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To investigate the role of vacuolar sulfate in seed yield and quality, we have targeted the single pea SULTR4 gene (PsSULTR4), which encodes a transporter homologous to Arabidopsis SULTR4;1 and 4;2 that allow sulfate efflux from the vacuole to the cytosol. By simulating the 3D structure of PsSULTR4, we observed that it is similar to that of SULTR4;1 in Arabidopsis. Furthermore, a phylogenetic analysis revealed a high level of conservation of SULTR4 protein motifs across land species. A fluorescent protein fusion experiment confirmed that PsSULTR4 localizes to the vacuolar membrane.Five sultr4 mutants were identified by TILLING (Targeting Induced Local Lesions IN Genomes), two of which showed a reduced seed yield under sulfur deficiency. These mutants are currently being characterized by studying SULTR4 mRNA and protein stability, and the effect of the mutations on the simulated 3D structure. Seed yield of these two mutants was not affected under sulfur-sufficient conditions but changes in seed protein composition were observed. In particular, less sulfur-rich PA1 albumins accumulated in mature seeds. This is likely to be associated with a reduced utilization of sulfate within the seeds, since the total seed sulfur content was unchanged compared to the wild-type, whereas the seed sulfate concentration was significantly higher in both mutants. This highlights the crucial role of vacuolar sulfate in maintaining seed quality, but also seed yield when sulfur is limiting.

Dans leur environnement naturel, les plantes doivent faire face à plusieurs stress biotiqueset abiotiques au cours de leur cycle de développement. Certains de ces stress peuventsurvenir au même moment – comme le stress hydrique et les carences nutritionnelles – etpeuvent avoir des effets synergiques, antagonistes ou additifs sur les réponses moléculairesdes plantes. Chez le pois (Pisum sativum), il a été observé que l’effet de la carence en soufre(S) sur la composition protéique des graines peut être atténué lorsque cette carence estcombinée à un stress hydrique (Henriet et al., 2019). Afin de mieux caractériser les réponsesmoléculaires du pois au stress hydrique et/ou à la carence en S, une analyse multi-omiques(transcriptomique, protéomique, métabolomique, ionomique) a été réalisée à partir defeuilles récoltées à différents jours post-floraison, sur différents lots de plantes ayant subi ounon un stress hydrique, une carence en S, ou une combinaison des deux stress. Nos analysesont révélé que 21% des ARNm, protéines, métabolites et ions qui répondentsignificativement aux stress, ont une réponse accentuée lorsque les deux types de stresssont combinés, suggérant des effets synergiques ou additifs. En utilisant une approcheintégrative de réseau de co-expression, nous avons mis en évidence des gènes fortementrégulés par les changements de concentration en S dans les feuilles. La comparaison desdonnées de transcriptomique et protéomique nous permet de formuler des hypothèsesquant au niveau de régulation (transcriptionnel ou post-transcriptionnel) des gènes deréponse aux stress identifiés. Enfin, ce travail nous permet de proposer des listes de gènescandidats qui pourraient jouer un rôle dans la tolérance des plantes aux situations de multistress.

Grain legumes have a key role to play in both agroecological and food transitions. Indeed, these plants are able to accumulate large amounts of proteins in their seeds even in the absence of nitrogen fertilization thanks to symbiotic N2 fixation in the root nodules. However, legumes are exposed to abiotic stresses, including nutrient deficiencies, making it important to optimize nutrient use efficiency for maintaining seed protein content and quality. Seed protein quality refers to the ability of the seed proteins to meet the body’s requirements for essential amino acids. It strongly depends on the amino acid balance, which determines protein digestibility. In pea (Pisum sativum) seeds, methionine and cysteine are, with tryptophan, the most limiting essential amino acids. Although the literature suggests that sulfate assimilation during seed development may be a source of sulfur amino acids for the synthesis of sulfur-rich storage proteins, work is still needed to understand how sulfate transport and metabolism regulate seed protein composition.Sulfate ions are transported in plant tissues by sulfate transporters (SULTR) encoded by the SULTR gene family. These ions are taken up from the rhizosphere by high-affinity transporters of group 1 (SULTR1) and their root-to-shoot transport is ensured by SULTR2 and SULTR3 transporters, which have also been shown to play a role in the translocation of sulfate within developing siliques or seeds in Arabidopsis. In the sink organs, sulfate can be stored in the vacuoles and remobilized via SULTR4 transporters, which allow sulfate efflux for further assimilation. Sulfate assimilation starts by a reaction catalyzed by ATP sulfurylase (ATPS), which produces Adenosine 5’-PhosphoSulfate (APS). APS can be reduced into sulfite ion (SO32-) and sulfide (S2-) by APS reductase (APR) and sulfite reductase (SiR), respectively. Sulfide can be incorporated into O-acetylserine (OAS) to provide cysteine, which is the precursor for the synthesis of methionine and glutathione (GSH). Here, we focused on the only gene encoding a SULTR4 transporter in pea to investigate its role in providing sulfate for the synthesis of sulfur-rich storage proteins (2S albumins, 11S globulins) in seeds. Two mutants of this gene were identified through a screening of the TILLING (Targeting Induced Local Lesions IN Genomes) population developed using the pea ‘Caméor’ cultivar. The first mutant, called W/-, has a nonsense mutation at position W78. The second mutant, called E/K, has a missense mutation leading to a substitution of a glutamate to a lysine at position 586 (E586K) in the STAS domain (for Sulfate Transporter and Anti-Sigma antagonist), which is essential for the transport function [1]. These mutants and their corresponding wild-type lines (same genetic background) were phenotyped under sulfur-sufficient and sulfur-deficient conditions. Seed yield of the sultr4 mutants was unchanged compared to the wild-type when the plants were cultivated under sulfur sufficiency, suggesting that sultr4 mutants use sulfate absorbed by roots and/or sulfur metabolites instead of vacuolar sulfate to maintain seed production. Nevertheless, seed yield of sultr4 mutants was significantly reduced under sulfur deficiency, suggesting a pivotal role for the remobilization of vacuolar sulfate to maintain protein biosynthesis in seeds when external sulfate supply is low. The results also revealed significant changes in seed protein composition of the sultr4 mutants, even under sulfur sufficiency. In this condition, the relative abundance of the sulfur-rich PA1 albumins was lower in mutant seeds. Moreover, sulfate content of these mature mutant seeds was two times higher than that of wild-type seeds, but seed sulfur content (and sulfur quantity per seed) were unchanged. Altogether, the data uncover a defect in the utilization of vacuolar sulfate within the developing mutant seeds. Based on these findings, we suggest that vacuolar sulfate remobilization during seed development is a vital sulfur source for the synthesis of sulfur-rich 2S albumins in seeds even under ample external sulfur supply. We studied the kinetics of seed development in the mutant and wild-type lines by measuring the fresh weight, dry weight and water content of seeds collected under sulfur-sufficient conditions. There was no change in these parameters kinetics compared to the wild-type until around 400 degree-days after pollination (that is to say that 400 is cumulative sum of daily mean temperature in °C between pollination day and seed sampling day). This result indicates that both sultr4 mutants, E/K and W/-, seem to have a normal early seed development. After this stage, water content of the W/- mutant seeds decreased faster than that of wild-type seeds, indicating that a complete loss of function of the SULTR4 transporter accelerates seed maturation. We further investigated the contribution of vacuolar sulfate to seed protein accumulation by focusing on the stages where no change in the seed development kinetics was observed. Wild-type and mutant seeds were harvested at 257 degree-days (embryogenesis) and 312 degree-days (early seed filling). We studied, by qRT-PCR, the expression of genes encoding enzymes of sulfur metabolism and seed storage proteins. For both mutants, the expression of genes involved in sulfate reduction (ATPS1 and APR3) increased during embryogenesis compared to the wild type, whereas the expression of several genes encoding sulfur-rich storage proteins (PA1 and PA2 albumins, and legumin) decreased at the early filling stage. This suggests that vacuolar sulfate availability could regulate storage protein production and consequently influence nutritional seed quality. Based on these data, we present a hypothetical model of the impact of vacuolar sulfate in fine-tuning sulfur metabolism and storage protein synthesis during pea seed development. Further experiments are currently performed to confirm this model.

