Multi-omics analyses of sid2 mutant reflect the need of isochorismate synthase ICS1 to cope with sulfur limitation in Arabidopsis thaliana.

The SID2 (SA INDUCTION-DEFICIENT2) gene that encodes ICS1 (isochorismate synthase), plays a central role in salicylic acid biosynthesis in Arabidopsis. The sid2 and NahG (encoding a bacterial SA hydroxylase) overexpressing mutants (NahG-OE) have currently been shown to outperform wild type, presenting delayed leaf senescence, higher plant biomass and better seed yield. When grown under sulfate-limited conditions (low-S), sid2 mutants exhibited early leaf yellowing compared to the NahG-OE, the npr1 mutant affected in SA signaling pathway, and WT. This indicated that the hypersensitivity of sid2 to sulfate limitation was independent of the canonical npr1 SA-signaling pathway. Transcriptomic and proteomic analyses revealed that major changes occurred in sid2 when cultivated under low-S, changes that were in good accordance with early senescence phenotype and showed the exacerbation of stress responses. The sid2 mutants displayed a lower sulfate uptake capacity when cultivated under low-S and lower S concentrations in their rosettes. Higher glutathione concentrations in sid2 rosettes under low-S were in good accordance with the higher abundance of proteins involved in glutathione and ascorbate redox metabolism. Amino acid and lipid metabolisms were also strongly modified in sid2 under low-S. Depletion of total fatty acids in sid2 under low-S was consistent with the fact that S-metabolism plays a central role in lipid synthesis. Altogether, our results show that functional ICS1 is important for plants to cope with S limiting conditions.

Leaf drought adaptive response in winter oilseed rape is altered at the onset of senescence: a study combining NMR relaxometry, multi-omics and microscopy.

Climate change is bringing more frequent and intense droughts, reducing overall water availability and adversely affecting crops. There is a need to improve our understanding of the tissular and cellular adaptation mechanisms that are critical for plant water conservation strategies. Here, we have used NMR relaxometry in combination with microscopy and multi-omic analysis to study the effects of progressive soil drought on winter oilseed rape (WOSR, Brassica napus L., cv. Aviso) leaves. This study reveals the structural and metabolic adjustments these leaves operate to maintain cell homeostasis. Our results are original in showing that the adaptive responses are altered in leaves at the onset of senescence, associated with changes in metabolic plasticity and mesophyll structures. Thus, long-term responses in young leaves involving osmotic adjustment were combined with the maintenance of tissue hydration and cell growth, contributing to high survival and recovery capacity. For the first time, short-term responses observed in early senescent-old leaves were associated with early drought-induced dehydration of the spongy layer. However, this dehydration was not followed by osmotic adjustment and did not allow maintenance of leaf tissue turgor. These findings open further studies on the genetic variability of drought responses related to identified short- and long-term structural and metabolic plasticity traits in Brassica species.

Evidence of a significant role of glutathione reductase in the sulfur assimilation pathway.

With the objective of studying the role of glutathione reductase (GR) in the accumulation of cysteine and methionine, we generated transgenic tobacco and Arabidopsis lines overexpressing the cytosolic AtGR1 and the plastidic AtGR2 genes. The transgenic plants had higher contents of cysteine and glutathione. To understand why cysteine levels increased in these plants, we also used gr1 and gr2 mutants. The results showed that the transgenic plants have higher levels of sulfite, cysteine, glutathione and methionine, which are downstream to adenosine 5' phosphosulfate reductase (APR) activity. However, the mutants had lower levels of these metabolites, while the sulfate content increased. A feeding experiment using 34 SO42- also showed that the levels of APR downstream metabolites increased in the transgenic lines and decreased in gr1 compared with their controls. These findings, and the results obtained from the expression levels of several genes related to the sulfur pathway, suggest that GR plays an essential role in the sulfur assimilation pathway by supporting the activity of APR, the key enzyme in this pathway. GR recycles the oxidized form of glutathione (GSSG) back to reduce glutathione (GSH), which serves as an electron donor for APR activity. The phenotypes of the transgenic plants and the mutants are not significantly altered under non-stress and oxidative stress conditions. However, when germinating on sulfur-deficient medium, the transgenic plants grew better, while the mutants were more sensitive than the control plants. The results give substantial evidence of the yet unreported function of GR in the sulfur assimilation pathway.

