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Proteomic analysis of nitrogen stress-responsive

proteins in two rice cultivars differing in N
utilization efficiency

Article · February 2011

DOI: 10.5584/jiomics.v1i1.22

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 JIOMICS | VOL 1 | ISSUE 1 | FEBRUARY 2011 | 78-87

JOURNAL OF INTEGRATED OMICS

Journal of Integrated Omics
A METHODOLOGICAL JOURNAL
HTTP://WWW.JIOMICS.COM

ORIGINAL ARTICLE | DOI: 10.5584/jiomics.v1i1.22

Proteomic analysis of nitrogen stress-responsive proteins in two rice

cultivars differing in N utilization efficiency .
Chen Song1, Fanrong Zeng1, Wu Feibo1, Wujun Ma2, Guoping Zhang*1.
1
Agronomy Department, Huajiachi Campus, Zhejiang University, Hangzhou 310029, China. 2Western Australian Department of Agriculture
and Food; Centre for Comparative Genomics and State Agriculture Biotechnology Centre, Murdoch University and Perth, WA 6150, Austral-
ia.

Received: 15 June 2010 Accepted: 9 September 2010 Available Online: 13 September 2010

ABSTRACT
Plant nitrogen utilization efficiency (NUE) has become critical important in modern agriculture, not only for crop growth and yield but also
for reducing production cost. Moreover, one of the major negative environmental impacts of agricultural activities is associated with excessive
nitrogen application. Improving NUE will ensure lower level of N fertilizer usage thus reduce environmental contamination. In order to un-
derstand the NUE mechanism of rice, the largest food crop in the world, a systematic proteomic study of investigating the nitrogen stress-
responsive proteins in two rice cultivars differing in NUE is conducted. Four leaf-old seedlings were treated with normal nutrition solution and
N-free solution for 12 h, 3 d and 7 d. Total proteins of leaves were extracted and separated by two-dimensional gel electrophoresis. Although
more than 1000 protein spots were reproducibly detected, only a very small proportion of spots showed differential expression, including 10
and 24 up-regulated, 2 and 12 down-regulated in the two cultivars Chunyou 58 and Yongyou 6, respectively. This indicates that relatively
simply biochemical pathways maybe involved with NUE thus the NUE as a trait maybe efficiently manipulated. Mass spectrometry based
peptide mass fingerprinting (PMF) procedure identified 31 protein spots. Six stress-induced proteins were found, including DegP2, harpin
binding proteins, Heat shock-related proteins, glutathione S-transferase GSTF14, Fibrillin-like protein and Glyceraldehyde-3-phosphate dehy-
drogenase. Apart from the stress related proteins, the other differential proteins identified were mainly these involved in the regulation of the
main leaf biological function, photosynthesis metabolism, such as Rubisco activase, RuBisCo large subunit, etc. The study also detected two
novel proteins, harpin binding protein and oryzains gamma precursor. The current study reveals new insights into N stress response and theo-
retical bases for improving NUE of rice crop.

Keywords: Mass spectrometry, rice (Oryza sativa L.), nitrogen utilization efficiency, Two-dimensional gel electrophoresis.

1. Introduction
A key element in modern agriculture is the application of field was absorbed and utilized by crops; the majority of them
nitrogen fertilizer, which has dramatically increased the crop was lost to the atmosphere or leached into groundwater, lakes
yield [1]. In order to meet the food demand of the increased and rivers, causing increasingly severe pollutions to the envi-
world population, application of nitrogen fertilizer in the ronments [4]. Therefore, development of crop cultivars with
world has been increased by 10 folds in the last half century, high N utilization is essential for agricultural sustainability
It was predicted that the increase trend will continue in this and environmental protection.
century, from 87 million tones in 2000 to 236 million tones in The improvement of N fertilizer utilization could be real-
2050 [2]. Meanwhile, most of the high yield varieties of the ized by enhancing the ability of N uptake and/or increasing N
major crops developed in the last several decades had high utilization efficiency (NUE). For the former, a crop or a spe-
nitrogen demand for the realization of yield potential [3]. On cial cultivar has high ability of N uptake from the soils with
the other hand, less than half of the N fertilizers applied to the low N concentration, which is referred as high uptake effi-

*Corresponding author: Guoping Zhang. Agronomy Department, Huajiachi Campus, Zhejiang University, Hangzhou 310029, China. Email Address:
[email protected]