Les graines de pois constituent un aliment sain et nourrissant, notamment de par leur forte teneur en protéines (environ 25%). La qualité nutritionnelle de ces protéines est néanmoins limitée par leur faible proportion en acides aminés soufrés (AAS) : cystéine et méthionine. L’objectif était d’étudier la contribution du stock de sulfate contenu dans les vacuoles à l’élaboration du rendement et de la qualité des graines, notamment la synthèse des AAS. Nous avons ciblé l’unique gène SULTR4 de pois qui code un transporteur permettant l’efflux de sulfate de la vacuole vers le cytosol. Après avoir confirmé sa localisation à la membrane vacuolaire, nous avons recherché des mutants de pois pour ce gène par la méthode TILLING (Targeting Induced Local Lesions IN Genomes). Parmi les cinq mutants obtenus, deux ont un rendement en graines moindre lorsqu’ils sont cultivés en condition de carence en soufre. L’un porte une mutation induisant l’arrêt de la séquence protéique et l’autre modifie un résidu extrêmement conservé dans les gènes SULTR4 chez les plantes terrestres. Le rendement en graines de ces deux mutants reste inchangé comparé au génotype sauvage lorsque les plantes sont bien alimentées en soufre. De manière intéressante, la composition protéique des graines est modifiée : la quantité relative en albumines PA1, protéines très riches en AAS, est plus faible comparé aux graines du génotype sauvage. Le gène SULTR4 s’exprime dans la graine de pois en développement, particulièrement au début de la phase de remplissage. Les graines des mutants sultr4 accumulent la même quantité de soufre que le type sauvage, mais la quantité de sulfate est plus élevée dans les graines de ces mutants. Ces résultats suggèrent un défaut d’utilisation du sulfate à l’intérieur même des graines. L’expression préférentielle de SULTR4 dans les téguments et l’albumen des graines suggère que ces tissus sont une source de sulfate pour la synthèse des AAS dans l’embryon.

Plants in their physical environment face multiple biotic and abiotic stresses duringtheir life cycle. In nature, environmental stresses often co-occur – such as water deficit andnutrient deficiencies – and can have synergistic, antagonistic or additive effects on the plantmolecular responses. In pea (Pisum sativum), combination of water stress (WS) and sulfur (S)deficiency showed a mitigation effect on the seed protein composition, as compared to Sdeficiency occurring alone (Henriet et al., 2019). To better understand how pea responds toWS and/or S deficiency, a multi-omics (transcriptomics, proteomics, metabolomics, ionomics)analysis has been performed from leaf samples collected during a time course, on differentlots of plants subjected to either WS, S deficiency, or to the combination of the two stressors.Our analyses revealed that 21% of the stress-responsive mRNAs, proteins, metabolites andions showed a stronger response when S deficiency occurs with WS, suggesting synergisticor additive effects. Using a weighted-gene co-expression network approach, we evidencedgenes strongly regulated by changes in the S concentration in leaves. The comparisonbetween our transcriptomics and proteomics data allows us to formulate hypotheses on thelevel of regulation (transcriptional or post-transcriptional) of these genes. We also proposecandidate genes that might help plants to better tolerate combinatorial stresses.

