Mécanismes de l'accumulation des bétaïnes en réponse au stress osmotique chez Sinorhizobium meliloti en culture libre et lors de la symbiose avec Medicago sativa (importance du transporteur de bétaïnes, BetS)

Les recherches menées au laboratoire portent sur l'adaptation de la symbiose S. meliloti-M. sativa aux contraintes osmotiques en considérant à la fois le partenaire bactérien et la plante hôte. Il est bien établi que la croissance de S. meliloti, fortement inhibée en milieu de forte osmolarité, est restaurée de manière spectaculaire par un apport exogène de glycine bétaïne (GB) ou de proline bétaïne (PB). Au cours de mon travail de thèse, je me suis attaché à identifier et à caractériser un transporteur de bétaïnes, nommé BetS. Ce système, de type BCCT (Betaine Choline Carnitine Transporter), est un symport Na+-dépendant, exprimé de façon constitutive et soumis à une régulation post-traductionnelle par l'osmolarité du milieu. Le phénotype d'un mutant betS a été analysé. Il apparaît clairement que BetS joue un rôle majeur dans les premières étapes de l'adaptation de S. meliloti à un stress osmotique (1). Une série de mutants de délétions des extrémités N- et C- terminales cytoplasmiques du transporteur a été réalisée afin de déterminer le rôle de ces régions dans la perception du stress osmotique. Les résultats indiquent que l'extrémité C-terminale joue un rôle primordial dans la structure et/ou le fonctionnement du système. En parallèle, les mécanismes d'adaptation de la luzerne au stress salin ont été analysés. Cette légumineuse, soumise à une salinité élevée (NaCl 0,2 M) accumule dans les différents organes (tige, racine et nodosité) de la proline et de la PB. La PB est synthétisée, dans les tiges, à partir de la proline et son catabolisme est fortement inhibé à forte osmolarité. Des modifications importantes de la structure du symbiosome et de la teneur en Na+ et K+ sont observées dans le bactéroïde qui accumule la proline et la PB en condition de stress. La PB est transportée à travers la membrane péribactéroïdienne et par les bactéroïdes via le système BetS qui contribue à l'accumulation de cette bétaïne (2). L'utilisation d'une fusion betS-lacZ et des mesures de transport réalisées sur des bactéroïdes isolés ont montré que le gène betS est exprimé au cours des différentes étapes du développement de la nodosité et que le transporteur BetS est fonctionnel dans les nodosités matures. Un mutant du gène betS n'est cependant pas altéré dans son phénotype symbiotique. En revanche, des bactéroïdes issus d'une souche surexprimant le gène betS accumulent la PB plus rapidement que ceux issus de la souche sauvage. Ce résultat est corrélé au maintien de l'activité fixatrice d'azote à un niveau élevé après 7 jours de stress, alors que cette activité chute de 50 % chez les bactéroïdes issus de la souche sauvage. Cependant, la surproduction n'apporte plus d'avantage après 14 jours de stress. Ces résultats indiquent un rôle bénéfique important de la PB, mais limité à la phase initiale de réponse au stress. En outre, le gène betS a été introduit chez Bradyrhizobium japonicum, souche d'intérêt agronomique nodulant le soja (Glycine max), mais très sensible au stress salin. L'expression du gène betS confère à B. japonicum la capacité d'accumuler la GB et la PB et de croître en présence de 80 mM de NaCl, lorsque ces composés sont disponibles dans le milieu, alors que la croissance de la souche sauvage est complètement inhibée dans les mêmes conditions. Des systèmes d'efflux de cette bétaïne permettent spontanément de relarguer la GB lors d'un retour à des conditions de faible osmolarité. Cependant, l'introduction du gène betS ne restaure pas la croissance de la souche complémentée lorsque la concentration en NaCl est de 100 mM. L'hypothèse d'une toxicité de l'ion Na+, liée à l'absence apparente d'échangeur Na+/H+, est discutée (3).NICE-BU Sciences (060882101) / SudocSudocFranceF

Exploring legume-rhizobia symbiotic models for waterlogging tolerance

Unexpected and increasingly frequent extreme precipitation events result in soil flooding or waterlogging. Legumes have the capacity to establish a symbiotic relationship with endosymbiotic atmospheric dinitrogen-fixing rhizobia, thus contributing to natural nitrogen soil enrichment and reducing the need for chemical fertilization. The impact of waterlogging on nitrogen fixation and legume productivity needs to be considered for crop improvement. This review focuses on the legumes-rhizobia symbiotic models. We aim to summarize the mechanisms underlying symbiosis establishment, nodule development and functioning under waterlogging. The mechanisms of oxygen sensing of the host plant and symbiotic partner are considered in view of recent scientific advances

Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation

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Which role for nitric oxide in symbiotic N2-fixing nodules: toxic by-product or useful signaling/metabolic intermediate?

The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship characterized by the formation of new organs called nodules, in which bacteria have the ability to fix atmospheric nitrogen (N2) via the nitrogenase activity. Significant nitric oxide (NO) production was evidenced in the N2-fixing nodules suggesting that it may impact the symbiotic process. Indeed, NO was shown to be a potent inhibitor of nitrogenase activity and symbiotic N2 fixation. It has also been shown that NO production is increased in hypoxic nodules and this production was supposed to be linked - via a nitrate/NO respiration process - with improved capacity of the nodules to maintain their energy status under hypoxic conditions. Other data suggest that NO might be a developmental signal involved in the induction of nodule senescence. Hence, the questions were raised of the toxic effects versus signaling/metabolic functions of NO, and of the regulation of NO levels compatible with nitrogenase activity. The present review analyses the different roles of NO in functioning nodules, and discusses the role of plant and bacterial (flavo)hemoglobins in the control of NO level in nodules

Nitric Oxide Metabolic Pathway in Drought-Stressed Nodules of Faba Bean (Vicia faba L.)

