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.

Repeated heat stress events during the reproductive phase impact the dynamic development of seeds in Brassica napus L.

Many studies pointed out the deleterious effects of high temperatures events during the crop reproductive phase on seed yield and quality. However, plant responses to repeated stressing events remain poorly understood, while the increased frequency of extreme abiotic constraints, such as spring and summer heat waves, has been proven as one feature of the on-going and future climate change. The responses of oilseed rape plants subjected to three heat stress sequences that differed in the intensity, the timing of application, the duration and the frequency of the high temperature events were investigated throughout the seed development and maturation phases under controlled conditions. Seed yield and components were measured in three different harvest dates. Biochemical and histological analyses of seeds were carried out in order to monitor the evolution of the main storage compounds (fatty acids, proteins, sugars) involved in seed nutritional quality. Although the effects of heat stress were not significant on total yield, differences in seed number and weight highlighted the strong compensation capacity in indeterminate growth species. Heat stress induced significant decreases and increases in seed oil and protein content respectively, to different extent according to the age of the pods. Soluble sugars concentrations were impacted by heat during seed development, but not when the seeds reached physiological maturity, thus indicating compensatory mechanisms that set up after the stress exposure. Our results led to conclude that the effects of repeated heat stresses on seed yield and quality were tightly related to (i) the optimal temperature of a given compound biosynthesis process, and (ii) the synchrony between the temperature event and the period of biosynthesis of the targeted storage compound. These results highlight the complexity to design thermo-sensitizing protocols to maintain or even improve the various seed quality related criteria, especially in species with indeterminate growth.

Biostimulant impacts of Glutacetine® and derived formulations (VNT1 and VNT4) on the bread wheat grain proteome.

Nitrogen (N) fertilizer is essential to ensure grain yield and quality in bread wheat. Improving N use efficiency is therefore crucial for wheat grain protein quality. In the present work, we analysed the effects on the winter wheat grain proteome of biostimulants containing Glutacetine® or two derived formulations (VNT1 and 4) when mixed with urea-ammonium-nitrate fertilizer. A large-scale quantitative proteomics analysis of two wheat flour fractions produced a dataset of 4369 identified proteins. Quantitative analysis revealed 9, 39 and 96 proteins with a significant change in abundance after Glutacetine®, VNT1 and VNT4 treatments, respectively, with a common set of 11 proteins that were affected by two different biostimulants. The major effects impacted proteins involved in (i) protein synthesis regulation (mainly ribosomal and binding proteins), (ii) defence and responses to stresses (including chitin-binding protein, heat shock 70 kDa protein 1 and glutathione S-transferase proteins), (iii) storage functions related to gluten protein alpha-gliadins and starch synthase and (iv) seed development with proteins implicated in protease activity, energy machinery, and the C and N metabolism pathways. Altogether, our study showed that Glutacetine®, VNT1 and VNT4 biostimulants positively affected protein composition related to grain quality. Data are available via ProteomeXchange with identifier PXD021513. SIGNIFICANCE: We performed a large-scale quantitative proteomics study of the total protein extracts from flour samples to determine the effect of Glutacetine®-based biostimulants treatment on the protein composition of bread wheat grain. To our knowledge, only a few studies in the literature have applied proteomic approaches to study bread wheat grains and in particular to investigate the effect of biostimulants on the grain proteome of this cereal crop. In addition, most approaches used fractional extraction of proteins to target reserve proteins followed electrophoresis which leads to low identification rate of proteins. We identified and quantified a large protein dataset of 4369 proteins and determined ontological class of proteins affected by biostimulants treatments. Our proteomics investigation revealed the important role of these new biostimulants in achieving significant changes in protein synthesis regulation, storage functions, protease activity, energy machinery, C and N metabolism pathways and responses to biotic and abiotic stresses in grain.

Glutacetine® Biostimulant Applied on Wheat under Contrasting Field Conditions Improves Grain Number Leading to Better Yield, Upgrades N-Related Traits and Changes Grain Ionome.

