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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.

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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National audience