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First published online June 13, 2008; 10.1104/pp.108.116905 Plant Physiology 147:1830-1844 (2008) © 2008 American Society of Plant Biologists A Proteomic Profiling Approach to Reveal a Novel Role of Brassica napus Drought 22 kD/Water-Soluble Chlorophyll-Binding Protein in Young Leaves during Nitrogen Remobilization Induced by Stressful Conditions1UMR INRA/UCBN 950 Ecophysiologie Végétale, Agronomie et Nutritions NCS, IFR 146 ICORE, Institut de Biologie Fondamentale et Appliquée, Université de Caen Basse-Normandie, F–14032 Caen, France (M.D., L.D., P.E., J.B., A.O., J.-C.A.); UMR INRA/Agrocampus Rennes/Université de Rennes 1 118 Amélioration des Plantes et Biotechnologies, Campus de Beaulieu, 35042 Rennes cedex, France (F.L.C.); and Department of Biomolecular Science, Toho University, Funabashi, Chiba 274–8510, Japan (H.S.)
Despite its water-soluble chlorophyll-binding protein (WSCP) function, the putative trypsin inhibitor (TI) activity of the Brassica napus drought 22 kD (BnD22) protein and its physiological function in young leaves during leaf nitrogen (N) remobilization promoted by stressful conditions remains an enigma. Therefore, our objectives were to determine (1) if BnD22 is related to the 19-kD TI previously detected in B. napus young leaves, and (2) if the levels of BnD22 transcripts, BnD22 protein, and TI activity in young leaves are associated with plant responses to stress conditions (N starvation and methyl jasmonate [MeJA] treatments) that are able to modulate leaf senescence. Compared to control, N starvation delayed initiation of senescence and induced 19-kD TI activity in the young leaves. After 3 d with MeJA, the 19-kD TI activity was 7-fold higher than the control. Using two-dimensional electrophoresis gel, TI activity, and electrospray ionization liquid chromatography tandem mass spectrometry analysis, it was demonstrated that two 19-kD proteins with isoelectric points 5.0 and 5.1 harboring TI activity correspond to BnD22 perfectly. BnD22 gene expression, TI activities, and BnD22 protein presented similar patterns. Using polyclonal anti-WSCP antibodies of Brassica oleracea, six polypeptides separated by two-dimensional electrophoresis were detected in young leaves treated with MeJA. Electrospray ionization liquid chromatography tandem mass spectrometry analysis of six polypeptides confirms their homologies with WSCP. Results suggest that BnD22 possesses dual functions (WSCP and TI) that lead to the protection of younger tissues from adverse conditions by maintaining metabolism (protein integrity and photosynthesis). By sustaining sink growth of stressed plants, BnD22 may contribute to a better utilization of recycling N from sources, a physiological trait that improves N-use efficiency.
To maintain or increase crop yields while benefiting the environment through reductions in nitrogen (N) fertilizer inputs, it will be necessary to improve both N-use efficiency (Good et al., 2004  ) and especially the recycling of endogenous N from vegetative organs to the growing tissues. When the N demand for growing vegetative or reproductive tissues exceeds the mineral N availability, the extent of endogenous N mobilization, particularly from senescing leaves, may lead to higher N-use efficiency (Aerts and Chapin, 2000; Yasumura et al., 2007). Proteolysis is one of the most important processes of N remobilization (Hörtensteiner and Feller, 2002; Zimmermann and Zentgraf, 2005). Moreover, the effective recycling of the N compounds from source leaves to sink growing tissues requires a fine coordination between sink demand and the processes of proteolysis. This is particularly important when plants are confronted by stresses that could lead to strong modification of the source/sink relationships. Therefore, understanding the mechanisms and associated regulation of (1) leaf proteolysis that presides over efficient protein-N remobilization and (2) maintenance of sink strength is a prerequisite for improving N recycling and subsequently N-use efficiency.