Do nitrogen- and sulphur-remobilization-related parameters measured at the onset of the reproductive stage provide early indicators to adjust N and S fertilization in oilseed rape (Brassica napus L.) grown under N- and/or S-limiting supplies?

MAIN CONCLUSION: Specific combinations of physiological and molecular parameters associated with N and S remobilization measured at the onset of flowering were predictive of final crop performances in oilseed rape. Oilseed rape (Brassica napus L.) is a high nitrogen (N) and sulphur (S) demanding crop. Nitrogen- and S-remobilization processes allow N and S requirements to reproductive organs to be satisfied when natural uptake is reduced, thus ensuring high yield and seed quality. The quantification of physiological and molecular indicators of early N and S remobilization could be used as management tools to correct N and S fertilization. However, the major limit of this corrective strategy is to ensure the correlation between final performances-related variables and early measured parameters. In our study, four genotypes of winter oilseed rape (OSR) were grown until seed maturity under four nutritional modalities combining high and/or low N and S supplies. Plant final performances, i.e., seed production, N- and S-harvest indexes, seed N and S use efficiencies, and early parameters related to N- or S-remobilization processes, i.e., photosynthetic leaf area, N and S leaf concentrations, leaf soluble protein and leaf sulphate concentrations, and leaf RuBisCO abundance at flowering, were measured. We demonstrated that contrasting final performances existed according to the N and S supplies. An optimal N:S ratio supply could explain the treatment-specific crop performances, thus justifying N and S concurrent managements. Specific combinations of early measured plant parameters could be used to predict final performances irrespective of the nutritional supply and the genotype. This work demonstrates the potential of physiological and molecular indicators measured at flowering to reflect the functioning of N- and S-compound remobilization and to predict yield and quality penalties. However, because the predictive models are N and S independent, instant N and S leaf analyses are required to further adjust the adequate fertilization. This study is a proof of a concept which opens prospects regarding instant diagnostic tools in the context of N and S mineral fertilization management.

Arabidopsis UMAMIT24 and 25 are amino acid exporters involved in seed loading.

Phloem-derived amino acids are the major source of nitrogen supplied to developing seeds. Amino acid transfer from the maternal to the filial tissue requires at least one cellular export step from the maternal tissue prior to the import into the symplasmically isolated embryo. Some members of UMAMIT (usually multiple acids move in an out transporter) family (UMAMIT11, 14, 18, 28, and 29) have previously been implicated in this process. Here we show that additional members of the UMAMIT family, UMAMIT24 and UMAMIT25, also function in amino acid transfer in developing seeds. Using a recently published yeast-based assay allowing detection of amino acid secretion, we showed that UMAMIT24 and UMAMIT25 promote export of a broad range of amino acids in yeast. In plants, UMAMIT24 and UMAMIT25 are expressed in distinct tissues within developing seeds; UMAMIT24 is mainly expressed in the chalazal seed coat and localized on the tonoplast, whereas the plasma membrane-localized UMAMIT25 is expressed in endosperm cells. Seed amino acid contents of umamit24 and umamit25 knockout lines were both decreased during embryogenesis compared with the wild type, but recovered in the mature seeds without any deleterious effect on yield. The results suggest that UMAMIT24 and 25 play different roles in amino acid translocation from the maternal to filial tissue; UMAMIT24 could have a role in temporary storage of amino acids in the chalaza, while UMAMIT25 would mediate amino acid export from the endosperm, the last step before amino acids are taken up by the developing embryo.

Three cytosolic glutamine synthetase isoforms localized in different-order veins act together for N remobilization and seed filling in Arabidopsis.

Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the gln1;1-gln1;2-gln1;3 triple-mutation affecting the three GLN1;1, GLN1;2, and GLN1;3 genes significantly reduced N remobilization, total seed yield, individual seed weight, harvest index, and vegetative biomass. The triple-mutant accumulated a large amount of ammonium that could not be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and was related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein, and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they were all expressed in the phloem companion cells but in veins of different order. Our results demonstrate that glutamine biosynthesis for N-remobilization occurs in veins of all orders (major and minor) in leaves, it is mainly catalysed by the three major GS1 isoforms (GLN1;1, GLN1;2, and GLN1;3), and it is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.

Increases in activity of proteasome and papain-like cysteine protease in Arabidopsis autophagy mutants: back-up compensatory effect or cell-death promoting effect?

Autophagy is essential for protein degradation, nutrient recycling, and nitrogen remobilization. Autophagy is induced during leaf ageing and in response to nitrogen starvation, and is known to play a fundamental role in nutrient recycling for remobilization and seed filling. Accordingly, ageing leaves of Arabidopsis autophagy mutants (atg) have been shown to over-accumulate proteins and peptides, possibly because of a reduced protein degradation capacity. Surprisingly, atg leaves also displayed higher protease activities. The work reported here aimed at identifying the nature of the proteases and protease activities that accumulated differentially (higher or lower) in the atg mutants. Protease identification was performed using shotgun LC-MS/MS proteome analyses and activity-based protein profiling (ABPP). The results showed that the chloroplast FTSH (FILAMENTATION TEMPERATURE SENSITIVE H) and DEG (DEGRADATION OF PERIPLASMIC PROTEINS) proteases and several extracellular serine proteases [subtilases (SBTs) and serine carboxypeptidase-like (SCPL) proteases] were less abundant in atg5 mutants. By contrast, proteasome-related proteins and cytosolic or vacuole cysteine proteases were more abundant in atg5 mutants. Rubisco degradation assays and ABPP showed that the activities of proteasome and papain-like cysteine protease were increased in atg5 mutants. Whether these proteases play a back-up role in nutrient recycling and remobilization in atg mutants or act to promote cell death is discussed in relation to their accumulation patterns in the atg5 mutant compared with the salicylic acid-depleted atg5/sid2 double-mutant, and in low nitrate compared with high nitrate conditions. Several of the proteins identified are indeed known as senescence- and stress-related proteases or as spontaneous cell-death triggering factors.

Fluxomics links cellular functional analyses to whole-plant phenotyping.

Fluxes through metabolic pathways reflect the integration of genetic and metabolic regulations. While it is attractive to measure all the mRNAs (transcriptome), all the proteins (proteome), and a large number of the metabolites (metabolome) in a given cellular system, linking and integrating this information remains difficult. Measurement of metabolome-wide fluxes (termed the fluxome) provides an integrated functional output of the cell machinery and a better tool to link functional analyses to plant phenotyping. This review presents and discusses sets of methodologies that have been developed to measure the fluxome. First, the principles of metabolic flux analysis (MFA), its 'short time interval' version Inst-MFA, and of constraints-based methods, such as flux balance analysis and kinetic analysis, are briefly described. The use of these powerful methods for flux characterization at the cellular scale up to the organ (fruits, seeds) and whole-plant level is illustrated. The added value given by fluxomics methods for unravelling how the abiotic environment affects flux, the process, and key metabolic steps are also described. Challenges associated with the development of fluxomics and its integration with 'omics' for thorough plant and organ functional phenotyping are discussed. Taken together, these will ultimately provide crucial clues for identifying appropriate target plant phenotypes for breeding.

Evidence for proteomic and metabolic adaptations associated with alterations of seed yield and quality in sulfur-limited Brassica napus L.

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase ß-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of ß-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

The contrasting N management of two oilseed rape genotypes reveals the mechanisms of proteolysis associated with leaf N remobilization and the respective contributions of leaves and stems to N storage and remobilization during seed filling.