Wheat is one of the most important cereals for human nutrition, but nitrogen (N) losses during its cultivation cause economic problems and environmental risks. In order to improve N use efficiency (NUE), biostimulants are increasingly used. The present study aimed to evaluate the effects of Glutacetine®, a biostimulant sprayed at 5 L ha-1 in combination with fertilizers (urea or urea ammonium nitrate (UAN)), on N-related traits, grain yield components, and the grain quality of winter bread wheat grown at three field sites in Normandy (France). Glutacetine® improved grain yield via a significant increase in the grain number per spike and per m2, which also enhanced the thousand grain weight, especially with urea. The total N in grains and the NUE tended to increase in response to Glutacetine®, irrespective of the site or the form of N fertilizer. Depending on the site, spraying Glutacetine® can also induce changes in the grain ionome (analyzed by X-ray fluorescence), with a reduction in P content observed (site 2 under urea nutrition) or an increase in Mn content (site 3 under UAN nutrition). These results provide a roadmap for utilizing Glutacetine® biostimulant to enhance wheat production and flour quality in a temperate climate.

Biostimulant Effects of Glutacetine® and Its Derived Formulations Mixed With N Fertilizer on Post-heading N Uptake and Remobilization, Seed Yield, and Grain Quality in Winter Wheat.

Biostimulants could play an important role in agriculture particularly for increasing N fertilizer use efficiency that is essential for maintaining both yield and grain quality in bread wheat, which is a major global crop. In the present study, we examined the effects of mixing urea-ammonium-nitrate fertilizer (UAN) or urea with five new biostimulants containing Glutacetine® or its derivative formulations (VNT1, 2, 3, and 4) on the physiological responses, agronomic traits, and grain quality of winter wheat. A first experiment under greenhouse conditions showed that VNT1, VNT3, and VNT4 significantly increased the seed yield and grain numbers per ear. VNT4 also enhanced total plant nitrogen (N) and total grain N, which induced a higher N Harvest Index (NHI). The higher post-heading N uptake (for VNT1 and VNT4) and the acceleration of senescence speed with all formulations enabled better nutrient remobilization efficiency, especially in terms of N mobilization from roots and straw toward the grain with VNT4. The grain ionome was changed by the formulations with the bioavailability of iron improved with the addition of VNT4, and the phytate concentrations in flour were reduced by VNT1 and VNT4. A second experiment in three contrasting field trials confirmed that VNT4 increased seed yield and N use efficiency. Our investigation reveals the important role of these new formulations in achieving significant increases in seed yield and grain quality.

Advances in Plant Sulfur Research.

As an essential nutrient required for plant growth and development, sulfur (S) deficiency in productive systems limits yield and quality. This special issue hosts a collection of original research articles, mainly based on contributions from the 11th International Plant Sulfur Workshop held on 16-20 September 2018 in Conegliano, Italy, focusing on the following topics: (1) The germinative and post-germinative behaviour of Brassica napus seeds when severe S limitation is applied to the parent plants; (2) the independence of S deficiency from the mRNA degradation initiation enzyme PARN in Arabidopsis; (3) the glucosinolate distribution in the aerial parts of sel1-10, a disruption mutant of the sulfate transporter SULTR1;2, in mature Arabidopsis thaliana plants; (4) the accumulation of S-methylcysteine as its γ-glutamyl dipeptide in Phaseolus vulgaris; and (5) the role of ferric iron chelation-strategy components in the leaves and roots of maize, have provided new insights into the effect of S availability on plant functionality. Moreover, the role of S deficiency in root system functionality has been highlighted, focusing on (6) the contribution of root hair development to sulfate uptake in Arabidopsis, and (7) the modulation of lateral root development by the CLE-CLAVATA1 signaling pathway under S deficiency. The role of S in plants grown under drought conditions has been investigated in more detail focusing (8) on the relationship between S-induced stomata closure and the canonical ABA signal transduction machinery. Furthermore, (9) the assessment of S deficiency under field conditions by single measurements of sulfur, chloride, and phosphorus in mature leaves, (10) the effect of fertilizers enriched with elemental S on durum wheat yield, and (11,12) the impact of elemental S on the rhizospheric bacteria of durum wheat contributed to enhance the scientific knowledge on S nutrition under field conditions.