We report on the interplay between water deficit and sulfur deficiency, two constraints that are increasingly faced by crops due to climate change and low-input agricultural practices. In particular, we aim at better understanding the role of sulfur nutrition in the trade-off between seed quality establishment and plant stress tolerance in pea (Pisum sativum L.), a grain legume crop which has a pivotal role to play in both agroecological and food transitions. Like other legumes, pea is able to accumulate large amounts of seed proteins even in the absence of nitrogen fertilizers thanks to its symbiosis with N2-fixing soil bacteria. In this study, we deprived pea plants (cv. Caméor) of sulfur from the mid-vegetative stage and applied a moderate water deficit for 9 days starting at flowering. Individual stresses and control conditions (well-watered, non-limiting sulfur conditions) were applied in parallel for comparison. Phenotypic measurements up to maturity revealed that the combination of the stresses impeded reproductive processes in a synergistic manner, reducing one-seed weight and seed number, thus highlighting the paramount importance of sulfur for maintaining seed yield components when plants encounter a moderate water deficit [1]. Focusing on seed quality attributes, water deficit mitigated the negative effect of sulfur deficiency on the accumulation of sulfur-rich globulins (11S) in mature seeds. Patterns of plant nutrient allocation, including the quantity of sulfur and nitrogen per seed, reflected a lower seed sink strength for nitrogen in the double-stressed plants compared to sulfur-deficient plants. This may readjust the nitrogen to sulfur ratio in the double-stressed seeds, thus rebalancing the amount of 7S (sulfur-poor) and 11S globulins. To uncover the molecular processes underlying the interplay between the two stresses, seeds and leaves, which are respectively sink and source of nutrients, were collected at different time points before, during and after the double stress period and subjected to multi-omics profiling studies. Inference of a protein network using the seed proteomic data identified a cluster of antioxidant proteins (including a glutathione S-transferase, a methionine sulfoxide reductase, and a thioredoxin) that may maintain redox homeostasis in early developing seeds and prevent cellular damage under stress conditions. Integration of these proteomic data with transcriptomic data at the transition to seed filling revealed transcriptional events associated with the accumulation of these antioxidant proteins [2]. This transcriptional defense response mainly involves genes of sulfate homeostasis and assimilation, which is reminiscent to our previous work in Arabidopsis showing that sulfate remobilization in seeds is likely to contribute to the seed's defense against oxidative stress [3]. This study thus provides candidates for targeted studies aimed at dissecting the signaling cascade linking sulfate metabolism to antioxidant processes in developing seeds. These results also highlight the importance of addressing the adaptive mechanisms used by plants to regulate sulfur homeostasis under abiotic constraints. Analysis of the proteomic and transcriptomic data obtained from the leaf samples revealed profound changes in response to water deficit and/or sulfur deficiency, especially when the two stresses were combined. Integration of these data with ionomics data obtained from the same leaf samples, using a weighted-gene co-expression network approach, highlighted genes associated with changes in leaf nutrient concentration, including sulfur concentration. The comparison between leaf transcriptomic and proteomic data allows us to formulate hypotheses on the level of regulation (transcriptional or post-transcriptional) of these genes. We also propose candidate genes that might help plants to better tolerate combinatorial stresses by contributing to adjust nutrient homeostasis in leaves.

Legumes have a key role to play in both agroecological and food transitions due to their ability to accumulate large amountsof seed proteins without nitrogen fertilization thanks to symbiotic N2 fixation in the root nodules. However, in agroecologicalsystems, legumes are more exposed to nutrient deficiencies, including sulfur deficiency, than in conventional systems, makingit important to optimize nutrient use efficiency for maintain seed protein quality, in particular the level of (semi) essentialamino acids like methionine and cysteine. These sulfur-containing amino acids are synthetized through the sulfur metabolicpathway starting from sulfate reduction. Sulfate is taken up from the soil and transported in plant parts by sulfate transporters(SULTR) and can be stored in the vacuoles for further remobilization by SULTR4 transporters when sulfur availability remainsscarce. Here, we investigated the contribution of vacuolar sulfate to seed yield and quality by using two mutants of the onlySULTR4 gene that exists in pea (Pisum sativum). Seed yield of the two mutants was significantly reduced in response to sulfurdeficiency, highlighting the prominent role of this transporter in stabilizing seed yield when sulfate is limiting. Interestingly,under sulfur-sufficient conditions, the mature sultr4 seeds accumulate lower amounts of sulfur-rich proteins but displayed ahigher sulfate concentration compared to the wild-type seeds. These data and the unchanged sulfur content of the mature sultr4seeds suggest that vacuolar sulfate remobilization within developing seeds contributes to storage protein synthesis. A geneexpression analysis, by qRT-PCR, revealed an up-regulation of genes involved in sulfate reduction and a down-regulation ofgenes encoding sulfur-rich storage proteins in both mutant seeds. Based on these results, we present a hypothetical model ofthe impact of vacuolar sulfate in fine-tuning sulfur metabolism and storage protein synthesis during pea seed development.Further experiments are currently performed to confirm this model.

Les légumineuses sont capables d’accumuler des quantités importantes de protéines dans leurs graines même en l’absence d’engrais azoté, ce qui fait d’elles des espèces à haut potentiel pour relever les défis alimentaires et accompagner la transition agroécologique. Des avancées génétiques ont permis de réduire la présence de certains facteurs « anti-nutritionnels » dans les graines de pois et de féverole (exemple des variétés pauvres en tanins, en inhibiteurs trypsiques, ou en vicine et convicine). Afin de promouvoir l’utilisation de ces graines en alimentation humaine, il est nécessaire d’aller plus loin dans l’amélioration de leur valeur nutritionnelle, en termes de teneur et composition en protéines, d’équilibre en acides aminés, de goût, mais aussi de résistance vis-à-vis des insectes, notamment les bruches qui se nourrissent des cotylédons. A cet effet, le levier de la sélection peut s’appuyer sur une diversité génétique naturelle et/ou induite pour ces caractères, révélée dans des travaux récents. Cette variabilité sera présentée, ainsi que les outils mobilisables pour mieux adapter les graines de légumineuses aux usages alimentaires. Parmi ces outils, est le déploiement des approches translationnelles pour l’étude de la conservation des déterminants génétiques et le transfert d’acquis entre espèces légumineuses (Medicago truncatula, pois, féverole, lentille).