Among the factors able to modulate N-use efficiency, the inhibitors of proteases may have a pivotal role in both mechanisms, i.e. rate of protein-N remobilization in source leaves and maintenance of metabolism in young leaves. Indeed, a few studies (Sugawara et al., 2002
Oilseed rape (Brassica napus) is a particularly interesting plant for studying the involvement of protease inhibitors in leaves and their contribution to N-use efficiency. Indeed, this important crop plant requires high amounts of mineral N but is characterized by low N-use efficiency as only 50% of fertilizer N is recovered by the crop at harvest date (Schjoerring et al., 1995
Compared to its WSCP function, the putative protease inhibitor activity of BnD22 remains an enigma. Therefore, assuming that a protease inhibitor may have a central role in the maintenance of metabolism in young leaves, leading to the modulation of N-use efficiency, our objectives are (1) to determine if BnD22 is related to the 19-kD TI previously detected in oilseed rape young leaves, and (2) to verify if the levels of the BnD22 transcript and protein as well as TI activity in young leaves are associated with the plant tolerance response to stressful conditions such as N starvation and methyl jasmonate (MeJA). Proteomic approaches using high-resolution two-dimensional electrophoresis (2-DE) and identification of the protein by electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC MS/MS) revealed that (1) a TI activity could be detected after 2-DE gel separation, (2) TI activity was harbored by BnD22, and (3) BnD22 cross-reacted with the antibodies that recognized a WSCP of Brassica oleracea (Nishio and Satoh, 1997
Effects of N Starvation and MeJA on Oilseed Rape Leaves In mature leaves, N starvation led to precocious senescence symptoms as indicated by visible yellowing (data not shown) and a large decrease in chlorophyll content (Table I ). The young leaves of N-deprived oilseed rape had no visible senescence symptoms (Fig. 1A ). The chlorophyll content in laminae of young leaves remained high throughout the experiment and was not affected by N starvation (Fig. 1B). The decline in the soluble protein concentration in laminae of mature leaves occurred earlier in N-deprived plants than control plants (data not shown). In contrast, the soluble protein concentration in laminae of young leaves decreased between day 21 and day 28 in both treatments (Fig. 1C). To further investigate whether nitrate availability led to a modification of protein patterns in young leaves, SDS-PAGE was performed (Fig. 1D). However, protein at 19 kD that was present at day 14 in both treatments disappeared after 21 d in control plants and 28 d in N-deprived plants.
In response to MeJA treatment, leaf senescence was accelerated in mature leaves in comparison to control plants (Table I). Young leaves of MeJA-treated plants showed yellowing at day 7 when compared to control oilseed rape (Fig. 2A ). There was also a decline in chlorophyll content in young leaves of MeJA-treated plants between day 3 and day 7 (Fig. 2B) and the concentration of soluble proteins at day 3 (–70% as compared to control; Fig. 2C). SDS-PAGE profiles revealed a decrease in both subunits of Rubisco after 3 d of MeJA treatment (Fig. 2D). In comparison to the controls, a protein of 19 kD was specifically expressed in young leaves of plants treated with MeJA since day 1 and was strongly accumulated at days 3 and 7 to reach a maximum of 9% of total soluble proteins (Fig. 2D).
Changes in TI Activities In laminae of young leaves of control plants (Fig. 3, A and C ), 19-kD TI activity was weakly present until day 14 and disappeared at day 21 (Fig. 3B). Nitrate starvation induced TI activity in the laminae of young leaf after 14 and 21 d (Fig. 3, A and B). The laminae of young leaves from plants treated with MeJA had significantly higher TI levels than control plants on all dates of treatment (Fig. 3C). For instance, after 3 d of MeJA treatment, TI activity levels were 7-fold higher than in control plants (Fig. 3D). This elevated TI level was maintained until day 7.