78-87: 78
 Chen Song et al., 2010 | Journal of Integrated Omics

ciency. For the latter, a crop or a cultivar may make the best [14-16].
use of N nutrient that the plant absorbed from soil for In general, a low or zero nitrogen application causes
producing biomass or harvest organs, which is evaluated by nutritional imbalance. Plants can perceive the stress signals
grain yield or biomass production per unit nitrogen and transmit them to the cellular machinery to activate
amount[1], expressed as NUEY (N utilization efficiency of adaptive responses. The adaptation is generally completed by
yield) and NUEB (N utilization efficiency of biomass), regulating gene expressions. Proteome dynamics under the
respectively. Although NUEY is affected by many stress conditions reflects the regulatory gene expressions. In
physiological processes, including nitrogen contribution to the current study, in order to understand the NUE
spikelet production during early panicle formation stage, and mechanism of rice, the largest food crop of the world, we
contribution to sink size by decreasing the number of adopted a systematic proteomic approach to investigate the
degenerated spikelets and increasing hull size during the late nitrogen stress-responsive proteins in two rice cultivars
panicle formation stage [5], the fact that higher rice yield is differing in NUE.
achieved mainly due to greater biomass production [6]
2. Material and methods
provided the possibility to explore the relationship between
NUEP and NUEY. 2.1 Plant materials and stress treatments
As an essential plant macronutrient, nitrogen is required
Seeds of two rice cultivars, Chunyou 58 (high NUE) and
for a variety of physiological processes. It comprises 1.5–2%
Yongyou 6 (Low NUE), were germinated and grown
of plant dry matter and approximately 16% of total plant
hydroponically in nutrient solution containing 2.9 mM
protein [4]. For rice, the leaf N, about 75% of total plant N, is
NH4NO3, 0.32 mM NaH2PO4, 1.0 mM K2SO4, 1.0 mM CaCl2,
associated with chloroplasts, which are physiologically
1.7 mM MgSO4
7H2O, 9.1 μM MnCl2
4H2O, 0.52 μM
important in dry matter production through photosynthesis
(NH4)6Mo7O24
4H2O, 18 μM H3BO3, 0.15 μM ZnSO4
7H2O,
[7]. It is also an important constituent of many important
0.16 μM CuSO4
5H2O, 36 μM FeCl3
6H2O. The pH value of
compounds, including amino acids, proteins (enzymes),
the solution was adjusted to 5.5 using 1 M HCl or NaOH
nucleic acids, chlorophyll and several plant hormones.
solution as required [17]. Half concentration of the nutrient
NUE is considered as the function of N in carbohydrate
solution was applied for the first 3 days and then changed to
production, which is closely related to the C/N balance. For
full nutrient solution. At the emergence of the fourth leaf, the
plants, N and C metabolism is tightly linked in most
seedlings were transferred into either a nutrient solution
biochemical pathways, which involve in carbon fixation,
without N supply as stress treatment or a nutrient solution
nitrogen transfer and utilization etc. Although roots play a
with the normal N concentration as control. Nutrient
dominant role of nitrogen uptake, leaf is the major organ for
solutions were renewed every four days. The upper expanded
carbon and nitrogen metabolism. N drives plant dry matter
leaves were harvested after 12 hours, 3 days, and 7 days after
production through the control of both leaf area index (LAI)
the treatment, and kept frozen in liquid nitrogen and kept at -
and leaf photosynthesis [8]. Moreover, the photosynthetic
80 ℃.
NUE (PNUE), which is dependent on the level of CO2
saturation of Rubisco, is another important factor to consider
2.2 Sample preparation
when NUE is compared among different genotypes. At low N
level, greater PNUE and NUE were found in C3 plants Leaf proteins were extracted by phenol extraction coupled
relative to C4 plants, whereas at high N level, the opposite is with ammonium acetate precipitation [18]. Three separate
true [9]. Consequently, identification of the regulatory extractions were conducted from three leaf samples of each
elements controlling the balance between N available to treatment. Briefly, 1) 0.4 g frozen plant tissue with 30 mg
maintain photosynthesis and the reallocation of the PVPP was grinded into a fine powder using cold pestle and
remobilized N to sink organs such as developing young leaves mortar. 2) Suspend the powder in cold phenol extraction
is of major importance, particularly when N supply is buffer (0.7 M sucrose; 0.1 M KCl; 0.5 MTris-HCl, pH 7.5 and
restricted. Therefore, the complex regulators of N related to 50 mM EDTA, 1% w/v DTT, pH 7.5; complete protease
primary CO2 assimilation, the photo-respiratory processes, inhibitor cocktail (Roche Applied Science)), then add an
and as storage pool need further investigation for optimizing equal volume of phenol saturated with Tris-HCl, pH 7.5, and
NUE under low N level [10-12]. In addition, the recent after 30 min shake the mixture at 4℃. 3) Centrifuge at 5,000 g
finding that synthesis, turnover, and degradation of Rubisco for 30 min at 4℃, then collect the upper phenolic phase;
are subjected to a complex interplay of regulations renews the discard the lower aqueous phase. 4) Add extraction buffer to
concept of the importance of N use and recycling by the the collected phenolic phase; repeat steps 5-6 and then repeat
plants [13]. Attempts have also been made to identify some of Step 5 again. 5) Add 5 volumes of cold 0.1 M ammonium
the components responsible for the physiological control of acetate in methanol to the collected phenol phase; stored at –
the ‘stay-green’ phenotype particularly in relation to NUE. 20℃ overnight. 6) Centrifuge the sample for 30 min at 5000 g
For example, in both sorghum and maize, delayed leaf at 4℃ and carefully remove the supernatant with a pipette
senescence allowed photosynthetic activity to be prolonged, and discard. 7) Add 2 volumes (based on the volume of the
which had a positive effect on N uptake capacity of the plants last collected phenolic phase) of ice-cold methanol to wash