L'utilisation de légumineuses comme source de protéines dans les produits alimentaires transformés est souvent limitée par le goût « vert » désagréable qu'elles peuvent conférer. Parmi les composés source de mauvais goût figurent les saponines, des triterpènes glycosylés qui contribuent à l'amertume, et les composés organiques volatiles (COV), notamment l’héxanal, qui proviennent de l’oxydation des acides gras insaturés présents dans les graines par des lipoxygénases, et qui donnent un goût « vert, herbeux » au produit fini. Les méthoxypyrazines se trouvent également en petites quantités dans les graines et pourraient être aussi impliquées dans la détermination de la note verte, bien que leur origine et leur biosynthèse soient moins claires. En outre, certains de ces composés, notamment les saponines, peuvent jouer un rôle de défense contre des agents pathogènes ou des ravageurs.Lors des processus de transformation des légumineuses, ces composés indésirables sont actuellement éliminés par des extractions sélectives ou par des traitements thermiques, ou leur goût est masqué par l'ajout d'autres produits chimiques. Si une approche génétique pouvait être utilisée, ces procédures énergivores et coûteuses pourraient être évitées.Des travaux récents réalisés dans le cadre du projet LEG’UP ont montré qu’il était possible d’exploiter la variabilité induite par TILLING (Targeting Induced Local Lesions IN Genomes) pour diminuer l’accumulation de ces composés «indésirables» dans les graines de pois. Le TILLING est une technique non OGM qui permet le criblage à grande échelle de collections de mutants induits chimiquement, pour repérer des mutations dans un gène donné. Ces populations ont été exploitées afin de rechercher des mutations dans un gène clé de la voie de biosynthèse des saponines, la β-amyrine synthase PsBAS1, ainsi que dans deux lipoxygénases majeures de la graine PsLOX2 et PsLOX3. Ces travaux ont conduit à l’identification de lignées présentant des teneurs en saponines des graines réduites à plus de 97% (Vernoud et al., 2021), ou une activité lipoxygénase réduite de 80%. Ces lignées sont maintenant disponibles pour des analyses de perception organoleptique et de composés organiques volatiles dans des farines, fractions protéiques ou ingrédients dérivés.

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National audience; Dans le contexte du changement climatique, des épisodes de stress hydrique plus fréquents sont attendus, entraînant des modifications de nutrition, de croissance des plantes, et donc des pertes de rendements. Chez les plantes légumineuses, telles que le pois protéagineux, un stress hydrique du sol diminue drastiquement l’acquisition de l’azote (N) par la fixation symbiotique du N2 atmosphérique, conduisant à une carence azotée de la plante et pouvant diminuer le rendement de 30 à 60% suivant les variétés. Il apparait donc nécessaire de sélectionner des génotypes de pois mieux adaptés à la sécheresse. Dans cette étude, les réponses architecturales, physiologiques et transcriptionnelles des racines et des nodosités de pois à un épisode de stress hydrique, suivi d'une période de récupération, ont été étudiées. Nous explorerons les potentielles relations entre traits d’architecture racinaire et tolérance au stress hydrique, via l’analyse d’une diversité de génotypes de pois soumise à un stress hydrique. Puis, nous nous focaliserons sur un nombre de génotypes plus restreint afin d’étudier soit la réponse physiologique du pois à des niveaux de disponibilité en eau, soit les bases moléculaires associées durant la phase de stress et la période de récupération lors d’un réarrosage.

International audience; Water stress and sulfur-deficiency are two constraints increasingly faced by crops due to climatechange and low-input practices. To investigate their interplay in the grain legume pea (Pisum sativumL.), sulfate was depleted at mid-vegetative stage and a moderate 9-day water stress period was imposedduring the early reproductive phase. The combined stress accelerated seed production, lowering yield,one-seed weight and seed number per plant, but rebalanced seed protein composition. In fact, themoderate water stress mitigated the negative effect of sulfur-deficiency on the accumulation of sulfurrichproteins in seeds, probably due to a lower seed sink strength for nitrogen, enabling a readjustmentof the sulfur-poor/sulfur-rich globulin ratio. These results uncovered the importance of sulfur forstabilizing seed yield in pea facing short and moderate water stress episodes, and showed that theadaptive response of sulfur-deprived plants to water stress is much more complicated than a simpleadditive response. Developing seeds were subjected to shotgun proteomics and transcriptomics toadvance our knowledge of the seed response to the single or double stresses. Among the results thatwill be presented is the building of a protein network together with a differential analysis, highlightinghubs potentially implicated in seed development and stress response.

National audience; The use of pulses as ingredients for the production of food products rich in plant proteins is increasing. However, protein fractions prepared from pea or other pulses contain significant amounts of saponins, glycosylated triterpenes which can impart a bitter taste to the final food product. Bitter flavours are currently either removed by energy-requiring physico-chemical treatments or masked by additives. We are in the process of identifying and characterizing the genes involved in saponin biosynthesis during pea seed development, with the objective of identifying mutants in which seed saponins no longer accumulate. To do this we have applied a saponin extraction protocol to follow the biosynthesis of these compounds during the development of pea seeds, and have identified mutants in a Pea TILLING population (1) that correspond to key steps in the saponin biosynthetic pathway. A mutation in a previously identified β-Amyrin Synthase gene (2), which is highly expressed in maturing pea seeds, reduced mature seed saponin content by 97% (3). Acknowledgement: This study is funded under the LEG'UP FUI (Unique Interministerial Fund) project (AAP No. 18)

International audience; Water stress and sulfur-deficiency are two constraints increasingly faced by crops due to climate change and low-input practices. To investigate their interplay in the grain legume pea (Pisum sativum L.), sulfate was depleted at mid-vegetative stage and a moderate 9-day water stress period was imposed during the early reproductive phase. The combined stress accelerated seed production, lowering yield, one-seed weight and seed number per plant, but rebalanced seed globulin composition. In fact, the moderate water stress mitigated the negative effect of sulfur-deficiency on the accumulation of sulfur-rich globulins in seeds, probably due to a lower seed sink strength for nitrogen, enabling a readjustment of the sulfur-poor/sulfur-rich globulin ratio. These results uncovered the importance of sulfur for stabilizing seed yield in pea facing short and moderate water stress episodes, and showed that the adaptive response of sulfur-deprived plants to water stress is much more complicated than a simple additive response. A transcriptome analysis of developing seeds at the end of the combined stress period clearly advanced our knowledge of the seed response to the single or double stresses. Among the results that will be presented is a specific up-regulation of a set of genes in response to the combined stress, indicating the establishment of unique regulatory processes when sulfur-deficiency is combined with water stress and highlighting candidate genes to fine-tune the regulation of globulin synthesis under stressed conditions.

