On the Nature of Carbon Isotope Discrimination in C 4 Species

Nature Plants

Stable isotopes are commonly used to study the diffusion of CO inside photosynthetic tissues of plants. The standard method to interpret the observed preference for the lighter carbon isotope in C 3 photosynthesis involves the model by Farquhar, O'Leary and Berry, which relates carbon isotope discrimination to physical and biochemical processes inside the leaf. However, under many conditions the model returns unreasonable results for mesophyll conductance to CO 2 diffusion (g m), especially when rates of photosynthesis are low. Here we re-derive the carbon isotope discrimination model using modified assumptions related to the isotope effect of mitochondrial respiration. In particular, we treat the carbon pool associated with respiration as separate from the pool of the primary assimilates. We experimentally test the model by comparing g m values measured with different CO 2 source gases varying in their isotopic composition and show that our new model returns matching g m values that are much more reasonable than those obtained with the old model. We use our results to discuss CO 2 diffusion properties inside the mesophyll.

PLANT PHYSIOLOGY, 1980

A mathematical model is developed which can be used to predict in vivo carbon isotope fractionations associated with carbon fixation in plants in terms of diffusion, CO2 hydration, and carboxylation components. This model also permits calculation of internal CO2 concentration for comparison with results of gas-exchange experments. The isotope fractionations associated with carbon fixation in Kaawchoi daigremontiana and Bryophylbun tubiflorm have been measured by isolation of malc acid following dark fixation and enzymic determination of the isotopic composition of carbon4 of this material. Corrections are made for residual malc acid, fumarase activity, and respiration. Comparison of these data with calculawww.plant.org on April 14, 2016 -Published by www.plantphysiol.org Downloaded from

Planta, 1988

Carbon-isotope ratios were examined as c513C values in several C3, C~, and C3-C4 F l a v e r i a species, and compared to predicted ~3 C values generated from theoretical models. The measured cI13C values were within 4%~ of those predicted from the models. The models were used to identify factors that contribute to C3-1ike 5t3C values in C3-C4 species that exhibit considerable CA-cycle activity. Two of the factors contributing to C3-1ike fi13C values are high CO2 leakiness from the C4 pathway and pi/pa values that were higher than C4 congeners. A marked break occurred in the relationship between the percentage of atmospheric CO2 assimilated through the C4 cycle and the c5 13 C value. Below 50% C4-cycle assimilation there was no significant relationship between the variables, but above 50% the 613C values became less negative. These results demonstrate that the level of C4-cycle expression can increase from 0 to 50% with little integration of carbon transfer from the C4 to the C3 cycle. As expression increases above 50%, however, increased integration of C3-and C4-cycle co-function occurs.

Journal of Experimental Botany, 2014

Crop species with the C 4 photosynthetic pathway are generally characterized by high productivity, especially in environmental conditions favouring photorespiration. In comparison with the ancestral C 3 pathway, the biochemical and anatomical modifications of the C 4 pathway allow spatial separation of primary carbon acquisition in mesophyll cells and subsequent assimilation in bundle-sheath cells. The CO 2 -concentrating C 4 cycle has to operate in close coordination with CO 2 reduction via the Calvin-Benson-Bassham (CBB) cycle in order to keep the C 4 pathway energetically efficient. The gradient in CO 2 concentration between bundle-sheath and mesophyll cells facilitates diffusive leakage of CO 2 . This rate of bundle-sheath CO 2 leakage relative to the rate of phosphoenolpyruvate carboxylation (termed leakiness) has been used to probe the balance between C 4 carbon acquisition and subsequent reduction as a result of environmental perturbations. When doing so, the correct choice of equations to derive leakiness from stable carbon isotope discrimination (Δ 13 C) during gas exchange is critical to avoid biased results. Leakiness responses to photon flux density, either short-term (during measurements) or long-term (during growth and development), can have important implications for C 4 performance in understorey light conditions. However, recent reports show leakiness to be subject to considerable acclimation. Additionally, the recent discovery of two decarboxylating C 4 cycles operating in parallel in Zea mays suggests that flexibility in the transported C 4 acid and associated decarboxylase could also aid in maintaining C 4 /CBB balance in a changing environment. In this paper, we review improvements in methodology to estimate leakiness, synthesize reports on bundle-sheath leakiness, discuss different interpretations, and highlight areas where future research is necessary. RT. 1997. Carbon isotope discrimination during C 4 photosynthesis: insights from transgenic plants. Australian Journal of Plant Physiology 24, 487-494. Weiner H, Burnell JN, Woodrow IE, Heldt HW, Hatch MD. 1988. Metabolite diffusion into bundle sheath cells from C 4 plants-relation to C 4 photosynthesis and plasmodesmatal function. Plant Physiology 88, 815-822.

