genes to sulfate starvation, low
sulfur induced (LSU; Hubberten et al. 2012a), is induced under all three nutrient
conditions compared here. LSU has been postulated to be involved in autophagy
(Zientara-Rytter et al. 2011), a degradatory process, which mobilises nutrients from
cellular macromolecules, even organelles (Fig. 8.3b) during senescence and nutri-
ent deficiency rescue programmes (Wu et al. 2012). See also the chapter of
Collados-Rodriguez et al. in this book.
The transcriptome responses to N, P, and S starvation overlap substantially
(Fig. 8.5; Watanabe et al. 2012). Among the 1,240 genes induced at least twofold
under phosphate starvation, 12 % are shared with sulfate and 43 % with nitrate
starvation, and from 1,602 sulfate starvation induced genes, 39 % are shared with
nitrate and 9 % with phosphate starvation. When assigning such expression profiles
to functional categories (Fig. 8.6) using MAPMAN annotations (Thimm
et al. 2004), complex patterns of up- and down-regulation within individual cate-
gories and even within gene families are observed which are not easy to interpret. It
becomes obvious that certain categories are over-represented, e.g. genes of photo-
synthesis and carbohydrate biosynthesis are down-regulated and that this is a
common trend for all nutrient starvations and also senescence (Fig. 8.6). Further-
more, the strength of the response mirrors the severity of symptoms (Figs. 8.5a and
8.6) as nitrogen starvation is usually stronger than phosphate, followed by sulfate
starvation (Wulff-Zottele et al. 2010). The molecular basis and the physiological
218 S.J. Whitcomb et al.

a Comparison of transcript changes during nutrient starvations

> 2-fold < 0.5-fold

S deficiency N deficiency S deficiency N deficiency

1602 genes 942 511 2037 3084 genes 874 genes 748 98 1085 1816 genes
106 24
43 430 4 609
P deficiency 661 P deficiency 177
1240 genes 814 genes

b Senescence associated genes (SAGs)

> 2-fold
SAGs (827 genes)
100
S deficiency N deficiency
80
< 0.5 53 genes 13 9 188 376 genes
% altered

60
0.5-2 25
40 6 154
20 >2 P deficiency 86
271 genes
0
S N P

Fig. 8.5 Transcriptomic responses to sulfate, nitrate and phosphate (S, N and P) deficiencies. (a)
Transcriptome data of Arabidopsis seedlings exposed to nitrate, phosphate and sulfate starvation
were compared. The Venn diagrams show the overlap respectively, the specificity between the
data sets in terms of genes induced or reduced by a factor of 2, respectively. The data sets were
obtained from the following publications: S, low-sulfate for 7 days + sulfate-0 mM for 2 days
(Bielecka, unpublished data); N, full nutrient for 7 days + nitrate-0 mM for 2 days (Scheible
et al. 2004); P, low-phosphate for 7 days + phosphate-0 mM for 1 day (Morcuende et al. 2007). (b)
Nutrient starvation induces senescence related responses (NuDIS) and the changes of the
subfraction of senescence-associated genes (SAGs) are displayed. The percentage of SAGs
(total 827 genes) with >2-fold and <0.5-fold changes is calculated (Buchanan-Wollaston
et al. 2003). The Venn diagram shows the overlap in SAGs between data sets, sulfate, nitrate
and phosphate deficiencies at a threshold of >2-fold. This depicts partial communalities between
the responses recruiting shared response mechanisms

consequences of the observed responses to nutrient deficiency are far from under-
stood. Under all three nutrient starvation conditions genes involved in the light
reactions, the Calvin-Benson cycle and photorespiration are downregulated. This
provides molecular support of previous findings on the effect of nutrient starvation
on photosynthesis. Nutrients are direct constituents of the photosynthetic apparatus,
such as N and S in protein and N and Mg in chlorophyll, which result in the fact that
24 % of leaf total N is located in the thylakoid membranes (Terashima and Evans
1988). Fe and S are part of the iron-sulfur clusters of the photosynthetic machinery.
P is obviously an essential part of photosynthetic reactions due to the centrality of
energy-rich, phosphate-containing organic compounds (ATP, sugar phosphates). In
addition to direct involvement in photosynthesis and carbohydrate synthesis, nutri-
ent depletion usually results in retarded growth because carbohydrate backbones
cannot be utilised for the biosynthesis of amino acids and proteins, vitamins and
cofactors or energy-rich compounds containing P such as ATP (Hawkesford 2000,
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 219

2012; Nikiforova et al. 2005; Davidian and Kopriva 2010). As a result, despite the
negative effect of nutrient depletion on photosynthesis (Fig. 8.6) photosynthesis-
derived carbohydrates might accumulate in the leaves. Reduced sink strength due to
reduced growth or seed development leads to a reduction of the carbohydrate export
from the leaf further contributing to carbohydrate accumulation (Rao et al. 1990;
Stitt 1991). This lack of sink strength and carbohydrate accumulation eventually
leads to oxidative stress under high light conditions, as excess energy cannot be
fully dissipated as heat. Consequently, nutrient starvation leads to photo oxidation,
which further damages the remaining photosynthetic apparatus (Cakmak and
Marschner 1992; Groot et al. 2003) and induction of senescence (Rolland
et al. 2006), resulting in feedback down-regulation of photosynthesis (Pieters
et al. 2001). A similar effect is caused when phloem loading is impaired leading

Fig. 8.6 A comparison of major metabolic pathway gene expression using MapMan. Data for
senescence is from ATGE experiments. Ratio (senescent leaf/green leaf) is shown in log2 scale.
Green leaf: L6 (ATGE_14A-C) and L8 (ATGE_15A-C). Senescent leaf: ATGE experiment
senescent leaf (ATGE_25A-C; Buchanan-Wollaston et al. 2005). Data for sulfate, nitrate and
phosphate deficiencies are from the same references as Fig. 8.5. Fold changes relative to the
nutrient sufficient control are shown in log2 scale. Red and Blue squares indicate genes induced or
repressed, respectively. First, different nutrient depletions show common response patterns, even
with the senesce data, especially for light reactions, Calvin cycle, and photorespiration. Further,
the strength of response to nutrient starvations affect plant metabolism in the order nitrogen,
phosphate, and sulfate
220 S.J. Whitcomb et al.

Fig. 8.6 (continued)

8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 221

Fig. 8.6 (continued)

to photosynthate accumulation in the leaf accompanied by chlorosis and necrosis

(von Schaewen et al. 1990). As shown in Figs. 8.5 and 8.6, established nutrient
starvation overlaps with developmental senescence, hence the term NuDIS.
The plant response to nutrient stress is not only complex with respect to specific
versus common responses, to the plant species and to specific nutrients but is
additionally a dynamic process. Likewise, the ability of plants to positively respond
to nutrient re-supply by fertilisation or if the plant manages to access nutrients is
strictly dependent on the developmental status of the plant (Zörb et al. 2012;
Steinfurth et al. 2012). Knowledge of the processes during alleviation of starvation
responses is incomplete at the molecular level. To understand these processes and
their molecular and regulatory bases it will be necessary to compare the responses
of plant (model systems and crops) at various stages and severities of starvation or
replenishment and also with combinations of mineral nutrient depletions, as exem-
plified in the chapter of Forieri et al. of this book. Furthermore, it will be helpful to
correlate existing knowledge with improved profiling and phenotyping tools and to
employ systems biology approaches such as transcriptomics, proteomics and
metabolomics. It will also be necessary to devise novel bioinformatics and
222 S.J. Whitcomb et al.

modelling tools. In order to disentangle these complex networks, it will probably be

necessary to specify and subgroup the NuDIS-associated Genes (NAGs) and SAGs
into functional categories, or response modules. One such effort has successfully
been made in identifying the OAS-module as part of the sulfate starvation response
(Hubberten et al. 2012a).

Conclusions and Expectations for Plant Mineral Nutrient

Systems Research
The challenge facing twenty-first century agriculture is multifaceted. Growth
of the world’s population will necessitate producing significantly more food,
while food production is increasingly in competition with non-food plant
commodities for the world’s finite arable land. Furthermore, food production
is becoming increasingly vulnerable in the face of a rapidly changing climate.
In addition to the climate-associated factors water, heat, and light, mineral
nutrient availability is a critical determinant of agricultural yield. In fact,
increased use of fertilisers in the twentieth century is one of the major factors
contributing to the “green revolution”. However, awareness is growing that
short-term solutions in the quest for higher yield, such as copious application
of fertiliser, need to be replaced with environmentally sustainable agricultural
production. Modern cultivars were selected for their elite performance under
“luxury” nutrient regimes. Therefore, development of new varieties that more
efficiently convert soil nutrients into harvestable yield will be absolutely
critical if global agriculture is to be able to feed the world sustainably.
One approach contributing to these goals is to understand the molecular
bases of plant response programs to various nutrient conditions, especially to
nutrient levels lower than typically used in intensive agriculture. Time
resolved systems biology analyses on single and multiple nutrient stresses
and nutrient replenishment will yield information about processes and can-
didate genes or alleles. Obviously, such analysis is currently still largely
based on research employing model organisms, as much at the screening
level as at the proof of concept level. To transfer this knowledge, field studies
will be necessary to prove the validity of the concepts, particularly as
different crop species follow different strategies (i.e. have different ecolog-
ical niches). Systems biology-level tools will be needed for field experiments
and breeding populations to ensure development of elite cultivars. This
necessitates novel approaches at the analytical but also at the bioinformatics
and modelling levels. Finally, systems level analysis needs to be extended
beyond the individual plant into the ecophysiological level, as crop plants
exist in a complex interaction web with geochemistry, climate, and the
surrounding biome.
Additionally, renewable resources must provide the commodities for a
bio-based chemistry. To this end, biodiversity can be exploited by crossing

(continued)
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 223

elite cultivars with wild members of the same species or even with more
distant relatives. Biodiversity can also be generated through mutagenesis.
Targeted engineering by transgenic approaches will allow breeders to exploit
knowledge and gene pools, even across species borders. Agricultural prac-
tices such as mixed cropping, improved fertilisation regimes, and improved
harvesting and storage procedures might also marginally contribute. With
respect to plant mineral nutrients, the ultimate breeding goal is to improve
uptake and internal use efficiency in novel crop cultivars in order to reduce
losses, reduce costs and maintain high yields and high quality of the harvested
material. All approaches have to take into account the paradigm of retaining
the very high yield potential of modern crop cultivars or the goal of providing
more food for a growing population on less land, environmentally sustainably
and affordably, will not be met.

References

Amtmann A, Armengaud P (2009) Effects of N, P, K and S on metabolism: new knowledge gained

from multi-level analysis. Curr Opin Plant Biol 12:275–283
Amtmann A, Blatt MR (2009) Regulation of macronutrient transport. New Phytol 181:35–52
Armengaud P, Breitling R, Amtmann A (2004) The potassium-dependent transcriptome of
Arabidopsis reveals a prominent role of jasmonic acid in nutrient signaling. Plant Physiol
136:2556–2576
Astolfi S, Zuchi S, Hubberten H-M, Pinton R, Hoefgen R (2010) Supply of sulphur to S-deficient
young barley seedlings restores their capability to cope with iron shortage. J Exp Bot 61:799–
806
Batten GD, Wardlaw IF (1987) Senescence and grain development in wheat plants grown with
contrasting phosphorus regimes. Austr J Plant Phys 14:253–265
Batten GD, Wardlaw IF, Aston MJ (1986) Growth and the distribution of phosphorus in wheat
developed under various phosphorus and temperature regimes. Austr J Agric Res 37:459–469
Bogard M, Jourdan M, Allard V, Martre P, Perretant MR, Ravel C, Heumez E, Orford S, Snape J,
Griffiths S, Gaju O, Foulkes J, Le Gouis J (2011) Anthesis date mainly explained correlations
between post-anthesis leaf senescence, grain yield, and grain protein concentration in a winter
wheat population segregating for flowering time QTLs. J Exp Bot 62:3621–3636
Bouguyon E, Gojon A, Nacry P (2012) Nitrate sensing and signaling in plants. Sem Cell Dev Biol
23:648–654
Breeze E, Harrison E, McHattie S, Hughes L, Hickman R, Hill C, Kiddle S, Kim YS, Penfold CA,
Jenkins D, Zhang CJ, Morris K, Jenner C, Jackson S, Thomas B, Tabrett A, Legaie R, Moore
JD, Wild DL, Ott S, Rand D, Beynon J, Denby K, Mead A, Buchanan-Wollaston V (2011)
High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a
distinct chronology of processes and regulation. Plant Cell 23:873–894
Buchanan B, Gruissem W, Jones RL (2007) Biochemistry & molecular biology of plants.
New York
Buchanan-Wollaston V, Earl S, Harrison E, Mathas E, Navabpour S, Page T, Pink D (2003) The
molecular analysis of leaf senescence – a genomics approach. Plant Biotechnol J 1:3–22
Buchanan-Wollaston V, Page T, Harrison E, Breeze E, Lim PO, Nam HG, Lin JF, Wu SH,
Swidzinski J, Ishizaki K, Leaver CJ (2005) Comparative transcriptome analysis reveals
224 S.J. Whitcomb et al.

significant differences in gene expression and signalling pathways between developmental and
dark/starvation-induced senescence in Arabidopsis. Plant J 42:567–585
Cakmak I, Marschner H (1992) Magnesium deficiency and high light intensity enhance activities
of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant
Physiol 98:1222–1227
Cassin G, Mari S, Curie C, Briat JF, Czernic P (2009) Increased sensitivity to iron deficiency in
Arabidopsis thaliana overaccumulating nicotianamine. J Exp Bot 60:1249–1259
Chapin LJ, Jones ML (2009) Ethylene regulates phosphorus remobilization and expression of a
phosphate transporter (PhPT1) during petunia corolla senescence. J Exp Bot 60:2179–2190
Davidian JC, Kopriva S (2010) Regulation of sulfate uptake and assimilation – the same or not the
same? Mol Plant 3:314–325
De Groot CC, Van Den Boogaard R, Marcelis LF, Harbinson J, Lambers H (2003) Contrasting
effects of N and P deprivation on the regulation of photosynthesis in tomato plants in relation to
feedback limitation. J Exp Bot 54:1957–1967
Doerner P (2008) Phosphate starvation signaling: a threesome controls systemic P(i) homeostasis.
Curr Opin Plant Biol 11:536–540
Dolferus R, Ji X, Richards RA (2011) Abiotic stress and control of grain number in cereals. Plant
Sci 181:331–341
Drew MC, Saker LR (1975) Nutrient supply and the growth of the seminal root system of barley.
II. Localized, compensatory increases in lateral root growth and rates of nitrate uptake when
nitrate supply is restricted to only part of the root system. J Exp Bot 26:79–90
Dyson T (1999) World food trends and prospects to 2025. Proc Natl Acad Sci U S A 96:5929–5936
Feller U, Anders I, Mae T (2008) Rubiscolytics: fate of rubisco after its enzymatic function in a
cell is terminated. J Exp Bot 59:1615–1624
Galili G, Hoefgen R (2002) Metabolic engineering of amino acids and storage proteins in plants.
Metab Eng 4:3–11
Gamuyao R, Chin JH, Pariasca-Tanaka J, Pesaresi P, Catausan S, Dalid S, Slamet-Loedin I,
Tecson-Mendoza EM, Wissuwa M, Heuer S (2012) The protein kinase Pstol1 from traditional
rice confers tolerance of phosphorus deficiency. Nature 488:535–541
Gojon A, Nacry P, Davidian JC (2009) Root uptake regulation: a central process for NPS
homeostasis in plants. Curr Opin Plant Biol 12:328–338
Gong YH, Zhang J, Gao JF, Lu JY, Wang JR (2005) Slow export of photoassimilate from stay-
green leaves during late grain-filling stage in hybrid winter wheat (Triticum aestivum L.). J
Agron Crop Sci 191:292–299
Gregersen PL, Holm PB, Krupinska K (2008) Leaf senescence and nutrient remobilisation in
barley and wheat. Plant Biol 10:37–49
Gregersen PL, Culetic A, Boschian L, Krupinska K (2013) Plant senescence and crop productivity.
Plant Mol Biol 82:603–622
Gruber BD, Giehl RF, Friedel S, von Wirén N (2013) Plasticity of the Arabidopsis root system
under nutrient deficiencies. Plant Physiol 163:161–179
Guo FQ, Crawford NM (2005) Arabidopsis nitric oxide synthase1 is targeted to mitochondria and
protects against oxidative damage and dark-induced senescence. Plant Cell 17:3436–3450
Guo Y, Gan S (2012) Convergence and divergence in gene expression profiles induced by leaf
senescence and 27 senescence-promoting hormonal, pathological and environmental stress
treatments. Plant Cell Environ 35:644–655
Guo Y, Cai Z, Gan S (2004) Transcriptome of Arabidopsis leaf senescence. Plant Cell Environ
27:521–549
Haensch R, Mendel RR (2009) Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe,
Ni, Mo, B, Cl). Curr Opin Plant Biol 12:259–266
Hawkesford MJ (2000) Plant responses to sulphur deficiency and the genetic manipulation of
sulphate transporters to improve S-utilization efficiency. J Exp Bot 51:131–138
Hawkesford MJ (2003) Transporter gene families in plants: the sulphate transporter gene family –
redundancy or specialization? Physiol Plant 117:155–163
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 225

Hawkesford MJ (2012) Sulfate uptake and assimilation – whole plant regulation. In: De Kok LJ,
Tausz M, Hawkesford MJ, Hoefgen R, McManus MT, Norton RM, Rennenberg H, Saito K,
Schnug E, Tabe L (eds) Sulfur metabolism in plants: mechanisms and application to food
security, and responses to climate change. Dordrecht Heidelberg London New York, pp 11–24
He ZL, Yang XE, Stoffella PJ (2005) Trace elements in agroecosystems and impacts on the
environment. J Trace Elem Med Bio 19:125–140
Himelblau E, Amasino RM (2001) Nutrients mobilized from leaves of Arabidopsis thaliana
during leaf senescence. J Plant Physiol 158:1317–1323
Hirel B, Le Gouis J, Ney B, Gallais A (2007) The challenge of improving nitrogen use efficiency in
crop plants: towards a more central role for genetic variability and quantitative genetics within
integrated approaches. J Exp Bot 58:2369–2387
Hoefgen R, Hesse H (2008) Sulfur and cysteine metabolism. In: Jez J (ed) Sulfur: a missing link
between soils, crops and nutrition, vol 50. American Society of Agronomy, Madison, pp 83–
104
Hoefgen R, Hesse H, Galili G (2004) Amino acid metabolism. In: Christou P, Klee H, (eds) Plants –
quantitative and qualitative features. Handbook of plant biotechnology. New Jersey pp 577–608
Hörtensteiner S (2012) Update on the biochemistry of chlorophyll breakdown. Plant Mol Biol
82:505–517
Hubberten H-M, Hesse H, Hoefgen R et al (2009) Lateral root growth in sulfur enriched patches.
In: Sirko A, De Kok LJ, Haneklaus S, Hawkesford MJ, Rennenberg H, Saito K, Schnug E,
Stulen I (eds) Sulfur metabolism in plants: regulatory aspects, significance of sulfur in the food
chain, agriculture and the environment. Backhuys Publishers/Margraf Publishers, Leiden/
Weikersheim, pp 105–108
Hubberten H-M, Drozd A, Tran BV, Hesse H, Hoefgen R (2012a) Local and systemic regulation of
sulfur homeostasis in roots of Arabidopsis thaliana. Plant J 72:625–635
Hubberten H-M, Klie S, Caldana C, Degenkolbe T, Willmitzer L, Hoefgen R (2012b) An
additional role of O-acetylserine as a sulphur status independent regulator during plant growth.
Plant J 70:666–677
Ishida H, Yoshimoto K, Izumi M, Reisen D, Yano Y, Makino A, Ohsumi Y, Hanson MR, Mae T
(2008) Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the
vacuole by an ATG gene-dependent autophagic process. Plant Physiol 148:142–155
Izumi M, Wada S, Makino A, Ishida H (2010) The autophagic degradation of chloroplasts via
rubisco-containing bodies is specifically linked to leaf carbon status but not nitrogen status in
Arabidopsis. Plant Physiol 154:1196–1209
Jaggard KW, Qi A, Ober ES (2010) Possible changes to arable crop yields by 2050. Philos Trans R
Soc London, Ser B 365:2835–2851
Jordan DR, Hunt CH, Cruickshank AW, Borrell AK, Henzell RG (2012) The relationship between
the stay-green trait and grain yield in elite Sorghum hybrids grown in a range of environments.
Crop Sci 52:1153–1161
Kato Y, Sakamoto W (2010) New insights into the types and function of proteases in plastids. Int
Rev Cell Mol Biol 280:185–218
Kertesz MA (1999) Riding the sulfur cycle – metabolism of sulfonates and sulfate esters in Gram-
negative bacteria. FEMS Microbiol Rev 24:135–175
Kertesz MA, Mirleau P (2004) The role of soil microbes in plant sulphur nutrition. J Exp Bot
55:1939–1945
Klatte M, Schuler M, Wirtz M, Fink-Straube C, Hell R, Bauer P (2009) The analysis of
Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron
loading and iron deficiency responses. Plant Physiol 150:257–271
Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic
rearrangements and regulatory networks. J Exp Bot 63:1593–1608
Lauer MJ, Blevins DG, Sierzputowska-Gracz H (1989) 31P-Nuclear magnetic resonance determi-
nation of phosphate compartmentation in leaves of reproductive soybeans (Glycine max L.) as
affected by phosphate nutrition. Plant Physiol 89:1331–1336
226 S.J. Whitcomb et al.