National audience; Pea (Pisum sativum) produces seeds rich in proteins. However, protein content and seed quality can be impacted by environmental factors including drought. Accumulation of seed storage proteins (SSP) during seed filling is a highly regulated process. While proteomics of SSP deposition in pea seeds is well documented, knowledge of the underlying regulatory gene networks, either in control or stress conditions, is lacking. In this study, a RNA-seq based transcriptome analysis of seed development in control and water stress condition was carried out. Developing seeds were collected at six time points, from late embryogenesis to early maturation. RNA-seq was performed on an Illumina Hiseq3000 and reads were mapped on Pisum sativum v1a genome version. A complete RNA-seq workflow analysis including (i) the annotation of new genes using StringTie, (ii) the analysis of unmapped reads (which could represent up to 24% of the total reads in some samples) using RepeatExplorer and Trinity and (iii) the differential expression analysis using Deseq2, will be presented. These data will help us to decipher the transcriptional regulatory network that controls pea seed development, notably during SSP deposition, and how these processes are impacted by drought. This study is funded under the REGULEG ANR Project (ANR-15-CE20-0001-02) coordinated by J. Buitink (INRA, Angers, France)

National audience; Le pois (Pisum sativum) possède la capacité de fixer l’azote atmosphérique via une symbiose avec des bactéries du sol, au sein de structures racinaires appelées nodosités, permettant ainsi de s’affranchir de l’apport d’engrais azotés pour sa culture. Cependant, la fixation symbiotique de l’azote est un processus très sensible au stress hydrique qui l’affecte négativement. Bien que la capacité d’une plante à récupérer après un stress hydrique puisse être déterminante pour sa survie et l’élaboration de son rendement, les mécanismes enclenchés lors de cette phase restent peu connus. Afin d’évaluer la capacité du pois à récupérer après un stress hydrique, notamment en termes de reprise de croissance et d’acquisition d’azote, et d’identifier les processus écophysiologiques sous-jacents à cette récupération, deux génotypes contrastés pour leur architecture racinaire et pour leur capacité à récupérer après un stress hydrique ont été sélectionnés. Ils ont été cultivés au sein de la plateforme de phénotypage à haut-débit 4PMI de Dijon et ont subi un stress hydrique suivi d’une période de ré-arrosage durant lesquelles une cinétique de prélèvements a été réalisée. Les processus écophysiologiques impliqués dans la récupération après le stress et en lien avec les flux d’eau, de carbone et d’azote au sein de la plante ont été analysés en utilisant un cadre d’analyse de type structure-fonction. L’efficacité des deux stratégies de récupération ainsi que les mécanismes associés ont ainsi pu être caractérisés et seront présentés.

Water stress and sulfur (S) deficiency are two constraints increasingly faced by crops due to climate change and low-input agricultural practices. To investigate their interaction in the grain legume pea (Pisum sativum), sulfate was depleted at the mid-vegetative stage and a moderate 9-d water stress period was imposed during the early reproductive phase. The combination of the stresses impeded reproductive processes in a synergistic manner, reducing seed weight and seed number, and inducing seed abortion, which highlighted the paramount importance of sulfur for maintaining seed yield components under water stress. On the other hand, the moderate water stress mitigated the negative effect of sulfur deficiency on the accumulation of S-rich globulins (11S) in seeds, probably due to a lower seed sink strength for nitrogen, enabling a readjustment of the ratio of S-poor (7S) to 11S globulins. Transcriptome analysis of developing seeds at the end of the combined stress period indicated that similar biological processes were regulated in response to sulfur deficiency and to the combined stress, but that the extent of the transcriptional regulation was greater under sulfur deficiency. Seeds from plants subjected to the combined stresses showed a specific up-regulation of a set of transcription factor and SUMO ligase genes, indicating the establishment of unique regulatory processes when sulfur deficiency is combined with water stress., Moderate water stress combined with sulfur deficiency during seed development in pea synergistically affects reproductive processes, but mitigates the negative impact of the deficiency on the seed transcriptome and rebalances seed globulin composition.

International audience; Pea (Pisum sativum), like other legumes, has the unique ability to fix atmospheric dinitrogen (N2) via symbiosis with soil bacteria known as rhizobia in root nodules. This particular feature makes the pea crop an essential component of sustainable cropping systems because of the reduction of nitrogen fertilizers it affords. However symbiotic nitrogen fixation (SNF) is very susceptible to abiotic stresses and particularly to water deficit, which is becoming an increasingly common threat in the current context of climate change. Water deficit impacts negatively SNF (Prudent et al., 2016), affecting both nodule number and growth (i.e. structural components of SNF) and their N-fixing efficiency (i.e. fonctional component of SNF). Such negative effects can even remain when optimal water conditions are restored. Although the ability of a plant to recover after a water deficit period may determine both its survival and its yield at harvest, the mechanisms occurring during the rewatering phase in terms of nitrogen nutrition and growth recovery remain poorly addressed. The aim of this study was to evaluate the capacity of pea genotypes to recover after a water deficit and assess which processes, whether structural or functional, underlie this recovery. Two pea genotypes, Kayanne and Puget, were selected based on their contrasted nodulated root system architecture and their ability to recover after a water deficit period. Plants were cultivated in 4PMI2 (Dijon – France) and subjected to a two-week water deficit period during the vegetative stage, followed by an optimal rewatering until physiological maturity. During the water deficit period and the two first weeks of re-watering, a sampling time-series was performed. Physiological mechanisms potentially involved in plant stress tolerance and in its recovery were analysed by using a structure-function based ecophysiological framework. This analysis was complemented by a study of the carbon and nitrogen fluxes within the soil-plant-atmosphere continuum. Both genotypes responded similarly during the water deficit period. Plant growth was negatively affected, and among plant compartments, nodule growth was particularly impacted resulting in a decrease of the nodule biomass over nodulated root biomass ratio and leading to a reduction of plant nitrogen nutrition. Conversely the two pea genotypes displayed contrasted recovery strategies during the re-watering period. Kayanne increased carbon allocation to the nodule compartment allowing a restoration of the nodule biomass over nodulated root biomass ratio. Puget also favored nodules for carbon allocation but at the expense of the roots leading to an overcompensation of the nodule biomass over nodulated root biomass ratio compared to the control. Differences in the kinetics of nitrogen acquisition recovery were also observed, with Kayanne initiating a nitrogen uptake recovery earlier than Puget during rewatering. Interestingly, Puget’s yield but not Kayanne’s was significantly reduced in response to water deficit suggesting that Kayanne’s strategy during rewatering after a water deficit could be more efficient. Perspectives of this work are to use transcriptomics and metabolomics to identify key genes and metabolic pathways underlying these contrasted recovery strategies. This study will generate key knowledges allowing us to contribute to designing pea ideotypes which are better adapted to fluctuating water constraints.

