Metabolic responses to waterlogging differ between roots and shoots and reflect phloem transport alteration in medicago truncatula

Root oxygen deficiency that is induced by flooding (waterlogging) is a common situation in many agricultural areas, causing considerable loss in yield and productivity. Physiological and metabolic acclimation to hypoxia has mostly been studied on roots or whole seedlings under full submergence. The metabolic difference between shoots and roots during waterlogging, and how roots and shoots communicate in such a situation is much less known. In particular, the metabolic acclimation in shoots and how this, in turn, impacts on roots metabolism is not well documented. Here, we monitored changes in the metabolome of roots and shoots of barrel clover (Medicago truncatula), growth, and gas-exchange, and analyzed phloem sap exudate composition. Roots exhibited a typical response to hypoxia, such as γ-aminobutyrate and alanine accumulation, as well as a strong decline in raffinose, sucrose, hexoses, and pentoses. Leaves exhibited a strong increase in starch, sugars, sugar derivatives, and phenolics (tyrosine, tryptophan, phenylalanine, benzoate, ferulate), suggesting an inhibition of sugar export and their alternative utilization by aromatic compounds production via pentose phosphates and phosphoenolpyruvate. Accordingly, there was an enrichment in sugars and a decline in organic acids in phloem sap exudates under waterlogging. Mass-balance calculations further suggest an increased imbalance between loading by shoots and unloading by roots under waterlogging. Taken as a whole, our results are consistent with the inhibition of sugar import by waterlogged roots, leading to an increase in phloem sugar pool, which, in turn, exert negative feedback on sugar metabolism and utilization in shoots.This research was funded by the Région Pays de la Loire and Angers Loire Métropole, via the grant Connect Talent Isosee

Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. 15NH4 labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized 15N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while 15N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt

Etude de l'impact de la fumure potassique sur la fetuque elevee (Festuca arundinacea L.) et le Trefle blanc (Trifolium repens L.) en monocultures ou en association et sur l'evolution du potassium assimilable dans le sol

SIGLECNRS T 58451 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Métabolisme azoté au cours de la germination et de la croissance post-germinative de Medicago truncatula (implications de la glutamate déshydrogenase dans la réponse au stress anoxique et à l'excès d'ammonium)

Le métabolisme azoté au cours de la germination et de la croissance post-germinative de la légumineuse modèle Medicago truncatula a été caractérisé, révélant un métabolisme très actif de l'ammonium et des acides aminés (biosynthèses de novo). Parmi les enzymes des métabolismes C et N étudiées au cours de ces phases de développement précoce, la glutamate déshydrogénase (GDH) a été retenue pour une étude plus approfondie. Nos résultats montrent que l'expression du gène codant pour la GDH est surtout localisée dans les tissus vasculaires (phloème) et la protéine présente la particularité d'être cytosolique alors qu'en général, cette enzyme est mitochondriale. Les mesures d'activité in vivo, par utilisation de l'isotope 15N, a révélé qu'en condition normale, la GDH a un rôle catabolique, constituant le principal fournisseur d'ammonium au cycle GS/GOGAT en l'absence d'assimilation primaire d'azote (réduction du nitrate). Un des résultats les plus originaux de notre travail est d'avoir montré que la GDH contribue à la réponse adaptative de plantules de Medicago truncatula au stress anoxique. En situation de manque d'oxygène, l'activité GDH bascule dans le sens de l'amination permettant alors l'oxydation du NADH et la régénération du NAD, vitale au maintien de la glycolyse et par conséquent, à la survie des plantes.Nitrogen metabolism was characterized during germination and post-germinative growth in the model legume Medicago truncatula. The study revealed active ammonium and amino acid metabolism (de novo biosynthesis). Among enzymes of C and N metabolism studied during these early developmental stages, glutamate dehydrogenase (GDH) was further characterized. In situ hybridisation and immunocytochemical study using transmission electron microscopy showed that GDH gene expression was localized in vascular tissues (phloem) and the protein was cytosolic while GDH is generally mitochondrial. Measurements of in vivo activity by 15N labelling revealed that GDH plays a catabolic role, providing ammonium to GS/GOGAT cycle in the absence of primary nitrogen assimilation (nitrate reduction). The most striking result was the involvement of GDH in the adaptive response of Medicago truncatula seedlings to anoxic stress. Under low oxygen, GDH activity is oriented towards the amination of oxoglutarate allowing the oxidation of NADH and NAD regeneration. The regeneration of NAD from NADH is thought to be vital to plants submitted to anoxia stress because in the absence of NAD glycolysis ceases.ANGERS-BU Lettres et Sciences (490072106) / SudocSudocFranceF