Lers A, Sonego L, Green PJ, Burd S (2006) Suppression of LX ribonuclease in tomato results in a
delay of leaf senescence and abscission. Plant Physiol 142:710–721
Li H, Chen Z, Hu M, Wang Z, Hua H, Yin C, Zeng H (2011) Different effects of night versus day
high temperature on rice quality and accumulation profiling of rice grain proteins during grain
filling. Plant Cell Rep 30:1641–1659
Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58:115–136
Long SP, Ort DR (2010) More than taking the heat: crops and global change. Curr Opin Plant Biol
13:241–248
Lopez-Bucio J, Acevedo-Hernandez G, Ramirez-Chavez E, Molina-Torres J, Herrera-Estrella L
(2006) Novel signals for plant development. Curr Opin Plant Biol 9:523–529
Lu C, Hawkesford MJ, Barraclough PB, Poulton PR, Wilson ID, Barker GL, Edwards KJ (2005)
Markedly different gene expression in wheat grown with organic or inorganic fertilizer. Proc
Royal Soc London – Series B: Biol Sci 272:1901–1908
Lynch J, White JW (1992) Shoot nitrogen dynamics in tropical common bean. Crop Sci 32:392–
397
Marschner P (2012) In: Marschner P (ed) Marschner’s mineral nutrition of higher plants, 3rd edn.
Elsevier, Amsterdam
Martinez DE, Costa ML, Gomez FM, Otegui MS, Guiamet JJ (2008) ‘Senescence-associated
vacuoles’ are involved in the degradation of chloroplast proteins in tobacco leaves. Plant J
56:196–206
Masclaux-Daubresse C, Chardon F (2011) Exploring nitrogen remobilization for seed filling using
natural variation in Arabidopsis thaliana. J Exp Bot 62:2131–2142
Masclaux-Daubresse C, Carrayol E, Valadier MH (2005) The two nitrogen mobilisation- and
senescence-associated GS1 and GDH genes are controlled by C and N metabolites. Planta
221:580–588
Masclaux-Daubresse C, Daniel-Vedele F, Dechorgnat J, Chardon F, Gaufichon L, Suzuki A (2010)
Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and
productive agriculture. Ann Bot 105:1141–1157
McAllister CH, Beatty PH, Good AG (2012) Engineering nitrogen use efficient crop plants: the
current status. Plant Biotechnol J 10:1011–1025
Morcuende R, Bari R, Gibon Y, Zheng W, Pant BD, Bläsing O, Usadel B, Czechowski T, Udvardi
MK, Stitt M, Scheible W-R (2007) Genome-wide reprogramming of metabolism and regula-
tory networks of Arabidopsis in response to phosphorus. Plant Cell Environ 30:85–112
Mueller ND, Gerber JS, Johnston M, Ray DK, Ramankutty N, Foley JA (2012) Closing yield gaps
through nutrient and water management. Nature 490:254–257
Naruoka Y, Sherman JD, Lanning SP, Blake NK, Martin JM, Talbert LE (2012) Genetic analysis
of green leaf duration in spring wheat. Crop Sci 52:99–109
Navabpour S, Morris K, Allen R, Harrison E, A-H-Mackerness S, Buchanan-Wollaston V (2003)
Expression of senescence-enhanced genes in response to oxidative stress. J Exp Bot 54:2285–
2292
Nikiforova VJ, Gakière B, Kempa S, Adamik M, Willmitzer L, Hesse H, Hoefgen R (2004)
Towards dissecting nutrient metabolism in plants: a systems biology case study on sulphur
metabolism. J Exp Bot 55:1861–1870
Nikiforova VJ, Kopka J, Tolstikov V, Fiehn O, Hopkins L, Hawkesford MJ, Hesse H, Hoefgen R
(2005) Systems re-balancing of metabolism in response to sulfur deprivation, as revealed by
metabolome analysis of Arabidopsis plants. Plant Physiol 138:304–318
Parrott DL, Martin JM, Fischer AM (2010) Analysis of barley (Hordeum vulgare) leaf senescence
and protease gene expression: a family C1A cysteine protease is specifically induced under
conditions characterized by high carbohydrate, but low to moderate nitrogen levels. New
Phytol 187:313–331
Pieters AJ, Paul MJ, Lawlor DW (2001) Low sink demand limits photosynthesis under P
(i) deficiency. J Exp Bot 52:1083–1091
8 Mineral Nutrient Depletion Affects Plant Development and Crop Yield 227

Rao M, Fredeen AL, Terry N (1990) Leaf phosphate status, photosynthesis, and carbon
partitioning in sugar beet. III. Diurnal changes in carbon partitioning and carbon export.
Plant Physiol 92:29–36
Richards RA (2000) Selectable traits to increase crop photosynthesis and yield of grain crops. J
Exp Bot 51:447–458
Robson PRH, Donnison IS, Wang K, Frame B, Pegg SE, Thomas A, Thomas H (2004) Leaf
senescence is delayed in maize expressing the Agrobacterium IPT gene under the control of a
novel maize senescence-enhanced promoter. Plant Biotechnol J 2:101–112
Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signaling in plants: conserved
and novel mechanisms. Annu Rev Plant Biol 57:675–709
Scheible WR, Morcuende R, Czechowski T, Fritz C, Osuna D, Palacios-Rojas N, Schindelasch D,
Thimm O, Udvardi MK, Stitt M (2004) Genome-wide reprogramming of primary and second-
ary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure
of Arabidopsis in response to nitrogen. Plant Physiol 136:2483–2499
Schmalenberger A, Hodge S, Hawkesford MJ, Kertesz MA (2009) Sulfonate desulfurization in
Rhodococcus from wheat rhizosphere communities. FEMS Microbial Ecol 67:140–150
Semenov MZ, Halford NG (2009) Identifying target traits and molecular mechanisms for wheat
breeding under a changing climate. J Exp Bot 60:2791–2804
Shinmachi F, Buchner P, Stroud JL, Parmar S, Zhao FJ, McGrath SP, Hawkesford MJ (2010)
Influence of sulfur deficiency on the expression of specific sulfate transporters and the
distribution of sulfur, selenium, and molybdenum in wheat. Plant Physiol 153:327–336
Steinfurth D, Zörb C, Braukmann F, Mühling KH (2012) Time-dependent distribution of sulphur,
sulphate and glutathione in wheat tissues and grain as affected by three sulphur fertilization
levels and late S fertilization. Plant Physiol 169:72–77
Stitt M (1991) Rising CO2 levels and their potential significance for carbon flow in photosynthetic
cells. Plant Cell Environ 14:741–762
Stitt M, Müller C, Matt P, Gibon Y, Carillo P, Morcuende R, Scheible W-R, Krapp A (2002) Steps
towards an integrated view of nitrogen metabolism. J Exp Bot 53:959–970
Tanksley SD, McCouch SR (1997) Seed banks and molecular maps: unlocking genetic potential
from the wild. Science 277:1063–1066
Terashima I, Evans JR (1988) Effects of light and nitrogen nutrition on the organization of the
photosynthetic apparatus in spinach. Plant Cell Phys 29:143–155
Tester M, Langridge P (2010) Breeding technologies to increase crop production in a changing
world. Science 327:818–822
Thimm O, Blaesing O, Gibon Y, Nagel A, Meyer S, Krüger P, Selbig J, Müller LA, Rhee SY, Stitt
M (2004) MAPMAN: a user-driven tool to display genomics data sets onto diagrams of
metabolic pathways and other biological processes. Plant J 37:914–939
Thomas H (2013) Senescence, ageing and death of the whole plant. New Phytol 197:696–711
Tohge T, Nishiyama Y, Hirai MY, Yano M, Nakajima J, Awazuhara M, Inoue E, Takahashi H,
Goodenowe DB, Kitayama M, Noji M, Yamazaki M, Saito K (2005) Functional genomics by
integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an
MYB transcription factor. Plant J 42:218–235
Tschoep H, Gibon Y, Carillo P, Armengaud P, Szecowka M, Nunes-Nesi A, Fernie AR, Koehl K,
Stitt M (2009) Adjustment of growth and central metabolism to a mild but sustained nitrogen-
limitation in Arabidopsis. Plant Cell Environ 32:300–318
Von Liebig J (1840) Die organische Chemie in ihrer Anwendung auf Agrikultur und Physiologie.
Vieweg, Braunschweig
von Schaewen A, Stitt M, Schmidt R, Sonnewald U, Willmitzer L (1990) Expression of a yeast-
derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of
carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of
transgenic tobacco plants. EMBO J 9:3033–3044
228 S.J. Whitcomb et al.

Wada S, Ishida H, Izumi M, Yoshimoto K, Ohsumi Y, Mae T, Makino A (2009) Autophagy plays a
role in chloroplast degradation during senescence in individually darkened leaves. Plant
Physiol 149:885–893
Walch-Liu P, Ivanov I, Filleur S, Gan Y, Remans T, Forde BG (2006) Nitrogen regulation of root
branching. Ann Bot 97:875–881
Wang R, Okamoto M, Xing X, Crawford NM (2003) Microarray analysis of the nitrate response in
Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to
glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol 132:556–567
Watanabe M, Hubberten H-M, Saito K, Hoefgen R (2010) General regulatory patterns of plant
mineral nutrient depletion as revealed by serat quadruple mutants disturbed in cysteine
synthesis. Mol Plant 3:438–466
Watanabe M, Hubberten H.-M, Hoefgen R (2012) Plant response to mineral ion availability:
transcriptome responses to sulfate, selenium and iron. In: De Kok LJ, Tausz M, Hawkesford
MJ, Hoefgen R, McManus MT, Norton RM, Rennenberg H, Saito K, Schnug E, Tabe, L (eds)
Sulfur metabolism in plants: mechanisms and application to food security, and responses to
climate change. Dordrecht Heidelberg London New York, pp 123–134
Watanabe M, Balazadeh S, Tohge T, Erban A, Giavalisco P, Kopka J, Mueller-Roeber B, Fernie
AR, Hoefgen R (2013) Comprehensive dissection of spatio-temporal metabolic shifts in
primary, secondary and lipid metabolism during developmental senescence in Arabidopsis
thaliana. Plant Physiol 162:1290–1310
Wu XY, Kuai BK, Jia JZ, Jing HC (2012) Regulation of leaf senescence and crop genetic
improvement. J Integr Plant Biol 54:936–952
Wulff-Zottele C, Gatzke N, Kopka J, Orellana A, Hoefgen R, Fisahn J, Hesse H (2010) Photo-
synthesis and metabolism interact during acclimation of Arabidopsis thaliana to high irradi-
ance and sulphur depletion. Plant Cell Environ 33:1974–1988
Xu G, Fan X, Miller AJ (2012) Plant nitrogen assimilation and use efficiency. Annu Rev Plant Biol
63:153–118
Yamada K, Matsushima R, Nishimura M, Hara-Nishimura I (2001) A slow maturation of a
cysteine protease with a granulin domain in the vacuoles of senescing Arabidopsis leaves.
Plant Physiol 127:1626–1634
Yan JQ, He CX, Wang J, Mao ZH, Holaday SA, Allen RD, Zhang H (2004) Overexpression of the
Arabidopsis 14-3-3 protein GF14 lambda in cotton leads to a “Stay-Green” phenotype and
improves stress tolerance under moderate drought conditions. Plant Cell Physiol 45:1007–1014
Yoshimoto N, Saito K (2012) Molecular and cellular regulation of sulfate transport and assimi-
lation. In: De Kok LJ, Tausz M, Hawkesford MJ, Hoefgen R, McManus MT, Norton RM,
Rennenberg H, Saito K, Schnug E, Tabe L (eds) Sulfur metabolism in plants: mechanisms and
application to food security, and responses to climate change. Dordrecht Heidelberg London
New York, pp 25–33
Zientara-Rytter K, Lukomska J, Moniuszko G, Gwozdecki R, Surowiecki P, Lewandowska M,
Liszewska F, Wawrzynska A, Sirko A (2011) Identification and functional analysis of Joka2, a
tobacco member of the family of selective autophagy cargo receptors. Autophagy 7:1145–1158
Zörb C, Steinfurth D, Gödde D, Niehaus V, Möhling KH (2012) Metabolite profiling of wheat flag
leaf and grains during grain filling phase as affected by sulfur fertilisation. Funct Plant Biol
39:156–166
Zuchi S, Cesco S, Varanini Z, Pinton R, Astolfi S (2009) Sulfur deprivation limits Fe-deficiency
responses in tomato plants. Planta 230:85–94
Chapter 9
Nutrient Use and Nutrient Use Efficiency
of Crops in a High CO2 Atmosphere

Sabine Tausz-Posch, Roger Armstrong, and Michael Tausz

Abstract Atmospheric CO2 concentrations [CO2] are continually increasing and

are predicted to reach ~550 μmol mol
1 by 2050, about a 40 % increase from 2013
levels. Such a large increase in one of the key resources for plant growth will have
significant effects on all plants, as carbon assimilation and, consequently, growth
and yield is stimulated by the so-called ‘CO2 fertilisation effect’. The one sided
increase in carbohydrate acquisition leads to changes in the chemical composition
of plants: despite decreases in nutrient concentrations in plant tissues, the greater
biomass developed by crops under elevated [CO2] could lead to increased nutrient
demand. Nutrient use efficiency in terms of yield divided by available nutrient may
improve, but grains or vegetative plant parts have decreased protein and mineral
nutrient concentrations, which can diminish market and nutritious value. A number
of hypotheses have been proposed to explain the decreases in nutrient concentra-
tions, among them: (1) Dilution by increased biomass, (2) decreased mass flow,
(3) changes in root architecture and function, (4) decreased nitrate reduction, and
(5) changes in nutrient allocation and remobilisation. In addition, elevated [CO2] is
likely to change soil processes, including nutrient supply. The extent to which some
or all of these contribute to changes in crop nutrition and yield quality is currently
unknown because most have not been sufficiently tested under relevant field
conditions. This chapter gives an overview of the changes in plant nutrition and
trade-offs under elevated [CO2] to point out that current and future efforts towards
improved plant nutrient efficiency should explicitly take into consideration rising
[CO2]. In particular, field testing of putative nutrient use efficiency traits and
nutrient management strategies should include elevated [CO2] as a relevant factor
in suitable exposure systems such as Free Air CO2 Enrichment (FACE) technology.

S. Tausz-Posch (*)
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne,
Creswick, VIC 3363, Australia
e-mail: [email protected]
R. Armstrong
Department of Environment and Primary Industries, The University of Melbourne,
Horsham, VIC 3402, Australia
M. Tausz
Department of Forest and Ecosystem Science, The University of Melbourne, Creswick,
VIC 3363, Australia

© Springer International Publishing Switzerland 2014 229

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_9
230 S. Tausz-Posch et al.

Keywords FACE (free air carbon dioxide enrichment) • Carbon dioxide • Climate
change • NUE (nitrogen use efficiency) • Metabolism • Nitrogen • Photosynthesis
• Nutrient content • Roots • Soils

Introduction

Carbon exchange between large reservoirs in the atmosphere, land and oceans
constitutes the complex global carbon cycle. The carbon involved in this cycle is
in a state of dynamic equilibrium, with as much as 400 gigatonnes of carbon
exchanged each year (Kitchen 2014). Since the industrial revolution in the nine-
teenth century, emission of anthropogenic greenhouse gases, predominantly in the
form of carbon dioxide, has disturbed this equilibrium (Kitchen 2014). In 2012,
human activities caused the release of more than 10 gigatonnes additional carbon
emissions into the atmosphere, a number that has grown from the past and will grow
with future global economic development.
Surpluses of released carbon have led to the increase in atmospheric CO2 concen-
trations [CO2] from a pre-industrial concentration of ~278 μmol mol
1 to a current
concentration of ~395 μmol mol
1. Atmospheric [CO2] is predicted to reach
~550 μmol mol
1 by 2050 according to the IPCC scenario A1B (Carter et al. 2007).
This would mean that every terrestrial plant will be exposed to at least a 40 % greater
concentration in one of the key resources for plant growth compared to present
conditions. Effects on climate aside, such a large change, especially if it is not matched
by similar changes in other plant nutrients, will also have considerable effects on plant
production (Ziska 2008). Arguably therefore any crop improvement efforts must
account for the direct effects of increasing atmospheric [CO2] on plant metabolism
and crop growth (Ainsworth et al. 2008; Hatfield et al. 2011; Tausz et al. 2013).
Since the 1980s, significant research efforts have been undertaken to understand
how plants will perform and grow under atmospheric [CO2] enrichment. Method-
ological approaches range from controlled environments such as laboratory growth
chambers and glasshouses to closed-top and open-top field chambers and Free Air
Carbon dioxide Enrichment (FACE) systems. Early enclosure experiments mark
the basic knowledge of our understanding of plant responses to CO2 enrichment but
they also include potential limitations. For example, results from enclosure exper-
iments may include “chamber effects”, possibly exaggerating the effects of ele-
vated [CO2]. Enclosure studies also often include changed radiation conditions,
greater than normal growing temperatures, changed microclimate factors such as
wind speed or relative humidity and/or restricted root growth due to the use of pots
or containers (Ainsworth and Long 2005; Amthor 2001). In contrast, FACE sys-
tems allow the effects of CO2 enrichment on plant metabolism and growth to be
studied under natural and fully open air conditions. Although some concerns have
been raised about the rapid fluctuations of [CO2] in FACE systems (Bunce 2012),
advantages such as the ability to grow crops in their natural microclimate probably
outweigh any such disadvantages. FACE systems might also produce misleading
data if they are used in climates and soils atypical for the plants investigated,
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 231

highlighting the importance to establish FACE experiments in major and

representative cropping areas (Amthor 2001; Tausz et al. 2013). There are a number
of large-scale (10–20 m diameter plots) FACE facilities in agricultural systems
currently in operation globally (Tausz et al. 2013).

Plant Metabolism Changes Under Elevated [CO2]

CO2 enrichment affects C3 plants primarily through increases in photosynthesis

rate (A) and a reduction in stomatal conductance (gs); all other effects of CO2
enrichment on plant metabolism and growth are linked to changes in these pro-
cesses (Fig. 9.1; Ainsworth and Rogers 2007). Increases in A under CO2 enrichment
occur because of particular properties of the key carbon fixation reaction catalysed
by Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Firstly, Rubisco
has a low affinity for CO2 as a substrate for carboxylation, which means that the
reaction is not saturated at current atmospheric [CO2]. Consequently, a rise in
[CO2] increases the carboxylation rate in this reaction, resulting in greater A
(Drake et al. 1997). Secondly, Rubisco also catalyses the oxygenation of
Ribulose-1,5-bisphosphate (RubP) within the photorespiratory pathway. Photores-
piration is competitively inhibited by CO2 and increases in atmospheric [CO2] will
therefore suppress this pathway (Moore et al. 1999).
In contrast to photosynthesis, the mechanisms responsible for the reduction of gs
under CO2 enrichment remain vague. Ainsworth and Rogers (2007) conducted a
meta-analysis of stomatal density responses to CO2 enrichment and they found that
an average 5 % decrease in density was not statistically significant. They concluded
that rather than changes in stomatal density, changes in stomatal aperture are
responsible for decreased gs under high [CO2]. Stomatal aperture is determined

• Yield
• Biomass
• Resource Use Efficiency
• Photosynthesis

CO2
• Stomatal conductance
• Nutrients
• Proteins

Elevated CO2 Ambient CO2

Fig. 9.1 General responses of crops grown under elevated [CO2]. With rising [CO2] grain yield,
biomass, photosynthesis, nutrient and water use efficiency increase while stomatal conductance,
nutrient concentrations and protein concentrations decrease. The picture on the right shows wheat
(Triticum aestivum L.) grown within the Australian Grains Free Air Carbon dioxide Enrichment
(AGFACE) facility in Horsham, Victoria, Australia, either under an elevated CO2 concentration of
~550 μmol mol
1 (on the left) or under an ambient CO2 concentration of ~395 μmol mol
1 (on the
right). Elevated [CO2] grown wheat shows a significant increase in tiller number and therefore in
biomass and yield
232 S. Tausz-Posch et al.

by the turgor pressure in guard cells via ion and organic solute concentrations
(Messinger et al. 2006). Rising [CO2] may be linked to depolarising the membrane
potential in guard cells, resulting in stomatal closure (Ainsworth and Rogers 2007;
Drake et al. 1997; Messinger et al. 2006).
A meta-analysis on the responses of A and gs to CO2 enrichment under FACE
conditions confirmed that in C3 crops light-saturated CO2 uptake rates (Asat)
increased on average by ~13 % while stomatal conductance is reduced by ~25 %
(Ainsworth and Rogers 2007). Increases in A have stimulating effects on plant
growth and productivity (Fig. 9.1; Ziska et al. 2012) and, depending on the plant
species investigated, greater A leads to greater biomass accumulation and yield
(termed “CO2 fertilisation effect”). For example, C3 crops respond to high [CO2]
with ~20 % greater biomass and ~17 % greater yield (Ainsworth and Rogers 2007;
Tausz-Posch et al. 2012). It is noteworthy that in the same meta-analysis (Ains-
worth and Rogers 2007) C4 crops also showed a significant ~11 % increase in Asat
in response to CO2 enrichment. In general, C4 crops are more independent of
ambient [CO2] as they have the ability to concentrate CO2 in the leaf mesophyll.
Their carboxylation reaction is generally close to saturated under current ambient
[CO2]. The positive response of C4 plants to CO2 enrichment was linked to the
improved water status of the crops investigated (Ainsworth and Rogers 2007).
The initial stimulation of C3 photosynthesis may not be sustained when plants
are exposed to CO2 enrichment for long time periods (Kirschbaum 2011; Moore
et al. 1999). Plants grown under CO2 enrichment showed decreased A relative to
plants grown under ambient [CO2] when both were measured at ambient
[CO2]. This down-regulation of photosynthesis under CO2 enrichment is called
“photosynthetic acclimation” (Moore et al. 1999). Photosynthetic acclimation is
linked to reduced concentrations of Rubisco in the leaves, and this is hypothesised
to result from repression of Rubisco synthesis by accumulating carbohydrates
(Drake et al. 1997). If there are insufficient sinks for the increased amounts of
carbohydrates produced in photosynthesis, this situation is accentuated (Moore
et al. 1999; Stitt and Krapp 1999). Although such acclimation reduces the photo-
synthetic capacity of plants, it does normally not completely offset the stimulation
caused by CO2 enrichment (Ainsworth and Rogers 2007).

Mineral Nutrition of Crops Under Elevated [CO2]

With changes in growth and yield, plants grown under CO2 enrichment also show
significant changes in their chemical composition (Erbs et al. 2010). Loladze
reported in 2002 that CO2 enrichment will alter the stoichiometry (relative elemen-
tal composition) of plants leading to reduced concentrations of most macro and
micro elements in plant tissues. As a consequence there are potential negative
consequences for the nutritional value of crops (Loladze 2002). For example,
decreased tissue nitrogen (N) concentrations under elevated [CO2] have been
widely reported (Stitt and Krapp 1999). As a large proportion of N in plants is
used for protein synthesis, protein concentrations in vegetative parts as well as
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 233

seeds and grains tend to decrease under elevated [CO2]. In many crops, particularly
cereals such as wheat or rice, concentrations of protein and minerals in grains are
important determinants of the nutritional value and functional properties (Erbs
et al. 2010; Högy et al. 2009).
The lower tissue N concentrations in conjunction with proportionally increased
carbon assimilation and its subsequent incorporation into storage and structural
material of the plant results in increased carbon to nitrogen ratio (C/N). This leads
to increased nitrogen use efficiency of plants under CO2 enrichment (Fig. 9.1;
Drake et al. 1997). The same principle can be applied for other macro and
micronutrients, although less documented than for N. Although overall nutrient
concentrations in plant biomass are decreased under CO2 enrichment, total nutrient
removal via grains or plant biomass, for example such as N, can increase under CO2
enrichment (Conroy and Hocking 1993; Lam et al. 2012b). Such effects will have
significant consequences for food quality and the management of our agricultural
systems in a future high [CO2] world.

Nitrogen

N is the ecologically and economically most important mineral nutrient in cropping

systems globally, and has thus received intense attention so that knowledge of plant
N metabolism is comparably advanced relative to most other nutrients. It is there-
fore not surprising that the majority of research on interactions of nutrients with
high [CO2] in plants has focused on N. We will therefore discuss major principles of
plant nutrition changes under CO2 enrichment using N as the main example.