PLANT PHYSIOLOGY, 1989

Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO2 assimilation or oxygen evolution, over a wide range of CO2 concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO2 near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO2 concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HC03-, indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of 14C-incorporation from 14CO2 into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO2 occurs predominantly by direct CO2 fixation by ribulose 1,5bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO2 concentrations, permeability coefficients for CO2 flux into bundle sheath cells were determined for a range of C4 species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C3 plants. These results support the concept that a CO2 permeability barrier exists to allow the development of high CO2 concentrations in bundle sheath cells during C4 photosynthesis.

2009

Non-photosynthetic, or heterotrophic, tissues in C 3 plants tend to be enriched in 13 C compared with the leaves that supply them with photosynthate. This isotopic pattern has been observed for woody stems, roots, seeds and fruits, emerging leaves, and parasitic plants incapable of net CO 2 fixation. Unlike in C 3 plants, roots of herbaceous C 4 plants are generally not 13 C-enriched compared with leaves. We review six hypotheses aimed at explaining this isotopic pattern in C 3 plants: (1) variation in biochemical composition of heterotrophic tissues compared with leaves; (2) seasonal separation of growth of leaves and heterotrophic tissues, with corresponding variation in photosynthetic discrimination against 13 C; (3) differential use of day v. night sucrose between leaves and sink tissues, with day sucrose being relatively 13 C-depleted and night sucrose 13 C-enriched; (4) isotopic fractionation during dark respiration; (5) carbon fixation by PEP carboxylase; and (6) developmental variation in photosynthetic discrimination against 13 C during leaf expansion. Although hypotheses (1) and (2) may contribute to the general pattern, they cannot explain all observations. Some evidence exists in support of hypotheses (3) through to (6), although for hypothesis (6) it is largely circumstantial. Hypothesis (3) provides a promising avenue for future research. Direct tests of these hypotheses should be carried out to provide insight into the mechanisms causing within-plant variation in carbon isotope composition.

Functional Plant …, 2009

Non-photosynthetic, or heterotrophic, tissues in C 3 plants tend to be enriched in 13 C compared with the leaves that supply them with photosynthate. This isotopic pattern has been observed for woody stems, roots, seeds and fruits, emerging leaves, and parasitic plants incapable of net CO 2 fixation. Unlike in C 3 plants, roots of herbaceous C 4 plants are generally not 13 C-enriched compared with leaves. We review six hypotheses aimed at explaining this isotopic pattern in C 3 plants: (1) variation in biochemical composition of heterotrophic tissues compared with leaves; (2) seasonal separation of growth of leaves and heterotrophic tissues, with corresponding variation in photosynthetic discrimination against 13 C; (3) differential use of day v. night sucrose between leaves and sink tissues, with day sucrose being relatively 13 C-depleted and night sucrose 13 C-enriched; (4) isotopic fractionation during dark respiration; (5) carbon fixation by PEP carboxylase; and (6) developmental variation in photosynthetic discrimination against 13 C during leaf expansion. Although hypotheses (1) and (2) may contribute to the general pattern, they cannot explain all observations. Some evidence exists in support of hypotheses (3) through to (6), although for hypothesis (6) it is largely circumstantial. Hypothesis (3) provides a promising avenue for future research. Direct tests of these hypotheses should be carried out to provide insight into the mechanisms causing within-plant variation in carbon isotope composition.

was reduced from approximately 5 kPa O 2 to 1 to 2 kPa O 2 , becoming similar to that of C 3 plants. Therefore, the higher O 2 requirement for optimal C 4 photosynthesis is specifically associated with the C 4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O 2 than in the wild type. When CO 2 fixation by Rubisco is limited, an increase in the CO 2 concentration in bundle-sheath cells via the C 4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O 2 while increasing CO 2 leakage and overcycling of the C 4 pathway. These results indicate that in C 4 plants the investment in the C 3 and C 4 cycles must be balanced for maximum efficiency.

The 2 H/ 1 H ratio of carbon-bound H in biolipids holds potential for probing plant lipid biosynthesis and metabolism. The biochemical mechanism underlying the isotopic differences between lipids from C 3 and C 4 plants is still poorly understood. GC-pyrolysis-IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry) measurement of the 2 H/ 1 H ratio of leaf lipids from controlled and field grown plants indicates that the biochemical isotopic fractionation (ε 2 H lipid_biochem ) differed between C 3 and C 4 plants in a pathway-dependent manner: ε 2 H C4 > ε 2 H C3 for the acetogenic pathway, ε 2 H C4 < ε 2 H C3 for the mevalonic acid pathway and the 1-deoxy-D-xylulose 5-phosphate pathway across all species examined. It is proposed that compartmentation of photosynthetic CO 2 fixation into C 4 mesophyll (M) and bundle sheath (BS) cells and suppression of photorespiration in C 4 M and BS cells both result in C 4 M chloroplastic pyruvatethe precursor for acetogenic pathwaybeing more depleted in 2 H relative to pyruvate in C 3 cells. In addition, compartmentation in C 4 plants also results in (i) the transferable H of NADPH being enriched in 2 H in C 4 M chloroplasts compared with that in C 3 chloroplasts for the 1-deoxy-D-xylulose 5-phosphate pathway pathway and (ii) pyruvate relatively 2 H-enriched being used for the mevalonic acid pathway in the cytosol of BS cells in comparison with that in C 3 cells.

