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Nitrate (NO3-) is an essential mineral ion for plant growth and is the main source of nitrogen (N). In A. thaliana, NRT2.1, NRT2.4 and NRT2.5 are the three main transporters of the high affinity transport system (HATS) for the uptake of NO3- by roots. Among them, NRT2.1 is the major player in HATS. NRT2.1 is transcriptionally controlled by all the regulations that affect the transport of NO3- : i) induction by NO3-, ii) repression by products resulting from the assimilation of NO3- and iii) induction by light and sugars. In addition to these transcriptional regulations, recent approaches indicate that NRT2.1 is also subject to post-translational regulations. Concerning the other members of the NRT2s family, NRT2.4 and NRT2.5 have been characterized as transporters with very high affinity for NO3-. Their expression is induced by N deficiency, while light and sugars also induce NRT2.4. Despite the functional importance of these transporters for plant growth, the mechanisms involved in their regulation are still poorly understood, in particular for the regulation by light and sugars as well as post-translational regulations of NRT2.1. In this context, the objectives of my thesis were: i) to characterize the light/sugar signaling pathway involved in the regulation of NRT2.1 and NRT2.4, (ii) to characterize the role of new regulators of N signaling pathways and C identified in my team and (iii) to participate in the study of post-translational regulations of the NRT2.1 protein. One of the major results of my thesis work concerns the regulation of NRT2s by light and sugars. The work previously carried out by the team had made it possible to determine that sugar signaling involves the oxidative pathway of pentose phosphates (OPPP). The results that I obtained allowed: (i) to identify that G6PD, the first enzyme of the OPPP pathway, seems to be at the origin of the sugar signal and (ii) to hypothesize that NADPH produced by this enzyme as well as redox signaling are involved in this regulation. In parallel, I characterized the role of BHLH093 transcription factor, previously identified in the team. I demonstrated its role in the regulation of NRT2.4 by light and sugars and highlighted the existence of another signaling pathway that is probably independent of the OPPP. A second important part of my thesis work concerned the study of the regulation of NRT2.4 and NRT2.5 transporters by N deficiency. This allowed me to demonstrate: (i) that NO3- is the signal involved in the repression of these two transporters in non-N-deficient plants and (ii) that this regulation depends on the signaling pathway linked to NRT1.1/NPF6.3. In addition, I was able to place known regulators of the response to high N into the signaling pathway that regulates NRT2.4 and NRT2.5 in response to repression by NO3-. Finally, I could confirm the essential role of the phosphorylation of the Ser501 residue, located in the C-terminal part, to repress the activity of NRT2.1 under repressive conditions, by studying its post-translational regulation. The hypothesis of a cleavage of the C-terminal part of NRT2.1 also led me to initiate an approach aiming to determine if the peptide released after cleavage and which contains the phosphorylated Ser501 site, could have a role in signaling NO3-.; Le Nitrate (NO3-) est un ion minéral indispensable pour la croissance des plantes et constitue la principale source d’azote (N). Chez A. thaliana, le système de transport à forte affinité (HATS) pour l'absorption de NO3- par les racines dépend principalement de trois transporteurs : NRT2.1, NRT2.4 et NRT2.5. Parmi eux, NRT2.1 constitue l’acteur majoritaire du HATS. NRT2.1 est la cible au niveau transcriptionnel de l’ensemble des régulations qui affectent le transport de NO3- : i) une induction par le NO3-, ii) une répression exercée par les produits issus de l’assimilation de NO3- et iii) une induction par la lumière et les sucres. En plus de ces régulations transcriptionnelles, des approches récentes indiquent que NRT2.1 est également soumis à des régulations post-traductionnelles. Concernant les autres membres de la famille NRT2s, NRT2.4 et NRT2.5 ont été caractérisé comme des transporteurs à très forte affinité pour le NO3- dont l’expression est induite par la carence en N et également par la lumière et les sucres pour NRT2.4. Malgré l’importance fonctionnelle de ces transporteurs pour la croissance des plantes, les mécanismes impliqués dans leur régulation restent encore mal connus, en particulier en ce qui concerne la régulation par la lumière et les sucres et les régulations post-traductionnelles de la protéine NRT2.1. Dans ce contexte les objectifs de ma thèse étaient : i) de caractériser la voie de signalisation lumière/sucre impliquée dans la régulation de NRT2.1 et NRT2.4, (ii) de caractériser le rôle de nouveaux régulateurs des voies de signalisation N et C identifiés dans mon équipe et (iii) de participer à l’étude des régulations post-traductionnelles de la protéine NRT2.1. Un des résultats majeurs de mon travail de thèse concerne la régulation des NRT2s par la lumière et les sucres. Les travaux réalisés dans l’équipe avait permis de déterminer que la signalisation sucre implique la voie oxydative des pentoses phosphates (OPPP). Les résultats que j’ai obtenus ont permis : (i) d’identifier que la première enzyme de la voie OPPP, la G6PD, semble être à l’origine du signal sucre et (ii) de faire l’hypothèse que le NADPH produit par cette enzyme ainsi que la signalisation redox sont impliqués dans cette régulation. En parallèle, la caractérisation du rôle de BHLH093, préalablement identifié dans l’équipe, a permis de démontrer le rôle de ce facteur de transcription dans la régulation de NRT2.4 par la lumière et les sucres et a mis en évidence l’existence d’une autre voie de signalisation sans doute indépendante de l’OPPP. Un deuxième volet important de mon travail de thèse a concerné l’étude de la régulation des transporteurs NRT2.4 et NRT2.5 par la carence en N. Ceci m’a permis de démontrer : (i) que le NO3- est le signal impliqué dans la répression de ces deux transporteurs chez des plantes non carencées en N et (ii) que cette régulation dépend de la voie de signalisation liée à NRT1.1/NPF6.3. De plus, l’étude de régulateurs connus pour être impliqués dans la réponse au fort N m’a permis de replacer certains de ces facteurs de transcription dans la voie de signalisation qui régule NRT2.4 et NRT2.5 en réponse à la répression par le NO3-. Enfin, l’étude de la régulation post-traductionnelle de la protéine NRT2.1 a permis de confirmer le rôle essentiel de la phosphorylation du résidu Ser501, située dans la partie C-terminale, pour désactiver l’activité de NRT2.1 en conditions répressives. L’hypothèse d’un clivage de la partie C-terminale de NRT2.1 m’a également amené à initier une approche visant à déterminer si le peptide libéré après clivage et qui contient le site Ser501 phosphorylé, pouvait avoir un rôle dans la signalisation NO3-.