BACKGROUND: Oilseed rape is the third largest oleaginous crop in the world but requires high levels of N fertilizer of which only 50% is recovered in seeds. This weak N use efficiency is associated with a low foliar N remobilization, leading to a significant return of N to the soil and a risk of pollution. Contrary to what is observed during senescence in the vegetative stages, N remobilization from stems and leaves is considered efficient during monocarpic senescence. However, the contribution of stems towards N management and the cellular mechanisms involved in foliar remobilization remain largely unknown. To reach this goal, the N fluxes at the whole plant level from bolting to mature seeds and the processes involved in leaf N remobilization and proteolysis were investigated in two contrasting genotypes (Aviso and Oase) cultivated under ample or restricted nitrate supply. RESULTS: During seed filling in both N conditions, Oase efficiently allocated the N from uptake to seeds while Aviso favoured a better N remobilization from stems and leaves towards seeds. Nitrate restriction decreased seed yield and oil quality for both genotypes but Aviso had the best seed N filling. Under N limitation, Aviso had a better N remobilization from leaves to stems before the onset of seed filling. Afterwards, the higher N remobilization from stems and leaves of Aviso led to a higher final N amount in seeds. This high leaf N remobilization is associated with a better degradation/export of insoluble proteins, oligopeptides, nitrate and/or ammonia. By using an original method based on the determination of Rubisco degradation in the presence of inhibitors of proteases, efficient proteolysis associated with cysteine proteases and proteasome activities was identified as the mechanism of N remobilization. CONCLUSION: The results confirm the importance of foliar N remobilization after bolting to satisfy seed filling and highlight that an efficient proteolysis is mainly associated with (i) cysteine proteases and proteasome activities and (ii) a fine coordination between proteolysis and export mechanisms. In addition, the stem may act as transient storage organs in the case of an asynchronism between leaf N remobilization and N demand for seed filling.

Differential CO2 effect on primary carbon metabolism of flag leaves in durum wheat (Triticum durum Desf.).

C sink/source balance and N assimilation have been identified as target processes conditioning crop responsiveness to elevated CO2 . However, little is known about phenology-driven modifications of C and N primary metabolism at elevated CO2 in cereals such as wheat. Here, we examined the differential effect of elevated CO2 at two development stages (onset of flowering, onset of grain filling) in durum wheat (Triticum durum, var. Sula) using physiological measurements (photosynthesis, isotopes), metabolomics, proteomics and (15) N labelling. Our results show that growth at elevated CO2 was accompanied by photosynthetic acclimation through a lower internal (mesophyll) conductance but no significant effect on Rubisco content, maximal carboxylation or electron transfer. Growth at elevated CO2 altered photosynthate export and tended to accelerate leaf N remobilization, which was visible for several proteins and amino acids, as well as lysine degradation metabolism. However, grain biomass produced at elevated CO2 was larger and less N rich, suggesting that nitrogen use efficiency rather than photosynthesis is an important target for improvement, even in good CO2 -responsive cultivars.

A profiling approach of the natural variability of foliar N remobilization at the rosette stage gives clues to understand the limiting processes involved in the low N use efficiency of winter oilseed rape.

Oilseed rape, a crop requiring a high level of nitogen (N) fertilizers, is characterized by low N use efficiency. To identify the limiting factors involved in the N use efficiency of winter oilseed rape, the response to low N supply was investigated at the vegetative stage in 10 genotypes by using long-term pulse-chase (15)N labelling and studying the physiological processes of leaf N remobilization. Analysis of growth and components of N use efficiency allowed four profiles to be defined. Group 1 was characterized by an efficient N remobilization under low and high N conditions but by a decrease of leaf growth under N limitation. Group 2 showed a decrease in leaf growth under low N supply that was associated with a low N remobilization efficiency under both N supplies despite a high remobilization of soluble proteins. In response to N limitation, Group 3 is characterized by an increase in N use efficiency and leaf N remobilization compared with high N that is not sufficient to sustain the leaf biomass production at a similar level to non-limited plants. Genotypes of Group 4 subjected to low nitrate were able to maintain leaf growth to the same level as under high N. The profiling approach indicated that enhancement of amino acid export and soluble protein degradation was crucial for N remobilization improvement. At the whole-plant level, N fluxes revealed that Group 4 showed a high N remobilization in source leaves combined with a better N utilization in young leaves. Consequently, an enhanced N remobilization limits N loss in fallen leaves, but this remobilized N needs to be efficiently utilized in young leaves to improve N use efficiency.