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International audience; Given their ability to fix atmospheric nitrogen, legumes are pivotal to the development of sustainable agriculture in Europe as a source of protein for food and feed. Pea (Pisum sativum) is currently the leading grain legume crop in France and major efforts are being made to reintroduce legumes as protein crops in Europe. However, instability of seed yield and quality due to environmental fluctuations still represent a real barrier for the development of these cultures, and breeding for stable yields is needed. In pea, drought stress occurring during the reproductive phase can greatly affect seed yield and quality. We investigated the response of pea plants (var. Caméor) subjected to water stress during the seed filling period, a phase associated with massive remobilization of nutrients from the vegetative organs (including leaves) to sustain the seed’s high-nitrogen demand. Transcriptomic profiling of leaf response to water stress by hybridization of a 40k pea micro-array revealed metabolic and regulatory pathways affected by drought and enabled the selection of candidate genes for drought resistance. One of these genes, named RAMOSUS1, encodes a carotenoid cleavage dioxygenase involved in strigolactone biosynthesis. Interestingly, phenotyping of the corresponding mutant showed it to have increased sensitivity to drought compared to the wild-type, suggesting that strigolactones could act as positive regulators for crop abiotic stress resistance, as already shown for Arabidopsis.

National audience

National audience; Pea (Pisum sativum L.) produces seeds rich in proteins but yield and quality remain unstable across years due to various stresses. Sulfur (S) deficiency and water stress are two abiotic stresses that interact in the current context of climate change and low-input practices, and recent studies suggest an important role for sulfate transport and metabolism in the plant response to water stress. To investigate the interplay between sulfur nutrition and the water stress response, sulfate-deprived pea plants were subjected to a 9-days period of water stress during the early reproductive phase. While water stress did not impact seed yield, sulfur deficiency alone or combined with water stress decreased it by 38% and 63% respectively. Analysis of seed protein composition by one-dimensional electrophoresis revealed differences in the accumulation of sulfur-rich (11S) and sulfur-poor (7S) globulins in response to individual or combined stresses. To decipher the strategy used by plants to regulate the 11S/7S ratio in mature seeds, carbon, nitrogen (N) and sulfur partitioning between the different plant compartments was studied. Next, to pinpoint the mechanisms by which seeds adjust their metabolism under multi-stress conditions, early developing seeds were collected at three time points during drought and subjected to quantitative proteomics (shotgun). A total of 3184 proteins were identified and quantified, and 97 proteins were differentially accumulated in response to these stresses allowing the identification of candidate proteins that could control seed development or detoxification mechanisms.

International audience

National audience

BAP EA GEAPSI DOCT INRA; International audience

International audience; The use of pulses as ingredients for the production of food products rich in plant proteins is increasing. However, protein fractions prepared from pea or faba beans contain significant amounts of saponins, glycosylated triterpenes which can impart a bitter taste to the final food product. In addition, saponins have also been described to be involved in plant responses to biotic and abiotic stresses1. In this study, we identified and characterized the genes involved in saponin biosynthesis during pea seed development2, and optimized a saponin extraction protocol to follow the biosynthesis of these compounds during the development of pea and faba bean seeds. The identification of mutants affecting the function of key genes of the saponin biosynthetic pathway is currently underway in pea3. This study is funded under the LEG'UP FUI (Unique Interministerial Fund) project (AAP No. 18).

AGAP : équipe GE2popAGAP : équipe GE2pop; Seeds of legumes such as pea (Pisum sativum L.) provide proteins for animal feed and human nutrition. Although the so-called ²antinutritional factors² have been reduced in these seeds, offering globally good nutritional value, amino acid balance needs to be improved and stabilized, notably regarding sulfur amino acid content. By integrating omics data, notably from protein quantity loci and genome-wide association studies (GWAS using the HapMap platform[1]) on the abundance of storage proteins in Medicago truncatula seeds, we provide a repertory of genes involved in transcription, post-translational modifications, transport, or targeting of globulins to storage vacuoles [2]. Inference of a gene co-expression network between GWAS-derived transcription factors and globulin genes revealed key regulators of seed protein composition. A systematic search for orthologous sequences in the pea gene atlas[3] enabled us to transfer the knowledge to the target pea crop. The potential of this translational genomics approach for revealing genes important for seed nutritional quality improvement will be presented through pea TILLING lines[4] for selected candidate genes that exhibit seed protein profiles with increased abundance of sulfur-rich proteins. Because sulfur nutrition enables sulfur-rich protein accumulation[5], we further studied the leaf transcriptome of pea subjected to sulfur deficiency during the reproductive period. Genes involved in the supply of sulfur for accumulation of sulfur-rich proteins have been characterized by analysis of TILLING mutants.

EABAPGEAPSI DOCT INRA; Le pois (Pisum sativum) possède la capacité de fixer l’azote atmosphérique via une symbiose avec des bactéries du sol, permettant ainsi de s’affranchir d’engrais azotés. Cependant, ce processus est très sensible au stress hydrique, et reste affecté même lorsque les conditions hydriques redeviennent optimales. Or, la capacité d’une plante à récupérer après un stress hydrique peut être déterminante pour l’élaboration de son rendement. Pourtant, les mécanismes enclenchés lors de la phase de récupération restent peu connus. Le but de cette thèse est d’évaluer la capacité de la plante à récupérer après un stress hydrique et d’identifier les processus écophysiologiques et moléculaires ainsi que les signaux intra-plante sous-jacents à cette récupération. Pour cela, deux génotypes contrastés pour leur architecture racinaire et pour leur capacité à récupérer après un stress hydrique ont été sélectionnés. Les processus écophysiologiques impliqués dans la récupération après le stress seront analysés en utilisant un cadre d’analyse structure-fonction associé à une étude des flux d’eau, de carbone et d’azote au sein du continuum sol-plante-atmosphère. Les processus moléculaires seront étudiés via des analyses transcriptomiques et métabolomiques afin d’identifier respectivement les gènes clés et les principales voies métaboliques impliqués. Cette combinaison d’approches permettra de contribuer à la conception d’idéotypes de pois mieux adaptés à des conditions hydriques fluctuantes.