Biogenic amines are allergenic compounds and also molecules that can alter the organoleptic profile of wines. Lactic acid bacteria in wine, including Oenococcus oeni, the main strain in malolactic fermentation, are able to produce these molecules from nitrogenous precursors. In order to reduce the biogenic amines content, it is necessary to understand which viticultural and oenological factors can contribute to this production.In the course of this work, we focused on the two main axes of control of the biogenic amines production. Firstly, the relationship between the concentration of precursor amino acids and the concentration of biogenic amines was studied along two axes: the role of the initial assimilable nitrogen content in grapes and the impact of the addition of oenological nitrogen nutrient products. Secondly, the control of the producing microbial flora was the central subject of this work by addressing the interest of co-inoculation with selected lactic acid bacteria and the bioprotection of grapes with S. cerevisiae and T. delbrueckii.The results of this work show how multifactorial is the biogenic amines subject. The grape variety, the process and the vintage are important factors in the variation of biogenic amine contents and variations within biogenic amines. For example, since the experiment on which this dissertation is based was conducted over two years (2019 and 2020), we were able to observe that the 2019 vintage was marked by higher levels of histamine, whereas for the 2020vintage it was putrescine that proved to be predominant. Furthermore, it has been established that the presence of biogenic amines in wines, even in very small quantities, has a significant impact on their sensory perception.Thus, the data in this dissertation underline the great effectiveness of co-inoculation with selected lactic acid bacteria in order to limit the peak production of these molecules, which occurs between the end of alcoholic and malolactic fermentation. However, the interest of bioprotection remains to be proven since in our experiment it did not permit to limit the final contents of biogenic amines. The effects of assimilable nitrogen amount and nitrogen additions over biogenic amines content has also been discussed and invites further studies on the subject since it is a crucial subject for the sector.; Les amines biogènes sont des composés allergènes et également des molécules pouvant altérer le profil organoleptique des vins. Les bactéries lactiques du vin, dont Oenococcus oeni, le principal acteur de la fermentation malolactique, sont capables de produire ces molécules, à partir de précurseurs azotés. Afin de réduire la teneur en amines biogènes, il est nécessaire de comprendre quels facteurs viticoles et oenologiques peuvent contribuer à cette production.Au cours de ce travail de mémoire ingénieur agronome, nous nous sommes intéressés aux deux axes de contrôle de la production des amines biogènes. Premièrement, la relation entre concentration en acides aminés précurseurs et concentration en amines biogènes a été étudiée selon deux axes : le rôle de la teneur en azote assimilable initial sur raisin et l’impact des ajouts de produits oenologiques nutritifs azotés. Secondement, le contrôle de la flore microbienneproductrice a constitué le sujet central de ce mémoire en abordant l’intérêt de la co-inoculation avec des bactéries lactiques sélectionnées et la bioprotection des raisins avec une S. cerevisiae et une T. delbrueckii.Les résultats de ce mémoire montrent à quel point la problématique des amines biogènes est un sujet plurifactoriel. Le cépage, le process et le millésime constituent des facteurs de variations importants des teneurs en amines biogènes et des variations au sein des amines biogènes. Par exemple, l’expérimentation sur laquelle ce mémoire est basée ayant été conduite sur deux années (2019 et 2020) nous avons pu remarquer que le millésime 2019 était marqué par desteneurs en histamine plus importantes alors que pour le millésime 2020 c’est la putrescine qui s’est montrée prédominante. De plus, il a été établi que la présence d’amines biogènes dans les vins, même en très faibles quantités, impacte significativement la perception sensorielle de ceux-ci.Ainsi, les données de ce mémoire soulignent la grande efficacité de la co-inoculation avec des bactéries lactiques sélectionnées afin de limiter le pic de production de ces molécules qui se situe entre la fin de la fermentation alcoolique et la fermentation malolactique. L’intérêt de la bioprotection reste cependant à prouver puisque dans notre expérimentation elle n’a pas permis de limiter les teneurs finales en amines biogènes. Le lien entre azote assimilable/ajouts azotésa également été abordé et invite à des études plus poussées sur le sujet puisqu’il constitue un sujet crucial pour la filière.