A novel method for determination of the (15) N isotopic composition of Rubisco in wheat plants exposed to elevated atmospheric carbon dioxide.

Although ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is mostly known as a key enzyme involved in CO2 assimilation during the Calvin cycle, comparatively little is known about its role as a pool of nitrogen storage in leaves. For this purpose, we developed a protocol to purify Rubisco that enables later analysis of its (15) N isotope composition (δ(15) N) at the natural abundance and (15) N-labeled plants. In order to test the utility of this protocol, durum wheat (Triticum durum var. Sula) exposed to an elevated CO2 concentration (700 vs 400 µmol mol(-1) ) was labeled with K(15) NO3 (enriched at 2 atom %) during the ear development period. The developed protocol proves to be selective, simple, cost effective and reproducible. The study reveals that (15) N labeling was different in total organic matter, total soluble protein and the Rubisco fraction. The obtained data suggest that photosynthetic acclimation in wheat is caused by Rubisco depletion. This depletion may be linked to preferential nitrogen remobilization from Rubisco toward grain filling.

Legume adaptation to sulfur deficiency revealed by comparing nutrient allocation and seed traits in Medicago truncatula.

Reductions in sulfur dioxide emissions and the use of sulfur-free mineral fertilizers are decreasing soil sulfur levels and threaten the adequate fertilization of most crops. To provide knowledge regarding legume adaptation to sulfur restriction, we subjected Medicago truncatula, a model legume species, to sulfur deficiency at various developmental stages, and compared the yield, nutrient allocation and seed traits. This comparative analysis revealed that sulfur deficiency at the mid-vegetative stage decreased yield and altered the allocation of nitrogen and carbon to seeds, leading to reduced levels of major oligosaccharides in mature seeds, whose germination was dramatically affected. In contrast, during the reproductive period, sulfur deficiency had little influence on yield and nutrient allocation, but the seeds germinated slowly and were characterized by low levels of a biotinylated protein, a putative indicator of germination vigor that has not been previously related to sulfur nutrition. Significantly, plants deprived of sulfur at an intermediary stage (flowering) adapted well by remobilizing nutrients from source organs to seeds, ensuring adequate quantities of carbon and nitrogen in seeds. This efficient remobilization of photosynthates may be explained by vacuolar sulfate efflux to maintain leaf metabolism throughout reproductive growth, as suggested by transcript and metabolite profiling. The seeds from these plants, deprived of sulfur at the floral transition, contained normal levels of major oligosaccharides but their germination was delayed, consistent with low levels of sucrose and the glycolytic enzymes required to restart seed metabolism during imbibition. Overall, our findings provide an integrative view of the legume response to sulfur deficiency.

Characterization of vegetative storage protein (VSP) and low molecular proteins induced by water deficit in stolon of white clover.

In stolon of white clover (Trifolium repens L.), the 17.3 kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS-PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28 days of water-deficit. Water deficit led to decrease protein concentration. SDS-PAGE revealed that two major proteins of 17.3 and 16 kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3 kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3 kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress.

Leaf senescence and nitrogen remobilization efficiency in oilseed rape (Brassica napus L.).