Autophagy Controls Sulphur Metabolism in the Rosette Leaves of Arabidopsis and Facilitates S Remobilization to the Seeds.

Sulphur deficiency in crops became an agricultural concern several decades ago, due to the decrease of S deposition and the atmospheric sulphur dioxide emissions released by industrial plants. Autophagy, which is a conserved mechanism for nutrient recycling in eukaryotes, is involved in nitrogen, iron, zinc and manganese remobilizations from the rosette to the seeds in Arabidopsis thaliana. Here, we have compared the role of autophagy in sulphur and nitrogen management at the whole plant level, performing concurrent labelling with 34S and 15N isotopes on atg5 mutants and control lines. We show that both 34S and 15N remobilizations from the rosette to the seeds are impaired in the atg5 mutants irrespective of salicylic acid accumulation and of sulphur nutrition. The comparison in each genotype of the partitions of 15N and 34S in the seeds (as % of the whole plant) indicates that the remobilization of 34S to the seeds was twice more efficient than that of 15N in both autophagy mutants and control lines under high S conditions, and also in control lines under low S conditions. This was different in the autophagy mutants grown under low S conditions. Under low S, the partition of 34S to their seeds was indeed not twice as high but similar to that of 15N. Such discrepancy shows that when sulphate availability is scarce, autophagy mutants display stronger defects for 34S remobilization relative to 15N remobilization than under high S conditions. It suggests, moreover, that autophagy mainly affects the transport of N-poor S-containing molecules and possibly sulphate.

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.

Transcriptional Reprogramming of Pea Leaves at Early Reproductive Stages.

Pea (Pisum sativum L.) is an important source of dietary proteins. Nutrient recycling from leaves contributes to the accumulation of seed proteins and is a pivotal determinant of protein yields in this grain legume. The aim of this study was to unveil the transcriptional regulations occurring in pea leaves before the sharp decrease in chlorophyll breakdown. As a prelude to this study, a time-series analysis of 15N translocation at the whole plant level was performed, which indicated that nitrogen recycling among organs was highly dynamic during this period and varied depending on nitrate availability. Leaves collected on vegetative and reproductive nodes were further analyzed by transcriptomics. The data revealed extensive transcriptome changes in leaves of reproductive nodes during early seed development (from flowering to 14 days after flowering), including an up-regulation of genes encoding transporters, and particularly of sulfate that might sustain sulfur metabolism in leaves of the reproductive part. This developmental period was also characterized by a down-regulation of cell wall-associated genes in leaves of both reproductive and vegetative nodes, reflecting a shift in cell wall structure. Later on, 27 days after flowering, genes potentially switching the metabolism of leaves toward senescence were pinpointed, some of which are related to ribosomal RNA processing, autophagy, or transport systems. Transcription factors differentially regulated in leaves between stages were identified and a gene co-expression network pointed out some of them as potential regulators of the above-mentioned biological processes. The same approach was conducted in Medicago truncatula to identify shared regulations with this wild legume species. Altogether the results give a global view of transcriptional events in leaves of legumes at early reproductive stages and provide a valuable resource of candidate genes that could be targeted by reverse genetics to improve nutrient remobilization and/or delay catabolic processes leading to senescence.

Seed Yield Components and Seed Quality of Oilseed Rape Are Impacted by Sulfur Fertilization and Its Interactions With Nitrogen Fertilization.