Nitrogen Concentrations in Plant Tissues Under CO2

Enrichment

Dry mass based tissue concentrations of nitrogen (Nm) are generally lower in high
[CO2] grown plants, as confirmed by a number of recent synthesis papers (Ains-
worth and Long 2005; Leakey et al. 2009; Stitt and Krapp 1999; Taub and Wang
2008; Wang et al. 2013). For example, leaf Nm decreased by about 13 % across a
number of FACE experiments (Ainsworth and Long 2005), or by an average of 9 %
for wheat across experiments with different CO2 exposure systems and CO2
concentrations (Wang et al. 2013). This decrease is somewhat smaller on a leaf
area basis, only about 4 % across a number of FACE experiments (Leakey
et al. 2009). The decrease in leaf N is consistently related to a decrease in total
leaf protein, specifically, and perhaps exclusively, to a decrease in the amount of
Rubisco protein (Leakey et al. 2009). Decreased Rubisco content in leaves is linked
to photosynthetic downward acclimation under prolonged exposure to increased
[CO2] and may thus reflect changes in N allocation patterns.
234 S. Tausz-Posch et al.

160

140
Total amino acids (mmol kg-1 DW)
120

100

80

60

40

20

0
Oct6 Oct28 Nov19 Dec12

Fig. 9.2 Total free amino acid concentrations in wheat flag leaves from heading (Oct 6) to
maturity (Dec 12). Plants were grown either under ambient CO2 (closed symbols) or an elevated
CO2 concentration of ~550 ppm (open symbols) within the AGFACE facility, Horsham, Australia.
Values are means (standard errors) with four replications in each

For wheat, a decrease in leaf N was also related to decreases in leaf chlorophyll
across a number of experiments (by 8 % according to a synthesis by Wang
et al. 2013), but such changes in chlorophyll are not consistently found. High
[CO2] can also lead to decreases of leaf nitrate concentrations (e.g. for wheat reported
in Hocking and Meyer 1991). There is little information on free amino acids, but
recent FACE data indicate about 20–30 % decreases in wheat leaves (Fig. 9.2).
There is quite a range in responses between different experiments, environments
or species. Most consistent among such variability is that decreases in leaf Nm are
less in legumes than in non-N fixing species, perhaps because legumes can channel
excess carbohydrates towards greater nitrogen fixation rates (Rogers et al. 2009).
Decreases in grain protein contents in the final harvest are commonly
unfavourable for food or feed quality, and of particular concern in cereals such as
wheat, where bread making and marketing quality is largely determined by protein
content (Loladze 2002; Taub et al. 2008). Grain N decreased under CO2 enrichment
by between 0 % and 20 % in cereals (wheat, rice, barley; as reviewed by Högy and
Fangmeier 2008; Högy et al. 2013; Taub et al. 2008) and by less than 5 % (but still
significantly so) in soybean (Taub et al. 2008).

Can Greater N Supply Restore Leaf and Grain N?

It is uncertain to what extent N supply determines the extent to which Nm decreases

in leaves or grains under CO2 enrichment: Some evidence suggested that Nm
decreases were most pronounced in N-deficient and less marked or even absent in
well fertilised plants (Stitt and Krapp 1999), including in FACE experiments
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 235

(Sinclair et al. 2000). This is in line with the suggestion that photosynthetic
downward acclimation, which is related to leaf N and (Rubisco) protein, is less
pronounced or absent in well-fertilised plants. However, recent synthesis papers
suggest that even if high N supply counteracts decreases in N to some extent, Nm
remains significantly lower in leaves (Wang et al. 2013) and grains (Taub
et al. 2008).
Whilst decreases in tissue concentrations of N are reasonably consistent and well
established, results on changes of overall N demand and uptake of the crop per m2
ground area are variable. N uptake into (above ground) biomass depends on the
relative magnitude of biomass increase and decrease in N tissue concentrations.
Most analyses suggest that biomass and yield stimulation are relatively greater than
the decrease in tissue N concentration, which would lead to greater N demands of
bigger crops under CO2 enrichment (Lam et al. 2012a, b, c) despite decreased N
concentrations in tissues. However, others suggest that N uptake remains
unchanged (Wang et al. 2013).
It is noteworthy that the plant growth response to elevated [CO2] in natural
ecosystems, which do not receive extra N input, may decrease over time, and this
was ascribed to a depletion of N (and other nutrient) reserves (¼ ‘progressive
nitrogen limitation’ (PNL) hypothesis, e.g. Hungate et al. 2003).

Adjustment of Nmcrit Under CO2 Enrichment

It has been suggested early on that critical tissue concentrations of N (and other
mineral nutrients) must be adjusted under high [CO2] (Conroy and Hocking 1993;
Fig. 9.3). Tissue concentrations of nutrients such as N are often used as a diagnostic
tool to assess a plant’s nutritional status. Critical tissue (such as leaf) concentration
of N (Nmcrit) would indicate sufficient N supply. Nmcrit is the value of Nm that is
needed for optimum growth, assuming otherwise non-limiting conditions. At leaf N
greater than Nmcrit growth will not increase further (Fig. 9.3). Conroy and Hocking
(1993) suggested that critical concentrations need adjustment towards lower values
under high [CO2] (Fig. 9.3). As such results were derived from pot experiments,
field evaluation in cropping systems will be important.

Does CO2 Enrichment Change N Response of Plants?

Similarly to other essential nutrients, insufficient N supply to crops can limit the
[CO2]-driven growth and yield enhancement; N-limited crops sometimes do not
show a significant effect of CO2 enrichment on biomass, or the relative yield and
biomass increase is less than in well fertilised crops (Stitt and Krapp 1999). As a
consequence, the N response curve may change under elevated [CO2]. N response
curves (Fig. 9.4) are derived from fertiliser experiments (fertiliser added at different
236 S. Tausz-Posch et al.

Fig. 9.3 Scheme illustrating the determination of critical N concentration (Nmcrit) in leaves and
proposed changes under elevated [CO2]. Concrete results reported in Conroy and Hocking (1993)

Fig. 9.4 Hypothetic nitrogen response curves (Mitscherlich curves) of crops grown under ambient
and high [CO2]. Nsat indicates sufficient N supply to achieve 90 % of maximum growth or yield. Yi
indicates yield without additional (fertiliser) N, Yii denotes yield after a certain fertiliser applica-
tion (arrow). It is not resolved whether and under which conditions Nsat changes under elevated
[CO2]. Agronomic efficiency of fertiliser application can be calculated as the difference between
yields with and without fertiliser (Yii-Yi) divided by the fertiliser applied (Table 9.1). In this
hypothetic system, agronomic efficiency of the fertiliser applied (arrow) would be greater under
elevated than ambient [CO2], because the yield difference is greater at the higher N application
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 237

rates), and are an important tool to assess N limitations and requirements of crops
(Hawkesford 2011). Figure 9.4 illustrates the important questions: (1) Is the amount
of N required for maximum growth different between high and ambient [CO2]?
(2) Is the N response (either in % yield increase per kg N or in kg yield increment
per kg N) greater under high [CO2] than under ambient [CO2]? Work on potted
wheat under relatively high CO2 enrichment concluded that the total amount of N
required for maximum growth remains unchanged, and N-responsiveness both in
absolute (g biomass per g added N) and relative terms (in % biomass per g added N)
was greater under high [CO2] (Conroy and Hocking 1993). Whilst this is in line
with the notion of generally greater resource use efficiency of plants under CO2
enrichment, there are few, if any, reports of field data (such as full N response
curves under free air conditions).

Nitrogen Use Efficiencies Under CO2 Enrichment

Changes in tissue concentrations in conjunction with biomass and yield changes

lead to changes in Nitrogen Use Efficiency (NUE) in their various definitions and its
components (Good et al. 2004; Hawkesford 2011; Table 9.1). A frequently used
definition for NUE is the ratio of biomass accumulation or yield over the amount of
available N, and according to this definition NUE is closely related to yield
(Hawkesford 2011). As yield increases under CO2 enrichment, so will by definition
NUE of the crop, provided available N remains unchanged. NUE as defined above
can be divided into Nitrogen Uptake Efficiency (NUpE, N taken up into the biomass
as a proportion of total available N) and Nitrogen Utilisation Efficiency (NUtE, the
ratio of yield or biomass over the N taken up (Hawkesford 2011). Depending on the
relative magnitude of changes in N uptake, growth and yield, and N concentrations
in biomass, the different components of NUE may change (Table 9.1 for an
overview).
On a physiological level, changes in Photosynthetic Nitrogen Use Efficiency
(PNUE) are the result of lower concentrations of leaf N in conjunction with greater
assimilation rates under CO2 enrichment leading per definition to greater PNUE,
where PNUE is defined as carbon assimilation rate (expressed in μmol C fixed per
m2 leaf area and s) per unit leaf N (expressed as g N per m2 leaf area; Narea). A
review of FACE experiments confirmed an increase in PNUE, and this increase was
mainly due to the large increase in assimilation rate, and to a minor extent to a
decrease in Narea (Leakey et al. 2009).
An important aspect of NUE in agricultural systems is the proportion of N
applied to crops (as fertiliser) that is utilised by the target crop. A significant
proportion of the applied N can either remain in the soil (although potentially
available to subsequent crops) or can be lost from the soil-plant systems by several
processes, including denitrification, volatilisation and leaching. For example, in
Australian wheat production systems actual fertiliser N recovered in the wheat plant
itself ranged from approximately 20–60 % (Chen et al. 2008). Recent
238 S. Tausz-Posch et al.

Table 9.1 Effects of elevated [CO2] on different aspects of plant nutrient (Nut) metabolism and
plant nutrient (Nut) use efficiencies. Nutrient use efficiency definitions after (Hawkesford 2011)
Major trends under elevated
Symbol Parameter Explanation [CO2]
Nutm Dry mass based g or mol nutrient per g Decrease in vegetative biomass
tissue tissue dry weight; e. g. mg and grains, most consistent for N
concentration g
1 or μmol g
1 but common also for many other
nutrients
Nutarea Leaf area based g or mol nutrient per m2 Usually decreases, perhaps to a
nutrient content leaf area lesser extent than Nutm
NutUp Nutrient uptake g per m2 plot surface area Commonly increases if biomass
(into biomass), stimulation is greater than Nutm
also nutrient reduction, or remains unchanged
demand
NutYield Nutrient yield g in harvested fraction per Commonly increases due to
plot surface area yield stimulation
NutUE Nutrient use Yield per available Increases due to increase in yield
efficiency nutrient
NutUpE Nutrient uptake Amount of nutrient taken Commonly increases due to
efficiency up into biomass per avail- greater increase in biomass than
able nutrient decrease in Nutm; may remain
unchanged
NutUtE Nutrient utilisation Yield per amount of nutri- Increase or no change
efficiency ent in biomass
NutHI Nutrient harvest Fraction of total nutrient in Decreases for N with ample N
index or nutrient (above ground) biomass supply; may remain unchanged
partitioning that ends up in harvest (e. g. at low N supply)
quotient
PNutUE Photosynthetic Carbon assimilation rate Increases for nitrogen (and other
nutrient use per amount of nutrient in nutrients) mainly due to stimu-
efficiency leaf lation of assimilation
FUE Fertiliser use Amount of nutrient taken Appears unchanged for N, but
efficiency up per amount of nutrient little information available
applied
NutAE Agronomic nutri- Yield increase per nutrient Possibly increases for N, but
ent efficiency applied with fertiliser only few field experiments use
more than 2 nutrient application
rates

experimentation using 15N labelled granular N (urea) fertiliser in dryland grain

systems in southern Australia under FACE found that although the biomass, plant
N content and the amount of N absorbed from the soil by wheat was increased,
elevated [CO2] did not affect the proportion of fertiliser N taken up by the plant (Lam
et al. 2012a). However, in order to meet the greater overall demand for N by the crop,
the authors suggested that greater amounts of N (as fertiliser) would be required in
future climates. Interestingly, elevated [CO2] did increase the rate of N2O emissions,
a potent greenhouse gas, from these wheat systems. This could contribute to
radiative forcing in the atmosphere, although the absolute amount of N lost from
the system (as N2O) would be negligible in terms of N budget (Lam et al. 2013).
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 239

Other Mineral Nutrients

Whilst effects of CO2 enrichment on plant N are relatively consistent and well
documented, there is little consensus about most other mineral nutrients. It is
important to recognise that different mineral nutrients are not independent of
each other: they can share or co-regulate common uptake and metabolic pathways,
and are regulated in an interdependent manner by plant growth or assimilate
availability. We have more detailed knowledge on interactions between P and N
or S and N (Hawkesford and De Kok 2007; White and Hammond 2008), but
interactions apply to other mineral nutrients as well. Although not investigated in
great detail under high [CO2], it is safe to assume that such interactions play an
important role in modifying the CO2 response of selected mineral elements and
their metabolism.
Recent reviews mostly suggest that elevated [CO2] also leads to decreased tissue
concentrations of mineral nutrients other than N, but results are inconsistent: For
example, one recent synthesis paper found significant decreases in 11 (if N is
excluded) elements in elevated [CO2] grown plant tissues, including Mg, K, P, S,
Fe, Ca, Mn, and Zn (McGrath and Lobell 2013). In apparent contrast, Duval
et al. (2012) found only a few significant changes in leaf mineral concentrations
(excluding N) in crops (excluding legumes) and N-fixers: in that analysis, among all
investigated mineral nutrients, only leaf Mg concentrations were significantly
decreased under CO2 enrichment in both functional groups. In legume leaves, B
and Fe decreased as well, whereas Mn even increased significantly. However,
results for grasses were different again, and perhaps most surprisingly, this study
found no significant effects of elevated [CO2] on grain nutrients (Duval et al. 2012).
In several other studies, which focused on the effects on grains rather than vegeta-
tive plant parts, there seems to be overall agreement that the elements N, S, Mg, Ca,
Zn, and Na are significantly reduced under CO2 enrichment while contrasting
results are reported for K, Mn, P and Fe (Fangmeier et al. 1999; Fernando
et al. 2012; Högy and Fangmeier 2008; Högy et al. 2013; Manderscheid et al. 1995).
Some of these discrepancies may be due to different nutrient supply conditions,
which is important for some micronutrients. Apparently contradictory results may
also point out some in principle problems with many synthesis papers, where the
analysed data set may be biased by particular exposure or growing systems, or
factors analysed independently (such as, for example, crop type) may be strongly
confounded by other factors. For example, it is common that data from different
crop types or plant functional groups come from separate groups of experiments
(for example, different large scale FACE experiments), and are therefore poten-
tially confounded by factors such as climate or soil. Further, environmental grow-
ing conditions can mediate nutrient responses to elevated [CO2]: in one FACE
study, variations in rainfall and/or temperature had a significant effect on the
response on macro and micro mineral concentrations in wheat grains to CO2
enrichment (Fernando et al. 2012).
240 S. Tausz-Posch et al.

The interactions between elevated [CO2] and mineral nutrition may be markedly
different for different nutrients: in contrast to N, where critical nutrient concentra-
tions are reduced (Fig. 9.2), critical concentrations of P generally remain the same
or are increased when C3 plants are grown under elevated [CO2] (Ghannoum
et al. 2007). Increases in critical P concentrations of C3 plants grown under elevated
[CO2] have been attributed to changes in competition for P. As photosynthetic
carbon fixation is preferred over the photorespiratory cycle, more P is needed for
the energy carrier ATP (Ghannoum et al. 2007). Similar to N, several studies have
noted that growth (and yield) response to high [CO2] depends on P nutrition. The
relative importance of this interaction between P and high [CO2] however varies
with species. Lam et al. (2012c) found that high [CO2] increases biomass of two
pulse species, chick pea (Cicer arietinum) by 18–64 % and field pea (Pisum
sativum) by 24–57 %, as well as the pasture legume barrel medic (Medicago
trunculata), and that this effect was greater when P supply was non-limiting.

Proposed Mechanisms for Decreased Nutrient

Concentrations in Elevated [CO2]

The mechanisms responsible for and controlling decreased nutrient concentrations

under elevated [CO2] are not completely understood, and there are a number of
hypotheses under consideration. Most of these hypotheses are connected to studies
conducted on N but, depending on the nutrient, similar principles may apply for
other nutrients. An overview is given in Fig. 9.5.

Dilution by Increased Biomass and Carbohydrate Production

CO2 enrichment increases C fixation and dry matter accumulation in plants.

Increased dry matter is mostly derived from greater carbohydrate (C, H and O)
accumulation and concentrations of other macro and micro nutrients will decrease
in biomass if their uptake is not increased (Taub and Wang 2008). For example,
Poorter et al. (1997) investigated the chemical composition and construction costs
of leaves of 27 wild and agricultural species at ambient and elevated [CO2]. They
found that the strongest response of plants to CO2 enrichment in respect to their
chemical composition is the increase in concentrations of total non-structural
carbohydrates (TNC) resulting from stimulated photosynthesis under high
[CO2]. The next strongest responses to CO2 enrichment were decreases in protein
and mineral concentrations leading the authors to conclude that a dilution effect
caused by accumulating TNC significantly contributes to decreasing nutrient con-
centrations under CO2 enrichment. Similar conclusions were made by Taub and
Wang (2008), who used graphical vector analyses to study biomass dilution effects
under CO2 enrichment.
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 241

Fig. 9.5 Schema of plant based mechanisms that potentially contribute to decreases in mineral
nutrient (symbolized by black circles) concentrations in crops grown under elevated
[CO2]. 1 Dilution in increased biomass by increased carbohydrate supply. 2 Decreased mass
flow due to decreased stomatal conductance. 3 Changes in root architecture and function. More
root mass in top soil may improve access to nutrients, but this may come at a cost of access to
deeper layers. Uptake physiology may change. 4 Decreased rate of nitrate (and possibly sulfate)
reduction. 5 Adverse changes in remobilisation from leaves and translocation to grains

Biomass dilution, however, does not entirely account for the total decrease in
nutrient concentrations. For example, when Poorter et al. (1997) cross-checked
decreasing nutrient concentrations for their direct dependence on increased TNC,
the minerals and proteins expressed on a TNC-free biomass basis remained signif-
icantly reduced, even if less so than on a total dry weight basis. This suggests that
processes other than dilution by TNC contribute to decreased nutrient concentra-
tions under CO2 enrichment. Simple dilution by greater (structural) biomass pro-
duction remains a possibility, but if biomass dilution is exclusively responsible for
decreasing nutrient concentrations under CO2 enrichment then all nutrients would
decrease equally in concentration. However, as reported earlier, decreases in
macro- and micronutrients can vary greatly among each other and across studies
(between 0.7 and 19.5 % or 3.7 and 18.3 %) (Fangmeier et al. 1999; Fernando
et al. 2012; Högy and Fangmeier 2008; Högy et al. 2009; Manderscheid et al. 1995),
and there seems to be no general relationship with growth stimulation by elevated
[CO2]. It has been repeatedly suggested that factors relating to nutrient uptake
efficiency and metabolism are involved in decreased nutrient concentrations under
high [CO2]. It has to be acknowledged, however, that an exact quantification of
possible biomass dilution effects within experiments, particularly under free air
growth conditions, is still missing.
242 S. Tausz-Posch et al.

Reduced Mass Flow

Transpiration is the evaporation of water into the atmosphere from the leaves and
stems of plants. It is a passive process mainly governed by the vapour pressure
deficit of the atmosphere and the soil moisture content, but mediated by the variable
resistance of the stomata. CO2 enrichment decreases stomatal conductance leading
to decreased transpiration in plants (Ainsworth and Rogers 2007). Transpiration has
several functions, and one of its functions is to drive the mass flow of nutrients in
the soil to the rhizosphere and root surfaces (Cramer et al. 2009). Mass flow driven
by transpiration contributes significantly to the delivery of nutrients to the plant,
and then the translocation within the plant. For example, Barber (1995) reported
that mass flow contributed by more than 70 % for N, S, Mg and Ca to the nutrient
concentrations in Zea mays with the remaining percentages taken up by diffusion
and interception of roots. The effectiveness of mass flow depends on factors that
influence transpiration. Decreased transpiration rates under CO2 enrichment will
reduce the mass flow of nutrients in the rhizosphere and hence decrease nutrient
availability for plant uptake, as well as decrease the delivery rate of nutrients to the
above ground plant parts. It was hypothesised early that this decreases mineral
nutrition of plants (Conroy and Hocking 1993; McGrath and Lobell 2013).
Evidence that reduced mass flow is partially responsible for decreases in nutrient
concentrations can be provided by comparing the extent to which individual
nutrients decrease under high [CO2]. For example, the largest decrease in concen-
tration was found for mobile nutrients such as N, Mg or Ca that are supplied to the
rhizosphere by mass flow. Conversely, the concentration of less mobile nutrients,
e.g. P, and those that are mainly dependent on physiological uptake processes was
decreased to a lesser extent by CO2 enrichment (Högy and Fangmeier 2008; Högy
et al. 2013; Taub and Wang 2008). A first conclusion might be that biomass dilution
leads to a decrease in concentration of all nutrients while concentrations of mobile
nutrients are further decreased via decreased transpiration mass flow. A recent
analysis aimed at testing the mass flow hypothesis on synthesised literature data
had to resort to pairing mass flow and nutrient uptake data from different studies,
because “No published studies that simultaneously examined mass flow and nutri-
ent concentration for plants grown in elevated [CO2] were available” (McGrath and
Lobell 2013), thus pointing out a major gap in experimental verification.

Root Architecture and Function

Roots are the first plant organs receiving nutrients from the soil and root morphol-
ogy and architectural traits such as length, depth, branching and curving determine
a plant’s access to nutrients (and other resources such as water) in the soil. Despite
their importance to plant growth, most studies on impacts of climate change vari-
ables such as elevated [CO2] have focused on above ground traits rather than roots
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 243

(Benlloch-Gonzalez et al. 2014). The basic development of a root system is given

by its genetic predisposition but conditions such as, for example, soil nutrient
availability or soil moisture content will greatly affect root growth as soon as
growth commences (Rich and Watt 2013).
All C in roots comes from the re-allocation of photosynthates from
photosynthesising above ground biomass with up to 50 % of all the C fixed being
transported below ground during early development (Gregory et al. 1996). Consid-
ering that C3 crops increase their light–saturated CO2 uptake rates on average by
~13 % when grown under CO2 enrichment (Ainsworth and Rogers 2007), the
amount of fixed C allocated to roots increases in plants growing under high CO2
and this can lead to changes in root architectural traits (Pritchard and Rogers 2000).
For example, high [CO2] significantly increases the root biomass and roots often
undergo the greatest relative dry weight gain among all plant organs (recently
reviewed by Madhu and Hatfield 2013). CO2 enrichment also changes the vertical
distribution of roots in a way that more roots accumulate in the top soil layers as
compared to the deeper layers in the soil. This leads to a shallower root system with
greater root density in the top layers (Pritchard and Rogers 2000; Pritchard
et al. 2006; VanVuuren et al. 1997). Although this may increase access to nutrients
which are generally concentrated in topsoils (e.g. Ho et al. 2005), it may also have
undesirable consequences where rooting access to subsoil water is important (Lilley
and Kirkegaard 2011).
Changes in root architectural traits will affect the spatial patterns of soil nutrient
exploitation. A dense root system with many lateral branches and root hairs is
considered to be better for nutrient acquisition as compared to plants with a sparse
root system because of their greater root surface (Kong et al. 2013). Total nutrient
uptake should therefore benefit from increased root length and mass under CO2
enrichment. However, it has been reported that root physiological activity often is
reduced in crops grown under CO2 enrichment (Fitter 1996; Bassirirad et al. 1996),
with potential repercussions on specific uptake capacity of roots. Uptake systems
for nutrients such as N in the form of nitrate and ammonium (Miller and Chapman
2011), or S in the form of sulphate (Hawkesford and De Kok 2006), are now well
characterised and the specific transport systems are subject to physiological regu-
lation. Whilst it is likely that changes in the carbon to nutrient balance in plants
affect such regulation, it is unknown how growth under elevated [CO2] affects
nutrient transporters and their regulation.