Despite its worldwide economic importance for food (oil, meal) and non-food (green energy and chemistry) uses, oilseed rape has a low nitrogen (N) use efficiency (NUE), mainly due to the low N remobilization efficiency (NRE) observed during the vegetative phase when sequential leaf senescence occurs. Assuming that improvement of NRE is the main lever for NUE optimization, unravelling the cellular mechanisms responsible for the recycling of proteins (the main N source in leaf) during sequential senescence is a prerequisite for identifying the physiological and molecular determinants that are associated with high NRE. The development of a relevant molecular indicator (SAG12/Cab) of leaf senescence progression in combination with a (15)N-labelling method were used to decipher the N remobilization associated with sequential senescence and to determine modulation of this process by abiotic factors especially N deficiency. Interestingly, in young leaves, N starvation delayed senescence and induced BnD22, a water-soluble chlorophyll-binding protein that acts against oxidative alterations of chlorophylls and exhibits a protease inhibitor activity. Through its dual function, BnD22 may help to sustain sink growth of stressed plants and contribute to a better utilization of N recycled from senescent leaves, a physiological trait that could improve NUE. Proteomics approaches have revealed that proteolysis involves chloroplastic FtsH protease in the early stages of senescence, aspartic protease during the course of leaf senescence, and the proteasome ß1 subunit, mitochondria processing protease and SAG12 (cysteine protease) during the later senescence phases. Overall, the results constitute interesting pathways for screening genotypes with high NRE and NUE.

Sulphur limitation provokes physiological and leaf proteome changes in oilseed rape that lead to perturbation of sulphur, carbon and oxidative metabolisms.

BACKGROUND: The decline in industrial emissions of sulphur (S) has led to a sulphate depletion in soil resulting in an alteration of crop performance. In oilseed rape, an S deficiency dramatically reduced the seed yield and/or quality. Paradoxically, little is known about the impact of sulphate limitation on oilseed rape leaf metabolism, despite it being a key determinant of growth. In order to identify the metabolic processes involved in the oilseed rape response to S restriction, an analysis of the young leaf proteome combined with a physiological study was carried out at the vegetative stage. RESULTS: S limitation does not significantly reduce the total shoot biomass but inhibits growth and photosynthesis of young leaves. This photosynthesis decline is not due to a decrease in chlorophyll content, which remains similar to Control. The increase in anthocyanins and H(2)O(2) content in young leaves of S-limited plants suggests that S restriction leads to an oxidative stress. Proteomic analysis at 35 d of S limitation also revealed the induction of 12-oxophitodienoate reductase and ACC synthase, respectively involved in jasmonate and ethylene biosynthesis, two phytohormones that could be implicated in oxidative stress. Proteins involved in photosynthesis and carbon metabolism were also modulated by S restriction. In particular, the decrease in plastocyanin and ferredoxin-NADP reductase suggests that H(2)O(2) accumulation is associated with perturbation of the photosynthetic electron transport chain. The accumulation of chloroplastic Cu-Zn SOD reinforces the idea that an oxidative stress probably occurs in the chloroplast. Proteomic results suggest that the maintenance of chlorophyll in S-limited conditions is related to an accumulation of Water Soluble Chlorophyll binding Proteins, involved in the protection of chlorophyll against ROS. The accumulation of the catalytic α-subunit of chloroplastic ATP synthase suggests that energy production is maintained. CONCLUSION: S limitation leads to photosynthesis and carbon metabolism disturbances that could be responsible for the oxidative stress observed in the young leaves of oilseed rape. Despite this, induction of proteins involved in oxidative stress resistance and energy production shows that the leaf capacity to capture and use photosynthetic active radiations for ATP production remains efficient for as long as possible.

Arabidopsis thaliana ASN2 encoding asparagine synthetase is involved in the control of nitrogen assimilation and export during vegetative growth.