International audience; Aim: Nitrogen is a major element conditioning grapevine growth, yield and aromatic profiles of berries and wines. Different tools can be used in order to detect differences in N status of the plant, including direct measurements of soil, plant nitrogen status (eg. petiole; must), or indirect observations of plant nutritional status such as leaf transmittance or reflectance (eg. SPAD; NDVI). However, the relationships between these indicators of nitrogen status and the overall plant functioning over vintages remain poorly known. The present study aimed at quantifying key vegetative and reproductive responses to plant nitrogen status over two successive seasons under different nitrogen supply levels. Methods and results: Potted plants of Sauvignon Blanc grafted onto SO4 were grown outdoors in 2017 and 2018 with no water limitation. Four mineral nitrogen fertilization levels (equivalent to 0 kg of N ha-1 or 0U, 20U, 40U, 80U) and one organic nitrogen fertilization level (40U) were imposed in 2017. These treatments were doubled in 2018 to increase the degree of nitrogen supply and consequently, the range of observed effects on plant growth and yield. Plant nitrogen status (SPAD) was monitored weekly during both growing cycles. Yield components were determined over the two seasons. Lastly, plant carbon status was addressed through dynamic measurement of plant development and photosynthesis, and destructive measurement of dry matter accumulation and carbon storage in annual and perennial organs at flowering, veraison and harvest. The SPAD values progressively decreased under lower N supply (0N) during the first year (from 31 to 16) and they were more than halved between the maximum and the minimum N treatments straight after budburst in year two (40 for 160N and 19 for 0N). Then, the differences in SPAD values among treatments were maintained up to harvest (2018). The gradient of N status resulted in a gradient of berry numbers per inflorescence (from 180 to 34 berries/inflorescence for 80N and 0N, respectively in 2018) and of individual berry dry matter at harvest (from 0.13 to 0.41 g for 160N and 0N, respectively in 2018). Quantitative relationships between N status and the relative reductions (% of reduction per %SPAD decrease) in terms of C gain (leaf area, photosynthesis), C growth (shoot, berry, trunk and root dry matter) and C storage (trunk and root) were fitted at flowering, veraison and harvest. The reduction in C gain under lower N supply was mainly related to the decrease in total leaf area before flowering (-1.64%). Although the photosynthesis rate tended to decrease under N deficiency over the season, it only poorly contributed to the reduction in C gain. The whole plant C growth was inhibited when N status decreased (-1.13% at harvest), due to the inhibition of shoot dry matter before veraison (-1.81%) and to a lower extent, to the lower dry matter in berries (-0.80%), trunks (-0.42%) and roots (-0.84%) at harvest. Part of the reduction in root dry matter was related to the lower starch reserves (-0.31%) at harvest. Interestingly, starch reserves tended to be higher under organic N supply than mineral N supply. Conclusion: The present results provided a general framework of carbon gain and use over time (within and between seasons) as impacted by N supply levels and form. Such a framework will be useful when building a model of the pluri-annual dynamics of carbon balance related to yield elaboration in grapevines.

Background After uptake from soil into the root tissue, distribution and allocation of nitrate throughout the whole plant body, is a critical step of nitrogen use efficiency (NUE) and for modulation of plant growth in response to various environmental conditions. In legume plants nitrate distribution is also important for the regulation of the nodulation process that allows to fix atmospheric N (N2) through the symbiotic interaction with rhizobia (symbiotic nitrogen fixation, SNF). Results Here we report the functional characterization of the Lotus japonicus gene LjNPF2.9, which is expressed mainly in the root vascular structures, a key localization for the control of nitrate allocation throughout the plant body. LjNPF2.9 expression in Xenopus laevis oocytes induces 15NO3 accumulation indicating that it functions as a nitrate importer. The phenotypic characterization of three independent knock out mutants indicates an increased shoot biomass in the mutant backgrounds. This phenotype is associated to an increased/decreased nitrate content detected in the shoots/roots. Furthermore, our analysis indicates that the accumulation of nitrate in the shoot does not affect the nodulation and N-Fixation capacities of the knock out mutants. Conclusions This study shows that LjNPF2.9 plays a crucial role in the downward transport of nitrate to roots, occurring likely through a xylem-to-phloem loading-mediated activity. The increase of the shoot biomass and nitrate accumulation might represent a relevant phenotype in the perspective of an improved NUE and this is further reinforced in legume plants by the reported lack of effects on the SNF efficiency. Electronic supplementary material The online version of this article (10.1186/s12870-019-1978-5) contains supplementary material, which is available to authorized users.

International audience; Senescence associated gene (SAG) 12, which encodes a cysteine protease isconsidered to be important in nitrogen (N) allocation to Arabidopsis thaliana seeds.A decrease in the yield and N content of the seeds was observed in the ArabidopsisSAG12 knockout mutants (sag12) relative to the wild type (Col0) under limited nitrogennutrition. However, leaf senescence was similar in both lines. To test whether SAG12is involved in N remobilization from organs other than the leaves, we tested whetherroot N could be used in N mobilization to the seeds. Root architecture, N uptakecapacity and 15N partitioning were compared in the wild type and sag12 under eitherhigh nitrogen (HN) or low nitrogen (LN) conditions. No differences in root architectureor root N uptake capacity were observed between the lines under HN or LN. However,under LN conditions, there was an accumulation of 15N in the sag12 roots comparedto the wild type with lower allocation of 15N to the seeds. This was accompanied by anincrease in root N protein contents and a significant decrease in root cysteine proteaseactivity. SAG12 is expressed in the root stele of the plants at the reproductive stage,particularly under conditions of LN nutrition. Taken together, these results suggest anew role for SAG12. This cysteine protease plays a crucial role in root N remobilizationthat ensures seed filling and sustains yields when nitrogen availability is low.

National audience; Pea seeds have interesting nutritional properties through their protein, vitamin and mineral contents. However, environmental conditions such as soil water availability during plant growth influence both final yield and seed quality in pea. Soil water conditions not only modulates pea nitrogen acquisition whatever the mode of nitrogen nutrition: mineral nitrogen acquisition by roots and atmospheric di-nitrogen fixation by nodules in interaction with rhizobia, but also the overall plant mineral nutrition. The aim of this study is to get a better understanding of soil water deficit impact on nutrient acquisition, storage and distribution inside the plant according to the mode of nitrogen nutrition. In this study the first experimentation aims at characterizing 13 individual mineral deficiencies on nutrient uptake and remobilisation into different plant organs, allowing to design an ionomic fingerprint specific to each mineral deficiency. A second experimentation consists in implementing single (at vegetative or reproductive stage) or repeated water deficits to pea plants grown under both modes of nitrogen nutrition. This experimentation will allow us to analyse how water deficit influences nutrient uptake, storage and remobilisation via (1) ecophysiological analysis by using a structure-function framework including water and mineral fluxes at the whole plant level, (2) transcriptomic analysis of nodules and roots. We intend to identify the main regulatory genes involved in either single water deficit response or water deficit memory stress response. This study will allow us to provide new insights towards a better management of preventive or/and curative mineral fertilization solution in the field, in order to prevent pea from mineral deficiencies and improve its yield and seed quality under fluctuating water environment.