Detection of TI Activity after 2-DE and Protein Identification The TI activity in young leaves treated during 7 d with MeJA was also detected after separation of soluble proteins by 2-DE (Fig. 4A ). A 2-DE gel stained with silver nitrate (Fig. 4B) indicated that TI activity corresponded to two spots with molecular mass of 19 kD and pI of 5.0 and 5.1, respectively. These two spots were analyzed using ESI-LC MS/MS, and the identified peptides A and B (Fig. 4C) revealed 100% homology to the amino acid sequence of BnD22 (accession no. X65637; Fig. 4C).
Identification of BnD22 after Separation of Total Proteins from Laminae by 2-DE The identification of BnD22 in laminae of young leaves of N-deprived and MeJA-treated plants was confirmed by 2-DE analysis of total proteins (Figs. 5 and 6 ). After silver staining of total proteins from control and N-deprived plants during 21 d, approximately 950 individual protein spots could be separated by 2-DE across a pI range of 4 to 7 (Fig. 5, A and B). Among the proteins presenting a different pattern between the control and N-deprived plants, two proteins (spot nos. 1 and 2) were also observed at 19 kD and pI 5.0 and 5.1 in laminae of young leaves submitted to N starvation (Fig. 5B). Partial sequences from these two spots, identified by ESI-LC MS/MS, were identical to the peptides A and B previously identified by ESI-LC MS/MS after separation of soluble proteins by 2-DE (Fig. 4C). Consequently, database searches revealed that both polypeptides in Figure 5 also presented 100% homology to BnD22 (Fig. 4C).
Figure 6 shows representative images of 2-DE gels obtained from total proteins of laminae of young leaves from control or MeJA-treated plants after 7 d of experimentation. The total number of protein spots on 2-DE gels as well as the total protein content (data not shown) declined when plants were treated with MeJA (975 spots in control; Fig. 6A) versus 850 spots in MeJA treatment (Fig. 6B). Two spots with a molecular mass of 19 kD and pI of 5.0 and 5.1 were largely accumulated in the MeJA treatment (Fig. 6B). These two proteins (spot nos. 1 and 2) were analyzed using ESI-LC MS/MS (Fig. 4C). The obtained partial sequences were strictly identical to peptides A and B and consequently showed 100% homology to BnD22 as described above after 2-DE of soluble proteins (Fig. 4C).
BnD22 is also considered to be a class II WSCP (Nishio and Satoh, 1997 Identification by ESI-LC MS/MS revealed that polypeptides of 19 kD and pI 5.0 and 5.1 (spot nos. 1 and 2) were identical to BnD22 (Table II ). Spot number 3 at 23 kD and pI 5.6 matched with WSCP2 of B. oleracea (accession no. AB078330; Table II). Using the Progenesis SameSpots software, image gel analysis revealed that BnD22 was induced 12-fold by MeJA, whereas WSCP2 was increased 4-fold (Table II). Spot numbers 4, 5, and 6 were induced 10-, 4-, and 6-fold, respectively (Table II). After BLASTp analysis (National Center for Biotechnology Information database), spot numbers 4, 5, and 6 at 19 kD and pI 5.7, 5.8, and 6.4, respectively, were identified as heat stress-induced protein (HSIP; accession no. AJ007414) or WSCP1 (accession no. AB012699) of B. oleracea. Multiple alignments of amino acid sequences (Fig. 7 ) allowed determination of the degree of homology between BnD22 and the three other proteins recognized by antibodies against WSCP (Fig. 6). Thus, HSIP and WSCP1 were strictly identical and shared 96% homology to BnD22 (Fig. 7). Consequently, these data suggest that WSCP1 and HSIP were the same protein and were different to BnD22. WSCP2 identified in spot number 3 at 23 kD and pI 5.6 showed only 54% homology to BnD22 and 55% homology to WSCP1.