78- 87: 79
 JIOMICS | VOL 1 | ISSUE 1 | FEBRUARY 2011

the pellet, centrifuge the sample for 10 min at 5,000 g at 4℃; 2.4 In-gel digestion and MALDI-TOF MS analysis
repeat step 7 two more times to remove ammonium acetate
Spot detection was realized without spot editing. The spots
and phenol, lipids and pigments and repeat step 9 twice again
were quantified using the % volume criterion. Only those
using acetone instead of methanol to replace. 8) Dry the pellet
with significant and reproducible changes were considered to
gently in a fume hood, and store the clear supernatants in
be differentially accumulated proteins. Protein spots were
aliquots at -80℃ until analyzed. The protein concentrations
excised from the Silver-stained gels and transferred into 0.2
were measured by a Bradford assay using bovine serum al-
mL Eppendorf tubes. Each spot was washed twice in milli-Q
bumin as standard (Bio-Rad, Hercules, CA, USA).
water (Millipore), destained by washing with 50% MeOH/
50mM NH4HCO3 for 30 min. The gels were then washed
2.3 Two-dimensional electrophoresis twice in milli-Q water, dehydrated by addition of ACN (ace-
tonitrile, Fisher A/0626/17), and dried in a SpeedVac (Ther-
Two-DE was performed according to the manual obtained mo Savant, Holbrook, NY, USA) for 30 min. Subsequently,
from GE Healthcare Life Sciences (Little Chalfont, United the gel in each tube was rehydrated in 3 μl of proteomics
Kingdom). Extracellular protein preparation containing 150 grade trypsin (Sigma) solution (20 ng/mL 40 mM NH4HCO3
μg protein was separated by 2-DE using 24 cm immobilized in 9% ACN) and incubated at 37℃ for 16 h. Peptides were
pH gradient strips pH4-7 (GE Healthcare, Milwaukee, WI, extracted twice by adding 40 μl of solution containing 50%
USA). Briefly, sample was diluted with rehydration solution can and 5% TFA (trifluoroacetic acid, GE HealthCare). The
(8 M Urea, 2 M Thiourea, 4% w/v CHAPS, 20 mM w/v DTT, extracted solutions were concentrated to 5-10 μl in a lyophi-
0.5% v/v IPG buffer pH 4–7, 0.002% w/v bromophenol blue) lizer (Virtis, Gardiner, NY, USA). Peptide mixtures were
to 0.5-1 mg protein per 100 μl. Immobiline DryStrip gels (pH mixed with an equal volume of 10 mg/mL CHCA (Sigma)
4-7, 24 cm; GE Healthcare Life Sciences) were then rehydrat- saturated with 50% ACN in 0.1% TFA (Sigma) and analyzed
ed with 450 mL of mixture solutions in 17 cm strip holders with a Voyager-DE STR MALDI-TOF-TOF mass spectrome-
and electrofocused with the GE Healthcare Life Sciences ter (ABI4700 System, USA) using a delayed ion extraction
IPGphor. Initially, all protein extracts were subjected to 2- and ion mirror reflector (Applied Biosystems, Foster city, CA,
DE. Among the three biological replicates of each leaf- USA). MS analysis was conducted with a MALDI-TOF/TOF
treatment sample, the one with best 2-DE quality were cho- mass spectrometer 4700 Proteomics Analyzer (Applied Bio-
sen to run two times more 2-DE. About 100 mg of protein systems, Framingham, MA, USA). Data were analyzed using
were loaded using in-gel rehydration. The focusing protocol GPS Explorer software (Applied Biosystem) and MASCOT
was as follows: 50 mA per strip at 20 ℃; (i) rehydration with software (Matrix Science, London, UK). Parameters were set
30 V for 12 h;(ii) 500 V for 1 h (step and hold);(iii) 1000 V for to Variable Modification - Oxidation, 1 Allowed Missed
1 h (step and hold); and (iv) 8000 V for 10 h (step and hold) Cleavage. NCBInr and Oryza sativa (rice) was selected as the
was applied until the total Vh reached 100 kVh. After IEF, the database and taxonomy, respectively.
strips were equilibrated twice with gentle shaking for 15 min
in SDS equilibration buffer. 3. Results and Discussion
The first step was performed in a equilibration solution
Physiological response to nitrogen stress
containing 6 M urea, 30% w/v glycerol, 2% w/v SDS, 1% w/v
DTT, 50 mM Tris-HCl buffer, pH 8.8 and 0.002% w/v bro- Four-leaf age seedlings were exposed to N-free nutrient so-
mophenol blue. The second step was performed in a solution lution and sampled at different times. Obvious nitrogen stress
modified by the replacement of DTT with 2.5% w/v iodoa- symptoms were observed in the seedlings, such as yellow
cetamide. When the equilibration was finished, the strips leaves and impaired tillers. Highly significant difference in
were loaded onto vertical SDS PAGE (12.5% T constant). The nitrogen content and accumulation were also found between
second dimension SDS electrophoresis was run using an Et- the N stress treatment and the control (Table 1). In normal
tan DALTsix electrophoresis Unit (Amersham Biosciences). condition with sufficient N supply, cultivar Yongyou 6 had
A denaturing solution (0.5% Agarose in running buffer) was higher dry weight than Chunyou 58, which was consistent
loaded onto the gel strips and electrophoresis was performed with the difference in nitrogen accumulation between the two
in a Laemmli running buffer (25 mM Tris–HCl pH 8.3, 192 cultivars. However, when the seedlings were exposed to N
mM glycine, 0.1% SDS). The gels were run at 2-2.5 W per gel stress, Yongyou 6 showed higher loss of dry weight than
for the first 40 min and followed by 17 W per gel for 6 h until Chunyou 58. The two cultivars both showed significant de-
the dye front reached the bottom of the gel. For quantitative cline of nitrogen content and accumulation under N stress
analysis of protein abundance profiles, gels were stained by relative to the normal condition.
silver-staining according to the manufacturer’s instructions
2-DE analysis of leaf proteins in nitrogen stressed rice
(GE Healthcare, Milwaukee, WI, USA). The stained gels were
scanned in an ImageScanner (PowerLook1100 scanner, Total proteins in the fully-expanded leaves were extracted
UMAX) and were analyzed with ImageMaster 2D Elite soft- and separated by 2-DE using pH 4–7 IPG strips in IEF. More
ware. The three technical replicates of each biological sample than 1,000 protein spots were reproducibly detected on gels
were pooled and averaged. by ImageMaster 2D Elite software. Spots with biological