Master

Les légumineuses ont un rôle majeur à jouer dans la transition vers une agriculture plus durable, en réduisant notamment le recours aux intrants azotés du fait de leurs propriétés spécifiques vis-à-vis du cycle de l’azote. Pourtant peu de références sont disponibles pour une diversité d’espèces quant à leurs performances agronomiques et aux flux d’azote qu’elles induisent. Une étude comparative sur dix espèces de légumineuses à graines a été menée afin i) de quantifier la fixation symbiotique, ii) d’évaluer sa réponse à la présence d’azote minéral du sol, et iii) de quantifier l’effet des légumineuses sur le rendement d’un blé suivant (non fertilisé) en lien avec la minéralisation de leurs résidus de culture. Pour neuf espèces sur dix la fixation symbiotique a permis d’assurer une croissance et une nutrition azotée des plantes aussi bonnes qu’avec une nutrition basée sur le prélèvement d’azote minéral. Des différences d’inhibition de la fixation symbiotique par l’azote minéral ont été mises en évidence et ont été partiellement expliquées par des différences d’efficience de prélèvement de l’azote minéral entre les espèces. Ces différences ont été corrélées à des différences de vitesse d’expansion latérale des racines entre les espèces. La grande majorité des légumineuses a engendré des rendements de blé supérieurs à ceux de blés cultivés après des céréales. La variabilité des rendements du blé a en partie pu être reliée à la minéralisation de l’azote des résidus, variable entre espèces selon leur rapport carbone / azote. Cependant l’azote fourni au sol par les légumineuses peut être perdu pendant la période d’interculture, avant que cet azote ne soit prélevé par la culture suivante. Ce risque augmente pour les espèces de légumineuses récoltées en été contrairement au début automne, du fait d’une interculture plus longue. Abstract: Symbiotic nitrogen fixation and pre-crop effect : are all grain legumes the same? Legumes should play a key role in the transition towards a more sustainable agriculture by allowing a reduction of nitrogen inputs due to their specific properties relative to the nitrogen cycle. However, there is a lack of references for a wide diversity of species regarding their agronomic performances and the nitrogen fluxes they induce. A comparative study on ten grain legumes was conducted to i) quantity symbiotic nitrogen fixation, ii) evaluate its response to inorganic soil nitrogen, and iii) quantify the effect of legumes on the yield of the subsequent wheat (unfertilised) as related to the N mineralisation of their crop residues. For nine species out of ten, symbiotic fixation was able to ensure plant growth and nitrogen acquisition at levels similar to those achieved by nutrition based on inorganic nitrogen. Differences in symbiotic fixation inhibition were observed and were partially explained by differences in inorganic nitrogen uptake efficiency among species. Those differences were related to differences in root lateral expansion rate among species. The great majority of legumes led to higher wheat yields compared to those of wheat cultivated after cereals. Wheat yield variability was partially related to differences in crop residue nitrogen mineralisation among species according to their carbon /nitrogen, Legumes should play a key role in the transition towards a more sustainable agriculture by allowing a reduction of nitrogen inputs due to their specific properties relative to the nitrogen cycle. However, there is a lack of references for a wide diversity of species regarding their agronomic performances and the nitrogen fluxes they induce. A comparative study on ten grain legumes was conducted to i) quantity symbiotic nitrogen fixation, ii) evaluate its response to inorganic soil nitrogen, and iii) quantify the effect of legumes on the yield of the subsequent wheat (unfertilised) as related to the N mineralisation of their crop residues. For nine species out of ten, symbiotic fixation was able to ensure plant growth and nitrogen acquisition at levels similar to those achieved by nutrition based on inorganic nitrogen. Differences in symbiotic fixation inhibition were observed and were partially explained by differences in inorganic nitrogen uptake efficiency among species. Those differences were related to differences in root lateral expansion rate among species. The great majority of legumes led to higher wheat yields compared to those of wheat cultivated after cereals. Wheat yield variability was partially related to differences in crop residue nitrogen mineralisation among species according to their carbon /nitrogen ratio. However, nitrogen supplied to the soil by legumes can be lost during the fallow period before nitrogen is retrieved by the subsequent crop. This risk increases with legumes harvested in summer vs. beginning of autumn, because of a longer fallow period.