Decreased Nitrate Reduction

Another hypothesis regarding the decrease in (specifically) plant N nutrition was

recently put forward by Bloom et al. (2010). Their paper showed a significant
decrease of nitrate reduction under elevated [CO2] in wheat. The hypothesis was
that the decrease in photorespiration affected nitrate assimilation whereby a number
of mechanisms could contribute (Bloom et al. 2010). Yong et al. (2007) reported
244 S. Tausz-Posch et al.

photosynthetic acclimation to CO2 enrichment in rice plants with excessive N

supply, but not in those with lower N supply. It was speculated that C and N
reduction compete for electrons from the photosynthetic light reaction, and that this
leads to an inhibition in nitrate assimilation. Bloom et al. (2010) also suggested that
the inhibition of nitrate assimilation by elevated [CO2] might affect nitrate uptake
rates as a follow-on effect. If impaired nitrate reduction is a reason for declining
tissue N concentrations then meeting N needs through ammonium NH4+ could
alleviate the problem. It has been suggested that ammonium-based fertilisers or the
use of nitrification inhibitors (to avoid conversion of ammonia into nitrate in the
soil) could be considered, provided NH4+ toxicity and detrimental effects on soils
can be managed (Bloom 2009). A recent study seems to corroborate this hypothesis
because plants supplied with NH4+ as an N source showed better responses to
elevated [CO2] than those supplied with NO3
(Carlisle et al. 2012). However, this
approach has not yet been tested under realistic field conditions. A similar principle
could also apply to S reduction, but whether S reduction rates are affected by
elevated [CO2] has not been tested.

Remobilisation of Nutrients

The remobilisation patterns of nutrients from leaves to developing seeds have partic-
ular significance for grains of cereal crops (Gregersen 2011). For example, Palta and
Fillery (1993) investigated N remobilisation patterns in wheat and they found that N
acquisition from the soil stopped by anthesis and that ~69 % of the N allocated to the
spike was derived from remobilisation from the vegetative plant parts. Remobilisation
strategies are particularly relevant for low rainfall cropping systems where crops are
water-limited during the grain filling period and therefore rely heavily on the nutrients
already stored in vegetative plant parts pre-anthesis, with flag leaves representing the
strongest source of nutrients (Palta and Fillery 1993). Nutrient remobilisation is
closely linked to leaf senescence processes and for optimum remobilisation exact
timing of processes is crucial (Gregersen 2011; Yang and Zhang 2006).
Nutrient and carbohydrate remobilisation patterns are already of interest in the
quest for more nutrient efficient crops (Gregersen 2011), but little is known about
whether changes in remobilisation patterns contribute to the decrease in nutrient
concentrations, particularly in grains, under CO2 enrichment. In a study on barley it
was proposed that developing grains have a greater sink capacity for N, as they
receive ample supplies of carbohydrates. N reserves in the leaves may become
depleted faster, leading to accelerated senescence of flag leaves (Fangmeier
et al. 2000). One recent study on canola in a high rainfall system found that elevated
[CO2] significantly decreased N remobilisation to seeds (Franzaring et al. 2012):
The NHI (see definition in Table 9.1) decreased by as much as 65 % under ample N
supply. It seems that changes in nutrient remobilisation can be significant, but field
studies with elevated [CO2] under soil drying conditions, for example in low
rainfall systems, where the remobilisation of nutrients is relatively most important
(Palta et al. 1994; Yang et al. 2000), are not yet available.
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 245

Interactions of Soils with Elevated Atmospheric [CO2]

In many agricultural systems, production is limited either by nutrients or other factors

such as water. As a consequence, plant growth is not restricted by C availability
(Poorter and Perez-Soba 2001) until these other limitations are overcome. Nutrient
use efficiency (NutUE) reflects the ability of plants to access nutrients from soil
(and fertilisers) as well as the dynamics of the nutrient once it is absorbed.
In contrast to the rapid growth in knowledge of how high [CO2] alters the above
ground physiological responses of plants, much less is known about potential
interactions between high [CO2], different soil processes and plant responses. For
example, soil available N levels have been hypothesised to gradually decline under
high [CO2]. This so-called progressive nitrogen limitation (PNL, Luo et al. 2004),
will result in available soil N levels becoming increasingly limiting as N and C are
sequestered in plant biomass and soil organic matter. As C/N ratios of plant residues
increase (as indicated in previous sections), soil N mineralisation rates will decrease
(Prior et al. 2008). Consequently, an understanding of how high [CO2] will affect
NutUE of plants must account for the influence of different soil properties on both
the capacity to supply nutrients to plants as well as potentially influence the ability
of the plant to access available nutrients (and other critical resources such as water)
in the soil.
Strong interactions can exist between different mineral nutrients (especially N)
at both a physiological level (see above) as well as in a broader agronomic context.
For example, the old weathered soils that underpin many Australian grain produc-
tion systems are very deficient in P (Donald 1964) and McDonald (1989) noted the
importance of N and P interactions in controlling grain yield in cereal crops. Only a
relatively small proportion of P in the soil is in a chemical form that is immediately
‘available’ to plants. Consequently, farmers generally manage soil P deficiencies to
plants by applying P fertilisers. However, a relative large proportion – up to 95 %
(McBeath et al. 2012) – of the fertiliser P becomes unavailable for the crops. For
example, P can be strongly adsorbed onto soil particles, precipitated as insoluble
forms (normally associated with iron, aluminium or calcium) or immobilised by
soil biota. Considerable research has been dedicated to investigating the potential of
plants (at both an intra and inter species level) to access these ‘unavailable’ forms of
P as a strategy to improve NutUE (e.g. Armstrong et al. 1993; Osborne and Rengel
2002; Wang et al. 2010). Although high [CO2] can actually temporarily increase P
immobilisation (as organic P) in the rhizosphere of crops (Jin et al. 2013), there is
no evidence that high [CO2] alters the ability of plants to acquire P from different
sparingly soluble sources of P (Hocking and Barret 2003; Jin et al. 2014).
Differences in NutUE between plants depend on the ability of roots to access
nutrients in the soil and fertiliser; especially for relatively immobile nutrients such
as P, most nutrient acquisition is via root interception (Barber 1995). In many
environments, soils can exhibit a range of physicochemical properties such as high
aluminium, sodicity, salinity and high boron that can significantly restrict root
growth, and therefore uptake of soil nutrients and water (Adcock et al. 2007). It
has been hypothesised that high [CO2] may ameliorate the negative effect of some
246 S. Tausz-Posch et al.

of these constraints such as high salinity (Poorter and Perez-Soba 2001), supported
by the finding that high [CO2] generally increases assimilate allocation to below
ground processes including root growth and exudation (Lynch and St. Clair 2004).
However, Munns et al. (1999) found that whereas there was a positive interaction
between [CO2] and salinity at low CO2 levels, there was no CO2 fertilisation effect
at high [CO2].

Outlook: Traits and Practices to Improve Crop Nutrition

and Quality Under High [CO2]

Given the extent of nutrient deficiencies globally and the financial (e.g. steadily
increasing fertiliser prices) and environmental (e.g. eutrophication of water ways)
cost of managing nutrients in agricultural systems it is not surprising that consid-
erable research has been devoted to improving NutUE in crops especially that of N
and P in both low and high input systems (Wiesler et al. 2001; Hawkesford 2011).
This research has encompassed genetic solutions (Hirel et al. 2007), improved
fertiliser forms (e.g. Chen et al. 2008; McLaughlin et al. 2011) as well as systems
approaches such as better predictions of crop nutrient requirements through
improved soil testing and predictions of crop nutrient demand often via the use of
computer simulation models (e.g. Carberry et al. 2002; Moeller et al. 2009). As
shown in this chapter, elevated [CO2] shifts the relative resource availability
towards photosynthetically fixed C and therefore affects the trade-offs that are
key to optimising plant traits and crop management in practice (Sadras and
Calderini 2009). Recently, the argument that crop improvement should explicitly
take into account elevated [CO2] has gained some traction, particularly as it was
shown that traits selected by breeders over the past 100 years were not necessarily
beneficial under increased [CO2] (Ainsworth et al. 2008; Tausz et al. 2013; Ziska
et al. 2012). Similarly, management practices should be evaluated under elevated
[CO2], as plant growth changes in amount and timing, and this will affect nutrient
demand, uptake and utilisation.
Most aspects of plant NutUE will improve as a consequence of the CO2
fertilisation effect (Table 9.1), but the overall increased nutrient demand of crops
accumulating more biomass ensures that the quest for optimum plant nutrition and
NutUE remains of paramount importance. Elevated [CO2] will only accentuate the
“yield quality conundrum”, e.g. the fact that mineral nutrients or protein concen-
trations decrease as crop yields increase (Hawkesford 2011). The main challenge
will be how to supply sufficient N to maintain grain protein concentrations under
high [CO2], but the issues are similar for other nutrients, as their global supplies are
becoming limited (such as P), they play important role in grain quality (such as S),
or micronutrients such as Fe and Zn that are crucial to human health. Genetic
(breeding or biotechnological) and management improvements targeting the quality
yield conundrum particularly under elevated [CO2] are therefore a high priority in
agricultural research.
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 247

For the successful development of new improved plant varieties a number of

pre-requisites must be met: (i) there must be genetic variability available (ii) there
must be an ability to readily identify the genes associated with this phenotypic
variability and (iii) the trait must be readily inheritable (Foulkes et al. 2009).
Although genetic variation for nutrient efficiency has been identified in many arable
crops (e.g. Svečnjak and Rengel 2006 in canola), there are relatively few reports of
the development of new varieties with greater NutUE. There are also arguments
that rather than directly selecting for traits such as improved NutUE, breeding for
yield improvement alone (which many plant breeders indicate is their number one
priority) has also contributed to increased N harvest index and increased N uptake
(Sadras and Lawson 2013). However, as highlighted in this chapter, there may be a
number of opportunities to identify specific target traits, for example employing the
developing knowledge on the mechanisms underlying the decline of plant nutrients
under elevated [CO2].
Because a key challenge in developing new varieties is managing the complexity
of interactions between traits and environmental conditions, elevated [CO2] must be
taken into consideration in realistic field settings. FACE experiments can play a key
role in assessing breeding traits, but also evaluating nutrient management under the
future climate conditions (including drought). Current FACE experiments mostly
target [CO2] expected for 2040–2050 (550 μmol mol
1 air), which is timely for
selection efforts considering that the turnaround time from trait identification to a
new variety can be 10–20 years (Chapman et al. 2012). Whilst changes in crop
management practices, such as new fertiliser products, application rates and timing
strategies, may require less lead time than the development of new crop varieties,
they will be most effectively applied and tested in the right combination with the
right crop varieties under the relevant environmental conditions. Dealing with
below ground processes, especially those associated with roots and soils, poses
particular difficulties, and adds complexity to the system. This complexity cannot
be avoided, as shown by the importance of roots and soil processes in the interac-
tion of high [CO2] with plant nutrition. The use of 3-D functional simulation
modelling (Dunbabin et al. 2013) offers considerable promise, especially when
combined with new technology such as x-ray computer tomography that allows
better imaging of root growth within soils. More flexible exposure systems that
combine the advantages of a FACE system (free air without enclosure effects) with
the use of large soil cores (such as the “SoilFACE” system as part of the AGFACE
facilities; Butterly et al. 2012) or lysimeters and rhizotron access also offer oppor-
tunities to investigate relevant plant nutrition processes in the field.
In summary, it will be important to consider increasing [CO2] in further
improvements of crop nutrition and NutUE, either by genetic or managements
adaptations. Facilities that enable crop growth under elevated [CO2] will therefore
play an increasing role in such efforts, alongside technologies that control other key
factors such as water supply or temperature regimes.
248 S. Tausz-Posch et al.

Acknowledgements The Australian Grains Free Air CO2 Enrichment (AGFACE) facility and
related experiments are jointly run by The University of Melbourne and the Victorian State
Department of Environment and Primary Industries (DEPI) with funding from the Grains Research
and Development Corporation (GRDC), the Australian Government, and the Australian Research
Council (ARC). Data in Fig. 9.2 were derived from collaboration of STP with Malcolm
Hawkesford, Rothamsted Research, UK.

References

Adcock D, McNeill AM, McDonald GK, Armstrong RD (2007) Subsoil constraints to crop
production on neutral and alkaline soils in south-eastern Australia: a review of current
knowledge and management strategies. Aust J Exp Agr 47:1245–1261
Ainsworth EA, Long SP (2005) What have we learned from 15 years of free-air CO2 enrichment
(FACE)? A meta-analytic review of the responses of photosynthesis, canopy. New Phytol
165:351–371
Ainsworth EA, Rogers A (2007) The response of photosynthesis and stomatal conductance to
rising CO2: mechanisms and environmental interactions. Plant Cell Environ 30:258–270
Ainsworth EA, Beier C, Calfapietra C, Ceulemans R, Durand-Tardif M, Farquhar GD, Godbold
DL, Hendrey GR, Hickler T, Kaduk J, Karnosky DF, Kimball BA, Koerner C, Koornneef M,
Lafarge T, Leakey ADB, Lewin KF, Long SP, Manderscheid R, McNeil DL, Mies TA,
Miglietta F, Morgan JA, Nagy J, Norby RJ, Norton RM, Percy KE, Rogers A, Soussana JF,
Stitt M, Weigel HJ, White JW (2008) Next generation of elevated CO2 experiments with crops:
a critical investment for feeding the future world. Plant Cell Environ 31:1317–1324
Amthor JS (2001) Effects of atmospheric CO2 concentration on wheat yield: review of results from
experiments using various approaches to control CO2 concentration. Field Crop Res 73:1–34
Armstrong RD, Helyar KR, Prangnell R (1993) Direct assessment of mineral phosphorus avail-
ability to tropical crops using 32P labelled compounds. Plant Soil 150:278–287
Barber SA (1995) Soil nutrient bioavailability: a mechanistic approach. Wiley, New York
Bassirirad H, Tissue DT, Reynolds JF, Chapin FS (1996) Response of Eriophorum vaginatum to
CO2 enrichment at different soil temperatures: effects on growth, root respiration and PO43

uptake kinetics. New Phytol 133:423–430
Benlloch-Gonzalez M, Berger J, Bramley H, Rebetzke G, Paltra JA (2014) The plasticity of the
growth and proliferation of wheat root system under elevated CO2. Plant Soil 374:963–976
Bloom AJ (2009) As carbon dioxide rises, food quality will decline without careful nitrogen
management. Calif Agr 63:67–72
Bloom AJ, Burger M, Rubio-Asensio JS, Cousins AB (2010) Carbon dioxide enrichment inhibits
nitrate assimilation in wheat and Arabidopsis. Science 328:899–903
Bunce JA (2012) Responses of cotton and wheat photosynthesis and growth to cyclic variation in
carbon dioxide concentration. Photosynthetica 50:395–400
Butterly C, Armstrong R, Chen D, Mathers N, Tang C (2012) Effect of elevated CO2 and N level
on growth of wheat and field pea. In: Yunusa I (ed) Capturing opportunities and overcoming
obstacles in Australian agronomy. Proceedings of the 16th Australian agronomy conference
2012, Armidale
Carberry PS, Probert ME, Dimes JP, Keating BA, McCown RL (2002) Role of modelling in
improving nutrient efficiency in cropping systems. Plant Soil 245:193–203
Carlisle E, Myers S, Raboy V, Bloom A (2012) The effects of inorganic nitrogen form and CO2
concentration on wheat yield and nutrient accumulation and distribution. Frontiers Plant Sci
3:195
Carter TR, Jones RN, Lu X, Bhadwal S, Conde C, Mearns LO, O’Neill BC, Rounsevell MDA,
Zurek MB (2007) New assessment methods and the characterisation of future conditions.
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 249

In: Parry ML, Canziani OF, Palutikof JP, van der Linden PJ, Hanson CE (eds) Contribution of
working group II to the fourth assessment report of the intergovernmental panel on climate
change. Cambridge University Press, Cambridge, pp 133–171
Chapman SC, Chakraborty S, Dreccer MF, Howden SM (2012) Plant adaptation to climate
change-opportunities and priorities in breeding. Crop Pasture Sci 63:251–268
Chen D, Suter HC, Islam A, Edis R, Freney JR, Walker CN (2008) Prospects of improving
efficiency of fertiliser nitrogen in Australian agriculture: a review of enhanced efficiency
fertilisers. Aust J Soil Res 46:289–301
Conroy J, Hocking P (1993) Nitrogen nutrition of C-3 plants at elevated atmospheric CO2
concentrations. Physiol Plantarum 89:570–576
Cramer MD, Hawkins HJ, Verboom GA (2009) The importance of nutritional regulation of plant
water flux. Oecologia 161:15–24
Donald CM (1964) Phosphorus in Australian agriculture. J Aust Inst Agric Sci 6:75–105
Drake BG, GonzalezMeler MA, Long SP (1997) More efficient plants: a consequence of rising
atmospheric CO2? Annu Rev Plant Phys 48:609–639
Dunbabin VM, Postma JA, Schnepf A, Pagès L, Javaux M, Wu L, Leitner D, Chen YL, Rengel Z,
Diggle AJ (2013) Modelling root–soil interactions using three–dimensional models of root
growth, architecture and function. Plant Soil 372:93–124
Duval BD, Blankinship JC, Dijkstra P, Hungate BA (2012) CO2 effects on plant nutrient concen-
tration depend on plant functional group and available nitrogen: a meta-analysis. Plant Ecol
213:505–521
Erbs M, Manderscheid R, Jansen G, Seddig S, Pacholski A, Weigel HJ (2010) Effects of free-air
CO2 enrichment and nitrogen supply on grain quality parameters and elemental composition of
wheat and barley grown in a crop rotation. Agr Ecosyst Environ 136:59–68
Fangmeier A, De Temmerman L, Mortensen L, Kemp K, Burke J, Mitchell R, van Oijen M,
Weigel HJ (1999) Effects on nutrients and on grain quality in spring wheat crops grown under
elevated CO2 concentrations and stress conditions in the European, multiple-site experiment
‘ESPACE-wheat’. Eur J Agron 10:215–229
Fangmeier A, Chrost B, Högy P, Krupinska K (2000) CO2 enrichment enhances flag leaf
senescence in barley due to greater grain nitrogen sink capacity. Environ Exp Bot 44:151–164
Fernando N, Panozzo J, Tausz M, Norton RM, Fitzgerald GJ, Myers S, Walker C, Stangoulis J,
Seneweera S (2012) Wheat grain quality under increasing atmospheric CO2 concentrations in a
semi-arid cropping system. J Cereal Sci 56:684–690
Fitter AH (1996) Characteristics and functions of root systems. In: Waisel Y, Eshel A, Kafka U
(eds) Plant roots: the hidden half. Marcel Dekker, New York, pp 1–20
Foulkes MJ, Reynolds MP, Sylvester-Bradley R (2009) Genetic improvement of grain crops: yield
potential. In: Sadras VO, Calderini DF (eds) Crop physiology applications for genentic
improvement and agronomy. Academic, Amsterdam, pp 355–386
Franzaring J, Gensheimer G, Weller S, Schmid I, Fangmeier A (2012) Allocation and
remobilisation of nitrogen in spring oilseed rape (Brassica napus L. cv. Mozart) as affected
by N supply and elevated CO2. Environ Exp Bot 83:12–22
Ghannoum O, Searson MJ, Conroy JP (2007) Nutrient and water demands under global climate
change. In: Newton PCD, Carran RA, Edwards GR, Niklaus PA (eds) Agroecosystems in a
changing climate. Taylor & Francis, Boca Raton, pp 53–83
Good AG, Shrawat AK, Muench DG (2004) Can less yield more? Is reducing nutrient input into
the environment compatible with maintaining crop production? Trends Plant Sci 9:597–605
Gregersen PL (2011) Senescence and nutrient remobilisation in crop plants. In: Hawkesford MJ,
Barraclough P (eds) The molecular and physiological basis of nutrient use efficiency in crops.
Wiley, Hoboken, pp 83–102
Gregory PJ, Palta JA, Batts GR (1996) Root systems and root:mass ratio – carbon allocation under
current and projected atmospheric conditions in arable crops. Plant Soil 187:221–228
Hatfield JL, Boote KJ, Kimball BA, Ziska LH, Izaurralde RC, Ort D, Thomson AM, Wolfe D
(2011) Climate impacts on agriculture: implications for crop production. Agron J 103:351–370
250 S. Tausz-Posch et al.