Nature, 2002

Functional Plant Biology, 1986

Conventional gas-exchange techniques that measure the stomatal conductance and rate of CO2 assimilation of leaves were combined with measurements of the carbon isotope composition of CO2 in air passing over a leaf. Isotopic discrimination during uptake was determined from the difference in the carbon isotope composition of air entering and leaving the leaf chamber. Isotopic discrimination measured over the short term correlated strongly with that determined from combusted leaf material. Environmental conditions were manipulated to alter the relative influences of stomatal conductance and carboxylation on the discrimination of carbon isotopes by intact leaves. With C3 plants, discrimination increased as the gradient in partial pressure of CO2 across the stomata decreased. For C4 plants there was little change in discrimination despite substantial changes in the diffusion gradient across the sto- mata. These results are consistent with, and provide the first direct experimental suppor...

PLANT PHYSIOLOGY, 1974

The content of 13C varies in plants with Crassulacean acid metabolism. Differences up to 3.5/,, in the '3C/'2C ratios were observed between leaves of different age in the same plant of BryophyUum daigremontianum. Soluble and insoluble carbon in the same leaf differed up to 8%S,, the largest difference occurring in the leaves with the highest Crassulacean acid metabolism activity. Models to account for the isotope discrimination by C3, C4, and Crassulacean acid metabolism plants are proposed.

Phytochemistry Reviews, 2000

Carbon isotope discrimination during photosynthetic CO 2 assimilation has been extensively studied and rigorous models have been developed, while the fractionations during photorespiratory and dark respiratory processes have been less well investigated. Whilst models of discrimination have included specific factors for fractionation during respiration (e) and photorespiration (f ), these effects have been considered to be very small, i.e. not significantly modifying the net discrimination expressed in organic material. On this paper we consider the fractionation effects associated with specific reactions set against the overall discrimination which occurs during source-product transformations. We review the studies which have recently shown that discrimination occurs during respiration at night in intact C 3 leaves, leading to the production of CO 2 enriched in 13 C (i.e., e = −6 ), and modifying the signature of the remaining plant material. Under photorespiratory conditions (i.e. increased oxygen concentration and high temperature), the photorespiratory fractionation factor may be high (with f around +10 ), and significantly alters the observed net photosynthetic discrimination measured during gas exchange. Fractionation factors for both respiration and photorespiration have been shown to be variable among species and with environmental conditions, and we suggest that the term 'apparent fractionation' be used to describe the net effect for each process. In this paper we review the fractionations during photorespiration and dark respiration and the metabolic origin of the CO 2 released during these processes, and we discuss the ecological implications of such fractionations.

Australian Journal of Plant Physiology, 1998

Transpiration efficiency, W, the ratio of plant carbon produced to water transpired and carbon isotope discrimination of leaf dry matter, ∆ d , were measured together on 30 lines of the C 4 species, Sorghum bicolor, in the glasshouse and on eight lines grown in the field. In the glasshouse, the mean W observed was 4.9 mmol C mol -1 H 2 O and the range was 0.8 mmol C mol -1 H 2 O. The mean ∆ d was 3.0‰ and the observed range was 0.4‰. In the field, the mean W was lower at 2.8 mmol C mol -1 H 2 O and the mean ∆ d was 4.6‰. Significant positive correlations between W and ∆ d were observed for plants grown in the glasshouse and in the field. The observed correlations were consistent with theory, opposite to those for C 3 species, and showed that variation in ∆ d was an integrated measure of long-term variation in the ratio of intercellular to ambient CO 2 partial pressure, p i /p a . Detailed gas exchange measurements of carbon isotope discrimination during CO 2 uptake, ∆ A , and p i /p a were made on leaves of eight S. bicolor lines. The observed relationship between ∆ A and p i /p a was linear with a negative slope of 3.7‰ in ∆ A for a unit change in p i /p a . The slope of this linear relationship between ∆ A and p i /p a in C 4 species is dependent on the leakiness of the CO 2 concentrating mechanism of the C 4 pathway. We estimated the leakiness (defined as the fraction of CO 2 released in the bundle sheath by C 4 acid decarboxylations, which is lost by leakage) to be 0.2. We conclude that, although variation in ∆ d observed in the 30 lines of S. bicolor is smaller than that commonly observed in C 3 species, it also reflects variation in transpiration efficiency, W. Among the eight lines examined in detail and in the environments used, there was considerable genotype x environment interaction.

Annual Review of Plant Physiology and Plant Molecular Biology, 1989

Annu. Rev. Plant Physiol. Plant Mol. Bioi. 1989. 40:503-37 Copyright © 1989 by Annual Reviews Inc. All rights reserved ... GD Farquhar,l 1. R. Ehleringer,2 and KT Hubic ... IResearch School of Biological Sciences, Australian National University, Canberra, ACT 2601 Australia ...