Prod 2019-81EAéquipe BAPéquipe SPEGESTADGEAPSIAGROSUPINRADOCT; Legumes should play a key role in the transition towards a more sustainable agriculture by allowing areduction of nitrogen inputs due to their specific properties relative to the nitrogen cycle. However, thereis a lack of references for a wide diversity of species regarding their agronomic performances and thenitrogen fluxes they induce. A comparative study on ten grain legumes was conducted to i) quantitysymbiotic nitrogen fixation, ii) evaluate its response to inorganic soil nitrogen, and iii) quantify the effectof legumes on the yield of the subsequent wheat (unfertilised) as related to the N mineralisation of theircrop residues. For nine species out of ten, symbiotic fixation was able to ensure plant growth andnitrogen acquisition at levels similar to those achieved by nutrition based on inorganic nitrogen.Differences in symbiotic fixation inhibition were observed and were partially explained by differences ininorganic nitrogen uptake efficiency among species. Those differences were related to differences inroot lateral expansion rate among species. The great majority of legumes led to higher wheat yieldscompared to those of wheat cultivated after cereals. Wheat yield variability was partially related todifferences in crop residue nitrogen mineralisation among species according to their carbon /nitrogen ratio. However, nitrogen supplied to the soil by legumes can be lost during the fallow period beforenitrogen is retrieved by the subsequent crop. This risk increases with legumes harvested in summer vs.beginning of autumn, because of a longer fallow period.; Les légumineuses ont un rôle majeur à jouer dans la transition vers une agriculture plus durable, en réduisant notamment le recours aux intrants azotés du fait de leurs propriétés spécifiques vis-à-vis du cycle de l’azote. Pourtant peu de références sont disponibles pour une diversité d’espèces quant à leurs performances agronomiques et aux flux d’azote qu’elles induisent. Une étude comparative sur dix espèces de légumineuses à graines a été menée afin i) de quantifier la fixation symbiotique, ii) d’évaluer sa réponse à la présence d’azote minéral du sol, et iii) de quantifier l’effet des légumineuses sur le rendement d’un blé suivant (non fertilisé) en lien avec la minéralisation de leurs résidus de culture. Pour neuf espèces sur dix la fixation symbiotique a permis d’assurer une croissance et une nutrition azotée des plantes aussi bonnes qu’avec une nutrition basée sur le prélèvement d’azote minéral. Des différences d’inhibition de la fixation symbiotique par l’azote minéral ont été mises en évidence et ont été partiellement expliquées par des différences d’efficience de prélèvement de l’azote minéral entre les espèces. Ces différences ont été corrélées à des différences de vitesse d’expansion latérale des racines entre les espèces. La grande majorité des légumineuses a engendré des rendements de blé supérieurs à ceux de blés cultivés après des céréales. La variabilité des rendements du blé a en partie pu être reliée à la minéralisation de l’azote des résidus, variable entre espèces selon leur rapport carbone / azote. Cependant l’azote fourni au sol par les légumineuses peut être perdu pendant la période d’interculture, avant que cet azote ne soit prélevé par la culture suivante. Ce risque augmente pour les espèces de légumineuses récoltées en été contrairement au début automne, du fait d’une interculture plus longue. Abstract: Symbiotic nitrogen fixation and pre-crop effect : are all grain legumes the same? Legumes should play a key role in the transition towards a more sustainable agriculture by allowing a reduction of nitrogen inputs due to their specific properties relative to the nitrogen cycle. However, there is a lack of references for a wide diversity of species regarding their agronomic performances and the nitrogen fluxes they induce. A comparative study on ten grain legumes was conducted to i) quantity symbiotic nitrogen fixation, ii) evaluate its response to inorganic soil nitrogen, and iii) quantify the effect of legumes on the yield of the subsequent wheat (unfertilised) as related to the N mineralisation of their crop residues. For nine species out of ten, symbiotic fixation was able to ensure plant growth and nitrogen acquisition at levels similar to those achieved by nutrition based on inorganic nitrogen. Differences in symbiotic fixation inhibition were observed and were partially explained by differences in inorganic nitrogen uptake efficiency among species. Those differences were related to differences in root lateral expansion rate among species. The great majority of legumes led to higher wheat yields compared to those of wheat cultivated after cereals. Wheat yield variability was partially related to differences in crop residue nitrogen mineralisation among species according to their carbon /nitrogen

International audience

Réponse du grain de blé à la nutrition azotée et soufrée : intégration de données « omiques ». Journée Protéome vert

article accepté; In natural environments, nitrogen (N) concentration in the soil fluctuates greatly and is often limiting for plant growth and development. The ability of plants to respond to changes in N availability is therefore essential for adaptation. The response of plants to N variations consists in particular of adjusting root N uptake systems and root architecture. To do so, plants integrate local sensing and signaling of external N availability with systemic sensing and signaling of their internal N status, in order to tune the functional and structural properties of the root system in accordance with the N demand for growth of the whole plant. Transcriptional regulation of gene expression is one of the most important processes plants use to adapt the properties of the root system in response to local and long-distance N pathways. This review focuses on the mechanisms that give rise to transcriptional responses in Arabidopsis roots under N fluctuations, with an emphasis on those associated with the regulation of nitrate uptake and transport systems. We discuss the putative long-distance signals triggering the gene expression responses, as well as the molecular players that locally induce transcriptional changes. We also highlight several observations revealing the importance of adopting an integrative approach in the regulation of N signaling.

Grain legume pulse crops are of great interest to allow a production of seeds high nutritional value without any contribution of nitrate fertilizer. The nitrogen nutrition of legumes depends indeed mainly on the fixation in nodules of atmospheric dinitrogen through the plant-rhizobium symbiosis, and to a lesser extent, absorption by roots of soil mineral nitrogen.A better understanding has been obtained on the genetic control of the development of roots and nodules and on their impact on nitrogen nutrition. High genetic variability of these characters has been detected, and the existence of genetic correlations between them demonstrated. A quantitative genetic approach has identified several genomic regions that may be involved in their variations. The two different ways to improve nitrogen nutrition were also studied: the improvement of nitrogen acquisition by roots through a detailed study of a root architecture mutant, and the improvement of symbiosis via the study of the ability of peas to promote symbiotic associations with the most effective rhizobia.The results provide interesting bases for the design of a pea nitrogen-nutrition ‘ideotype’. Beyond the essential complementarity between the two pathways of nitrogen acquisition, it is necessary to optimize the interaction between the two symbiotic partners, which is a complex mechanism involving nodules formation and functioning in connection with complex signaling and trophic interactions between the partners and intra-plant., La culture de Légumineuses présente le double intérêt de permettre une production de graines à haute valeur nutritionnelle sans nécessité d’un apport d’engrais azoté. La nutrition azotée des légumineuses dépend en effet majoritairement de la fixation symbiotique de l’azote atmosphérique réalisée par des bactéries du sol, les rhizobia, au sein des nodosités, et dans une moindre mesure, de l’assimilation de l’azote minéral du sol par les racines.Une meilleure compréhension a été acquise sur le contrôle génétique de la mise en place des racines et des nodosités et sur leur impact sur la nutrition azotée. Une grande variabilité génétique pour ces caractères a été mise en évidence, ainsi que l’existence de corrélations génétiques entre eux. Une approche de génétique quantitative a permis d’identifier des régions génomiques pouvant être impliquées dans leurs variations. Deux pistes d’amélioration de la nutrition azotée ont aussi été étudiées : l’amélioration de l’acquisition d’azote par les racines à partir d’une étude détaillée d’un mutant de développement racinaire, et l’amélioration de la symbiose via l’étude de la capacité des pois à favoriser les associations symbiotiques avec les rhizobia les plus performants.Les résultats obtenus apportent des bases de réflexion concernant la conception d’un idéotype de nutrition azotée. Au-delà de la complémentarité indispensable entre les deux voies d’acquisition d’azote, il convient d’optimiser l’interaction entre les deux partenaires symbiotiques, mécanisme complexe mettant en jeu la formation et le fonctionnement des nodosités, en lien avec une signalétique et des interactions trophiques entre partenaires et intra-plante.