Hawkesford MJ (2011) An overview of nutrient use efficiency and strategies for crop improve-
ment. In: Hawkesford MJ, Barraclough P (eds) The molecular and physiological basis of
nutrient use efficiency in crops. Wiley, Hoboken, pp 5–19
Hawkesford MJ, De Kok LJ (2006) Managing sulphur metabolism in plants. Plant Cell Environ
29:382–395
Hawkesford MJ, De Kok LJ (2007) Sulfur in plants: an ecological perspective. Springer,
Dordrecht
Hirel B, Le Gouis J, Ney B, Gallais A (2007) The challenge of improving nitrogen use efficiency in
crop plants: towards a more central role for genetic variability and quantitative genetics within
integrated approaches. J Exp Bot 58:2369–2387
Ho MD, Rosas JC, Brown KM, Lynch JP (2005) Root architectural tradeoffs for water and
phosphorus acquisition. Funct Plant Biol 32:737–748
Hocking P, Barret D (2003) Elevated atmospheric CO2 does not increase the capacity of plants to
access fixed soil phosphorus from an oxisol. In: Second international symposium on P
dynamics in the soil-plant continuum, Uniprint, Perth, pp 100–101
Hocking PJ, Meyer CP (1991) Carbon-dioxide enrichment decreases critical nitrate and nitrogen
concentrations in wheat. J Plant Nutr 14:571–584
Högy P, Fangmeier A (2008) Effects of elevated atmospheric CO2 on grain quality of wheat.
J Cereal Sci 48:580–591
Högy P, Wieser H, Kohler P, Schwadorf K, Breuer J, Franzaring J, Muntifering R, Fangmeier A
(2009) Effects of elevated CO2 on grain yield and quality of wheat: results from a 3-year free-
air CO2 enrichment experiment. Plant Biol 11:60–69
Högy P, Brunnbauer M, Koehler P, Schwadorf K, Breuer J, Franzaring J, Zhunusbayeva D,
Fangmeier A (2013) Grain quality characteristics of spring wheat (Triticum aestivum) as
affected by free-air CO2 enrichment. Environ Exp Bot 88:11–18
Hungate BA, Dukes JS, Shaw MR, Luo Y, Field CB (2003) Nitrogen and climate change. Science
302:1512–1513
Jin J, Tang C, Armstrong R, Butterly C, Sale P (2013) Elevated CO2 temporally enhances
phosphorus immobilization in the rhizosphere of wheat and chickpea. Plant Soil 368:315–328
Jin J, Tang C, Hogarth TW, Armstrong R, Sale P (2014) Nitrogen form but not elevated CO2
alters plant phosphorus acquisition from sparingly soluble phosphorus sources. Plant Soil
374:109–119
Kirschbaum MUF (2011) Does enhanced photosynthesis enhance growth? Lessons learned from
CO2 enrichment studies. Plant Physiol 155:117–124
Kitchen LD (2014) Turning knowledge into action. In: Dudonis C (ed) Global climate change –
turning knowledge into action. Pearson Education Inc, NJ, pp 341–342
Kong LG, Wang FH, Lopez-Bellido L, Garcia-Mina JM, Si JS (2013) Agronomic improvements
through the genetic and physiological regulation of nitrogen uptake in wheat (Triticum
aestivum L.). Plant Biotech Rep 7:129–139
Lam SK, Chen DL, Norton R, Armstrong R, Mosier AR (2012a) Nitrogen dynamics in grain crop
and legume pasture systems under elevated atmospheric carbon dioxide concentration: a meta-
analysis. Glob Change Biol 18:2853–2859
Lam SK, Chen D, Norton R, Armstrong R (2012b) Nitrogen demand and the recovery of 15N-
labelled fertilizer in wheat grown under elevated carbon dioxide in southern Australia. Nutr
Cycl Agroecosys 92:133–144
Lam SK, Chen D, Norton R, Armstrong R (2012c) Does phosphorus stimulate the effect of
elevated [CO2] on growth and symbiotic nitrogen fixation of grain and pasture legumes?
Crop Past Sci 63:53–62
Lam SK, Chen D, Norton R, Armstrong R, Mosier AR (2013) Influence of elevated atmospheric
carbon dioxide and supplementary irrigation on greenhouse gas emissions from a spring wheat
crop in southern Australia. J Agric Sci 151:201–208
Leakey ADB, Ainsworth EA, Bernacchi CJ, Rogers A, Long SP, Ort DR (2009) Elevated CO2
effects on plant carbon, nitrogen, and water relations: six important lessons from FACE. J Exp
Bot 60:2859–2876
9 Nutrient Use and Nutrient Use Efficiency of Crops in a High CO2 Atmosphere 251

Lilley JM, Kirkegaard JA (2011) Benefits of increased soil exploration by wheat roots. Field Crop
Res 122:118–130
Loladze I (2002) Rising atmospheric CO2 and human nutrition: toward globally imbalanced plant
stoichiometry? Trends Ecol Evol 17:457–461
Luo Y, Su B, Currie WS, Dukes JS, Finzi A, Hartwig U, Hungate B, McMurtrie RE, Oren R,
Parton WJ, Pataki DE, Shaw RM, Zak DR, Field CB (2004) Progressive nitrogen limitation of
ecosystem response to rising atmospheric carbon dioxide. Bioscience 54:731–739
Lynch JP, St. Clair SB (2004) Mineral stress: the missing link in understanding how global climate
change will affect plants in real world soils. Field Crop Res 90:101–115
Madhu M, Hatfield JL (2013) Dynamics of plant root growth under increased atmospheric carbon
dioxide. Agron J 105:657–669
Manderscheid R, Bender J, Jäger HJ, Weigel HJ (1995) Effects of season long CO2 enrichment on
cereals. 2. Nutrient concentrations and grain quality. Agr Ecosyst Environ 54:175–185
McBeath TM, McLaughlin MJ, Kirby JK, Armstrong RD (2012) The effect of soil water status on
fertiliser, topsoil and subsoil phosphorus utilisation by wheat. Plant Soil 358:337–348
McDonald GK (1989) The contribution of nitrogen fertiliser to the nitrogennutrition of rainfed
wheat crops in Australia: a review. Aust J Exp Agr 29:455–481
McGrath JM, Lobell DB (2013) Reduction of transpiration and altered nutrient allocation con-
tribute to nutrient decline of crops grown in elevated CO2 concentrations. Plant Cell Environ
36:697–705
McLaughlin MJ, McBeath TM, Smernik R, Stacey SP, Ajiboye B, Guppy C (2011) The chemical
nature of P accumulation in agricultural soils – implications for fertiliser management and
design: an Australian perspective. Plant Soil 349:69–87
Messinger SM, Buckley TN, Mott KA (2006) Evidence for involvement of photosynthetic
processes in the stomatal response to CO2. Plant Physiol 140:771–778
Miller AJ, Chapman N (2011) Transporters involved in nitrogen uptake and movement. In:
Hawkesford MJ, Barraclough P (eds) The molecular and physiological basis of nutrient use
efficiency in crops. Wiley-Blackwell, Chichester, pp 193–210
Moeller C, Asseng S, Berger J, Milroy SP (2009) Plant soil available water at sowing in
Mediterranean environments – is it a useful criterion to aid nitrogen fertiliser and sowing
decisions? Field Crop Res 114:127–136
Moore BD, Cheng SH, Sims D, Seemann JR (1999) The biochemical and molecular basis for
photosynthetic acclimation to elevated atmospheric CO2. Plant Cell Environ 22:567–582
Munns R, Cramer GR, Ball MC (1999) Interactions between rising CO2, soil salinity and plant
growth. In: Luo Y, Mooney HA (eds) Carbon dioxide and environmental stress. Academic, San
Diego, pp 139–167
Osborne LD, Rengel Z (2002) Genotypic differences in wheat for uptake and utilisation of P from
iron phosphate. Aust J Agr Res 53:837–844
Palta JA, Fillery IR (1993) Nitrogen accumulation and remobilization in wheat of 15N-urea applied
to a duplex soil at seeding. Aust J Exp Agr 33:233–238
Palta JA, Kobata T, Turner NC, Fillery IR (1994) Remobilization of carbon and nitrogen in wheat
as influenced by postanthesis water deficits. Crop Sci 34:118–124
Poorter H, Perez-Soba M (2001) The growth response of plants to elevated CO2 under non-optimal
environmental conditions. Oecologia 129:1–20
Poorter H, VanBerkel Y, Baxter R, Den Hertog J, Dijkstra P, Gifford RM, Griffin KL, Roumet C,
Roy J, Wong SC (1997) The effect of elevated CO2 on the chemical composition and
construction costs of leaves of 27 C-3 species. Plant Cell Environ 20:472–482
Prior SA, Torbert HA, Runion GB, Rogers HH, Kimball BA (2008) Free-air CO2 enrichment of
sorghum: soil carbon and nitrogen dynamics. J Environ Qual 37:753–758
Pritchard SG, Rogers HH (2000) Spatial and temporal deployment of crop roots in CO2-enriched
environments. New Phytol 147:55–71
Pritchard SG, Prior SA, Rogers HH, Davis MA, Runion GB, Popham TW (2006) Effects of
elevated atmospheric CO2 on root dynamics and productivity of sorghum grown under
conventional and conservation agricultural management practices. Agr Ecosyst Environ
113:175–183
252 S. Tausz-Posch et al.

Rich SM, Watt M (2013) Soil conditions and cereal root system architecture: review and
considerations for linking Darwin and Weaver. J Exp Bot 64:1193–1208
Rogers A, Ainsworth EA, Leakey ADB (2009) Will elevated carbon dioxide concentration
amplify the benefits of nitrogen fixation in legumes? Plant Physiol 151:1009–1016
Sadras VO, Calderini DF (2009) Crop physiology: applications for genetic improvement and
agronomy. Academic Press, Elsevier, Burlington
Sadras VO, Lawson C (2013) Nitrogen and water-use efficiency of Australian wheat varieties
released between 1958 and 2007. Eur J Agron 46:34–41
Sinclair TR, Pinter PJ, Kimball BA, Adamsen FJ, LaMorte RL, Wall GW, Hunsaker DJ, Adam N,
Brooks TJ, Garcı́a RL, Thompson T, Leavitt S, Matthias A (2000) Leaf nitrogen concentration
of wheat subjected to elevated CO2 and either water or N deficits. Agr Ecosyst Environ
79:53–60
Stitt M, Krapp A (1999) The interaction between elevated carbon dioxide and nitrogen nutrition:
the physiological and molecular background. Plant Cell Environ 22:583–621
Svečnjak Z, Rengel Z (2006) Canola cultivars differ in nitrogen utilisation efficiency at vegetative
stage. Field Crop Res 97:221–226
Taub DR, Wang X (2008) Why are nitrogen concentrations in plant tissues lower under elevated
CO2? A critical examination of the hypotheses. J Integr Biol 50:1365–1374
Taub DR, Miller B, Allen H (2008) Effects of elevated CO2 on the protein concentration of food
crops: a meta-analysis. Global Chang Biol 14:565–575
Tausz M, Tausz-Posch S, Norton RM, Fitzgerald GJ, Nicolas ME, Seneweera S (2013) Under-
standing crop physiology to select breeding targets and improve crop management under
increasing atmospheric CO2 concentrations. Env Exp Bot 88:71–80
Tausz-Posch S, Seneweera S, Norton RM, Fitzgerald GJ, Tausz M (2012) Can a wheat cultivar
with high transpiration efficiency maintain its yield advantage over a near-isogenic cultivar
under elevated CO2? Field Crop Res 133:160–166
Van Vuuren MMI, Robinson D, Fitter AH, Chasalow SD, Williamson L, Raven JA (1997) Effects
of elevated atmospheric CO2 and soil water availability on root biomass, root length, and N, P
and K uptake by wheat. New Phytol 135:455–465
Wang X, Tang C, Guppy CN, Sale PWG (2010) Cotton, wheat and white lupin differ in
phosphorus acquisition from sparingly soluble sources. Env Exp Bot 69:267–272
Wang L, Feng Z, Schjoerring JK (2013) Effects of elevated atmospheric CO2 on physiology and
yield of wheat (Triticum aestivum L.): a meta-analytic test of current hypotheses. Agr Ecosyst
Environ 178:57–63
White PJ, Hammond JP (2008) The ecophysiology of plant-phosphorus interactions. Springer,
Dordrecht
Wiesler F, Behrens T, Horst WJ (2001) The role of nitrogen-efficient cultivars in sustainable
agriculture. Sci World 1(S2):61–69
Yang JC, Zhang JH (2006) Grain filling of cereals under soil drying. New Phytol 169:223–236
Yang JC, Zhang JH, Huang ZL, Zhu QS, Wang L (2000) Remobilization of carbon reserves is
improved by controlled soil-drying during grain filling of wheat. Crop Sci 40:1645–1655
Yong ZH, Chen GY, Zhang DY, Chen Y, Chen J, Zhu JG, Xu DQ (2007) Is photosynthetic
acclimation to free-air CO2 enrichment (FACE) related to a strong competition for the
assimilatory power between carbon assimilation and nitrogen assimilation in rice leaf?
Photosynthetica 45:85–91
Ziska LH (2008) Rising atmospheric carbon dioxide and plant biology: the overlooked paradigm.
DNA Cell Biol 27:165–172
Ziska LH, Bunce JA, Shimono H, Gealy DR, Baker JT, Newton PCD, Reynolds MP, Jagadish
KSV, Zhu CW, Howden M, Wilson LT (2012) Food security and climate change: on the
potential to adapt global crop production by active selection to rising atmospheric carbon
dioxide. Proc R Soc B Biol Sci 279:4097–4105
Chapter 10
Monitoring Plant Nutritional Status

Moez Maghrebi, Fabio Francesco Nocito, and Gian Attilio Sacchi

Abstract Methods and techniques effective in achieving yield objectives, opti-

mizing the use of resources and preventing environmental contamination are
defined as agronomic Best Management Practices (BMPs). Considering
fertilisations BMPs pursue the aims to match mineral nutrient supply with crop
requirements, minimizing their losses from the field. If spatial and temporal infor-
mation about crop needs were available, precision fertilisation approaches could be
planned in order to increase fertiliser use efficiency and to improve some economic
and environmental aspects related to the crop systems. The state of the art and the
research perspectives on the fine tuning of optical devices allowing proximal or
remote sensing at sub-field scale of crops traits related to plant nutritional status, as
well as on the exploitation of the gene fusion concept in developing transgenic
bioindicators monitoring the nutritional status of the plants are here reviewed and
discussed. Concerning the latter aspect particular attention is paid to the develop-
ment of synthetic promoters conferring to the biondicator nutrient specificity and
also able to target the expression of the associated reporter genes in organs in which
their signals should be early and easily detectable.

Keywords Agronomy • Nutrient monitoring • Fertilizers • Precision agriculture

• Critical concentrations • Bioindicators

Introduction

Mineral nutrient availability is one of the most important factors determining yield
in agriculture. Conventional farming requires a continuous and large supply of
fertilisers to the soil in order to replace the nutrients removed with plant harvest. In
the predicted scenario of a rising demand for food and energy for the expected
world population of about nine billion in 2050 (Godfray et al. 2010) a dramatic
increase in the use of fertilisers in the crop systems is foreseen. Since fertiliser
production and distribution have a high demand for energy, in the last decade the

M. Maghrebi • F.F. Nocito • G.A. Sacchi (*)

Department of Agricultural and Environmental Sciences – Production, Landscape,
Agroenergy, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
e-mail: [email protected]

© Springer International Publishing Switzerland 2014 253

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9_10
254 M. Maghrebi et al.

price of fertilisers, although fluctuating, have been burgeoning and it is highly

improbable that this trend will change in the next few years (http://faostat3.fao.org/
home/index.html).
Only a fraction of the nutrient provided to the soil as fertiliser is taken up from
the crops. This fraction, expressed as a percentage, is defined as the crop Apparent
Recovery (AR; Craswell and Godwin 1984):

AR ¼ 100ðNF  NnF Þ=F

where NF and NnF are the total amount of the nutrient absorbed by the crop, if
fertilised or not, respectively, and F is the amount of the nutrient added to the soil
with the fertiliser. For the main crops quite low average values of AR are reported:
about 35 % for N (Raun and Johnson 1999), 10–30 % for P (Malhi et al. 2002) and
seldom higher than 50 % for K (Rengel and Damon 2008). The low capacity of
crops in removing the nutritional elements added to the soil has significant envi-
ronmental implications and reflects limits in the management of the fertilisation
practices, the existence of constraints due to both chemical-physical and microbi-
ological soil properties and plant intrinsic biological limits. AR does not consider
yield traits. On the contrary, the Agronomic Efficiency (AE) of the fertiliser, defined
by the ratio dY/dF, where dY is the infinitesimal yield (Y) and dF is the infinitesimal
increase in the amount of the nutrient in the soil (F) due to the fertiliser application,
considers such traits. AE can in turn be expressed considering two components: the
crop Removal Efficiency (RE), defined as the ratio dNF/dF, where dNF is the
infinitesimal incremental amount of the nutrient taken up by plant after the
fertilisation, and the Physiologic Efficiency (PE), defined as the ratio dY/dNF:

AE ¼ RE  PE ¼ dN F =dF  dY=dN F ¼ dY=dF

In the field, both RE and PE, and then AE, depend on the interaction between
genetic and environmental factors. In other chapters of this book the molecular and
genetic aspects determining AE for some essential elements are extensively
reviewed. Here, in a perspective of precision farming (Pierce and Nowak 1999),
some strategies to improve AE by optimising dNF throughout the use of plant-based
sensor systems are presented and discussed.

Fertiliser Best Management Practices

Definitions

In order to limit the intrinsic risks of diffuse pollution due to intensive agriculture,
both local and supranational authorities are committed to the fine tuning of methods
or techniques found to be the most effective and practical means in achieving yield
objective optimisation and preventing contaminations of soils, water resources and
air. As a whole, these recommended measures are defined as agronomic Best
10 Monitoring Plant Nutritional Status 255

Management Practices (BMPs) and include: the choice of variety, planting date,
row spacing, seeding rates, integrated pest management, weed control, disease
control, and nutrient management. Focusing on the input into the field of inorganic
nutrients, BMPs are considered the management practices that foster the effective
and responsible use of fertiliser matching nutrients supply with crop requirements
and minimise their losses from the fields.

Toward a Fertiliser Precision Management

Fertiliser BMPs include the identification of the: (a) the right product by matching
the fertiliser characteristics to the crop site specific needs and soil properties; (b) the
right time by synchronising the presence of the nutrient with the moment of crop
maximum demand and uptake capacity; (c) the best rate by matching the amount of
fertiliser input to crop needs in order to avoid over-input leading to nutrient
leaching and other losses to the environment, as well as starvation conditions;
(d) the right place by making sure the presence of the nutrients where plants can
efficiently take up them.
Although in a field a relatively high spatial variability in the crop requirements
of a specific nutrient could exist, fertilisers are uniformly applied, to avoid yield
gaps and considering the less fertile portion of soil, where the crops have the
maximum demand. The amounts of nutrient provided in excess with the fertiliser
can be absorbed by the crops without resulting in any benefit in term of yield or
leached towards the underground water becoming a concern for the quality of the
environment.
If spatial and temporal information about crop needs of nutrients were available
site- and time-specific inputs of the nutrient could be planned, resulting in a
precision fertilisation approach, and in a win-win option to increase fertiliser
nutrient efficiency and to improve the economic and environmental sustainability
of the crop systems. In other words, the optimisation of dNF term, in the equation
defining AE, requires detailed information for decision support systems, allowing
farmers to adopt the minimal nutrient input for maximal return, according to a
Fertiliser Best Management Practices (FBMPs) approach.

Evaluating Plant Nutritional Status

Soil and Leaf Analyses

Within a field the spatial variability of the soil chemical-physical and biological
characteristics, including the amount of bioavailable forms of the mineral nutrients
essential for crops, can be pronounced. Mapping this variability and plant
256 M. Maghrebi et al.

nutritional status at high temporal and spatial resolution represents the first step
towards the setup of site-specific FBMPs. In the last years, several sensor-based
techniques to assess parameters indicative of the nutritional status of soil-plant
systems have been proposed to replace, or support traditionally used physical
measures and chemical analysis. Results on soil properties obtained from electro-
magnetic induction sensors along with those derived from the use of ground
conductivity meters and radiometers analysing canopy reflectance, appear to pro-
vide data which can be used to target N-fertilisation to specific field conditions
(Adamchuck et al. 2011). However, soil and plant chemical analyses are still widely
used in developing methods for the evaluation of the nutritional status of the crops.
In particular, the evaluation of elemental concentrations in plant tissues can be
helpful in diagnosing nutrient deficiency. This strategy is currently used to assess
nutrient availability and guide fertility programmes for many fruit tree crops.
Nevertheless, the usefulness of this approach in order to develop FBMPs for
herbaceous crops is rather debatable, since the concentration of a specific element
may change among the leaves of a single plant, and may also change over time
within a single leaf (Barker and Pilbeam 2007). Thus, plant sampling, in term of
timing and tissues to choose, is the most critical step (Kalra 1998). Moreover,
elemental analysis detects only severe and long-term deficiency since a plant’s
initial response to nutrient limitation is to activate mechanisms aimed at
maintaining the ionic homeostasis of their cells (Schatchtman and Shin 2007;
Gojon et al. 2009). Finally, conferring a diagnostic value to the concentration of a
single element could be misleading since complex cross-talk connections between
the regulatory mechanisms controlling the ionic homeostasis in plants exist
(Rouached et al. 2010).

Nutrient-Critical Concentration and Dilution Curve

The critical concentration of a nutrient (nc) stands for the concentration of the
nutrient in the shoots above which, in the absence of other growth limiting factors,
the plant is sufficiently supplied with the nutrient to achieve its maximum potential
yield. In other words, when the nc is achieved and maintained, further supplies of
the nutrient will not influence the growth of the plants and, in the absence of any
sort of demand-driven negative-regulation of its uptake, it could be uselessly
accumulated in the plant tissues. For some nutrients it is possible to define the
so-called toxicological value, which indicates the concentration above which fur-
ther nutrient accumulation induces damage on cell metabolism and structure. All
the concentrations of the nutrient between its critical and toxicological values
define the so-called luxury range (Fig. 10.1), which depends on the chemical
properties of the element and on its biochemical roles. The fine-tuning of the
application of fertiliser to maintain the concentration of the mineral nutrients in
the plant tissues as close as possible to their critical values represents a FBMP
approach.
10 Monitoring Plant Nutritional Status 257

Fig. 10.1 Relationship between the concentration of a nutrient in the shoot and the biomass
relative yield

The critical concentration of a nutrient is not a constant value since it depends on

genotype, environmental conditions, and the developmental stage of the plants
(Lemaire and Gastal 2009; Greenwood et al. 1986). For instance, in the above-
ground tissue of cereals the value of nc (as % on the DW basis) changes during the
plant developmental stages according to the dilution function:

nc ¼ aW b

where W is the maximum above-ground biomass in a specific stage of the plant

cycle, a represents the concentration of nc in the shoot when the crop mass is 1 Mg
DM ha1, and b represents the dilution coefficient.
The dilution curve of a specific cultivar is obtained by plotting the nc values, at a
given developmental stage and field situation determined by a set a fertilisation
experiments versus the accumulated plant shoot biomass (Fig. 10.2). Once the
dilution curve for a specific nutrient is known, it is possible to evaluate the
nutritional status of the crop by evaluating its nutritional index (NI) defined as the
ratio between the actual nutrient concentration in the shoots and the corresponding
value of nc. Indeed, if NI is lower than 1 the crop is in a suboptimal nutritional status
for a given nutrient and needs to be fertilised; if the NI is higher than 1 the crop is in
the luxury range.
The actual feasibility of such an approach for a sustainable management of N
fertilisation is conditioned by the limited availability of easy and low cost methods
to rapidly estimate the value of W in the field and, mainly by the actual concentra-
tions of the nutrient in the shoots over a representational cropping area. The value of
W can be extrapolated by the crop leaf area index (LAI) currently evaluated through
remote sensing approaches (Zheng and Moskal 2009). The evaluation of the actual
258 M. Maghrebi et al.

Fig. 10.2 Dilution curve for a generic essential nutrient as defined by six nc values determined for
six plant developmental stages

concentration of the nutrient, without adopting the traditional chemical analyses, is

the most difficult challenge to overcome.
Since nitrogen is the nutritional element that most often affects crop production
and the current world use of N fertilisers is approximately 90 million metric tons
(with an estimated cost of about $50 billion), it is reasonable that several research
efforts have been focused on the fine tuning of non-invasive methods for the
determination of N levels in shoots throughout the entire growth cycle of crops as
a guide to N-FBMPs (for an exhaustive review see Samborski et al. 2009). Assum-
ing nitrogen as an example, in the next paragraph we briefly summarize the
advances on the non-destructive approaches developed or under investigation for
monitoring the nutritional status of a crop.