78-87: 80
 Chen Song et al., 2010 | Journal of Integrated Omics

Table 1. Shoot dry weigh and N content and accumulation of the rice cultivars under the different N treatments.
Shoot dry weight (g/pot) N content (%) Shoot N accumulation(mg/pot)
Cultivar Treatment
12h 3d 7d 12h 3d 7d 12h 3d 7d
Chunyou 58 0-N 0.14a 0.21a 0.42b 6.24a 4.22b 2.04b 8.7a 8.9a 8.6b
Control 0.15a 0.22a 0.53a 6.34a 5.37a 4.54a 9.2a 11.8a 24.0a
Yongyou 6 0-N 0.28a 0.44b 0.58b 6.46a 4.21b 3.27b 18.1a 18.5b 18.9b
Control 0.30a 0.65a 0.82a 6.37a 5.14a 4.00a 19.1a 33.4a 32.8a

significance (ratio > 1.3) between the two treatments are trol samples, in different stages are shown in Figure 3. Quali-
showed in Figure 1. In order to investigate changes in protein tative changes of spots have been found. For example, Y-D6
accumulation profiles between the control and N-stressed was visible in all stages of the N stress treatment but invisible
rice plants, the ratio of differentially accumulated proteins in control samples, suggesting that it was induced under N
between N stress and the normal treatments was calculated, stress treatment. Some differentially accumulated proteins
and the proteins with the ratio of over 1.3 were further exam- showed quantitative changes in a time-dependent manner.
ined (Fig. 2). Apparently, there were more proteins, which For instance, C-U5, Y-U5, Y-U4 and Y-U16 showed smaller
showed significant and reproducible changes in Yongyou 6 difference between the treated and control samples in early
than in Chunyou 58. In addition, the two cultivars differed stages of N stress, such as at 12 h. Their abundance ratios
greatly in the number of differential proteins (up or down were greater at 3 d or 7 d (Fig. 3), indicating that the synthe-
regulation) over the time of treatment. Chunyou 58 reached ses of the proteins in the treated sample were enhanced. In
the maximum differential proteins in 3 d after the treatment addition, spot Y-U4 was observed with a dramatic increase in
of N stress, while Yongyou 6 did not show the obvious differ- the abundance in the treated sample at 3 d, while spot Y-D7
ence over the time of treatment. decreased dramatically and almost disappeared in the treated
There were 31 protein spots in the two cultivars that sample at 7 d.
showed reproducible changes during the treatment, and were
selected for MALDI-TOF MS analysis. Among them, 2 and N stress responsive proteins identified by MS
11 were down-regulated spots for Chunyou 58 (C-D1, C-D2) A total of 37 differentially accumulated protein spots were
(Fig. 2A) and Yongyou 6 (Y-D1-Y-D10) (Fig. 2B), respective- analyzed and identified by MALDI-TOF/ TOF MS with high
ly; and 8 and 16 were up-regulated spots for Chunyou 58 (C- probability (Table 2). “Spots view” of 15 protein spots of
U1–C-U6) (Fig. 2A) and Yongyou 6 (Y-U1-Y-U11) (Fig. 2B), time-dependent changes was shown in Figure 4 as examples.
respectively. The abundance ratios, i.e. the percentage vol- Four identified proteins were found in both varieties in all
umes in treated samples over the percentage volumes in con- times during the stress treatment (Table 2). Spots C-U1 and
Y-U6 were identified as the same protein, ribulose-1, 5-
bisphosphate carboxylase/oxygenase activase. However, they
were located at different positions on the gels, with different
100 Mr and pI (Fig. 2 A and B), indicating that they might be
90 isoforms of ribulose-1, 5-bisphosphate carboxylase/ oxygen-
80 ase activase. It can be assumed that the enzyme is up-
70 regulated under stress since its expression is enhanced with
decreased RuBisCo abundance which will reduce photosyn-
Spots No.

60
50 thesis. Spots C-D1 and Y-D10 were identified as rubisco large
40 subunit with similar Mr and pI. Spots C-U8 and Y-U15 were
30
identified as H protein subunit of glycine decarboxylase 3'-
20
partial. Spots C-D2 and Y-D5 were identified as putative
10
transposase.
Five proteins were involved in photosynthetic metabolism,
0
12h 3d 7d 12h 3d 7d
Chunyou 58 Yongyou 6 including ribulose-1,5-bisphosphate carboxylase/oxygenase
down-regulated up-regulated activase (C-U1 and Y-U6), type II light-harvesting chloro-
phyll a/b-binding protein (C-U4), carbonic anhydrases (C-
Figure 1. Number of spots whose abundance ratio of the differentially U5), rubisco large subunit (C-D1 and Y-D10), 23kDa poly-
accumulated proteins were over 1.3 after N stress treatment. The peptide of photosystem II (Y-U9), dTDP-glucose 4-6-
percentage volume was considered as the abundance of each spot. The
dehydratase-like protein (C-U7) and H protein subunit of
abundance ratio of each spot was calculated by percentage volume in
treated samples/ percentage volume in control samples as up- glycine decarboxylase 3'-partia (C-U8 and Y-U15). Six pro-
regulated spots (□), while the ones was calculated by percentage vol- teins were the stressor response to N stress i.e. DegP2(Y-D6),
ume in control sample/ percentage volume in treated sample as down- harpin binding proteins(Y-D11), Heat shock-related proteins
regulated spots (■).