La culture de Légumineuses présente le double intérêt de permettre une production de graines à haute valeur nutritionnelle sans nécessité d’un apport d’engrais azoté. La nutrition azotée des légumineuses dépend en effet majoritairement de la fixation symbiotique de l’azote atmosphérique réalisée par des bactéries du sol, les rhizobia, au sein des nodosités, et dans une moindre mesure, de l’assimilation de l’azote minéral du sol par les racines. Une meilleure compréhension a été acquise, par ce travail de thèse, sur le contrôle génétique de la mise en place des racines et des nodosités et sur leur impact sur la nutrition azotée. Une grande variabilité génétique pour ces caractères a été mise en évidence, ainsi que l’existence de corrélations génétiques entre eux. Une approche de génétique quantitative a permis d’identifier des régions génomiques pouvant être impliquées dans leurs variations. Deux pistes d’amélioration de la nutrition azotée ont aussi été étudiées : l’amélioration de l’acquisition d’azote par les racines à partir d’une étude détaillée d’un mutant de développement racinaire, et l’amélioration de la symbiose via l’étude de la capacité des pois à favoriser les associations symbiotiques avec les rhizobia les plus performants. Les résultats obtenus apportent des bases de réflexion concernant la conception d’un idéotype de nutrition azotée. Au-delà de la complémentarité indispensable entre les deux voies d’acquisition d’azote, il convient d’optimiser l’interaction entre les deux partenaires, mécanisme complexe mettant en jeu la formation et le fonctionnement des nodosités, en lien avec une signalétique et des interactions trophiques complexes entre partenaires symbiotiques et intra-plante. ABSTRACT Grain legume pulse crops are of great interest to allow a production of seeds high nutritional value without any contribution of nitrate fertilizer. The nitrogen nutrition of legumes depends indeed mainly on the fixation in nodules of atmospheric dinitrogen through the plant-rhizobium symbiosis, and to a lesser extent, absorption by roots of soil mineral nitrogen. A better understanding has been obtained on the genetic control of the development of roots and nodules and on their impact on nitrogen nutrition. High genetic variability of these characters has been detected, and the existence of genetic correlations between them demonstrated. A quantitative genetic approach has identified several genomic regions that may be involved in their variations. The two different ways to improve nitrogen nutrition were also studied: the improvement of nitrogen acquisition by roots through a detailed study of a root architecture mutant, and the improvement of symbiosis via the study of the ability of peas to promote symbiotic associations with the most effective rhizobia. The results provide interesting bases for the design of a pea nitrogen-nutrition ‘ideotype’. Beyond the essential complementarity between the two pathways of nitrogen acquisition, it is necessary to optimize the interaction between the two symbiotic partners, which is a complex mechanism involving nodules formation and functioning in connection with complex signaling and trophic interactions between the partners and intraplant, Grain legume pulse crops are of great interest to allow a production of seeds high nutritional value without any contribution of nitrate fertilizer. The nitrogen nutrition of legumes depends indeed mainly on the fixation in nodules of atmospheric dinitrogen through the plant-rhizobium symbiosis, and to a lesser extent, absorption by roots of soil mineral nitrogen. A better understanding has been obtained on the genetic control of the development of roots and nodules and on their impact on nitrogen nutrition. High genetic variability of these characters has been detected, and the existence of genetic correlations between them demonstrated. A quantitative genetic approach has identified several genomic regions that may be involved in their variations. The two different ways to improve nitrogen nutrition were also studied: the improvement of nitrogen acquisition by roots through a detailed study of a root architecture mutant, and the improvement of symbiosis via the study of the ability of peas to promote symbiotic associations with the most effective rhizobia. The results provide interesting bases for the design of a pea nitrogen-nutrition ‘ideotype’. Beyond the essential complementarity between the two pathways of nitrogen acquisition, it is necessary to optimize the interaction between the two symbiotic partners, which is a complex mechanism involving nodules formation and functioning in connection with complex signaling and trophic interactions between the partners and intraplant.