Non-destructive Monitoring of Crop Nutritional Status: The

Example of Nitrogen

Nitrogen availability affects chlorophyll content in leaves (Schlemmer et al. 2005)

and as a consequence the level of this pigment is considered a good indicator of the
nitrogen nutritional status of a crop (Samborski et al. 2009 and references therein).
Instruments analysing the spectral properties of leaf tissue to estimate their chlo-
rophyll content (optical chlorophyll meters) have been developed to evaluate the
need for agricultural N applications. Due to the pigment’s light absorption proper-
ties (in the visible wavelength range), the higher the chlorophyll content, the higher
the reflectance of the leaf (in the 525–680 nm range) and consequently, the higher
10 Monitoring Plant Nutritional Status 259

the amount of red light absorbed. Combining light absorbance measures at 660 nm
and near-infrared (NIR) light transmittance at 940 nm, which in turn depend on leaf
moisture content and thickness, a good estimation of chlorophyll per unit area has
been obtained in the major crop leaves. It has been proved that the chlorophyll
meter can detect the early signs of N stress not yet detectable by visual analysis
using a leaf colour chart (Debaeke et al. 2006).
Recently, it has been proposed a hand-held instrument (Dualex®), exploiting
chlorophyll as an internal sensor of photons, enables the user to contemporaneously
assess the level of both photosynthetic pigments in the mesophyll and flavonoids in
the epidermis of the leaf. Briefly, comparing the amount of chlorophyll fluores-
cence emitted under UV excitation (λ 380) with that emitted under visible light (λ
660) whether absorbed or not, the instrument is able to evaluate the level of
flavonoids absorbing in the UV range. Contemporaneously, by comparison of the
light transmittance at λ 720, in the range of chlorophyll absorption, and at λ 840, in the
range influenced by leaf structural properties but not by chlorophyll, a reliable
evaluation of the levels of the photosynthetic active pigment is obtained. Dualex®
thus allows measurement of a Nitrogen Balance Index (NBI®), which indicates the
ratios of both chlorophyll and flavonoids units and as a result is related to leaf N
content (Cartelat et al. 2005) since leaf flavonoids can be considered an indicator of
N availability. Indeed, in N-starved plants the concentration of carbon-based
secondary metabolites increase (Hamilton et al. 2001) and in particular, due to
the enhanced expression of specific transcription factors involved in controlling
their biosynthetic pathway, those of anthocyanin and flavonols (Lea et al. 2007).
Several experimental evaluations suggest that in the case of wheat and corn
Dualex® seems to furnish more reliable information about the N status of the
plants with respect to other hand-held optical systems (Tremblay et al. 2012).
Since leaf N status influences the quantum yield of PSII electron transport and
then the chlorophyll fluorescence parameters (Lu and Zhang 2000), canopy fluo-
rescence quenching analyses could be considered suitable for sensing crop N status
(Tremblay et al. 2012). In particular, the recent introduction of a hand-held fluo-
rimeter (Multiplex®) equipped with LEDs generating four wavelengths (λ375, λ450,
λ530, λ630) and detectors monitoring fluorescence at three wavelengths (λ447 or λ590
if the excitation at λ450 is used or not, respectively, λ665 and λ735) seems to be quite
promising for the in-season assessment of crop N status (Tremblay et al. 2012 and
references therein). Combining different excitation and emission bands the instru-
ment provides independent parameters related to chlorophyll, flavonoids and N
content of the plants (Tremblay et al. 2012).
The devices described above determine optical parameters for individual leaves
or, in the case of Multiplex®, at a typical distance of a few centimeters thus
monitoring circular canopy surfaces of not more than 10 cm in diameter. Conse-
quently, they are not particularly suitable in evaluating the N status of a crop at field
scale. Sensors, analysing canopy reflectance properties and thus its N status and
needs are also available (Erdle et al. 2011). They are classified as passive (Yara
N-Sensor®/Field Scan and FiledSpec® Portable Spectroradiometer) or active
(GreenSeeker® and Crop CircleTM) non-contact sensors depending on the sunlight
260 M. Maghrebi et al.

reflected by the canopy or on their own specific light sources in the visible (650 or
590 nm) and NIR (770 or 880 nm) range, respectively. Spectral data collected by
these devices allow the calculation of the so-called normalised differences vegeta-
tion index (NDVI) according to the formula:

NDVI ¼ ðNIR  VisÞ=ðNIR þ VisÞ

where NIR and Vis stand for the spectral reflectance measurements acquired in the
NIR or visible (red) regions, respectively. The NDVI value is about 0.5 when the
vegetation chlorophyll content and thus, in the absence of any other stress factors,
plant N status is optimal; conversely in sub-optimal conditions the value of NDVI is
much lower.
Several examples of the use of these portable proximal sensors (which can also be
mounted on tractors) in the fine-tuning of variable-rate technology for site-specific
N fertilisation exist (Solari et al. 2008; Diacono et al. 2013). The possibilities to
easily and efficiently translate the information on N crop status, obtained by the
hand-held or proxy sensor approaches described as above, in site- and time-specific
recommendations for FBMPs can be invalidated by a plethora of biotic and/or biotic
stressors, including a non-optimal availability of nutrients other than N, which
influence the chlorophyll content of the leaves. Therefore, the parameters and the
vegetation index obtained are usually validated by setting up standardisation pro-
cedures providing for plots of the same cultivar in the same environment at different
N availability. In this way genetic, environmental and agronomical factors can be
eliminated as potential sources of error and making the data obtained by the sensor-
based approaches more reliable (Samborski et al. 2009; Diacono et al. 2013).
Hyperspectral radiometers providing contemporaneous reflectance measure-
ments over a relatively narrow wavebands (<10 nm), should make it possible to
identify specific regions of the spectrum which could be used to develop new
indices, highly sensitive to plant N status and unaffected by other exogenous factors
(Hansen and Schjoerring 2003). Indeed, an increasing number of studies suggest
that field as well as airborne or spaceborne hyperspectral canopy radiometric data
can be useful for estimating plant nitrogen concentration in cultivated or natural
environments (Ollinger et al. 2008, Stroppiana et al. 2009), although recently some
criticisms about the remote sensing of leaf tissue constituents by hyperspectral data
have been raised (Knyazikhin et al. 2013).
Leaf chlorophyll concentration is also an indirect diagnostic symptom for N status
of the crop. However, it is important to take into account that reduced chlorophyll
biosynthesis is a relatively late response to N starvation which only becomes evident
after the plant has initiated other molecular and physiological responses for
maintaining N homeostasis (Schatchtman and Shin 2007; Gojon et al. 2009).
Unfortunately, non-destructive reliable monitoring approaches comparable with
those above described for N have not been developed for the other mineral nutrients
whose availability affects crop yield (in particular P, K and S). Thus, for these
nutrients the chance to adopt FBMPs is limited to the classic chemical evaluation of
plant tissues and soils.
10 Monitoring Plant Nutritional Status 261

Plant Bioindicator for Nutritional Status

Bioindication and Biomonitoring

The development of quick and inexpensive methods to determine changes in

nutrient bioavailability is required in order to monitor soil nutrient dynamics for
better fertiliser management for a variety of crops in different environmental
conditions. Developing bioassays based on the use of specific plant sentinels or
bioindicators, may represent a reliable and efficient strategy to obtain quick,
accurate and low-cost information about nutrient availability changes in a given
crop system. Thus, the use of these modern biotechnologies could allow the
non-destructive analysis of plants under field conditions. Development of these
kinds of tools represents a new and challenging area of research.
Plants respond to nutrient supply or shortage through a complex of physiolog-
ical, morphological, and developmental responses, which are under the control of
several gene pathways. Microarray technology is a convenient tool for rapid
analysis of plant gene expression patterns under a variety of environmental and
nutritional conditions. Genome-wide microarray analyses showed extensive
changes in the expression of several genes involved in primary and secondary
metabolism, nutrient transport, protein synthesis, regulation of gene expression
and cellular growth processes (Maruyama-Nakashita et al. 2003; Wang
et al. 2003; Bi et al. 2007; Li et al. 2010; Kant et al. 2010; Ma et al. 2012). Such
studies not only improved our general understanding on plant responses to nutrient
availability but also provided a reliable data from which to develop new molecular
strategies for real-time monitoring of plant nutritional status.
Recently, Yang et al. (2011) used multiple whole genome microarray experi-
ments to identify gene expression biomarkers capable of assessing plant responses
under limiting and sufficient nitrogen conditions. Using logistic regression statisti-
cal approaches, they identified a common set of genes in maize whose expression
profiles quantitatively assessed the extent of plant stress under different nitrogen
conditions. Interestingly, such a biomarker gene set is independent of maize
genotype, tissue type, developmental stage, and environment (including plants
grown under controlled conditions and in the field), and thus has the potential to
be used as an agronomic tool for real-time monitoring and to optimise nitrogen
fertiliser usage.

The Gene Fusion Concept Enables to Define a New Class

of Transgenic Bioindicators

The existence of gene pools, which specifically respond to the nutritional status of
the plant, has introduced a new class of bioindicators, based on the concept of gene
fusion (Fig. 10.3). A generic nutrient-responsive gene is formally considered as
262 M. Maghrebi et al.

Fig. 10.3 Gene fusion is a

DNA construction in which
the coding sequence from
one gene (reporter) is
transcribed and/or
translated under the
direction of the controlling
sequence of another gene
(controller). (a) Gene
structure. (b) Gene fusion

consisting of two parts: the promoter or controller that senses the nutritional status
of the plant and directs the synthesis of a new product from the second component,
the responder. By replacing the original sequence of the responder gene with a new
and easily studied gene, called a reporter gene, it should be possible to obtain
valuable information about the activity of the promoter. Such a molecular manip-
ulation should provide information about the nutritional status of the plant by
simply measuring the activity of the reporter protein.
Plant biologists to study how a particular gene is controlled when measurement
of the gene product is too difficult have extensively used the gene fusion concept.
The elective tool used in this type of studies is the GUS gene fusion system, which
uses uidA from E. coli as a reporter gene. This gene encodes a β-glucuronidase able
to hydrolyse a wide range of β-D-glucuronide substrates producing coloured or
other compounds in amounts proportional to enzyme activity (Jefferson 1989).
Assays for testing the activity of GUS in genetically modified plants are carried
out on plant material or plant extracts under laboratory conditions. A variety of
glucuronides is commercially available and can be used for fluorometric, spectro-
photometric, luminometric and histochemical GUS analyses, qualitative as well as
quantitative (Gallagher 1992).
Unfortunately, reporter systems based on the activity of β-glucuronidase (GUS)
have severe intrinsic limitations that preclude their application under field condi-
tions since they require the enzyme (GUS) and its substrate be brought together to
produce the hydrolytic products necessary for the analysis. Likewise, luciferin–
luciferase imaging systems have been used in plants, but their application in the
field is hampered by the low level of light emission and the need for sensitive
photon-counting cameras to detect signal (de Ruijter et al. 2003). Thus, new tools to
perform non-destructive analysis are essential to develop specific sentinel plants to
be directly used under field conditions.
In a pioneering paper, Jefferson (1993) listed some criteria useful to develop new
reporter systems suitable for agricultural molecular biology. Some of the key
criteria, such an in vivo reporter system are that it should be: (i) non-destructive;
(ii) non-disruptive to avoid physiological alteration of crop performance; (iii) useful
10 Monitoring Plant Nutritional Status 263

and functional in most crop species; (iv) inexpensive and capable of being used
everywhere; (v) simply to detect with little or no instrumentation; (vi) easy to use
under field conditions.
Naturally fluorescent proteins could offer a valuable alternative to the use of
GUS reporter systems since, in contrast to GUS, the detection of their expression
does not require the addition of a substrate. Green fluorescent protein (GFP), a
spontaneously fluorescent protein, was initially isolated from the luminescent
marine jellyfish (Aequorea victoria). GFP emits a highly and stable bright green
fluorescence after absorbing blue light (Tsien 1998). The wild type Aequorea
protein has a major excitation peak at 395 nm which is about three times higher
in amplitude than a minor peak at 475 nm. In normal solution, excitation at 395 nm
gives emissions peaking at 508 nm, whereas excitation at 475 nm gives a maximum
at 503 nm (Heim et al. 1994). Since its discovery GFP from Aequorea victoria has
become a frequently used tool in plant biology. The first studies on transgenic plants
expressing wild type GFP proved the usefulness of this protein as an in vivo and
real-time visible marker and encouraged researchers to modify it in order to obtain
new variants that could be more effectively synthesised in plant cells and macro-
scopically detectable at the whole plant level (Stewart 2001). One of these modified
versions of GFP is the mGFP5er variant that produces a stable protein targeted to
the endoplasmic reticulum as the result of the addition of a N-terminal Arabidopsis
basic chitinase fusion and a C-terminal HDEL fusion (Haseloff et al. 1997). The
coding sequence contains three mutations that enhance the folding of mGFP5er at
higher temperatures and allows excitation of the protein using either ultraviolet
(395 nm) or blue (473 nm) light (Siemering et al. 1996). In addition, new fluores-
cence colours have been created through mutagenesis of the natural protein giving
longer excitation and emission wavelengths and to enhance the fluorescence bright-
ness. The new colours range from blue and cyan (EBFP and ECFP) to yellow
(EYFP); such new proteins have excitation/emission peaks at 383/474, 434/472 and
514/527 nm, respectively (Spiess et al. 2005; Mena et al. 2006).
Fluorescent proteins have been largely used as visual genetic labels at the whole
plant, tissue and cell levels, since they offer a fast and easy-to-use non-destructive
tool with which the efficiency and timing of gene expression can be evaluated.
Detection and quantification of fluorescence at the whole-plant level normally
requires the use of complex and expensive laboratory instruments (scanning laser
and fluorescence imaging systems). Portable instruments, such as fibre optic probe
fluorometers, have recently been designed to assess GFP fluorescence under field
conditions (Harper and Stewart 2000; Millwood et al. 2003). However, because
bioindicators need to be disseminated over a wide area, a successful field applica-
tion of these plants also requires a cost-effective remote monitoring system pro-
viding real-time information about the nutritional status of a whole crop system.
Recently Adams et al. (2011) proposed an alternative method for crop monitoring
in which sentinel plants and sensing units are deployed in tandem at specific
locations. Ideally, such a system integrates biological and sensory technologies
with communication technologies to provide a practical field-deployable telemetry
system.
264 M. Maghrebi et al.

The gene fusion concept could be used to measure complex phenomena, even in
the absence of mechanistic knowledge of how that phenomenon works (Jefferson
1993). This technology is completely general and could be exploited to develop
transgenic bioindicators providing signals whose intensity is proportional to the
concentration of a given analyte in growing environment (i.e. mineral nutrients,
pollutants, water, etc.) or to the intensity of a biotic or abiotic stress that plants
could experience during their growth. Potential targeted traits to be monitored are
only limited by the availability of specific promoters (or controllers) driving the
reporter expression under a specific condition.
Recently these technologies have been applied in plants to develop model
transgenic bioindicators of the nutritional status to be used for laboratory purposes.
To date, reporter gene activity has been used to assess the phosphate, sulfate and
magnesium status in Arabidopsis and also to detect the level of nickel in the
growing medium (Hammond et al. 2003; Krizek et al. 2003; Maruyama-Nakashita
et al. 2006; Kamiya et al. 2012). In all these studies GUS, GFP and LUC have been
successfully used as reporter genes to indicate nutritional status under the control of
promoter sequences indirectly identified by microarray analyses.
Hammond and co-workers (2003) first proposed the creation of an Arabidopsis
transgenic bioindicator, able to monitor plant phosphorous status. They fused GUS
with the promoter of the phosphate starvation responsive gene SQD1 (a gene
involved in the synthesis of sulfolipids), obtaining an Arabidopsis transgenic line
in which GUS activity increased following P starvation. Interestingly, the reporter
responses to P withdrawal were much more rapid and quantitative than phenotypic
observations, showing this approach is particularly suitable for developing efficient
systems for monitoring plant P status. More recently Kamiya et al. (2012) used a
similar approach to establish a novel monitoring system for magnesium in plants. In
particular they obtained an Arabidopsis transgenic line that expressed luciferase
(LUC) under the control of the Mg deficiency-inducible CAX3 promoter. The
transgenic lines showed a clear response under low Mg conditions and the degree
of luminescence reflected the accumulation of endogenous CAX3 mRNA. How-
ever, CAX3 induction does not seem to be specific to low Mg, since the levels of
other ions (Ca2+ and Na+) or P starvation may influence transcription (Shigaki and
Hirschi 2000).
Notwithstanding some limitations Arabidopsis ‘smart’ plants could also be used
as tools in basic research aimed at isolating novel mutants disrupted in nutrient
homeostasis or identifying plants with enhanced nutrient use efficiency. For
instance, the key transcription factor, SLIM1, regulating the sulfur assimilatory
pathway has recently been identified by screening Arabidopsis mutants carrying a
fluorescent reporter gene under the control of the sulfur limitation-responsive
promoter of the SULTR1;2 sulfate transporter (Maruyama-Nakashita et al. 2006).
Using this approach it is possible to identify all the potential genes involved in
controlling the expression of SULTR1;2 under sulfur shortage, since in this condi-
tion the relative mutants will display altered fluorescence emissions as compared to
the wild type bioindicator.
10 Monitoring Plant Nutritional Status 265

Strategies to Enhance the Specificity of Bioindicators

In the future, the use of smart plant technology in crops would provide rapid
bioassay methods to obtain valuable information about nutrient availability in the
soil solution and/or the nutritional status of the plants allowing efficient temporal
and special application of fertilisers and the development of decision-making
systems for precision farming. To date the exploitation of these technologies is
limited, not only by the lack of telemetry systems suitable for plant monitoring
across a large area, but also by the lack of precise information to design a
transformation-cassette that would enable the nutrient-specific control of reporter
activity. Thus, the choice of a core promoter to confer specific transgene expres-
sion, represent the major challenge we have to face in order to develop the next
generation of bioindicators.
A typical plant promoter consists of CAAT and TATA boxes for recognition of
DNA-dependent RNA polymerase, several-tens of bp upstream of the transcription
initiation site (Yoshida and Shinmyo 2000). Specific DNA sequences, called cis-
elements, generally upstream of the core promoter, drive the cell- or organ-specific
expression of the downstream gene under certain environmental conditions. Spe-
cific factors, called trans-factors (or transcription factors), bind to the cis-elements
affecting RNA polymerase activity. Generally, multiple-cis-elements and trans-
factors work together to induce the full regulation of gene expression, since gene
expression is generally under the control of several factors (Yoshida and Shinmyo
2000; Venter 2007).
In recent years, a wide range of different promoters have been characterised and
extensively used for regulating the expression of transgenes in plant cells (Venter
2007). In several cases, the cis-elements that are necessary for transcriptional
regulation and the trans-factors that interact with these elements have been iden-
tified. From these studies has emerged a complex picture in which DNA sequence
cis-elements that are important for regulation are scattered over thousands of base
pairs, and these elements interact with trans-factors that can be either ubiquitous or
highly restricted in their distribution. In this way diverse expression patterns may be
achieved through combinations of a limited number of regulatory elements and
trans-acting factors. The knowledge of these combinatorial mechanisms should
allow the generation different transcription patterns by ‘cut and pasting’ the com-
ponents in different ways.
Analysis of the cauliflower mosaic virus (CaMV) 35S promoter has contributed
to the understanding of transcriptional regulatory mechanisms and has allowed the
design of inducible transgene expression cassettes. The 343 to 46 upstream
region relative to the site of initiation of transcription (+1) of the promoter is
responsible for the strength of transcription. Two regions, 343 to 208 and
208 to 90, are responsible for transcriptional activation, and the 90 to 46
region plays an accessory role by further increasing the transcriptional activity
(Odell et al. 1985; Fang et al. 1989). Artificial promoters are generally constructed
by a combinatorial design of different promoter elements, with the minimal core
266 M. Maghrebi et al.

Fig. 10.4 Schematic representation of a synthetic promoter useful for bioindication purposes. The
core region of the CaMV 35S promoter is fused with a combinatorial engineering of cis-elements
(blue boxes) which, following the interaction with specific transcription factors, drives the reporter
expression under particular conditions

DNA fragment (46 to +8 bp) of the CaMV 35S promoter as the main component
(Fig. 10.4). The core-promoter region contains a TATA-box necessary for
recruiting RNA polymerase II and the orchestrated assembly of general transcrip-
tion factors to form the pre-initiation complex (Novina and Roy 1996). The CaMV
35S core-promoter is ideal for transcription initiation and has been used in several
synthetic plant promoters in which combinatorial engineering of cis-element have
been introduced upstream of the core-promoter sequence.
The use of synthetic promoters allowing for targeted inducibility of a reporter
gene is of considerable interest to develop engineering strategies aimed at creating
plant bioindicators for real-time monitoring of nutritional status. For these purposes
promoter sequence domains or cis-elements conferring nutrient- and organ-
specificity should be combined in order to target the reporter expression in organs
(shoot and leaves) in which signals should be easily detectable.
Many different plant promoters have been described as able to restrict gene
expression to particular cells, tissues or organs. The GaMYB2 promoter is cotton
fibre- and Arabidopsis trichome-specific, and can drive gene expression specifically in
glandular cells (head cells) of glandular trichomes in transgenic tobacco (Shangguan
et al. 2008). Some cis-elements regulating tissue-specific gene expression have also
been identified. For instance, mesophyll expression module 1 (Mem1), a 41 bp
fragment of the ppcA1 promoter, directs mesophyll-specific expression. The
tetranucleotide sequence, CACT has been identified as a key component of Mem1
by evolutionary and functional studies (Gowik et al 2004). More recently, Ye
et al. (2012) identified a rice green tissue-specific expression gene, DX1, and described
two novel tissue-specific cis-elements (GSE1 and GSE2) within the DX1 promoter. In
particular, GSE1 acted as a positive regulator in all green tissues, whereas GSE2 acted
as a positive regulator only in sheath and stem tissues.
10 Monitoring Plant Nutritional Status 267

Obviously, nutrient-specific cis-elements are equally as important for reporter

expression as tissue-specific cis-elements. Nutrient-inducible plant promoters con-
tain multiple cis-acting elements, only some of which may specifically contribute to
nutrient inducibility. A number of potential nutrient responsive cis-elements have
recently been identified in the promoter of several nutrient responsive genes and
have been indicated as key regulatory factors of gene expression under different
nutritional conditions.
Sulfur-responsive elements (SUREs) have been identified in the promoter
regions of the Arabidopsis NIT3 nitrilase and β-subunit β-conglycinin gene from
soybean (Awazuhara et al. 2002; Kutz et al. 2002), although no consensus
sequences have been shown yet. However, an interesting study on Arabidopsis
sulfate transporter SULTR1;1 promoter demonstrates that a 5 bp sequence is
essential to promote sulfur response of SULTR1;1 (Maruyama-Nakashita
et al. 2005). Such a sequence also appears in the promoter regions of many
sulfur-responsive genes, suggesting its involvement in the transcriptional control
of a gene set required for adaptation to sulfur-limiting conditions. Deletion analysis
of the barley IDS2 (iron deficiency-specific clone no. 2) gene promoter allowed the
identification of two cis-acting elements, iron-deficiency-responsive element 1 and
2 (IDE1 and IDE2), which synergistically induced iron-specific expression in
tobacco roots. Finally, comparative analyses of several nitrite reductase gene pro-
moters from various higher plants have recently allowed identification of a con-
served sequence motif as nitrate-responsive cis-element (Konishi and Yanagisawa
2010).