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 JIOMICS | VOL 1 | ISSUE 1 | FEBRUARY 2011

(Y-U2), glutathione S-transferase GSTF14(Y-U4), Fibrillin- RCA (C-U1 and Y-U6) was strongly up-regulated by N stress
like protein(Y-U6) in Yongyou 6, and Glyceraldehyde-3- in most expanded leaves of both rice cultivars (Table 2). N
phosphate dehydrogenase (C-U3) in Chunyou 58. stress can reduce the photosynthesis either by impairing acti-
vation state of RuBisCo, which is often attributed to the loss
of RCA activity or by reducing the abundance of RuBisCo
proteins. The reduced RuBisCo was proved by a dramatic
down- regulation of RuBisCo large subunit (RLS) (C-D1 and
Y-D10) in N stress samples (Table 2). These results suggest
that the RCA was over-expressed as a feedback mechanism
for decrease of RCA activity or RuBisCo content in both rice
cultivars. However, different additional protective strategy
was found between Chunyou 58 and Yongyou 6. Type II
light-harvesting chlorophyll a/b-binding protein (LHCP) (C-
U4) and carbonic anhydrases (CA) (C-U5) were found in the
N treated Chunyou 58 and 23 kDa polypeptide of photosys-
tem II (PsbP-PSII) (Y-U9) was strongly up-regulated in treat-
ed samples of Yongyou 6. LHCP is an approximately 25,000-
D thylakoid membrane protein, which captures and transmits
the energy from the sunlight into biomass [22]. The increased
LHCP was found under N stress in this experiment. With the
reduced photosynthetic efficiency resulting from N stress, the
up-regulated LHCP in N stressed sample might be a compen-
sation for the low photosynthetic efficiency in order to cap-
ture and transform more energy to produce the carbohydrate
for plant growth. AC forms a family of enzymes, which cata-
lyze rapid conversion of carbon dioxide to bicarbonate and
protons. In plants, AC may increase CO2 concentration with-
in chloroplasts in order to enhance carboxylation rate of Ru-
BisCO [23]. Rengel (1995) found that higher photosynthetic
rate under Zn deficiency was related to higher CO2 availabil-
ity due to higher CA activity in some wheat genotypes [24]. It
may be assumed that the less reduction of biomass in N-
stressed Chunyou 58 comparing to Yongyou 6 might be at-
tributed to higher CA activity. PsbP-PSII is one of subunits of
Figure 2. Representative 2-DE maps of rice leaf proteins. Differen- the oxygen-evolving complex (OEC) of PSII. The 23-kDa
tially accumulated protein spots (Ratio>1.3) which appeared in all subunit allows PSII to evolve oxygen under both Ca2+ and Cl2-
time are selected for MALDI-TOF MS analysis and indicated by
label in the map. Two and twelve down-regulated spots for Chun- limiting conditions, suggesting that it acts as a concentrator
you58 (C-D1, C-D2) and Yongyou6 (Y-D1-Y-D10) are indicated by of these ions [10]. The dramatic up-regulation of PsbP-PSII
red marker. Ten and twenty four up-regulated spots for Chunyou58 was found in N-stressed samples of Yongyou 6, which may
(C-U1–C-U6) and Yongyou6 (Y-U1-Y-U11) are indicated on the act as a compensation for the decreased photosynthesis in-
map by green marker duced by N stress. The difference in regulated metabolisms
between the two rice cultivars may be one of the major causes
that lead to more biomass reduction for Yongyou 6 than for
Photosynthesis and photorespiration Chunyou 58.
Rubisco activase (RCA, spots C-U1 and Y-U6) is the key A marked increase in H protein subunit of glycine decar-
enzyme for the rapid formation of the critical carbamate in boxylase 3'-partial was found in N-stressed rice plants (H-
the active site of RuBisCo. It is modulated either by reaction GDC) (Chunyou 58, C-U8; Yongyou 6, Y-U15). H-protein is
with CO2 and Mg2+ to carbanylate a lysine residue in the cata- the lipoyl-protein component of the glycine decarboxylase
lytic site, or by the binding of inhibitors within the catalytic complex (GDC), which oxidizes glycine to support pho-
site [19]. A variable number of RCA genes have been report- torespiration [25]. GDC consists of four proteins, including
ed in different plant species. In monocotyledonous plants, H-protein that helps to receive the released CO2. It was re-
two genes have been detected [20]. Two mature RCA poly- ported that the expression of H-protein gene in leaf was
peptides, with molecular mass ranging between 41 kDa and stimulated by light [26]. In this experiment, more H-protein
47 kDa are present in most plants [21]. Increased 43 Kda/41 was found in N-stressed samples, indicating that photorespi-
KDa was found in low light intensity whereas the decreased ration might be enhanced when the plants are exposed to N
one was found in water-stress [20]. Our result showed that stress.