Although the impact of sulfur (S) availability on the seed yield and nutritional quality of seeds has been demonstrated, its impact coupled with nitrogen (N) availability remains poorly studied in oilseed rape. A deeper knowledge of S and N interactions on seed yield components and seed quality could improve S and N fertilization management in a sustainable manner. To address this question, our goals were to determine the effects of nine different S fertilization management strategies (i) in interaction with different levels of N fertilization and (ii) according to the timing of application (by delaying and fractionating the S inputs) on agronomic performances and components of seed yield. The impact of these various managements of S and N fertilizations was also investigated on the seed quality with a focus on the composition of SSPs (mainly represented by napins and cruciferins). Our results highlighted synergetic effects on S and N use efficiencies at optimum rates of S and N inputs and antagonistic effects at excessive rates of one of the two elements. The data indicated that adjustment of S and N fertilization may lead to high seed yield and seed protein quality in a sustainable manner, especially in the context of reductions in N inputs. Delaying S inputs improved the seed protein quality by significantly increasing the relative abundance of napin (a SSP rich in S-containing amino acids) and decreasing the level of a cruciferin at 30 kDa (a SSP with low content of S-amino acids). These observations suggest that fractionated or delayed S fertilizer inputs could provide additional insights into the development of N and S management strategies to maintain or improve seed yield and protein quality. Our results also demonstrated that the S% in seeds and the napin:30 kDa-cruciferin ratio are highly dependent on S/N fertilization in relation to S supply. In addition, we observed a strong relationship between S% in seeds and the abundance of napin as well as the napin:30 kDa-cruciferin ratio, suggesting that S% may be used as a relevant index for the determination of protein quality in seeds in terms of S-containing amino acids.

A Genotypic Comparison Reveals That the Improvement in Nitrogen Remobilization Efficiency in Oilseed Rape Leaves Is Related to Specific Patterns of Senescence-Associated Protease Activities and Phytohormones.

Oilseed rape (Brassica napus L.) is an oleoproteaginous crop characterized by low N use efficiency (NUE) that is mainly related to a weak Nitrogen Remobilization Efficiency (NRE) during the sequential leaf senescence of the vegetative stages. Based on the hypothesis that proteolysis efficiency is crucial for the improvement of leafNRE, our objective was to characterize key senescence-associated proteolytic mechanisms of two genotypes (Ténor and Samouraï) previously identified with contrasting NREs. To reach this goal, biochemical changes, protease activities and phytohormone patterns were studied in mature leaves undergoing senescence in two genotypes with contrasting NRE cultivated in a greenhouse under limiting or ample nitrate supply. The genotype with the higher NRE (Ténor) possessed enhanced senescence processes in response to nitrate limitation, and this led to greater degradation of soluble proteins compared to the other genotype (Samouraï). This efficient proteolysis is associated with (i) an increase in serine and cysteine protease (CP) activities and (ii) the appearance of new CP activities (RD21-like, SAG12-like, RD19-like, cathepsin-B, XBCP3-like and aleurain-like proteases) during senescence induced by N limitation. Compared to Samouraï, Ténor has a higher hormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes senescence, particularly under low N conditions, and this is correlated with the stronger protein degradation and serine/CP activities observed during senescence. Short statement: The improvement in N recycling during leaf senescence in a genotype of Brassica napus L. characterized by a high nitrogen remobilization efficiency is related to a high phytohormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes leaf senescence and is correlated with an increase or the induction of specific serine and cysteine protease activities.

Germinative and Post-Germinative Behaviours of Brassica napus Seeds Are Impacted by the Severity of S Limitation Applied to the Parent Plants.