What Do Bioindicators Sense? A Key Problem

Modification of promoter architecture necessary for manipulating gene reporter

activity requires accurate studies of the regulatory network involved in controlling
gene expression under different nutritional conditions. Unfortunately, for the most
part, these aspects are still largely unknown preventing the optimal design of a
synthetic nutrient-inducible promoter, particularly in cases where a mineral nutrient
undergoes complex assimilatory metabolisms (i.e. nitrate or sulfate) or interacts
with other nutrients. In all these cases the specific question to be answered is: what
do synthetic nutrient-specific promoters sense?
For example, the transcriptional regulatory mechanisms involved in sulfate
uptake and assimilation reasonably result from direct sensing of the plant nutri-
tional status rather than from the composition of the external soil solution
(Lappartient and Touraine 1997; Lappartient et al. 1999). This control involves
an inter-organ signaling mechanism in which key intermediates of the sulfate
assimilatory pathway may act as negative or positive signals in modulating the
expression of the sulfur-responsive genes. Adequate levels of sulfur compounds
would repress gene expression through a negative feedback loop preventing exces-
sive sulfate uptake and reduction; vice versa a contraction of the intermediates
268 M. Maghrebi et al.

along the assimilatory pathway would unrepress gene transcription allowing sulfate
to enter the pathway. A second regulatory loop, involving OAS as a key interme-
diate, would act in promoting gene unrepression when nitrogen and carbon supply
exceeds sulfur availability within the cells (Hawkesford 2000). In this context the
need to dissect the molecular mechanisms involved in the nutritional signal per-
ception and transduction is evident since, in several cases, the relationships existing
between gene expression and the levels of the signal-intermediates are not always
clear. Further research is needed to associate single gene expressions to a specific
nutritional signal or sulfur-nutritional status.
Genome-wide expression analyses have revealed that nitrate supply induces
changes in the expression of several genes, not only those involved in nitrate
reduction and assimilation. Such behaviour is likely both due to the direct effects
of nitrate itself and indirect effects caused by changes in nitrogen metabolite
content or nitrogen nutritional status. In fact, nitrate is thought to act as a signal
molecule influencing the expression of a number of genes, since their expression is
rapidly induced by nitrate even in mutants severely compromised for nitrate
reductase activity (Wang et al. 2004). In addition, it has been shown that nitrate-
inducible expression NADH/nitrate reductase mRNA in maize roots, scutella and
leaves also occurs in the presence of inhibitors of protein synthesis, suggesting that
the signal transduction system mediating this response is constitutively expressed in
plant cells, independently of the presence or the absence of nitrate in the growing
medium (Price et al. 2004). Results of these studies clearly shows that dissection
analyses of the signal transduction pathways controlling gene expression under
different nitrogen supply should provide important information to define smart
plants able to sense the cellular level of nitrite or the general nitrogen nutritional
status of a crop system.

Conclusions
Growing varieties with enhanced efficiency and/or modifying the environ-
ment in which the crop is grown can increase the nutrient use efficiency of a
crop production system. The selection of varieties with improved nutrient use
efficiency is a more generic approach, which necessarily requires deep
knowledge of the genetic variation and inheritance of these traits. On the
other hand, the improvement of the agricultural practices aimed at sustaining
the nutrient needs of a crop may provide more immediate advantages in terms
of cost and environmental quality.
Although much progress has been made in improving fertilisation prac-
tices there remain considerable uncertainties about the persistence of nutri-
ents in the soil and their actual availability to the plants. The development of
quick and inexpensive methods to determine changes in nutrient bioavail-
ability in the soil or the nutritional status of the plants are desirable for better
fertiliser management for different crops in a variety of environmental con-
ditions. It could be particularly important not only for areas of intensive
agriculture but also for agriculture in developing countries where accessibil-
ity to fertilisers could be a problem.

(continued)
10 Monitoring Plant Nutritional Status 269

The development of optical devices allowing the remote monitoring at

sub-field scale of traits specifically linked to molecular, biochemical or
physiological consequences of scarcity or excess, of specific essential ele-
ments other than nitrogen, is surely a challenge for future research. Following
on from the spectranomic approach developed by Asner and Martin (2008) as
the application of metabolomics for the assessment of biochemical traits at a
landscape scale, Brunetti et al. (2013) suggest an airborne hyperspectral
analysis whereby changes in absorbance and reflectance patterns of specific
chemical compounds within leaves could further improve detection of bio-
chemical phenotypes across a relatively large scale. A combination of these
approaches could define exciting new frontiers for the fine-tuning of contin-
uous and non-destructive methods of monitoring plant nutritional status in the
field. In the next years, whereby multidisciplinary studies, efforts have to be
planned to actually translate in field the knowledge accumulated that seem to
promise interesting deliverables.

References

Adamchuck VI, Viscarra Rossel RA, Sudduth KA, Lammers PS (2011) Sensor fusion for precision
agriculture. In: Thomas C (ed) Sensors fusion-foundation and applications. InTech Publishers,
Croatia, pp 27–40
Adams JP, Topsakal E, Yuceer C (2011) Fluorescing phytosensors: a speculated next step for
environmental monitoring. Plant Mol Biol Biotechnol 2:16–25
Asner GP, Martin RE (2008) Airborne spectranomics: mapping canopy chemical and taxonomic
diversity in tropical forests. Front Ecol Environ 7:269–278
Awazuhara M, Kim H, Goto DB, Matsui A, Hayashi H, Chino M, Kim SG, Naito S, Fujiwara T
(2002) A 235-bp region from a nutritionally regulated soybean seed-specific gene promoter can
confer its sulfur and nitrogen response to a constitutive promoter in aerial tissues of
Arabidopsis thaliana. Plant Sci 163:75–82
Barker AV, Pilbeam DJ (2007) In: Baker AV, Pilbeam DJ (eds) Handbook of plant nutrition. CRC
Publisher, Boca Raton, pp 3–18
Bi YM, Wang RL, Zhu T, Rothstein SJ (2007) Global transcription profiling reveals differential
responses to chronic nitrogen stress and putative nitrogen regulatory components in
Arabidopsis. BMC Genomics 8:281
Brunetti C, George RM, Tattini M, Field K, Davey MP (2013) Metabolomics in plant environ-
mental physiology. J Exp Bot 64:4011–4020
Cartelat A, Cerovic ZG, Goulas Y, Meyer S, Lelarge C, Prioul JL, Barbottien A, Jeuffroy MH,
Gate P, Agati G, Moja I (2005) Optically assessed contents of leaf polyphenolics and chloro-
phyll as indicators of nitrogen deficiency in wheat (Triticum aestivum L.). Field Crop Res
91:35–49
Craswell ET, Godwin DC (1984) The efficiency of nitrogen fertilizers applied to cereals grown in
different climates. In: Tinker PB, Luachli A (eds) Advances in plant nutrition, vol 1. Praeger
Publisher, New York, pp 1–55
de Ruijter NCA, Verhees J, van Leeuwen W, van der Krol AR (2003) Evaluation and comparison
of the GUS, LUC, and GFP reporter system for gene expression studies in plants. Plant Biol
5:103–115
270 M. Maghrebi et al.

Debaeke P, Rouet P, Justes E (2006) Relationship between the normalized SPAD index and the
nitrogen nutrition index: application to durum wheat. J Plant Nutr 29:75–92
Diacono M, Rubino P, Montemurro F (2013) Precision N management of wheat. A review. Agron
Sustain Dev 33:219–241
Erdle K, Mistele B, Schmidhalter U (2011) Comparison of active and passive spectral sensors in
discriminating biomass parameters and nitrogen status in wheat cultivars. Field Crop Res
124:74–84
Fang RX, Nagy F, Sivasubramaniam S, Chua NH (1989) Multiple cis regulatory elements for
maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants. Plant
Cell 1:141–150
Gallagher SR (1992) GUS protocols: using the GUS gene as a reporter of gene expression.
Academic, San Diego, pp 1–221
Godfray HCJ, Beddington JR, Crute IR, Haddad L, Lawrence D, Muir JF, Pretty J, Robinson S,
Thomas SM, Toulmin C (2010) Food security: the challenge of feeding 9 billion people.
Science 327:812–818
Gojon A, Nacry P, Davidian JC (2009) Root uptake regulation: a central process for NPS
homeostasis in plants. Curr Opin Plant Biol 12:328–338
Gowik U, Burscheidt J, Akyildiz M, Schlue U, Koczor M, Streubel M, Westhoff P (2004)
Cis-regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria
trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene. Plant Cell
16:1077–1090
Greenwood DJ, Neeteson JJ, Draycott A (1986) Quantitative relationships for the dependence of
growth rate of arable crops on their nitrogen content, dry weight and aerial environment. Plant
Soil 91:281–301
Hamilton J, Zangerl A, DeLucia E, Berenbaum M (2001) The carbon-nutrient balance hypothesis:
its rise and fall. Ecol Lett 4:86–95
Hammond JP, Bennett MJ, Bowen HC, Broadley MR, Eastwood DC, May ST, Rahn C, Swarup R,
Woolaway KE, White PJ (2003) Changes in gene expression in Arabidopsis shoots during
phosphate starvation and the potential for developing smart plants. Plant Physiol 132:578–596
Hansen PM, Schjoerring JK (2003) Reflectance measurement of canopy biomass and nitrogen
status in wheat crops using normalized difference vegetation indices and partial least squares
regression. Remote Sens Environ 86:542–553
Harper BK, Stewart CN (2000) Patterns of green fluorescent protein expression in transgenic
plants. Plant Mol Biol Report 18:141a–141i
Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and
subcellular localization of green fluorescent protein are required to mark transgenic
Arabidopsis plants brightly. Proc Natl Acad Sci U S A 94:2122–2127
Hawkesford MJ (2000) Plant responses to sulphur deficiency and the genetic manipulation of
sulphate transporters to improve S-utilization efficiency. J Exp Bot 51:131–138
Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation
of green fluorescent protein. Proc Natl Acad Sci U S A 91:12501–12504
Jefferson RA (1989) The GUS reporter gene system. Nature 342:837–838
Jefferson RA (1993) Beyond model systems: new strategies, methods, and mechanisms for
agricultural research. Ann N Y Acad Sci 700:53–73
Kalra YP (1998) Handbook of reference methods for plant analysis. CRC Press, Boca Raton
Kamiya T, Yamagami M, Hirai MY, Fujiwara T (2012) Establishment of an in planta magnesium
monitoring system using CAX3 promoter-luciferase in Arabidopsis. J Exp Bot 63:355–363
Kant S, Bi YM, Rothstein SJ (2010) Understanding plant response to nitrogen limitation for the
improvement of crop nitrogen use efficiency. J Exp Bot 62:1499–1509
Knyazikhin Y, Schull MA, Stenberg P, Mõttus M, Rautiainen M, Yang Y, Marshak A, Carmona
PL, Kaufmann RK, Lewis P, Disney MI, Vanderbilt V, Davis AB, Baret F, Jacquemoud S,
Lyapustin A, Myneni RB (2013) Hyperspectral remote sensing of foliar nitrogen content. Proc
Natl Acad Sci U S A 110:E185–E192
10 Monitoring Plant Nutritional Status 271

Konishi M, Yanagisawa S (2010) Identification of a nitrate-responsive cis-element in the

Arabidopsis NIR1 promoter defines the presence of multiple cis-regulatory elements for
nitrogen response. Plant J 63:269–282
Krizek BA, Prost V, Joshi RM, Stoming T, Glenn TC (2003) Developing transgenic Arabidopsis
plants to be metal-specific bioindicators. Environ Toxicol Chem 22:175–181
Kutz A, Muller A, Hennig P, Kaiser WM, Piotrowski M, Weiler EW (2002) A role for nitrilase 3 in
the regulation of root morphology in sulphur-starving Arabidopsis thaliana. Plant J 30:95–106
Lappartient AG, Touraine B (1997) Glutathione-mediated regulation of ATP sulfurylase activity,
SO42 uptake, and oxidative stress response in intact canola roots. Plant Physiol 114:177–183
Lappartient AG, Vidmar JJ, Leustek T, Glass AMD, Touraine B (1999) Inter-organ signalling in
plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots medi-
ated by phloem-translocated compound. Plant J 18:89–95
Lea US, Slimestad R, Smedvig P, Lillo C (2007) Nitrogen deficiency enhances expression of
specific MYB and bHLH transcription factors and accumulation of end products in the
flavonoids pathway. Planta 225:1245–1253
Lemaire G, Gastal F (2009) Quantifying crop responses to nitrogen deficiency and avenues to
improve nitrogen use efficiency. In: Sadras VO, Calderini DF (eds) Crop physiology: appli-
cations for genetic improvement and agronomy. Elsevier Publisher, Adelaide, pp 171–211
Li L, Liu C, Lian X (2010) Gene expression profiles in rice roots under low phosphorus stress.
Plant Mol Biol 72:423–432
Lu C, Zhang J (2000) Photosynthetic CO2 assimilation, chlorophyll fluorescence and
photoinhibition as affected by nitrogen deficiency in maize plants. Plant Sci 151:135–143
Ma TL, Wu WH, Wang Y (2012) Transcriptome analysis of rice root responses to potassium
deficiency. BMC Plant Biol 12:161
Malhi BS, Haderlein LK, Pauly DG, Johnston AM (2002) Improving fertilizer phosphorus use
efficiency. Better Crop 86:8–9
Maruyama-Nakashita A, Inoue E, Watanabe-Takahashi A, Yamaya T, Takahashi H (2003)
Transcriptome profiling of sulfur-responsive genes in Arabidopsis reveals global effects of
sulfur nutrition on multiple metabolic pathways. Plant Physiol 132:597–605
Maruyama-Nakashita A, Nakamura Y, Watanabe-Takahashi A, Inoue E, Yamaya T, Takahashi H
(2005) Identification of a novel cis-acting element conferring sulfur deficiency response in
Arabidopsis roots. Plant J 42:305–314
Maruyama-Nakashita A, Nakamura Y, Tohge T, Saito K, Takahashi H (2006) Arabidopsis SLIM1
is a central transcriptional regulator of plant sulfur response and metabolism. Plant Cell
18:3235–3251
Mena MA, Treynor TP, Mayo SL, Daugherty PS (2006) Blue fluorescent proteins with enhanced
brightness and photostability from a structurally targeted library. Nat Biotechnol 24:1569–
1571
Millwood RJ, Halfhill MD, Harkins D, Russotti R, Stewart CN (2003) Instrumentation and
methodology for quantifying GFP fluorescence in intact plant organs. Biotechniques 34:638–
643
Novina CD, Roy AL (1996) Core promoters and transcriptional control. Trends Genet 12:351–355
Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the
cauliflower mosaic virus 35S promoter. Nature 313:810–812
Ollinger SV, Richardson AD, Martin ME, Hollinger DY, Frolking SE, Reich PB, Plourd LC, Katul
GG, Munger JW, Oren R, Smith ML, Paw UKT, Bolstad PV, Cook BD, Day MC, Martin TA,
Monson RK, Schmid HP (2008) Canopy nitrogen, carbon assimilation, and albedo in temperate
and boreal forests: functional relations and potential climate feedbacks. Proc Natl Acad Sci U S
A 105:19336–19341
Pierce FJ, Nowak P (1999) Aspects of precision agriculture. In: Sparks DL (ed) Advances in
agronomy. Academic Press, New York, pp 1–85
Price J, Laxmi A, St Martin SK, Jang JC (2004) Global transcription profiling reveals multiple
sugar signal transduction mechanisms in Arabidopsis. Plant Cell 16:2128–2150
272 M. Maghrebi et al.

Raun WR, Johnson GV (1999) Improving nitrogen use efficiency for cereal production. Agron J
91:357–363
Rengel Z, Damon PM (2008) Crops and genotypes differ in efficiency of potassium uptake and
use. Physiol Plant 133:624–636
Rouached H, Secco D, Arpa BA (2010) Regulation of ion homeostasis in plants: current
approaches and future challenges. Plant Sign Behav 5:501–502
Samborski SM, Tremblay N, Fallon E (2009) Strategies to make use of plant sensors-based
diagnostic information for nitrogen recommendations. Agron J 101:800–816
Schatchtman DP, Shin R (2007) Nutrient sensing and signalling: NPKS. Annu Rev Plant Biol
58:47–69
Schlemmer MR, Francis DD, Shanahan JF, Schepers JS (2005) Remotely measuring chlorophyll
content in corn leaves with differing nitrogen levels and relative water content. Agron J
97:106–112
Shangguan XX, Xu B, Yu ZX, Wang LJ, Chen XY (2008) Promoter of a cotton fiber MYB gene
functional in trichomes of Arabidopsis and glandular trichomes of tobacco. J Exp Bot 59:3533–
3542
Shigaki T, Hirschi K (2000) Characterization of CAX-like genes in plants: implications for
functional diversity. Gene 257:291–298
Siemering KR, Golbik R, Sever R, Haseloff J (1996) Mutations that suppress the thermosensitivity
of green fluorescent protein. Curr Biol 6:1653–1663
Solari F, Shanahan JF, Ferguson R, Schepers JS, Gitelson A (2008) Active sensor reflectance
measurements of corn nitrogen status and yield potential. Agron J 100:571–579
Spiess E, Bestvater F, Heckel-Pompey A, Toth K, Hacker M, Stobrawa G, Feurer T, Wotzlaw C,
Berchner-Pfannschmidt U, Porwol T, Acker H (2005) Two-photon excitation and emission
spectra of the green fluorescent protein variants ECFP, EGFP and EYFP. J Microsc 217:200–
204
Stewart CN (2001) The utility of green fluorescent protein in transgenic plants. Plant Cell Rep
20:376–382
Stroppiana D, Boschetti M, Brivio PA, Bocchi S (2009) Plant nitrogen concentration in paddy rice
from field canopy hyperspectral radiometry. Field Crop Res 111:119–129
Tremblay N, Wang Z, Cerovic ZC (2012) Sensing crop nitrogen status with fluorescence indica-
tors. A review. Agron Sustain Dev 32:451–464
Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544
Venter M (2007) Synthetic promoters: genetic control through cis engineering. Trends Plant Sci
12:118–124
Wang R, Okamoto M, Xing X, Crawford NM (2003) Microarray analysis of the nitrate response in
Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to
glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol 132:556–567
Wang R, Tischner R, Gutiérrez RA, Hoffman M, Xing X, Chen M, Coruzzi G, Crawford NM
(2004) Genomic analysis of the nitrate response using a nitrate reductase-null mutant of
Arabidopsis. Plant Physiol 136:2512–2522
Yang XS, Wu J, Ziegler TE, Yang X, Zayed A, Rajani MS, Zhou D, Basra AS, Schachtman DP,
Peng M, Armstrong CL, Caldo RA, Morrell JA, Lacy M, Staub JM (2011) Gene expression
biomarkers provide sensitive indicators of in planta nitrogen status in maize. Plant Physiol
157:1841–1852
Ye R, Zhou F, Lin Y (2012) Two novel positive cis-regulatory elements involved in green tissue-
specific promoter activity in rice (Oryza sativa L ssp.). Plant Cell Rep 31:1159–1172
Yoshida K, Shinmyo A (2000) Transgene expression systems in plant, a natural bioreactor. J
Biosci Bioeng 90:353–362
Zheng G, Moskal LM (2009) Retrieving leaf area index (LAI) using remote sensing: theories,
methods and sensors. Sensors 9:2719–2745
Index

A Apparent recovery, 254

Abscisic acid (ABA), 100, 141 APR. See Adenosine 5’-phosphosulfate
O-Acetylserine (OAS), 52, 58, 60–63, 68, 71, reductase
72, 74, 76, 77, 222, 268 APS. See Adenosine 5’-phosphosulfate
O-Acetylserine thiol-lyase (OAS-TL), 58, Arabidopsis, 33, 35, 37–43, 56, 57, 59, 61–67,
60–63, 68, 71, 72, 75 69–72, 74–76, 80, 96, 97, 99–106, 112,
Acquisition efficiency, 8, 94, 113 117, 118, 135–139, 141–145, 147, 154,
Acrylamide, 53 156, 176, 177, 179–184, 187–189,
Adaptation, 15, 19, 29, 31, 35, 59, 75, 80, 192–194, 207, 209–212, 214, 215, 217,
98–100, 104, 105, 111, 112, 114, 117, 218, 263, 264, 266, 267
119, 148, 172, 213, 247, 267 Arabidopsis thaliana, 20, 40, 41, 51–80, 99,
Adenosine 5’-phosphosulfate (APS), 38, 105, 106, 135, 137, 138, 147, 179–184,
59–61, 68 207, 209–212, 217
reductase, 59, 60, 68 Artificial promoter, 265
Adenosine 5’-phosphosulfate kinase (APK), Assimilation, 3, 31, 32, 53, 59–71, 74–79, 134,
59–61, 68 135, 138, 146, 157, 172, 206, 233, 237,
Adenosine 5’-phosphosulfate reductase (APR), 238, 243, 244, 267, 268
59–61, 68–72, 75–77, 145 efficiency, 30
S-Adenosylmethionine (SAM), 60, 64, 65, 72, Atg8 (authophagy-related protein 8) 177, 178,
137, 217 181, 182, 185, 188–192, 195
Aequorea victoria, 263 Atmospheric sulfur nutrition, 13
Aglycone, 66 ATPase, 54, 115, 156, 174, 184
Agriculture, 2, 3, 7, 12, 14–16, 19, 30, 52, 53, ATP sulfurylase (ATPS), 59–61, 68–70, 75
80, 135, 205–207, 222, 253, 254, 268 Autophagosome, 175–178, 181, 185, 190, 195
Agroecosystems, 13, 14, 206 Autophagy, 171–196, 209, 217
Agronomic efficiency, 236, 254 Auxin, 69, 73, 74, 100, 106, 113, 118, 141
Agronomy, 3, 5
Alfalfa. See Medicago sativa
Amino acids, 31, 35, 53, 58, 59, 61, 62, 64, 66, B
67, 75, 76, 96, 134, 146, 172, 180, 186, Baking quality, 52
193, 195, 208, 210, 218, 234 Barley. See Hordeum vulgare
Animals, 62, 109, 143, 173, 175, 176, 178, 189, Basic Helix Loop Helix transcription
190, 193, 196 factors, 140
Antagonism, 162–164 Berseem clover, Trifolium alexandrinum, 163
APK. See Adenosine 5’-phosphosulfate kinase BHLH transcription factors, 115, 139, 141

© Springer International Publishing Switzerland 2014 273

M.J. Hawkesford et al. (eds.), Nutrient Use Efficiency in Plants,
Plant Ecophysiology 10, DOI 10.1007/978-3-319-10635-9
274 Index

Biodiversity, 110, 222, 223 Crop yield, 9, 12, 30, 52, 80, 94, 135, 160, 196,
Biofertilizer, 164 205–223, 246, 260
Bioindicators, 261–268 β-Cyanoalanine synthase, 62
nutritional status, 261–269 Cysteine biosynthesis, 62–64, 71, 72, 76
Biomass, 6–10, 12, 19, 32, 33, 35, 107–109, Cytokinin, 74, 76, 77, 142, 188
117, 172, 196, 206, 208, 209, 212–215, Cytoplasm, 108, 146, 174, 185, 195, 196
231–233, 235, 237, 238, 240–243, 245, Crop
246, 257 improvement, 80, 118, 119, 230, 246
dilution, 240–242 quality, 80, 209
production, 6, 9, 10, 12, 241 yield, 9, 12, 30, 52, 80, 94, 135, 160, 196,
Biomonitoring nutritional status, 261 205–223, 246, 260
Boron, 51, 153–165, 245 CSC. See Cysteine synthase complex
deficiency, 154, 156–159, 163 Cystathionine, 60, 65
toxicity, 163 Cystathionine γ-synthase (CGS), 60, 65, 72
transporter, 154, 155 Cysteine, 52, 53, 58, 60–65, 68, 70–72, 74, 76,
Breeding, 2, 3, 10, 11, 15–17, 19, 20, 29, 30, 134, 146, 189, 208
42–44, 46, 52, 53, 80, 94, 107, 110–113, Cysteine synthase complex (CSC), 62, 63, 68,
115, 119, 135, 205, 207, 209, 213, 216, 71, 72
222, 223, 246, 247