78-87: 82
 Chen Song et al., 2010 | Journal of Integrated Omics

10
9
8
7
6
12H

5
4
3
2
1
0
C-U1

C-U2

C-U3

C-U4

C-U5

C-U6

C-U7

C-U8

C-D1

C-D2

Y-U1

Y-U2

Y-U3

Y-U4

Y-U5

Y-U6

Y-U7

Y-U8

Y-U9

Y-U10

Y-U11

Y-U12

Y-U13

Y-U14

Y-U15

Y-U16

Y-D1

Y-D2

Y-D3

Y-D4

Y-D5

Y-D6

Y-D7

Y-D8

Y-D9

Y-D10

Y-D11
10
9
8
7
6
3D

5
4
3
2
1
0
C-U1

C-U2

C-U3

C-U4

C-U5

C-U6

C-U7

C-U8

C-D1

C-D2

Y-U1

Y-U2

Y-U3

Y-U4

Y-U5

Y-U6

Y-U7

Y-U8

Y-U9

Y-U10

Y-U11

Y-U12

Y-U13

Y-U14

Y-U15

Y-U16

Y-D1

Y-D2

Y-D3

Y-D4

Y-D5

Y-D6

Y-D7

Y-D8

Y-D9

Y-D10

Y-D11
10
9
8
7
7D

6
5
4
3
2
1
0
C-U1

C-U2

C-U3

C-U4

C-U5

C-U6

C-U7

C-U8

C-D1

C-D2

Y-U1

Y-U2

Y-U3

Y-U4

Y-U5

Y-U6

Y-U7

Y-U8

Y-U9

Y-U10

Y-U11

Y-U12

Y-U13

Y-U14

Y-U15

Y-U16

Y-D1

Y-D2

Y-D3

Y-D4

Y-D5

Y-D6

Y-D7

Y-D8

Y-D9

Y-D10

Y-D11
Figure 3. Abundance ratios of the differentially accumulated proteins after 12 h, 3 d and 7 d of N stress treatment. The percentage volume was
considered as the abundance of each spot. The abundance ratio of each spot was calculated by percentage volume in treated samples / percent-
age volume in control samples. The up-regulated proteins include C-U1 to C-U6 (A, D, G), and Y-U1 to Y-U11 (B, E, H); the down-regulated
proteins are C-D1 to C-D2 (A, D, G), and Y-D1 to Y-D10 (C, F, I). Spots with * means either the abundance ratio of differentially accumulated
protein was over 10,000 or the protein was absent in the treated or control sample.

N stress- induced proteins abiotic stress conditions. Yang (2006) reported that ABA
Many N stress-related proteins were identified in this treatment increased fibrillin accumulation, thus enhancing
study. In general, Yongyou 6 was more sensitive to N stress the tolerance of photosystem II to light stress-triggered
than Chunyou 58. There were 5 N stress-induced proteins in photo-inhibition in Arabidopsis [31]. In this study, fibrillin–
Yongyou 6, including two down-regulated ones: DegP2(Y- like protein were increased in N stressed plants of Yongyou 6,
D6) and harpin binding proteins(Y-D11), and three up- indicating that as a feedback mechanism for N deficiency, the
regulated ones: heat shock-related proteins(Y-U2), efficiency of photosynthesis was improved by inducing more
glutathione S-transferase GSTF14(Y-U4), and Fibrillin-like fibrillin proteins to protect the photosystem II. The harpin
protein(Y-U6). On the contrast, only one stress-related protein group, which is first found and identified by Wei et
protein was found in Chunyou 58, i.e. glyceraldehyde-3- al. (1992) in Erwinia amylovora [32], may elicit multiple plant
phosphate dehydrogenase (C-U3). DegP2 is a member of a responses, causing beneficial effects on crop improvement
large family of related Deg/Htr serine proteases found in [33]. The current results indicated the possible defense
most organisms, including bacteria [27], humans [28] and mechanism of rice plants in response to N stress by inducing
plants [29]. Bacterial DegP/HtrA protease has been harpin proteins, thus enhancing photosynthesis and nitrogen
implicated in tolerance to various stresses, including uptake. Heat shock-related proteins (HSP) are a class of
oxidation, salinity, pH and heat [28]. The current results functionally related proteins, whose expression is increased
showed that this protein was reduced under N stress (Table when cells are exposed to elevated temperatures or other
2), suggesting that the effect of DegP/HtrA protease on stresses [34]. The function of glutathione S-transferase
enhancing plant tolerance under the stress condition relies on GSTF14 protects cells from injury by a wide range of stresses
the nitrogen nutrition. Fibrillin-like protein is a glycoprotein, in plants [35]. A significant up-regulation of heat shock
which is essential for the formation of elastic fibers [30]. As related proteins and GSTF14 was found in N stressed
lipid-binding proteins of plastids, fibrillin are induced under Yongyou 6 (Table 2).

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 Chen Song et al., 2010 | Journal of Integrated Omics

Table 2. Differentially accumulated proteins identified by MS.