Figure 7 indicated that BnD22 and WSCP1 presented a similar N-signal peptide (cleaved between amino acid nos. 19 and 20) and C-signal peptide (cleaved between amino acid nos. 196 and 197), while WSCP2 harbored only an N-signal peptide that is cleaved between amino acid numbers 24 and 25. Interestingly, WSCP2 did not possess the specific motif of the Künitz-type protease inhibitor (Fig. 7), while this motif was present in BnD22 and WSCP1. Moreover, searches of predicted sites for phosphorylation of amino acids revealed 10 sites for BnD22 and WSCP1 and 16 sites for WSCP2 (Fig. 7).
Although proteins recognized by anti-WSCP antibodies were induced by MeJA, other proteins were also up-regulated by this hormone (Fig. 6). Among the induced proteins, three polypeptides were highly accumulated (at least 5-fold) and were identified by ESI-LC MS/MS (Table III
). The selected proteins correspond to a putative TI at 21 kD (spot no. 7 induced 11-fold; accession no. U18995), a WSCP2 at 25 kD (spot no. 8 induced 11-fold; accession no. AB078330), and a Jacalin-related lectin family protein at 49 kD (spot no. 9 induced 5-fold; accession no. NM_112518). This last result is in accordance with those reported in recent works using transcriptional approaches (Jiang et al., 2006
To determine whether TI activity was correlated with BnD22 gene expression, the level of BnD22 transcripts was studied by quantitative (Q)-PCR in the laminae of young leaves in the control, N-deprived, or MeJA-treated oilseed rape. Compared to day 14, the BnD22 transcript level in laminae of control plants decreased 5-fold at day 21 and 2-fold at day 28 (Fig. 8A ). Under N-deprived conditions, BnD22 expression increased in young laminae throughout the experiment and was always higher than the control at all time points.
Figure 8B presented the accumulation of BnD22 transcripts in young laminae from control and MeJA-treated plants. In control plants, the transcript level of BnD22 increased during the experiment. Compared to the control plants, the expression of BnD22 in plants treated by MeJA significantly increased at all time points. For instance, between days 0 and 3, the expression of the BnD22 gene increased 27-fold in MeJA-treated plants versus only 8-fold higher in control plants.
The goals of this study were to verify if BnD22 (1) was homologous to the 19-kD protease inhibitor previously detected by Etienne et al. (2007) in young leaves of oilseed rape, and (2) could be involved in N-use efficiency through the maintenance of physiological functions of sink tissues in plants submitted to stressful conditions. N starvation and MeJA treatment were used to induce leaf senescence (Rossato et al., 2002; Gombert et al., 2006) and TI activity (Cipollini and Sipe, 2001; Etienne et al., 2007).
Detection of TI activity after SDS-PAGE revealed that 19-kD TI activity was induced in young leaves of N-deprived and MeJA-treated plants compared to the controls. In young leaves treated with MeJA, TI activity was 7-fold higher than in controls after 3 d of treatment (Fig. 3D). To identify the proteins harboring TI activity, soluble protein extracts of young leaves treated with MeJA were analyzed by 2-DE gels and through detection of TI activity (Fig. 4). Two polypeptides that were common in both gels were identified precisely at a molecular mass of 19 kD and a pI of 5.0 and 5.1, respectively. ESI-LC MS/MS analysis (Table II) revealed that both polypeptides were homologous to BnD22, a protein previously identified in the youngest leaves of oilseed rape subjected to drought or salinity stress (Reviron et al., 1992
In a drought-resistant genotype of wheat (Triticum aestivum), the induction of a protein homologous to BnD22 identified in the thylakoid membrane showed a significant role for BnD22 during the stress response (Guseynova et al., 2006
Abiotic stresses such as drought, heat stress, and N starvation, which led to the accumulation of BnD22, also led to a delay of leaf senescence in young leaves (Reviron et al., 1992
Nishio and Satoh (1997)
Interestingly, BnD22 is a class II WSCP that is able to bind chlorophyll (Chl a/b) and chlorophyll precursors such as chlorophyllides a and b in tetrameric forms (Nishio and Satoh, 1997