EABAPGEAPSI DOCT INRA; Pea (Pisum sativum L.) produces seeds rich in proteins, but seed yield and quality remain unstable across years due to abiotic stresses occurring during the reproductive period. Drought and sulfur deficiency are two abiotic stresses that interact in the current context of climate change and lowinput practices, and recent studies suggest a role of sulfate transport and metabolism in the plant response to drought (Ernst et al., 2010; Chan et al., 2013; Gallardo et al., 2014; Ahmad et al., 2016). In this study, we investigated the impact of sulfur deficiency combined with drought on the pea seed proteome. Pea plants were subjected to sulfur-deficiency two weeks after sowing, and a water stress was applied from flowering to the beginning of seed filling. Seeds were collected during embryogenesis (5, 9 and 12 days after pollination) and at maturity. The one-dimensional protein profile of mature seeds revealed changes in seed protein composition in response to combined stresses. A shotgun proteomics approach was then used to study the metabolic response of early developing seeds to drought combined with sulfur deficiency. A total of 2104 proteins were identified and quantified, and almost all of them vary during development, or in response to drought and/or sulfur deficiency. These data will enable us to build the metabolic pathways involved in pea seed embryogenesis and to establish a model by which early developing seeds adjust their metabolism under multi-stress conditions.

International audience

BAP EA GEAPSI INRA; International audience; In the context of climate change, more frequent episodes of water stress are expected, which will negatively impact symbiotic N2 fixation and consequentely plant nitrogen nutrition, growth and productivity. This emphasizes the need to select drought tolerant pea genotypes. In this study, the physiological and transcriptional responses of both roots and nodules to a drought event, followed by a recovery period were investigated. The hybridization of a 40k pea microarray indicated that, as a result of drought, ~390 and ~380 genes were at least 2-fold differencially regulated in roots and nodules, respectively. After rewatering, most of these genes were regulated in an opposite manner to drought effect. This analysis allowed to identify common and specific metabolic regulatory processes involved in drought tolerance and recovery. The most highly deregulated genes in response to drought (including LEA family members, delta-1-pyrroline-5-carboxylate synthase, SWEET family members...) were subsequently analysed for their expression patterns in response to several drought events each followed by a recovery period. We will discuss the behavior of these genes in terms of kinetics and intensity of their expression. Acknowledgement: this study was supported by the Burgundy & Franche-Comté Region (FABER program), Terres Inovia, the FP7-ABSTRESS project and its grant FP7-613551, the FP7-LEGATO project and its grant agreement FP7-289562.

The use of pulses as ingredients for the production of food products rich in plant proteins is increasing. However, protein fractions prepared from pea or faba beans contain significant amounts of saponins, glycosylated triterpenes which can impart a bitter taste to the final food product. In addition, saponins have also been described to be involved in plant responses to biotic and abiotic stresses1. In this study, we identified and characterized the genes involved in saponin biosynthesis during pea seed development2, and optimized a saponin extraction protocol to follow the biosynthesis of these compounds during the development of pea and faba bean seeds. The identification of mutants affecting the function of key genes of the saponin biosynthetic pathway is currently underway in pea3. This study is funded under the LEG'UP FUI (Unique Interministerial Fund) project (AAP No. 18).

Seeds of legumes such as pea (Pisum sativum L.) provide proteins for animal feed and human nutrition. Although the so-called ²antinutritional factors² have been reduced in these seeds, offering globally good nutritional value, amino acid balance needs to be improved and stabilized, notably regarding sulfur amino acid content. By integrating omics data, notably from protein quantity loci and genome-wide association studies (GWAS using the HapMap platform[1]) on the abundance of storage proteins in Medicago truncatula seeds, we provide a repertory of genes involved in transcription, post-translational modifications, transport, or targeting of globulins to storage vacuoles [2]. Inference of a gene co-expression network between GWAS-derived transcription factors and globulin genes revealed key regulators of seed protein composition. A systematic search for orthologous sequences in the pea gene atlas[3] enabled us to transfer the knowledge to the target pea crop. The potential of this translational genomics approach for revealing genes important for seed nutritional quality improvement will be presented through pea TILLING lines[4] for selected candidate genes that exhibit seed protein profiles with increased abundance of sulfur-rich proteins. Because sulfur nutrition enables sulfur-rich protein accumulation[5], we further studied the leaf transcriptome of pea subjected to sulfur deficiency during the reproductive period. Genes involved in the supply of sulfur for accumulation of sulfur-rich proteins have been characterized by analysis of TILLING mutants.

International audience

BAP EA GEAPSI BAP EA GEAPSIBAPEAGEAPSI; Drought is a major environmental factor limiting the productivity of crop plants. In pea drought stress occurring during the reproductive phase can greatly affect seed yield and quality. We investigated the response of pea plants (var. Caméor) subjected to water stress during the seed filling period, a phase when massive remobilization from the vegetative organs occurs to sustain seed high-nitrogen demand. Pea plants were subjected to drought stress at the beginning of the seedfilling period of the first two nodes for 8 days. Total and one-seed biomass decreased by 35% and 20% respectively by this limited water stress. Nitrogen allocation to the different plant compartments was also affected, with an increased N allocation to the leaves from the vegetative nodes and a decrease to the root and seed compartments. Transcriptomic changes in water-stressed leaves from the vegetative nodes were analysed by hybridization of a 40k pea micro-array : 178 genes were at least two fold up-regulated in water-stressed samples compared to well-watered samples, whereas 55 genes were downregulated. Among the most strongly up-regulated genes were those encoding a glutamine amidotransferase, a sucrose transporter from the SWEET family, a phosphoenolpyruvate carboxykinase and a carotenoid cleavage dioxygenase involved in strigolactone biosynthesis. Acknowledgement: this project was supported by the Burgundy Region (AGRALE6, FABER program) and the FP7 LEGATO project.