L’azote (N) est un facteur majeur limitant la croissance des plantes. Sa disponibilité peut également avoir un impact sur la résistance des plantes aux pathogènes en régulant leur immunité. Afin de mieux comprendre les liens entre la nutrition azotée et les défenses de la plante, nous avons analysé l’impact de la disponibilité en N sur la résistance de Medicago truncatula à un pathogène racinaire, Aphanomyces euteiches, en prenant en compte la variabilité génétique de la plante. Cet oomycète est considéré comme un des facteurs limitant le plus la production des légumineuses. Deux conditions de nutrition azotée, non limitante ou carencée en N, et dix génotypes de M. truncatula ont été testés in vitro. Les résultats ont montré que la résistance est modulée par les conditions nutritionnelles, dépendament du génotype. Les analyses d’expression de gènes impliqués dans le métabolisme azoté et dans les réponses de défense ainsi que la quantification des teneurs en acides aminés et des composés métaboliques secondaires ont montré des réponses différentes selon les génotypes et la condition nutritive. Elles ont souligné en particulier le rôle potentiellement important de la glutamine dans ce pathosystème. De plus, nous avons mis en évidence l’importance de l’homéostasie du monoxyde d’azote (NO) dans la résistance de M. truncatula à A. euteiches et que la disponibilité en azote impactait l’homéostasie du NO en affectant les niveaux de S-nitrosothiols et l’activité de la S-nitrosoglutathion réductase dans les racines. Ces résultats soulignent l’importance du métabolisme azoté et de son interaction avec le génotype de la plante dans les réactions de défense chez M. truncatula., Nitrogen (N) is a major limiting factor for plant growth. N availability can also impact plant resistance to pathogens by regulating plant immunity. To better understand the links between N nutrition and plant defense, we analyzed the impact of N availability of plant on Medicago truncatula resistance to the root pathogen, Aphanomyces euteiches, taking into account plant genetic variability. This oomycete is considered as the most limiting factor for legume production. Two conditions of N nutrition, non-limiting or deprived in N, and ten plant genotypes were tested in vitro. The results showed that the resistance is modulated by nutritional conditions, depending on plant genotype. Analysis of the expression of genes involved in N metabolism and defense and quantification of different amino-acids contents and secondary metabolic compounds showed different responses of the genotypes and highlighted a potential role of glutamine in this pathosystem. Furthermore, our work underlined the importance of nitric oxide (NO) homeostasis for M. truncatula resistance to A. euteiches and that N availability impacts NO homeostasis by affecting S-nitrosothiol levels and S-nitrosoglutathione reductase activity in roots. These studies highlight, therefore, the importance of N metabolism and its interaction with plant genotype in defense responses in M. truncatula.

L’augmentation des rendements est un enjeu majeur chez les céréales. Dans cet objectif, il est nécessaire de maintenir la qualité du grain de blé, qui est principalement déterminée par sa teneur et sa composition en protéines de réserve. En effet, une forte relation négative existe entre le rendement et la teneur en protéines. Par ailleurs, la qualité du grain est fortement influencée par la disponibilité en azote et en soufre dans le sol. La limitation des apports d’intrants azotés à la culture et la carence en soufre récemment observée dans les sols représentent ainsi des difficultés supplémentaires pour maitriser cette qualité. Une meilleure connaissance des mécanismes moléculaires impliqués dans le contrôle du développement du grain et la mise en place de ses réserves protéiques en réponse à la nutrition azotée et soufrée est donc primordiale. L’objectif de cette thèse a ainsi été d’apporter de nouveaux éléments à la compréhension de ces processus de régulation, aujourd’hui peu connus. Pour cela, les approches -omiques sont apparues comme une stratégie de choix pour identifier les acteurs moléculaires mis en jeu. Le protéome nucléaire a été une cible importante dans les travaux menés. L’étude de ces protéines nucléaires a révélé certains régulateurs transcriptionnels qui pourraient être impliqués dans le contrôle de la mise en place des réserves du grain. Dans une approche combinant des données de protéomique, transcriptomique et métabolomique, une vision intégrative de la réponse du grain à la nutrition azotée et soufrée a été obtenue. L’importance d’un apport de soufre dans le contrôle de la balance azote/soufre du grain, déterminante pour la composition du grain en protéines de réserve, a été clairement vérifiée. Parmi les changements observés au niveau du métabolisme cellulaire, certains des gènes affectés par la modification de cette balance pourraient orchestrer l’ajustement de la composition du grain face à des situations de carences nutritionnelles. Ces nouvelles connaissances devraient permettre de mieux maitriser la qualité du grain de blé dans un contexte d’agriculture durable., Improving the yield potential of cereals represents a major challenge. In this context, wheat grain quality has to be maintained. Indeed, grain quality is mainly determined by the content and the composition of storage proteins, but there is a strongly negative correlation between yield and grain protein concentration. In addition, grain quality is strongly influenced by the availability of nitrogen and sulfur in soils. Nowadays, the limitation of nitrogen inputs, and also the sulfur deficiency recently observed in soils represent major difficulties to control the quality. Therefore, understanding of molecular mechanisms controlling grain development and accumulation of storage proteins in response to nitrogen and sulfur supply is a major issue. The objective of this thesis was to create knowledge on the comprehension of these regulatory mechanisms. For this purpose, the best strategy to identify molecular actors involved in these processes consisted of -omics approaches. In our studies, the nuclear proteome was an important target. Among these proteins, we revealed some transcriptional regulators likely to be involved in the control of the accumulation of grain storage compounds. Using an approach combining proteomic, transcriptomic and metabolomic data, the characterization of the integrative grain response to the nitrogen and sulfur supply was obtained. Besides, our studies clearly confirmed the major influence of sulfur in the control of the nitrogen/sulfur balance that determines the grain storage protein composition. Among the changes observed in the cell metabolism, some genes were disturbed by the modification of this balance. Thus these genes could coordinate the adjustment of grain composition in response to nutritional deficiencies. These new results contribute in facing the challenge of maintaining wheat grain quality with sustainable agriculture.