Despite N-starvation conditions having reduced the growth at whole-plant level, the young leaves showed a significant growth (+362.6 ± 0.056 mg of dry matter versus +553.1 ± 0.024 mg of dry matter in control between days 14 and 28; data not shown) and a similar chlorophyll content as compared to control (Fig. 1B). These data are in accordance with results recently reported by Etienne et al. (2007)
Using proteomics approaches, this study clearly reveals that the 19-kD TI activity induced in young leaves of oilseed rape in response to stress provoked by N starvation or MeJA is identical to BnD22, a protein that belongs to class II WSCPs. The level of gene expression, protein abundance, and TI activity of BnD22 observed in young leaves present similar patterns and are associated with the maintenance of growth, chlorophyll content, and concentration of soluble proteins. This leads to maintenance of sink strength and may have important consequences for N-use efficiency. The dual WSCP/TI functions of BnD22 suggest that this protein is strongly involved in the maintenance of metabolism (protein integrity and photosynthesis capacity) of young leaves especially when plants are confronted with adverse conditions. In a sense, it could be hypothesized that BnD22 may have an important role in plant resistance to diverse forms of stress. Future investigations using immunolocalization at subcellular level in oilseed rape leaf and/or GFP technologies in Arabidopsis (Arabidopsis thaliana) will attempt to determine the intracellular localization and confirm the physiological functions of BnD22 in the maintenance/protection of the young leaves of plants exposed to various stresses. Additionally, to estimate the contribution of BnD22 to N-use efficiency, it could be interesting to study genotypes of oilseed rape characterized by contrasting leaf N remobilization, leaf lifespan, or by their resistance to low mineral N availability.
Plant Material Seeds of oilseed rape (Brassica napus ‘Capitol’) were surface sterilized by exposure to 80% ethanol for 30 s followed by 20% sodium hypochlorite for 20 min. After 10 washes in demineralized water, seeds were germinated on foam rubber (Oasis growing pinpot; Agrimedia). Just after emergence of the third leaf, seedlings were transplanted into 2-L pots filled with attapulgite (one plant per pot) and were grown under greenhouse conditions with a thermoperiod of 20°C (day) and 18°C (night) and a photoperiod of 16 h. Natural light was supplemented with Neon tubes (Philips TLD 36W) supplying an average photosynthetically active radiation of 200 µmol photons m–2 s–1 at the top of the canopy. Plants were watered every 2 d with 100 mL of nutrient solution containing 2 mM KNO3, 3 mM CaCl2, 1 mM K2SO4, 0.5 mM MgSO4, 0.4 mM KH2PO4, 0.15 mM K2HPO4, 0.2 mM Fe-Na-EDTA, 14 µM H3BO3, 5 µM MnSO4, 3 µM ZnSO4, 0.7 µM (NH4)6Mo7O24, 0.7 µM CuSO4, and 0.1 µM CoCl2. After 85 d of growth corresponding to the rosette stage, plants were split into four sets for nitrate starvation and corresponding control or foliar spraying of MeJA and the corresponding control (water spraying).
Rosette plants grown with nutrient solution containing 3 mM KNO3 were split into two sets of plants. Plants were supplied every 2 d with nutrient solution without KNO3 (N starvation) and 3 mM of KNO3 (control plants). These mineral N treatments were applied during 28 d. Plants were sampled in triplicate after 14, 21, and 28 d of treatment. For MeJA treatment, one set of plants was sprayed with MeJA solution (100 µM MeJA, 0.05% Tween 20, 6.5 mL plant–1 d–1) applied to the leaves. The second set of plants, corresponding to the control, was sprayed with water (containing 0.05% Tween 20, 6.5 mL plant–1 d–1) on the same date. Control and MeJA-treated plants were sampled in triplicate on the first day (day 0 of experiment) and after 1, 3, and 7 d of treatment. For all treatments, each leaf rank was separated based on the date of their appearance from the oldest to youngest. The chlorophyll content was measured using a SPAD-502 chlorophyll meter (Minolta). Thereafter, leaves were weighed and the laminae was separated from the petiole, frozen in liquid N, and stored at –80°C until further analysis.