BAPGEAPSI; Legume seeds are increasingly in demand as an inexpensive source of protein for incorporation in foodstuffs. However, protein fractions prepared from pea (Pisum sativum) and faba bean (Vicia faba) seeds contain significant quantities of saponins, which may confer an undesired bitter flavor on the final food product. Saponins have also been implicated in abiotic and biotic stress responses, hence a further interest in studying their accumulation and genetic control in these species. We have identified pea genes putatively involved in saponin biosynthesis in developing pea seeds, and optimized an extraction method for use in following saponin accumulation and distribution in seeds of pea and faba bean. Acknowledgement. This project is supported by the FUI (Fonds Unique Interministériel) AAP no. 18 LEG’UP.

National audience; L’utilisation des légumineuses à graines en tant qu’ingrédients pour la fabrication de produits alimentaires riches en protéines végétales est en plein essor. Cependant les fractions protéiques préparées à partir de graines de pois ou de féveroles contiennent une quantité significative de saponines, des triterpènes de glycosides qui peuvent conférer un goût d’amertume au produit alimentaire final. En outre, les saponines ont été également décrites pour être impliquées dans les réponses aux stress biotiques et abiotiques. Dans cette étude, nous avons identifié et caractérisé les gènes impliqués dans la biosynthèse des saponines lors du développement de la graine de pois, et optimisé un protocole d’extraction des saponines afin de suivre la biosynthèse de ces composés lors du développement des graines de pois et de féveroles. L’identification de mutants affectant la fonction de gènes clé de la voie de biosynthèse est actuellement en cours chez le pois. Cette étude est financée dans le cadre du projet FUI (Fond Unique Interministériel) LEG’UP (AAP no. 18).

National audience; Dans un contexte de changements climatiques, la fréquence et l’intensité des évènements de sécheresse ne cessent d’augmenter. Une compréhension des mécanismes physiologiques à l’origine d’une meilleure tolérance à la sécheresse est donc nécessaire afin de pouvoir sélectionner des plantes de culture mieux adaptées à cet environnement fluctuant. L’objectif de cette étude est de déterminer si des différences de perception précoce du stress, notamment au niveau membranaire, peuvent expliquer les variations de tolérance au stress hydrique observées entre différents génotypes de la légumineuse modèle Medicago truncatula. Pour ce faire, une core-collection de 16 génotypes de Medicago à laquelle sont ajoutés trois génotypes, le génotype de référence Jemalong A17 et deux génotypes identifiés dans la littérature comme étant tolérants au stress hydrique, ont été étudiés. Ils ont été phénotypés pour leur réponse au PolyEthylène Glycol (PEG) dont le rôle est de mimer le manque d’eau. Les plantes ont été cultivées 15 jours dans des poches en plastique transparent contenant un papier filtre imbibé d’une solution nutritive, laissant ainsi le système racinaire apparent. Au bout d’une semaine, la moitié des plantes a reçu la solution nutritive seule (condition contrôle), tandis que l’autre moitié a reçu la solution nutritive additionnée de PEG (condition stress). Elles ont ensuite été scannées quotidiennement afin de pouvoir suivre le développement du système racinaire : vitesse de croissance, longueur du pivot, apparition des racines latérales, etc. A l’issue des 15 jours de culture, le contenu relatif en eau des feuilles, la surface foliaire et la biomasse des parties aériennes et racinaires ont été mesurées. Ces données ont permis d’identifier des génotypes contrastés pour leur réponse au PEG. En parallèle, trois types de signalisation précoce au niveau membranaire ont été étudiés sur le génotype de référence Jemalong A17 via l’utilisation de sondes fluorescentes en microscopie confocale : le taux d’endocytose avec la sonde FM4-64, le degré d’ordre avec la sonde di-4-ANEPPDHQ et la production de formes actives de l’oxygène (FAO) avec la sonde H2DCFDA. Une augmentation de l’endocytose et du degré d’ordre a été observée mais aucune production de FAO n’a pu être détectée. La prochaine étape de ce projet consistera à étendre ces analyses aux génotypes contrastés pour leur réponse au stress, afin de tester l’hypothèse selon laquelle il existerait un lien entre perception précoce et meilleure tolérance au stress hydrique.

Pea (Pisum sativum L.) produces seeds rich in proteins for human and animal nutrition and its cultivation enriches the soils in nitrogen, thus decreasing the need for nitrogen fertilization. Increasing pea cultivation and productivity is an agroecological challenge which requires to improve pea tolerance to environmental stresses. Drought and the lack of sulfur in soils are two abiotic stresses that interact in the current context of climate change and low-input practices. Products of sulfur metabolism, like glutathione, are known to play a protective role against many stresses but their interaction with the plant response to drought remains to be studied. A system biology approach will be used to study the influence of sulfur nutrition on the dynamics of gene and protein networks associated with the response of pea leaves to drought during the reproductive phase. This approach will provide metabolic regulation models connecting sulfur nutrition to the drought response. The integration of other data (e.g., physiological, yield components) will reveal regulatory factors potentially responsible for the physiological variations observed and/or for the modifications of agronomic traits under these environmental constraints. In addition to provide a better understanding of the role of sulfur in the plant’s response to drought, the project will lead to the identification of gene and protein candidates for stabilizing or improving the productivity and seed quality of pea.

International audience

International audience

National audience

L’utilisation des légumineuses à graines en tant qu’ingrédients pour la fabrication de produits alimentaires riches en protéines végétales est en plein essor. Cependant les fractions protéiques préparées à partir de graines de pois ou de féveroles contiennent une quantité significative de saponines, des triterpènes de glycosides qui peuvent conférer un goût d’amertume au produit alimentaire final. En outre, les saponines ont été également décrites pour être impliquées dans les réponses aux stress biotiques et abiotiques. Dans cette étude, nous avons identifié et caractérisé les gènes impliqués dans la biosynthèse des saponines lors du développement de la graine de pois, et optimisé un protocole d’extraction des saponines afin de suivre la biosynthèse de ces composés lors du développement des graines de pois et de féveroles. L’identification de mutants affectant la fonction de gènes clé de la voie de biosynthèse est actuellement en cours chez le pois. Cette étude est financée dans le cadre du projet FUI (Fond Unique Interministériel) LEG’UP (AAP no. 18).