Travaux relatifs aux bactéries d’intérêt alimentaire La nutrition azotée et sa régulation chez Lactococcus lactis Contexte scientifique et problématique de recherche : Signalomic des bactéries et des communautés bactériennes d’intérêt alimentaire

Competitive interactions among free living organisms such as plants and algae are known to be regulated by rates and ratios of environmental nutrient supplies1. Micro-organisms that rely on a host for their reproduction cycle hijack host metabolic machinery, as well as host resources in metabolites, enzymes and proteins. As the synthesis of these molecules requires nitrogen and phosphorus, altered host nutrients supplies have been hypothesized and thereafter shown to influence microbial dynamics, based on the response of single and a group of micro-organism species2. Yet, rates and ratios of nutrients supplied to hosts could also mediate the interactions among co-occurring micro-organisms. Understanding these effects would be an important advance because interactions among co-occuring microbes can alter both host population dynamics and epidemiological processes3.[br/] The objective of our study was to determine the outcomes of nitrogen and phosphorus addition to plant hosts on infection rates of and interactions among two plant virus species.

EAGeapsiCT2EJ2; Nutrition azotée et fonctionnement agrophysiologique spécifique des légumineuses Anne-Sophie Voisin et François Gastal La nutrition azotée des plantes de la famille des légumineuses (Fabacées) est assurée par deux voies complémentaires : absorption de l’azote minéral du sol par les racines, comme chez tous les végétaux supérieurs, et fixation de l’azote atmosphérique, propre à ces espèces, grâce à une symbiose* avec des bactéries du sol. Cette spécificité confère aux cultures de légumineuses une autonomie vis-à-vis de la disponibilité en azote du sol et de l’apport d’engrais azotés, et un rôle clé dans les flux azotés des systèmes de production végétale. La fixation symbiotique de l’azote étant un processus biologique et dynamique, connaître ses déterminants agrophysiologiques permet d’affiner les stratégies de gestion des cultures, pour bénéficier au mieux des aménités liées à l’utilisation de cet azote « écologique ». Après un rappel sur les mécanismes relatifs à la fixation symbiotique des plantes, ce chapitre visera à résumer les connaissances qualitatives et quantitatives sur la dynamique des flux azotés au sein des cultures et de leur environnement. Par ailleurs, rappelons que d’autres spécificités propres à ces plantes peuvent amener une gestion différente de ces cultures par rapport aux autres cultures et avoir des conséquences sur leur utilisation en agriculture : leur potentiel génétique (ressources génétiques et amélioration variétale actuelle), leur réactivité face à des composantes du milieu (comme la lumière, la température ou l’eau), leur plasticité en termes de composition de produits récoltés (notamment teneur en composants secondaires spécifiques). Ces autres spécificités seront décrites dans ce chapitre si elles ont une interaction avec la nutrition azotée ; sinon elles seront seulement évoquées et renverront aux autres chapitres de l’ouvrage

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

Resource-ratio theory has widely been used to explain outcomes of competitive interactions between species in communities of plants and other free-living organisms. Yet, we have little empirical data on the effects of nutrient supplies on the interactions among microbial organisms that use plants as hosts. A framework for understanding the mechanisms governing the assembly of plant microbial communities would be an important advance because plants often host a diversity of micro-organisms, and interactions among co-occurring microbes can alter both host and microbe population dynamics. To better understand the effects of nutrient supplies to hosts on microbe interactions, we singly inoculated and co-inoculated plants of one annual crop species (Avena sativa, Poaceae) with two systemic and phloem-restricted virus species, barley yellow dwarf virus-PAV (BYDV-PAV, Luteovirideae) and cereal yellow dwarf virus-RPV (CYDV-RPV, Luteovirideae). Host plants were grown across a factorial combination of N and P supply rates. P addition lowered CYDV-RPV prevalence. N addition altered the interaction strength among viruses. The co-inoculation of BYDV-PAV lowered CYDV-RPV infection rate, but this antagonistic interaction only occurred at low nutrient supply rates and disappeared at high N supply rate. These results provide insights on how nutrient-host-virus interactions might drive microbe community composition and increase the risk of co-infection, especially in light of the global scale of human alterations of biochemical cycles.

Modelling the distribution of leaf nitrogen is central to specify photosynthetic parameters and simulate canopy photosynthesis using leaf gas exchange models. Nitrogen distribution depends both on soil nitrogen availability and within-canopy light distribution. We propose the use of a set of empirical relationships with measurable parameters to dynamically account for it. The model was assessed on alfalfa canopies under contrasting N nutrition levels. It proved able to predict nitrogen distribution and daily patterns of photosynthesis from leaf to whole canopy for a large range of situations., Modelling the spatial and temporal distribution of leaf nitrogen (N) is central to specify photosynthetic parameters and simulate canopy photosynthesis. Leaf photosynthetic parameters depend on both local light availability and whole-plant N status. The interaction between these two levels of integration has generally been modelled by assuming optimal canopy functioning, which is not supported by experiments. During this study, we examined how a set of empirical relationships with measurable parameters could be used instead to predict photosynthesis at the leaf and whole-canopy levels. The distribution of leaf N per unit area (Na) within the canopy was related to leaf light irradiance and to the nitrogen nutrition index (NNI), a whole-plant variable accounting for plant N status. Na was then used to determine the photosynthetic parameters of a leaf gas exchange model. The model was assessed on alfalfa canopies under contrasting N nutrition and with N2-fixing and non-fixing plants. Three experiments were carried out to parameterize the relationships between Na, leaf irradiance, NNI and photosynthetic parameters. An additional independent data set was used for model evaluation. The N distribution model showed that it was able to predict leaf N on the set of leaves tested. The Na at the top of the canopy appeared to be related linearly to the NNI, whereas the coefficient accounting for N allocation remained constant. Photosynthetic parameters were related linearly to Na irrespective of N nutrition and the N acquisition mode. Daily patterns of gas exchange were simulated accurately at the leaf scale. When integrated at the whole-canopy scale, the model predicted that raising N availability above an NNI of 1 did not result in increased net photosynthesis. Overall, the model proposed offered a solution for a dynamic coupling of leaf photosynthesis and canopy N distribution without requiring any optimal functioning hypothesis.

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