We investigated the function of ASN2, one of the three genes encoding asparagine synthetase (EC 6.3.5.4), which is the most highly expressed in vegetative leaves of Arabidopsis thaliana. Expression of ASN2 and parallel higher asparagine content in darkness suggest that leaf metabolism involves ASN2 for asparagine synthesis. In asn2-1 knockout and asn2-2 knockdown lines, ASN2 disruption caused a defective growth phenotype and ammonium accumulation. The asn2 mutant leaves displayed a depleted asparagine and an accumulation of alanine, GABA, pyruvate and fumarate, indicating an alanine formation from pyruvate through the GABA shunt to consume excess ammonium in the absence of asparagine synthesis. By contrast, asparagine did not contribute to photorespiratory nitrogen recycle as photosynthetic net CO(2) assimilation was not significantly different between lines under both 21 and 2% O(2). ASN2 was found in phloem companion cells by in situ hybridization and immunolocalization. Moreover, lack of asparagine in asn2 phloem sap and lowered (15) N flux to sinks, accompanied by the delayed yellowing (senescence) of asn2 leaves, in the absence of asparagine support a specific role of asparagine in phloem loading and nitrogen reallocation. We conclude that ASN2 is essential for nitrogen assimilation, distribution and remobilization (via the phloem) within the plant.

Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction.

Although the mechanisms of nodule N(2) fixation in legumes are now well documented, some uncertainty remains on the metabolic consequences of water deficit. In most cases, little consideration is given to other organs and, therefore, the coordinated changes in metabolism in leaves, roots, and nodules are not well known. Here, the effect of water restriction on exclusively N(2)-fixing alfalfa (Medicago sativa L.) plants was investigated, and proteomic, metabolomic, and physiological analyses were carried out. It is shown that the inhibition of nitrogenase activity caused by water restriction was accompanied by concerted alterations in metabolic pathways in nodules, leaves, and roots. The data suggest that nodule metabolism and metabolic exchange between plant organs nearly reached homeostasis in asparagine synthesis and partitioning, as well as the N demand from leaves. Typically, there was (i) a stimulation of the anaplerotic pathway to sustain the provision of C skeletons for amino acid (e.g. glutamate and proline) synthesis; (ii) re-allocation of glycolytic products to alanine and serine/glycine; and (iii) subtle changes in redox metabolites suggesting the implication of a slight oxidative stress. Furthermore, water restriction caused little change in both photosynthetic efficiency and respiratory cost of N(2) fixation by nodules. In other words, the results suggest that under water stress, nodule metabolism follows a compromise between physiological imperatives (N demand, oxidative stress) and the lower input to sustain catabolism.

A specific method of 34S labelling provides evidence that sulphate assimilation occurs in developing seeds and pod walls of Brassica napus L. subjected to ample or limited S nutrition.

RATIONALE: Seeds from different species actively assimilate sulphur (S) from sulphate. This has never been proved for Brassica napus L., a high S demanding plant, especially with regard to S limitation. The role of pod walls in the assimilation and allocation of S in well-fed and sulphate-limited conditions also needs to be clarified. METHODS: Freshly harvested seeds and pod walls from plants well-supplied (HS) or limited with sulphate (LS) from the 'visible buds' stage were subjected to a nutrient solution containing (34)S-sulphate (10 atom% excess) for 24 h. The (34)S labelling of the sulphate and protein fractions was determined with an elemental analyser connected to a continuous flow isotope ratio mass spectrometer. The amino acid profiles of seeds and pod walls were also determined by ion-exchange chromatography. RESULTS: The 24 h of (34)S-sulphate feeding treatment leads to an important production of proteins in HS and LS seeds, associated with a decrease in numerous amino acid contents. The treatment also leads to an incorporation of (34)S in seeds and pod walls proteins in both HS and LS conditions. The incorporation of (34)S in proteins was not different between HS and LS seeds, but was lower in LS than in HS pod walls, related to a higher incorporation in the other organic S compounds. CONCLUSIONS: This study provides evidence that Brassica napus seeds and pod walls are able to assimilate sulphate in HS and LS conditions, and that the LS condition leads to enhancement of the sulphate assimilation capacity of pod walls, which may be of crucial importance for the allocation of S to developing seeds.