In oilseed rape (Brassica napus L.), sulphur (S) limitation leads to a reduction of seed yield and nutritional quality, but also to a reduction of seed viability and vigour. S metabolism is known to be involved in the control of germination sensu stricto and seedling establishment. Nevertheless, how the germination and the first steps of plant growth are impacted in seeds produced by plants subjected to various sulphate limitations remains largely unknown. Therefore, this study aimed at determining the impact of various S-limited conditions applied to the mother plants on the germination indexes and the rate of viable seedlings in a spring oilseed rape cultivar (cv. Yudal). Using a 34S-sulphate pulse method, the sulphate uptake capacity during the seedling development was also investigated. The rate of viable seedlings was significantly reduced for seeds produced under the strongest S-limited conditions. This is related to a reduction of germination vigour and to perturbations of post-germinative events. Compared to green seedlings obtained from seeds produced by well-S-supplied plants, the viable seedlings coming from seeds harvested on plants subjected to severe S-limitation treatment showed nonetheless a higher dry biomass and were able to enhance the sulphate uptake by roots and the S translocation to shoots.

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.

Sensitivity analyses for improving sulfur management strategies in winter oilseed rape.

Because sulfur (S) depletion in soil results in seed yield losses and grain quality degradation, especially in high S-demanding crops such as oilseed rape (Brassica napus L.), monitoring S fertilisation has become a central issue. Crop models can be efficient tools to conduct virtual experiments under different fertilisation management strategies. Using the process-based model SuMoToRI, we aimed to analyse the impact of different S fertilisation strategies coupled with the variablility observed in major plant characteristics in oilseed rape i.e. radiation use efficiency (RUE), carbon (C) allocation to the leaves (ß) and specific leaf area (SLA) on plant performance-driven variables encompassing total biomass (TDW), S in the photosynthetic leaves (QSmobile.GL) and leaf area index (LAIGL). The contrasting S supply conditions differed in the amount of S (5 levels), and the timing of application (at bolting and/or at flowering, which included a fractioned condition). For this purpose, we performed a global sensitivity analysis (GSA) and calculated two sensitivity indices i.e. the Partial Raw Correlation Coefficient (PRCC) and the Sobol index. The results showed that whatever the timing of S supply, TDW, LAIGL and QSmobile.GL increased as S input increased. For a given S supply, there was no difference in TDW, LAIGL and QSmobile.GL between a single and a fractioned supply. Moreover, delaying the supply until flowering reduced the TDW and LAIGL whereas QSmobile.GL increased. Results showed that RUE had the greatest impact on TDW under all levels of S supply and all application timings, followed by ß and SLA. RUE mostly impacted on QSmobile.GL, depending on S supply conditions, whereas it was the parameter with the least impact on LAIGL. Ultimately, our results provide strong evidence of optimised S fertilisation timings and plant characteristics that will guide producers in their agricultural practices by using specific varieties under constrained S fertilisation strategies.

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.

Proteomic Investigations of Proteases Involved in Cotyledon Senescence: A Model to Explore the Genotypic Variability of Proteolysis Machinery Associated with Nitrogen Remobilization Efficiency during the Leaf Senescence of Oilseed Rape.

Oilseed rape is characterized by a low nitrogen remobilization efficiency during leaf senescence, mainly due to a lack of proteolysis. Because cotyledons are subjected to senescence, it was hypothesized that contrasting protease activities between genotypes may be distinguishable early in the senescence of cotyledons. To verify this assumption, our goals were to (i) characterize protease activities in cotyledons between two genotypes with contrasting nitrogen remobilization efficiency (Ténor and Samouraï) under limiting or ample nitrate supply; and (ii) test the role of salicylic acid (SA) and abscisic acid (ABA) in proteolysis regulation. Protease activities were measured and identified by a proteomics approach combining activity-based protein profiling with LC-MS/MS. As in senescing leaves, chlorophyll and protein contents decrease in senescing cotyledons and are correlated with an increase in serine and cysteine protease activities. Two RD21-like and SAG-12 proteases previously associated with an efficient proteolysis in senescing leaves of Ténor are also detected in senescing cotyledons. The infiltration of ABA and SA provokes the induction of senescence and several cysteine and serine protease activities. The study of protease activities during the senescence of cotyledons seems to be a promising experimental model to investigate the regulation and genotypic variability of proteolysis associated with efficient N remobilization.

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.