Modulation du métabolisme azoté sous hypoxie racinaire, chez Medicago truncatula

Les plantes se développent dans un environnement dynamique, ce qui impose souvent des contraintes sur la croissance et le développement. Parmi les facteurs environnementaux adverses, fréquemment rencontrés par les plantes terrestres, l'inondation ou submersion temporaire qui impose une hypoxie racinaire (1-2% d'oxygène). La submersion du sol peut avoir un très fort impact sur la survie des plantes, et donc sur la production agricole ainsi que les écosystèmes naturels. La plupart des études antérieures sur les altérations biologiques et biochimiques chez les plantes, dues au stress hypoxique, ont porté sur le métabolisme des sucres. Toutefois, quelques données concernant les effets de l'hypoxie racinaire sur le métabolisme azoté (N) sont récemment devenues disponibles. Le but de notre travail est d'étudier les effets de l'hypoxie racinaire sur le métabolisme azoté chez la plante modèle "Medicago truncatula". Les résultats obtenus ont montré que l'hypoxie racinaire entraine une augmentation significative de la biomasse aérienne (MF et MS) avec allongement des tiges et augmentation du nombre de feuilles pendant une période transitoire allant jusqu'à 5 semaines avant d'engendrer la mort des plantes. Les effets sur le métabolisme primaire ont été suivis par des analyses métabolomiques (GC-MS), marquage à l'azote (15N) et expression de gènes impliqués dans le métabolisme azoté. Nos résultats montrent que si l'hypoxie racinaire entraine des modifications sommes toute attendues des métabolismes de l'azote et du carbone, elle entraine également des réarrangements de ces métabolismes dans la partie aérienne non soumise à l'hypoxie. La réponse des parties aériennes en termes de croissance et de modification métabolique a été obtenue même quand une partie minoritaire seulement du système racinaire a été soumise à l'hypoxie suggérant une communication racine - partie aérienne qui mériterait d'être plus étudiée dans l'avenir.Plants grow in a dynamic environment, which often, imposes constraints on growth and development. Among the adverse environmental factors commonly encountered by land plants, flooding or waterlogging which imposes a temporary root hypoxia (1-2% oxygen). Hypoxia stress jeopardizes plant survival, and therefore induces tremendous damage on the agricultural production and natural ecosystem. Most previous studies of biological and biochemical alterations in plants, due to hypoxic stress, have focused on sugar metabolism. However, few data, regarding the effect of root hypoxia on nitrogen (N) metabolism have recently become available. The aim of our work is to study the impact of root hypoxia on nitrogen metabolism in the model plant "Medicago truncatula." Our results showed that root hypoxia leads to a significant increase in shoot biomass (MF and MS) with increased stem elongation and number of leaves during a transitional period of almost 5 weeks before inducing plant death. Effects on primary metabolism were followed by metabolomic analysis (GC-MS), labeling of nitrogen (15N) and expression of genes involved in nitrogen metabolism. Root hypoxia induced the expected rearrangement of carbon and nitrogen in the root but interestingly it induced significant changes in C and N metabolisms in the aerated shoot. Hypoxia-induced changes in shoot biomass and metabolism were obtained in split-root experiment where only part of the root system was submitted to hypoxia. The response of the aerated shoot to root hypoxia suggests a communication between root and shoot upon hypoxia aiming at a coherent adaptive response at the whole plant level. The nature of this communication deserves to be more thoroughly investigated.ANGERS-BU Lettres et Sciences (490072106) / SudocSudocFranceF

Reconfiguration of N Metabolism upon Hypoxia Stress and Recovery: Roles of Alanine Aminotransferase (AlaAT) and Glutamate Dehydrogenase (GDH)

In the context of climatic change, more heavy precipitation and more frequent flooding and waterlogging events threaten the productivity of arable farmland. Furthermore, crops were not selected to cope with flooding- and waterlogging-induced oxygen limitation. In general, low oxygen stress, unlike other abiotic stresses (e.g., cold, high temperature, drought and saline stress), received little interest from the scientific community and less financial support from stakeholders. Accordingly, breeding programs should be developed and agronomical practices should be adapted in order to save plants’ growth and yield—even under conditions of low oxygen availability (e.g., submergence and waterlogging). The prerequisite to the success of such breeding programs and changes in agronomical practices is a good knowledge of how plants adapt to low oxygen stress at the cellular and the whole plant level. In the present paper, we summarized the recent knowledge on metabolic adjustment in general under low oxygen stress and highlighted thereafter the major changes pertaining to the reconfiguration of amino acids syntheses. We propose a model showing (i) how pyruvate derived from active glycolysis upon hypoxia is competitively used by the alanine aminotransferase/glutamate synthase cycle, leading to alanine accumulation and NAD+ regeneration. Carbon is then saved in a nitrogen store instead of being lost through ethanol fermentative pathway. (ii) During the post-hypoxia recovery period, the alanine aminotransferase/glutamate dehydrogenase cycle mobilizes this carbon from alanine store. Pyruvate produced by the reverse reaction of alanine aminotransferase is funneled to the TCA cycle, while deaminating glutamate dehydrogenase regenerates, reducing equivalent (NADH) and 2-oxoglutarate to maintain the cycle function