MS Protein Ratio
Rank Protein Name Accession No. Protein MW Protein PI
SPOT Score 12h 3d 7d
ribulose-1,5-bisphosphate
C-U1 carboxylase/oxygenase activase [Oryza gi|115486823 218 48127.9 5.85 1.40 1.52 2.55
sativa]
putative gypsy-type retrotransposon [Oryza
C-U2 gi|18071410 39 165471.2 9.53 1.36 2.36 1.40
sativa (japonica cultivar-group)]
glyceraldehyde-3-phosphate dehydrogenase
C-U3 gi|2331137 173 36707.0 9.55 1.74 1.66 1.75
[Oryza sativa]
type II light-harvesting chlorophyll a/b-
C-U4 binding protein [Oryza sativa Japonica gi|218174 54 28566.4 5.61 1.54 1.88 1.68
Group]
C-U5 carbonic anhydrase 3 [Oryza sativa] gi|5917783 134 29585.0 8.41 1.92 1.79 1.34
putative metalloproteinase [Oryza sativa
C-U6 gi|14165330 42 32250.9 6.36 1.75 1.38 1.72
(japonica cultivar-group)]
dTDP-glucose 4-6-dehydratase-like protein
C-U7 gi|18201659 40 26091.4 7.1 1.60 1.08 1.03
[Oryza sativa]
H protein subunit of glycine decarboxylase
C-U8 3'-partial [Oryza sativa (japonica cultivar- gi|10257441 67 7156.6 8.98 22.25 11.89 1.50
group)]
C-D1 rubisco large subunit gi|476752 79 45614.8 8.43 1.46 1.91 1.38
putative transposase [Oryza sativa
C-D2 gi|34015353 49 6.56 1.42 1.39 2.13
(japonica cultivar-group)]
ribulose-1,5-bisphosphate carboxylase
Y-U1 gi|13569643 273 21737.8 4.78 1.49 2.04 1.35
activase [Oryza sativa]
heat shock-related protein [Oryza sativa
Y-U2 gi|29367425 404 45014.5 5.02 1.69 1.48 1.52
(japonica cultivar-group)]
Os06g0176700 [Oryza sativa (japonica
Y-U3 gi|115466716 203 40022.5 5.16 1000000 1000000 1000000
cultivar-group)]
glutathione S-transferase GSTF14 [Oryza
Y-U4 gi|46276327 514 30766.5 7.77 4.52 6.38 2.00
sativa(japonica cultivar-group)]
putative protein kinase ADK1 [Oryza sativa
Y-U5 gi|52077492 41 26201.6 9.55 1.49538 1.37301 1.34866
Japonica Group]
fibrillin-like protein [Oryza sativa (japonica
Y-U6 gi|29367475 510 33923.7 5.04 1.51 1.72 2.47
cultivar-group)]
Putative wall-associated protein kinase
Y-U7 gi|14029040 40 53505.1 6.01 1.8159 1.55712 1.55934
[Oryza sativa (japonica cultivar-group)]
oryzain gamma precursor [Oryza sativa
Y-U8 gi|218185 51 39692.5 7.07 1.79 1.48 2.01
Japonica Group]
23kDa polypeptide of photosystem II [Ory-
Y-U9 gi|2570499 271 27173.9 9.06 1.82 2.74 t
za sativa]
Os08g0455800 [Oryza sativa (japonica
Y-U10 gi|115476734 240 21683.7 5.15 1.56 1.79 1.34
cultivar-group)]
hypothetical protein [Oryza sativa Japonica
Y-U11 gi|42407348 54 7766.8 10.96 1.44 2.02 2.00
Group]
Os08g0478200 [Oryza sativa (japonica
Y-U12 gi|115476908 371 19712.9 5.19 1.70 1.98 1.33
cultivar-group)]
Os10g0471300 [Oryza sativa (japonica
Y-U13 gi|115482468 161 18653.4 5.61 1.57 1.38 1.61
cultivar-group)]
hypothetical protein LOC_Os03g43310
Y-U14 gi|53370666 39 20432.4 10.86 1.45 1.87 1.94
[Oryza sativa (japonica cultivar-group)]
H protein subunit of glycine decarboxylase
Y-U15 gi|10257441 60 7156.6 8.98 1.37 4.58 1.88
3'-partial [Oryza sativa (japonica cultivar-

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 Chen Song et al., 2010 | Journal of Integrated Omics

group)]
Os06g0705100 [Oryza sativa (japonica
Y-U16 gi|115469830 298 24997.9 8.74 1000000 2.06 1.42
cultivar-group)]
putative chloroplast inner envelope protein
Y-D1 gi|10140720 812 108209.8 5.37 2.38 1.43 1.35
[Oryza sativa (japonica cultivar-group)]
putative SecA [Oryza sativa Japonica
Y-D2 gi|52075758 410 114899.0 5.78 2.47 1.49 1.54
Group]
Os03g0401300 [Oryza sativa (japonica
Y-D3 gi|115453437 72 93362.3 5.94 1.79 2.84 1.77
cultivar-group)]
Os02g0285800 [Oryza sativa (japonica
Y-D4 gi|115445587 360 74035.2 7.08 2.16 1.76 1.32
cultivar-group)]
putative transposase [Oryza sativa
Y-D5 gi|34015353 45 66527.4 6.56 1.51 1.94 1.37
(japonica cultivar-group)]
putative DegP2 protease [Oryza sativa
Y-D6 gi|51038169 37 65771.4 5.73 1.94 1.93 1.61
(japonica cultivar-group)]
Os06g0562600 [Oryza sativa (japonica
Y-D7 gi|115468554 268 59720.8 5.71 1.39 2.11 1000000
cultivar-group)]
eukaryotic initiation factor 4A [Oryza
Y-D8 gi|303844 207 47393.1 5.43 1.91 4.52 2.08
sativa Japonica Group]
hypothetical protein [Oryza sativa
Y-D9 gi|13236651 142 45168.2 5.27 2.90 1.43 1.35
(japonica cultivar-group)]
Y-D10 rubisco large subunit gi|476752 157 45614.8 8.43 1.31 1.70 1.63
harpin binding protein 1 [Oryza sativa
Y-D11 gi|38679325 78 28457.0 8.92 1.43 1.65 2.30
(indica cultivar-group)]

