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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.

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).

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The modification of histones by acetyl groups has a key role in the regulation of chromatin structure and transcription. The Arabidopsis thaliana histone acetyltransferase GCN5 regulates histone modifications as part of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) transcriptional coactivator complex. GCN5 was previously shown to acetylate lysine 14 of histone 3 (H3K14ac) in the promoter regions of its target genes even though GCN5 binding did not systematically correlate with gene activation. Here, we explored the mechanism through which GCN5 controls transcription. First, we fine-mapped its GCN5 binding sites genome-wide and then used several global methodologies (ATAC-seq, ChIP-seq and RNA-seq) to assess the effect of GCN5 loss-of-function on the expression and epigenetic regulation of its target genes. These analyses provided evidence that GCN5 has a dual role in the regulation of H3K14ac levels in their 5′ and 3′ ends of its target genes. While the gcn5 mutation led to a genome-wide decrease of H3K14ac in the 5′ end of the GCN5 down-regulated targets, it also led to an increase of H3K14ac in the 3′ ends of GCN5 up-regulated targets. Furthermore, genome-wide changes in H3K14ac levels in the gcn5 mutant correlated with changes in H3K9ac at both 5′ and 3′ ends, providing evidence for a molecular link between the depositions of these two histone modifications. To understand the biological relevance of these regulations, we showed that GCN5 participates in the responses to biotic stress by repressing salicylic acid (SA) accumulation and SA-mediated immunity, highlighting the role of this protein in the regulation of the crosstalk between diverse developmental and stress-responsive physiological programs. Hence, our results demonstrate that GCN5, through the modulation of H3K14ac levels on its targets, controls the balance between biotic and abiotic stress responses and is a master regulator of plant-environmental interactions.

Prod 2019-88t BAP GEAPSI INRA; National audience; Since 2004, two INRA teams (UMR Agroecology Dijon and IJBP, Versailles), both partners of PEAMUST, have created 3 EMS-induced mutant collections in the framework of European or French ANR projects. The genotypes chosen for these collections have specific agronomic traits according to the research subject. For example, the Terese rms3 and -rms4 branched genotypes (Rameau et al., 1997) are well suited to the study of shoot architecture in relation with strigolactone and the lines easy to grow and to backcross in glasshouse conditions. Caméor, an early cultivar with high seed protein content, is the reference genotype for the pea sequencing project. The 336/11 winter pea line with the Hr allele is very sensitive to photoperiod and cold tolerant, which enables the screening of genes contributing to winter pea performance. These 3 collections currently represent a total of 13500 mutated lines. The high-throughput detection of mutations in specific regions of the genome is critical to exploit allelic resources from these collections. Since 2014, mutation detection system has evolved toward NGS technologies and a new one has been implemented in INRA (IPS2) increasing consequently the sensitivity of the detection, allowing a better exploitation of these collections. Spanning the 3 collections mutation rate was assessed ranging from 10 to 40 thousand mutations per genome. As part of the PeaMUST project, about 15 kb of candidate gene sequences were sequenced across 81,000 families. 313 mutations were detected, of which 9% induce a stop codon or a splice defect and 70% induce amino acid changes. These results showed the very high potential of these collections for their exploitation in functional validation or creation of allelic variants. Rameau C, Bodelin C, Cadier D, Grandjean O, Miard F, Murfet IC (1997) New ramosus mutants at loci Rms1, Rms3 and Rms4 resulting from the mutation breeding program at Versailles. Pisum Genet 29: 7–12

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)

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International audience; In the context of highly pressured agricultural production using low levels of inputs, cropping strategies could take advantage of existing plant-microbiome interactions that can improve both plant growth and health. We believe that plant genetics can provide some clues to better understand the mechanisms underlying microbiome recruitment by the plant.The aims of this work were (i) to assess plant genotype effect on the rhizosphere bacterial communities in relation to the plant nutritional strategies for a core collection of 155 genotypes of Medicago truncatula and (ii) to highlight the genetic determinisms potentially associated in these interactions. To achieve these aims, we developed a multidisciplinary approach where the plant growth and nutritional strategies were characterized using an ecophysiological framework, the rhizosphere bacterial communities were described using massive parallel sequencing, and the genetic determinisms were analysed using Genome-Wide-Association Studies. We observed a significant effect of the plant genotype and identified different groups of plant genotypes according to their nutritional strategies and to the structure and composition of their associated rhizosphere bacterial communities. To go further, we also highlighted loci linked to microbiome recruitment by the plant.First, we showed that plant genetics may provide the levers to manipulate soil microflora through plant microbiome recruitment. Then, this work demonstrates the importance of combining approaches from different disciplines to better unravel plant-microbiome interactions.

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.

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Les légumineuses sont capables de produire des graines riches en protéines sans apport d’engrais azoté grâce à la symbiose racinaire avec des bactéries du genre rhizobium. Riches en lysine, ces protéines sont utilisées pour l’alimentation des animaux d’élevage et en nutrition humaine. Afin de promouvoir la culture des légumineuses, il est nécessaire d’optimiser et de stabiliser la qualité de cette fraction protéique. L’objectif de ma thèse est de mettre en évidence les déterminismes génétiques sous-jacents à la teneur et à la composition protéique des graines de légumineuses ainsi qu’à la plasticité de ces composantes vis-à-vis de l’environnement. Afin de répondre à cet objectif, l’approche choisie est en deux volets connectés, l’un utilisant l’espèce modèle Medicago truncatula comme tremplin pour l’identification de gènes candidats et l’autre se focalisant directement sur le pois. La méthode combine un protéotypage des graines de collections d’écotypes produites dans différentes conditions environnementales. A partir des données de protéotypage, une étude de génétique d’association à l’échelle du génome (GWAS) sera effectuée. Les modules de régulation géniques impliquant les gènes identifiés par GWAS seront mis en évidence en utilisant les ressources transcriptomiques associées au développement de la graine de M. truncatula. En utilisant la synténie avec le pois, nous rechercherons les orthologues des gènes candidats chez cette espèce cible. Cette approche permettra d’identifier des gènes candidats pour le contrôle de la composition protéique ainsi que de sa plasticité vis-à-vis de l’environnement chez le pois.

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