Frozen laminae samples (200 mg fresh weight) from each triplicate were ground in a mortar with liquid N in the presence of 150 mg polyvinylpolypyrrolidone. Soluble protein samples were extracted in citrate Na-phosphate buffer (20 mM citrate and 160 mM Na2HPO4, pH 6.8). The homogenate was centrifuged at 12,000g at 4°C for 1 h and the resulting supernatant was used for the determination of soluble protein concentration by protein-dye staining (Bradford, 1976
Frozen laminae samples (200 mg fresh weight) from each triplicate were ground in a mortar using liquid N and resuspended in 2 mL of cold acetone containing 10% TCA. After centrifugation at 16,000g for 3 min at 4°C, the supernatant was discarded and the pellet was rinsed as previously described by Wang et al. (2003)
For 2-DE of soluble proteins, 100 µL of protein was purified using a ReadyPrep 2-DE clean-up kit (Bio-Rad). The final pellet was resuspended in rehydration R2D2 buffer. The extract of total or soluble proteins was first separated according to charge in the electrofocusing PROTEAN IEF system (Bio-Rad) at 20°C, using 18-cm gel strips forming an immobilized linear pH gradient from 4 to 7 (GE Healthcare). Each strip was rehydrated at 50 µA/gel for 14 h in the presence of 330 µL of R2D2 buffer containing 125 µg of total or soluble proteins. Isoelectric focusing ran for 15 min at 250 V, 2 h at 500 V, and then until 50 kV at 10,000 V. After electrofocusing, the strips were immediately equilibrated in the equilibration buffer (75 mM Tris-HCl, 3% [w/v] SDS, 300 mM Tris base) containing dithiothreitol (65 mM), followed by a second incubation in equilibration buffer containing iodoacetamide (50 mM) and bromphenol blue (0.5%). 2-DE electrophoresis was carried out on 12% polyacrylamide (w/v) gels (20 x 20 cm) using an Investigator system (Millipore) at 300 mV. Gels were stained using the silver-staining procedure described by Blum et al. (1987)
Images of the 2-DE gels were acquired with the ProXPRESS 2D proteomic Imaging system and analyzed using the Progenesis SameSpots software v3.0 (Nonlinear Dynamics) according the manufacturer's protocol. Gels from three independent biological replicates were used. Spot detection, warping, and matching were performed automatically by the software. Matching was automatic but verified manually; artifacts, or spots that could not be confidently verified as true matches, were disregarded rather than manually edited, and misalignments were corrected by manual warping when appropriate. Molecular mass and pI were calculated using Samespots software calibrated with commercial molecular mass standards (precision protein standards unstained; Bio-Rad) run in separate marker lane on 2-DE gel.
For SDS-PAGE, one volume of each triplicate was mixed together and prepared in 2x lysis buffer without β-mercaptoethanol. SDS-PAGE was performed using a 5.5% polyacrylamide (w/v) stacking gel and an 18% polyacrylamide (w/v) resolving gel as described by Etienne et al. (2007)
The proteins samples were first separated by 2-DE as described above except that 250 µg of proteins was loaded in the strip. Electrophoretic transfer of polypeptides from 2-DE gels onto polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Proteigene) was conducted by semidry electroblotting (2.5 mA for 20 min; Milli Blot system; Proteigene), according to the protocol described by Towbin et al. (1979)
Spots of interest were manually excised from gels and destained as described by Gharahdaghi et al. (1999) Alignment analysis of assigned proteins was predicted using ClustalW (npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html). The research of predicted cleavage sites of signal peptides and phosphorylation sites was performed using SignalP 3.0 (www.cbs.dtu.dk/services/SignalP/) and NetPhos 2.0 (www.cbs.dtu.dk/services/NetPhos/), respectively.