International audienceDrought is an environmental stress that strongly impacts plants. It affects all stages of growth and induces profound disturbances that influence all cellular functions. Legumes can establish a symbiosis with Rhizobium-type bacteria, whose function is to fix atmospheric nitrogen in organs called nodules and to meet plant nitrogen needs. Symbiotic nitrogen fixation (SNF) is particularly sensitive to drought. We raised the hypothesis that, in drought-stressed nodules, SNF inhibition is partly correlated to hypoxia resulting from nodule structure compaction and an increased O2 diffusion barrier, and that the nodule energy regeneration involves phytoglobin-nitric oxide (Pgb-NO) respiration. To test this hypothesis, we subjected faba bean (Vicia faba L.) plants nodulated with a Rhizobium laguerreae strain to either drought or osmotic stress. We monitored the N2-fixation activity, the energy state (ATP/ADP ratio), the expression of hypoxia marker genes (alcohol dehydrogenase and alanine aminotransferase), and the functioning of the Pgb-NO respiration in the nodules. The collected data confirmed our hypothesis and showed that (1) drought-stressed nodules were subject to more intense hypoxia than control nodules and (2) NO production increased and contributed via Pgb-NO respiration to the maintenance of the energy state of drought-stressed nodules

Cross-Regulation between N Metabolism and Nitric Oxide (NO) Signaling during Plant Immunity

International audiencePlants are sessile organisms that have evolved a complex immune system which helps them cope with pathogen attacks. However, the capacity of a plant to mobilize different defense responses is strongly affected by its physiological status. Nitrogen (N) is a major nutrient that can play an important role in plant immunity by increasing or decreasing plant resistance to pathogens. Although no general rule can be drawn about the effect of N availability and quality on the fate of plant/pathogen interactions, plants' capacity to acquire, assimilate, allocate N, and maintain amino acid homeostasis appears to partly mediate the effects of N on plant defense. Nitric oxide (NO), one of the products of N metabolism, plays an important role in plant immunity signaling. NO is generated in part through Nitrate Reductase (NR), a key enzyme involved in nitrate assimilation, and its production depends on levels of nitrate/nitrite. NR substrate/product, as well as on L-arginine and polyamine levels. Cross-regulation between NO signaling and N supply/metabolism has been evidenced. NO production can be affected by N supply, and conversely NO appears to regulate nitrate transport and assimilation. Based on this knowledge, we hypothesized that N availability partly controls plant resistance to pathogens by controlling NO homeostasis. Using the Medicago truncatula/Aphanomyces euteiches pathosystem, we showed that NO homeostasis is important for resistance to this oomycete and that N availability impacts NO homeostasis by affecting S-nitrosothiol (SNO) levels and S-nitrosoglutathione reductase activity in roots. These results could therefore explain the increased resistance we noted in N-deprived as compared to N-replete M, truncatula seedlings. They open onto new perspectives for the studies of N/plant defense interactions

Proline Betaine Accumulation and Metabolism in Alfalfa Plants under Sodium Chloride Stress. Exploring Its Compartmentalization in Nodules

The osmoprotectant Pro betaine is the main betaine identified in alfalfa (Medicago sativa). We have investigated the long-term responses of nodulated alfalfa plants to salt stress, with a particular interest for Pro betaine accumulation, compartmentalization, and metabolism. Exposure of 3-week-old nodulated alfalfa plants to 0.2 m NaCl for 4 weeks was followed by a 10-, 4-, and 8-fold increase in Pro betaine in shoots, roots, and nodules, respectively. Isotope-labeling studies in alfalfa shoots indicate that [(14)C]Pro betaine was synthesized from l-[(14)C]Pro. [(14)C]Pro betaine was efficiently catabolized through sequential demethylations via N-methylPro and Pro. Salt stress had a minor effect on Pro betaine biosynthesis, whereas it strongly reduced Pro betaine turnover. Analysis of Pro betaine and Pro compartmentalization within nodules revealed that 4 weeks of salinization of the host plants induced a strong increase in cytosol and bacteroids. The estimated Pro betaine and Pro concentrations in salt-stressed bacteroids reached 7.4 and 11.8 mm, respectively, compared to only 0.8 mm in control bacteroids. Na(+) content in nodule compartments was also enhanced under salinization, leading to a concentration of 14.7 mm in bacteroids. [(14)C]Pro betaine and [(14)C]Pro were taken up by purified symbiosomes and free bacteroids. There was no indication of saturable carrier(s), and the rate of uptake was moderately enhanced by salinization. Ultrastructural analysis showed a large peribacteroid space in salt-stressed nodules, suggesting an increased turgor pressure inside the symbiosomes, which might partially be due to an elevated concentration in Pro, Pro betaine, and Na(+) in this compartment

Potassium uptake into growing barley leaf cells

Fricke W, Volkov V, Amtmann A, et al. Potassium uptake into growing barley leaf cells. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology. 2009;153(Suppl. 2):S186