D
C Deficiency, 7, 13, 18, 19, 30, 42, 51–53, 56, 59,
Calcium/proton exchanger3 (CAX3), 264 71, 73–79, 99, 102, 103, 105, 109, 110,
Calvin cycle, 157, 219 117–119, 135–142, 145–148, 154–161,
CaMV. See Cauliflower mosaic virus 163, 165, 171–196, 206, 207, 212, 213,
Candidate genes, 37–39, 42–43, 222 215, 217–219, 246, 256
Canopy reflectance, 256, 259 Deficiency-responsive element 2 (IDE2),
Carbohydrates, 7, 9, 65, 76, 98, 116, 118, 141, 267
157, 207, 212, 217–219, 232, 234, Degradation, 52, 64–66, 103, 105, 114, 119,
240–241, 244 137, 139, 172, 173, 175, 177, 184–187,
Carbon dioxide (CO2), 1, 4, 9, 10, 51, 77, 79, 190–196, 209–211, 213, 214, 216
141, 157, 172, 229–247 Depletion, 95, 98, 108, 113–115, 205–223, 235
fertilisation effect, 229, 232, 246 Deposition, dry, wet, 14
Carbon fixation, 6, 231, 240 Development, 3, 6, 8, 16, 46, 58, 60, 67, 101,
Cargo, 175–177, 183, 185, 187, 190–195 102, 109, 113, 118, 135, 143–146, 158,
Β-Carotene, 157 159, 165, 175, 179–181, 205–223, 230,
Carrot, Daucus carota, 156 243, 247, 257, 258, 261, 265, 268, 269
Cauliflower mosaic virus (CaMV), 265, 266 Diagnostic values, 108, 256
CAX3. See Calcium/proton exchanger3 Dilution curve of nutrients, 256–258
CGS. See Cystathionine γ-synthase Diversity, 10, 11, 13, 14, 20, 30, 31, 40, 97,
Chlorophyll, 134, 157, 188, 208–211, 215–218, 110, 111
234, 258–260 Drought, 13, 19, 35, 42–44, 78, 110, 180–182,
Chloroplast, 18, 57, 59, 61, 63, 96, 134, 189, 192, 209, 212, 213, 247
143–146, 157, 186, 209, 210 Dwarf cultivars, 15
Chlorosis, 79, 135, 137–139, 145, 211, Dwarfing genes, 16
215, 221 DX1 gene, 266
Cis-acting iron deficiency responsive element,
141, 267
Cis-elements, 97, 101, 116, 141, 144, 265–267 E
Climate change, 19, 205, 242 Ecophysiology, 20
CO2. See Carbon dioxide (CO2) Ecosystem services, 3
β-Conglycinin, 69, 267 γ-ECS. See γ-Glutamylcysteine synthase
Cotton, Gossypium, 157, 266 Efficient cultivars, 30, 113, 206
C4 plants, 11, 20, 232 EIL. See Ethylene insensitive-like (EIL)
Critical concentrations, 235, 240, 256, 257 transcription factors
Index 275

Elevated CO2, 229–247 Glutathione (GSH), 53, 60, 61, 64, 70, 74, 76, 77
Engineered plants, 78, 79 Glutathione synthetase (GSHS), 60, 64
Environmental factors, 3, 12, 15, 30, 61, 254 Glycine max, 21, 125, 132, 225
Epistasis, 34 Glycolipid, 109, 156
Ethylene, 62, 65, 68, 139–141 Glycoprotein, 156
Ethylene insensitive-like (EIL) transcription Grain, 8, 10, 11, 14, 19, 30, 33, 52, 53, 106–110,
factors, 68, 69 113, 115, 116, 118, 119, 162, 208, 215,
Eucalyptus oblique, 154 216, 231–235, 238, 239, 241, 244–246
Grape, Vitis vinifera, 44, 162
Green fluorescent protein (GFP), 57, 58, 96,
F 188, 263, 264
Fenton reaction, 134, 143 Green revolution, 15, 16, 222
FER-like iron deficiency-induced transcription Green tissue-specific cis-acting element1
factor, 139 (GSE1), 266
Ferric chelate reductase 2, 136 Green tissue-specific cis-acting element2
Ferritin, 134, 143, 144 (GSE2), 266
Fertilisation, 3, 6, 13–15, 94, 108, 110, 207, Groundnut, Arachis hypogaea, 159
213, 221, 223, 232, 246, 254–257, GUS reporter gene, 262–264
260, 268
Fertiliser, 5, 7, 13, 15, 16, 30, 33, 46, 51–53,
78, 94, 106–109, 111, 112, 119, 160, H
164, 206, 222, 235–238, 244–247, Haber-Bosch process, 15
253–256, 258, 261, 265, 268 Haplotype, 42
Fertiliser best management practices (FBMPs), Harvest index, 5, 8, 33, 238, 247
254–256, 260 Heme, 134, 147
Fe-S cluster(s), 18, 134, 143–148 Helianthus annuus, 156, 159, 162, 163
Flavonoids, 259 Heterogeneous Inbred Families (HIFs), 36–38
Food security, 16 Hill reaction, 157
Free air carbon dioxide enrichment (FACE), Homeostasis, 9, 72, 75–78, 80, 98, 99, 102,
230–234, 237–239, 247 117–119, 137, 141–148, 173, 175, 196,
Free amino acid(s), 35, 53, 146, 234 214, 256, 260, 264
Homocysteine, 60, 64, 65
Hordeum vulgare, 85, 121, 124, 129, 151, 226
G HY5. See Long hypocotyl 5
GaMYB2 promoter, 266 Hydroponics, 32
Gene(s), 16, 29, 53, 93, 135, 154, 172, 209, Hyperspectral radiometers, 260
247, 253
Gene fusion, 261–264
Genetic backgrounds, 31, 34, 37, 43, 44, I
80, 99 Ideotypes, 33
Genetic diversity, 30, 310, 311 Introgression lines (ILs), 34
Genome-wide association studies (GWAS), Iron (Fe), 18, 31, 32, 38, 51, 61, 133–148, 189,
41–43, 80 206, 218, 245, 267
Genomics, 34, 37, 44 deficiency, 18, 139, 140
Genotypic variation, 80, 99, 113–115 regulated transporter 1, 136, 137
GFP. See Green fluorescent protein strategy I, 135–141, 147
Global change, 226 strategy II, 135–138, 140, 141, 147
Global phosphorus cycle, 106 transporters, 138, 145, 206
Glucosinolates, 52, 60, 61, 65–67, 73–75, 210, Iron deficiency-responsive element 1 (IDE1),
212, 214 141, 267
aliphatic, 73 Iron deficiency specific clone no. 2 (IDS2),
indolic, 66, 73 141, 267
β-Glucuronidase (uidA, GUS), 262 Isothiocyanates, 65
γ-Glutamylcysteine synthase (γ-ECS), 60, 64 Isotope labeling, 32
276 Index

J Methionine (Met), 52, 60, 64–65, 72, 146, 208

Jasmonic acid, 74, 75, 142 Methionine synthase (MS), 60, 65
Methylthioadensone (MTA), 65
Micronutrients, 2, 14, 51, 118, 155, 160, 161,
K 164, 172, 206, 207, 233, 239–241, 246
Kinase(s), 59, 60, 65, 68, 72, 74, 104, 106, 115, Micronutrient use efficiency, 133–148
116, 118, 176–179, 181, 184, 192 microRNA395, 69
Mineral fertilizer, 106, 206
Mitochondria, 59, 62, 63, 134, 143–148,
L 185, 186
Law of diminishing returns, 16 Mitscherlich curve, 236
Law of the minimum, 15, 18, 206 Molybdenum transporters (MOT), 57–58
Law of the optimum, 18 Monitoring, 99, 185, 253–269
Leaf, 10, 33, 56, 79, 80, 96, 97, 135, 138, 139, MRT. See Mean residence time
157, 161–163, 180, 186, 188, 207–213, MTO1 region, 72
215, 216, 218, 219, 221, 232–235, Mugineic acids, 136–138
237–239, 244, 255–260 Multigenic inheritance, 33
Lentil, Lens culinaris, 162 Mutant(s), 31, 38, 54, 56, 57, 61, 63, 67–71,
Liebig’s Law of the minimum, 15, 206 74–76, 97, 99–104, 106, 113, 136–140,
Light use efficiency (LUE), 19 142–146, 179, 182, 184, 186–190, 192,
Linkage disequilibrium (LD), 42, 43, 80 193, 264, 268
Lipids, 105, 116, 134, 185, 190, 208, 210 MYB transcription factors, 73, 101
LIR motif, 190, 191, 193, 195 Mycorrhiza, 13, 97
Litter decomposability, 17 Myrosinase, 66
Long hypocotyl 5 (HY5), 71, 76
Luciferase (LUC), 144, 262, 264
Lysosome, 176, 187 N
Natural variation, 29–46, 70, 80
NBR1, 183, 184, 190, 192–194, 196
M Near-infrared light transmittance, 259, 260
Macronutrients, 2, 51–80, 94, 119, 206, 214 Near isogenic lines (NILs), 34, 35, 37, 38, 43
Macronutrient use efficiency, 51–80 Nicotianamine, 134, 136, 137, 147, 206
MAGIC populations, 35 Nicotiana tabaccum, 123, 168
Maize. See Zea mays Nitrate, 7, 13, 18, 19, 31, 33, 35, 39, 59, 62,
Male sterility, 158, 159, 181 75–77, 104, 156, 188, 210, 217–219,
Mammals, 64, 138, 173, 175, 184, 194 234, 241, 243–244, 267, 268
Mapping population, 34–36 leaching, 19
Mass flow, 13, 95, 241, 242 reductase, 76, 156, 268
Mean residence time (MRT), 6, 7, 17 reduction, 243–244, 268
Medicago, 97, 101, 105, 161, 162, 240 uptake, 7, 18, 76, 244
M. sativa, 97, 105, 161, 162 Nitrilase (NIT3), 69, 74, 267
Membrane(s), 18, 54, 55, 58, 63, 67, 96, 97, Nitrogen (N), 2, 6, 30–33, 35, 37, 51, 71,
102, 104, 105, 109, 135, 136, 138, 75–77, 117, 134, 135, 156, 184, 186,
143–145, 147, 156, 173, 175–178, 184, 188, 206, 208, 209, 213, 215, 217, 219,
185, 189, 191, 192, 195, 218, 231 232–238, 245, 258–261, 268, 269
Mesophyll expression module 1 (Mem1), 266 Nitrogen balance index (NBI), 259
Metabolism, 2, 8–10, 13, 20, 21, 30, 35, 53, Nitrogen response curve, 236
58–69, 71, 74, 76–79, 94, 104, 105, Nitrogen use efficiency (NUE/NitUE), 6–8, 14,
114–116, 118, 119, 134, 145–148, 18, 19, 76, 233, 237–238
156–158, 172, 196, 210, 214, 215, 219, Nondestructive analysis, 261, 262
230–233, 238, 239, 241, 256, 261, 267 Non-structural carbohydrates, 7, 240
Metabolome, 172, 217 Normalized differences vegetation index
Metabolomics, 20, 41, 43, 209–212, 221, 269 (NDVI), 260
Index 277

Nucleus, 106, 146, 195, 196 P

nutrient, 195 p62, 183, 190, 192–196
NuDIS, 212, 215, 217, 218, 221, 222 PAPS. See 3’-Phosphoadenosine
Nutrient 5’-phosphosulfate
availability, 6, 8, 12, 20, 31, 159, 206, 207, Partitioning, 115, 116, 135, 141,
214, 216, 222, 242, 243, 253, 256, 143–145, 238
261, 265 Phaseolus vulgaris, 161–163
content, 15, 32, 33, 161, 238 PB1, 192–194
critical concentration, 256–258 Peach, Prunus persica, 159
deficiency, 30, 141, 146, 171–196, 217, Peanut, Arachis hypogaea, 159
218, 246, 256 PCD. See Programmed cell death
interactions, 18, 135, 160, 161, 163–165, P forms, Phenolic acids, 157
233, 239, 240, 245 Phloem, 55–57, 64, 97, 104, 142, 147, 155,
limitation, 30–33, 147, 172, 256 158, 210, 219
monitoring, 253–269 Phosphate, 18, 59, 93–119, 178, 206, 264
reallocation, 8, 10 Phosphate response 1(PHR1), 77, 78, 97,
(re-)mobilization, 6, 8 101–104, 106, 113, 116
storage, 8, 11, 15 Phosphate transporters, 96–98, 100–103, 106,
uptake, 7, 13, 18, 19, 31, 32, 46, 159, 161, 114–116, 118, 119, 210
164, 212, 238, 241–243 3’-Phosphoadenosine 5’-phosphosulfate
Nutrient acquisition efficiency (NAcE), 5, 7, 19 (PAPS), 59–61
Nutrient assimilation efficiency (NAE), 30 O-Phosphohomoserine, 60, 65, 72
Nutrient demand and supply (NDS), 159, Phospholipids, 77, 94, 95, 105, 108, 109,
164, 165 118, 119
Nutrient depletion-induced senescence Phosphorus (P) 30, 31, 51, 77, 94–95,
(NuDIS), 212–216, 218, 221, 222 100, 106, 107, 114, 117–119, 135,
Nutrient depletion-induced stress, 212 189, 215
Nutrient productivity (NP), 6, 7, 9, 17 accumulation, 101–103, 105
Nutrient remobilisation efficiency (NRE), 30 acquisition, 94, 98, 108, 110, 111, 113, 114,
Nutrient-responsive gene(s), 261 117, 119
Nutrient uptake efficiency (NUpE), 30–33, acquisition efficiency (PAE), 94, 99, 104,
237, 238, 241 106–108, 111, 113, 119
Nutrient use efficiency (NUE), 1–21, 29–46, availability, 94, 101, 108, 112–114, 119
51–80, 133–148, 159–165, 205–207, pools, 108, 109
213, 216, 229–247, 264, 268 starvation, 96–106, 111–117, 119, 264
Nutrient utilization efficiency (NUtE), 5–7, 9, transport, 118
17–19, 30, 237 utilization efficiency (PUE), 94, 98, 99,
Nutritional index (NI), 257 104–112, 114–116, 118, 119
Nutritional status, 11, 13, 100, 117, 176, 235, Photorespiration, 10, 218, 219, 231, 243
253–269 Photosynthesis, 9, 10, 19, 59, 77, 134, 145,
157, 158, 186, 207, 214–219, 231,
232, 240
O Photosynthetic acclimation, 232, 244
OAS. See O-Acetylserine Photosynthetic rate, 9, 10, 157, 158
OAS-module, 222 PHR1. See Phosphate response 1
OAS-TL. See O-Acetylserine thiol-lyase Phytosiderophores, 138, 146
Omics, 32 Phytosulfokines (PSK), 65–67
Open reading frames (ORFs), 39 Plasmalemma, 162
Oryza sativa, 10, 11, 15, 35, 40, 54, 80, 96, 97, Posttranslational modifications, 172
99–103, 105, 106, 110, 114–118, 137, Precision agriculture, 16
138, 141, 142, 145, 147, 155, 159, 206, Programmed cell death (PCD), 173, 175,
207, 233, 234, 244, 266 180–183, 192
Oscillations, 2, 3, 14, 15 Progressive nitrogen limitation, 235, 245
278 Index

Promoter, 59, 68, 69, 79, 97, 101, 104, 115, ROS. See Reactive oxygen species
116, 141–144, 262, 264–267 Rubisco, 9, 10, 79, 186, 188, 209,
Proteasome, 173–175, 195, 196 231–233, 235
Protein
homeostasis, 196
turnover, 109, 172–175 S
Proteomic studies, 104 Salinity, 35, 36, 245, 246
Proton efflux, 102 Salt, 12, 36, 42, 109, 178, 180–182, 188, 189,
PSI genes (P starvation inducible genes), 99, 192, 193
102, 103 SAM. See.S-Adenosylmethionine
PSK. See Phytosulfokines Secretion, 67, 98, 104, 105, 112–114, 117, 138
Seed(s), 7, 10, 12, 30–32, 35, 41, 42, 52, 56, 58,
64, 80, 109, 117–119, 138, 143, 145,
Q 158–160, 164, 179, 188, 206–210,
QTL analysis, 34, 39, 40, 80, 112, 118, 119 212–215, 219, 233, 244
Quantitative trait loci (QTL), 32–40, 43, Seed filling, 31, 212, 215
44, 46, 80, 100, 106, 110, 112, 113, Senescence, 10, 11, 33, 144, 173, 179–183,
117–119 186, 188, 189, 206–219, 221, 244
Serine acetyltransferase (SAT, Serat), 60, 62,
63, 68
R Shoot, 13, 31, 32, 35, 36, 56, 69, 70, 78–80, 94,
Radiometers, 256, 260 96–98, 101–103, 106, 108, 110, 112, 114,
Radish, Raphanus sativus, 169 116–118, 136, 138, 140–142, 145, 155,
Reactive oxygen species (ROS), 64, 134, 162–164, 172, 208, 214, 256–258, 266
142–144, 147, 178, 182, 191, 209, Signaling, 78, 100–104, 141, 173, 176–184,
210, 215 188, 189, 267
Receptor(s), 67, 74, 118, 183, 184, 190, Signaling cascades, 106, 116–117
192–195 Single nucleotide polymorphism (SNP), 41,
Recombinant inbred lines (RILs), 34–37, 71, 42, 71
80, 118 SLIM1. See Sulfur limitation 1
Regulation, 53, 54, 58–59, 63, 65, 67–78, 80, Soil, 1, 30, 51, 93, 133, 153–155, 188, 206,
98–102, 106, 117, 135, 137, 139–144, 229, 253
154, 156, 157, 172, 173, 189, 196, 243, interactions, 245–246
261, 265 Solanum lycopersicum, 57, 97, 101, 103, 117,
Remobilisation, 30–33, 98, 103, 109, 115, 186, 135, 137, 139, 142, 146, 161–163,
188, 208, 209, 241, 244 192, 206
efficiency, 8, 32 Sorbitol, 77, 155
Remote sensing, 257, 260 Soybean. See Glycine max
Reporter gene(s), 262, 264, 266 Spectral reflectance, 260
Reproductive growth, 3, 157–159 Starch, 12, 15, 157, 186, 208, 209, 212
Resource mobilisation, 213 Starvation, 33, 56, 58, 69, 70, 74, 75, 79,
Resources, 3, 16, 29, 33, 38, 46, 79, 80, 94, 98, 96–106, 111–117, 172, 177–183, 186,
99, 109, 208, 209, 213–215, 222, 230, 188, 189, 191, 192, 196, 212–219, 221,
237, 242, 245, 246, 254 222, 255, 260, 264
Rhamnogalacturonan II (RG II), 156 STAS domain, 54, 58
Rice. See Oryza sativa Stomatal conductance, 11, 19, 157, 231, 232,
Rice green tissue-specific expression gene 241, 242
(DX1), 266 Stress, 13, 19, 40, 42, 43, 57, 58, 69, 73, 74, 99,
Roots 100, 102, 107, 112, 117, 144, 178–181,
architecture, 19, 31, 35, 100, 103, 106, 112, 184, 186–189, 191, 192, 196, 206,
241–243 209–212, 215, 217, 219, 221,
plasticity, 98, 100 259–261, 264
Index 279

Sugar alcohol, 155 Transporter of mugineic acids, 136, 138

Sugar metabolism, 115 Trehalose, 155
Sulfate Triticum aestivum, 9–11, 15, 19, 40, 52, 53, 79,
reduction, 38, 54, 59, 60, 70, 71, 74, 80, 94, 96, 97, 101, 103, 106, 109–111,
76, 241 113–118, 137, 147, 159, 163, 186, 206,
transporter(s), 53–60, 68, 69, 72, 74, 75, 207, 209, 212, 215, 231, 233, 234,
77–79, 147, 264, 267 237–239, 244, 259
uptake, 53, 54, 56, 58, 59, 68–70, 72, 74,
77–79, 212, 267
Sulfation, 60, 67 U
Sulfide, 52, 61–64, 67, 71, 76, 79, 134, 146, 189 Ubiquitin, 103, 106, 137, 172–175, 178, 185,
Sulfite oxidase (SO), 62 192, 194
Sulfite reductase (SiR), 60–62, 71, 78, 145 Ubiquitin-binding domain (UBA), 192,
Sulfur (S), 7, 30, 51–80, 134, 135, 145–147, 194–195
189, 208, 210, 212, 214, 216, 217, Ubiquitin proteasome system (UPS), 173–175,
264, 267, 268 189, 195
deficiency, 52, 53, 56, 71, 73, 74, 76, uidA reporter gene, 262
78–79, 103, 135, 156, 194, 217 Utilization efficiency, 5, 94, 100
Sulfur limitation1 transcription factor
(SLIM1), 68–70, 73, 78, 264
Sulfur responsive elements (SUREs), 69, 267 V
Sunflower. See Helianthus annuus Vacuole, 18, 39, 52, 53, 56–58, 108, 145, 147,
SURE element, 69 176–178, 185–187, 189, 190, 192, 209,
Sustainable agriculture, 205–207 212, 213
Systems biology, 207, 216–222

W
T Water use efficiency (WUE), 18, 19, 231
Targeting induced local lesions in genomes Wheat. See Triticum aestivum
(TILLING), 38 Whole plant physiology, 16, 158
Target of Rapamycin (TOR), 176–184 Wild-type, 31, 57, 61, 69, 74, 114, 144, 145,
Thiols, 52, 58, 60–62, 64, 68, 70 188, 189, 263, 264
Threonine synthase (TS), 60, 65, 72
Tomato. See Solanum lycopersicum
Trade-off X
non-physical, 20 Xylem, 13, 52, 56, 70, 102, 142, 147, 154,
physical, 18, 19 155, 161
Transcriptional profiling, 112
Transcription factors, 68, 71, 73, 77, 97, 100,
101, 104, 113, 115–117, 119, 142, 147, Y
217, 259, 264–266 Yeast, 54, 96, 101, 102, 116, 138, 143,
Transcriptome, 104, 105, 112, 116, 172, 173–176, 178, 179, 183–187, 189,
217, 218 190, 194
Transcriptomics, 20, 209–212, 218, 221 Yellow stripe, 136, 138
Trans-factors, 265 Yield, 3, 30, 51, 94, 135, 153–165, 171,
Transgenic bioindicators, 261–264 205–223, 229, 253
Transition metal, 134, 143, 207 Yield potential (Yp), 7, 12, 20, 205, 207, 223
Transpiration, 155, 158, 161, 212, 242
Transport, 18, 52–59, 63, 64, 68–70, 79, 97–99,
102, 113, 116, 118, 134, 136–138, 142, Z
143, 145, 147, 148, 154–156, 158, 162, Zea mays, 7, 8, 10, 13, 242, 259
178, 187, 188, 190, 192, 195, 196, 206, Zinc, 51, 133, 137, 138, 143, 145, 192,
212, 243, 259, 261 194, 207