Total RNA was extracted from 250 mg of laminae fresh matter. Frozen samples were ground to a powder with a pestle in a mortar containing liquid N. The resulting powder was suspended in 750 µL extraction buffer (0.1 M Tris, 0.1 M LiCl, 0.01 M EDTA, 1% SDS [w/v], pH 8) and 750 µL of hot phenol (80°C, pH 4). This mixture was vortexed for 30 s and after addition of 750 µL of chloroform/isoamylalcohol (24:1), the homogenate was centrifuged at 15,000g (5 min, 4°C). The supernatant was transferred into 4 M LiCl solution (w/v) and incubated overnight at 4°C. After centrifugation (15,000g, 30 min, 4°C), the pellet was suspended in 250 µL of sterile water. Fifty microliters of 3 M sodium acetate (pH 5.6) and 1 mL of 96% ethanol were added to precipitate the total RNA for 1 h at –80°C. After centrifugation (15,000g, 20 min, 4°C), the pellet was washed with 1 mL of 70% ethanol, then centrifuged at 15,000g for 5 min at 4°C. The resulting pellet was dried for 5 min at room temperature and resuspended in sterile water containing 0.1% SDS and 20 mM EDTA. Quantification of total RNA was performed by spectrophometre at 260 nm (BioPhotometer) before reverse transcription (RT) and Q-PCR analyses.
For RT, 1 µg of total RNA was converted to cDNA with an iScript cDNA synthesis kit using the manufacturer's protocol (Bio-Rad). For Q-PCR amplification, primers of BnD22 were designed after multiple alignment of nucleotidic sequences between BnD22 (accession no. X65637), encoding an oilseed rape TI, WSCP1 (accession no. AB012699) and WSCP2 (accession no. AB078330), encoding B. oleracea WSCP1 and WSCP2, respectively, and HSIP (accession no. AJ007414), encoding a B. oleracea HSIP. To target BnD22 cDNA, specific primers were designed in the nonconserved region. Thus, specific primers selected for the Q-PCR analysis were: BnD22 forward primer 5'-CCGGTTAGCTTCGGATATGA-3' and reverse primer 5'-AGCTATTTGGGGCCGTACTT-3'. For Q-PCR analysis, EF1-
Results are presented as mean values for the three plant material (triplicate) batches with SEs. The effects of mineral N starvation or MeJA treatment were assessed by ANOVA, and mean separation was performed using Fisher-Snedecor test. Statistical significance was postulated at P < 0.05. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers X65637, AB078330, AB012699, AJ007414, U18995, AB078330, NM112518, and DQ312264.
The authors would like to thank Dr. Laurent Coquet, Dr. Philippe Laîné, Dr. Aurélie Verneuil, and Dr. Vianney Pichereau for their valuable help in ESI-LC MS/MS analyses, and Sandrine Rezé for her technical help in Q-PCR analyses. We would like to thank the three anonymous reviewers for their valuable comments. Received January 26, 2008; accepted June 10, 2008; published June 13, 2008.
1 This work was supported by a Ph.D. grant from the Institut National de la Recherche Agronomique and the Conseil Régional de Basse-Normandie (to M.D.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jean-Christophe Avice (jean-christophe.avice{at}unicaen.fr). www.plantphysiol.org/cgi/doi/10.1104/pp.108.116905 * Corresponding author; e-mail jean-christophe.avice{at}unicaen.fr.
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ABSTRACT
RESULTS
a-Cortés et al., 1991
) or not (N starvation; 
) with nitrate during 28 d. Vertical bars indicate ±SE of the mean (n = 3) and fit within the plot if not visible. SDS-PAGE was obtained by loading a constant amount of 3 µg of soluble proteins per lane and using silver nitrate staining. Molecular mass markers (kD) are listed on the left of the gel. A protein at 19 kD with specific changes during N treatment is indicated on the right of the gel. LSU, Large subunit of Rubisco (55 kD); SSU, small subunit of Rubisco (14 kD).
gene (accession no. 