Glyceraldehyde 3-phosphate dehydrogenase (C-U3) Resolving the cause and effect relationship in plants subject
(GAPDH, EC 1.2.1.1) was up-regulated in N stressed Chun- to a nitrogen limitation is difficult because nitrogen stress
you 58. GAPDH plays important roles in various cellular initiates a series of complex physiological responses varied
processes. It is a central glycolytic protein with pivotal role in over the time and the stress degree. Many of the physiological
energy production, and is also an abundant and crucial en- metabolisms were directly or indirectly involved with the
zyme in glycolysis and gluconeogenesis in most plants [36]. stress effect. For example, the net photosynthesis rate in most
Moreover, GAPDH is a protein with multi-function, involv- extended leaves acclimate to N stress with highly up-
ing in the translational control of gene expression [37]. For regulated RCA in two cultivars as well as LHCP / CA and
the last decade, there were many reports that GAPDH works PsbP-PSII in Chunyou 58 and Yongyou 6, respectively. The
as a stressor associated with oxidative stress in cells that un- similar results were also reported by De Groot et al. (2003)
dergo apoptosis [38]. It may be suggested that over- that photosynthetic light-harvesting and electron-transport
expression of GAPDH in N stressed Chunyou 58 acts not activity acclimate to nitrogen stress so that the internal rela-
only as an oxidative signal to N deficiency, but also an energy tionships between electron transport by photosystems I and
production through its glycolytic function. II do not change; the linear relationship between PSII, and
PSI was not affected [40]. In protein profile, RCA could play
Membrane transporter
a role of a chaperone, either in helping target the thylakoid
Putative chloroplast inner envelop protein (Y-D1) and Se- membrane or in protection of translation machinery related
cA protein (Y-D2) were down-regulated in N stressed to thylakoid against abiotic stress [41]. Thus the up-regulated
Yongyou 6. Chloroplast inner envelop is highly specialized RCA could protect the photosynthesis machinery under N
with transport proteins, which involved in the movement of stress. Despite of the similar protection from the RCA, differ-
ions, small molecules, or macromolecules. SecA proteins were ent protective strategies were drown out from cultivars differ-
found in the thylakoid membrane as well as the cytoplasmic ing in NUE. LHCP / CA were up regulated in Chunyou 58
membrane, and they involved in protein translocation across resulting in the slightly biomass decrease subjected to a nitro-
the thylakoid membrane [39]. The current results showed gen limitation. Only PsbP-PSII was consistently up regulated
that the translocation across the chloroplast or thylakoid for carbon production metabolism over the stress period in
membrane was inhibited in Yongyu 6 under N stress. Yongyou 6. This may be considered as the major cause for the
biomass differentiation between the cultivars.
Different protective strategies under N stress between two cul-
tivars

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 Chen Song et al., 2010 | Journal of Integrated Omics

12H 3D 7D 12H 3D 7D 12H 3D 7D

Stressed
C-D1 Y-U1 Y-U15
CK

Stressed
C-U1 Y-U2 Y-D1
CK

Stressed
C-U3 Y-U4 Y-D2
CK

Stressed
C-U4 Y-U6 Y-D6
CK

Stressed
C-U5 Y-U8
CK Y-D10

Stressed
C-U8 Y-U9
CK
Y-D11

Figure 4. Time-dependent changes of the 15 of 48 differentially accumulated proteins. Proteins in leaves were extracted from both control and
stressed samples after 12h, 3d and 7d treatment and separated by 2-DE.

4. Concluding remarks

A systematic proteomic analysis of the leaf proteins in N thione S-transferase GSTF14, fibrillin-like protein and glycer-
stressed rice was carried out in this study. Of the six protein aldehyde-3-phosphate dehydrogenase. Two proteins, i.e.
spots involved in photosynthesis and photorespiration, three putative chloroplast inner envelop protein and SecA protein
were identified in the two rice cultivars at all times during the are related to membrane translocation, Moreover, two novel
treatment. These are: Rubisco activase, RuBisCo large subu- proteins, harpin binding protein and oryzains gamma pre-
nit, and H protein subunit of glycine decarboxylase 3'-partial. cursor, were found in the rice leaves under N stress. These
Six stress-induced proteins were identified, including DegP2, results provide useful information for further investigation of
harpin binding proteins, heat shock-related proteins, gluta- their functions using genetic or genomic approaches.

78-87: 86
 Chen Song et al., 2010 | Journal of Integrated Omics

19. Portis, A. R., Photosynthesis Research 2003, 75, 11-27.

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Younan Ouyang of Chinese Rice Research Institute for his 22. Keegstra, K., Olsen, L. J. and Theg, S. M., Annual Review of
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