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Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoter CaMV35S::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter Napin2S::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway.

Many physiological processes like stomata aperture, nutrient up-take, cellular elongation and cell signalling involve anion fluxes at the two main membranes, the plasma and vacuolar membranes of plant cells. Specialized membrane proteins form active and passive anion transport systems mediating and regulating anion fluxes. Ion channels are passive transport systems mediating ion fluxes across membranes along the electrochemical gradient. Whereas active transporters work against the electrochemical gradient of the transported ion allowing its accumulation into a cellular compartment. In plant cells, the H⁺ gradient is the main energy source of antiporters and symporters that couple the transport of anions like NO₃⁻ and Cl⁻ to the transport of H⁺. In the presents work, we aimed at analysing anion and H⁺ fluxes at two levels. First, we used an electrophysiological approach to study the functional properties of two anion transport systems acting at the vacuolar membrane, AtCLCc and AtCLCg. We also expressed a biosensor, clopHensor in A. thaliana to dynamically measure in vivo the [NO₃⁻] and pH of the cytosol. We chose stomata guard cells as a cellular model to study these fluxes. Our results illustrate the in vivo dynamics of cytosolic [NO₃⁻] and pH variations in the cytosol of guard cells. Our data show that in guard cells the cytosolic [NO₃⁻] is highly influenced by the extracellular [NO₃⁻]. At last, clopHensor’s expression in plants KO for the vacuolar NO₃⁻/H⁺ antiporter AtCLCa and for the plasma membrane anion channel SLAC1 allowed us to dissect the role of the two membranes in controlling the variation of cytosolic [NO₃⁻] and pH. This work enabled to visualize the activity of an anion channel (SLAC1) and of a NO₃⁻/H⁺ antiporter (AtCLCa) in vivo and to quantify the impact of anion and proton fluxes on cytosolic homeostasis of guard cells.; De nombreux processus physiologiques tels que les mouvements stomatiques, l’absorption des nutriments, l’élongation cellulaire et la signalisation cellulaire impliquent des flux d’anions entre les membranes plasmique et vacuolaire des cellules végétales. Ces flux ioniques sont régulés par des canaux et transporteurs membranaires. Les canaux ioniques transportent passivement les ions au travers des membranes selon le gradient électrochimique. Les transporteurs actifs permettent le transport contre le gradient électrochimique de l’ion transporté induisant son accumulation dans un compartiment cellulaire. Dans les cellules végétales, le gradient de H+ entre différents compartiments constitue la principale source d’énergie couplée par les symports et les antiports au transport de NO₃⁻ et Cl⁻. Au cours de ma thèse, j’ai analysé ces flux ioniques avec deux approches. Une première approche a consisté en l’étude fonctionnelle par électrophysiologie de deux protéines membranaires, AtCLCc et AtCLCg impliquées dans le transport d’anions. Dans une deuxième approche, un biosenseur, clopHensor a été exprimé chez A. thaliana et a permis de mesurer simultanément la [NO₃⁻] et le pH cytosoliques in vivo. Les cellules de garde ont été choisies comme modèle cellulaire pour l’étude de la dynamique in vivo de la [NO₃⁻]cyt et du pH. Nous avons mis en évidence que la [NO₃⁻]cyt est influencée par les conditions extracellulaires dans ces cellules. Enfin l’expression de clopHensor en plantes KO pour un antiport NO₃⁻/H⁺ vacuolaire, AtCLCa, et d’un canal anionique de la membrane plasmique, SLAC1, nous a permis d’étudier la contribution de deux membranes dans la régulation de [NO₃⁻] et du pH cytosolique. Les travaux menés ont permis de visualiser l’activité de canaux et de transporteurs d’anions et H⁺ in vivo et de quantifier leur impact sur l’homéostasie du cytosol.

Metabolomics of leaf laminae and veins during ageing reveal tissue specificities and different senescence programmes., Leaf senescence is a long developmental process important for nutrient management and for source to sink remobilization. Constituents of the mesophyll cells are progressively degraded to provide nutrients to the rest of the plant. Up to now, studies on leaf senescence have not paid much attention to the role of the different leaf tissues. In the present study, we dissected leaf laminae from the midvein to perform metabolite profiling. The laminae mesophyll cells are the source of nutrients, and in C3 plants they contain Rubisco as the most important nitrogen storage pool. Veins, rich in vasculature, are the place where all the nutrients are translocated, and sometimes interconverted, before being exported through the phloem or the xylem. The different metabolic changes we observed in laminae and midvein with ageing support the idea that the senescence programme in these two tissues is different. Important accumulations of metabolites in the midvein suggest that nutrient translocations from source leaves to sinks are mainly controlled at this level. Carbon and nitrogen long-distance molecules such as fructose, glucose, aspartate, and asparagine were more abundant in the midvein than in laminae. In contrast, sucrose, glutamate, and aspartate were more abundant in laminae. The concentrations of tricarboxylic acid (TCA) compounds were also lower in the midvein than in laminae. Since nitrogen remobilization increased under low nitrate supply, plants were grown under two nitrate concentrations. The results revealed that the senescence-related differences were mostly similar under low and high nitrate conditions except for some pathways such as the TCA cycle.

Editeur : Alain Gojon (INRA); As a result of climate changes, land use and agriculture have to adapt to new demands. Agriculture is responsible for a large part of the greenhouse gas (GHG) emissions that have to be urgently reduced in order to protect the environment. At the same time, agriculture has to cope with the challenges of sustainably feeding a growing world population. Reducing the use of the ammonia-nitrate fertilizers that are responsible for a large part of the GHGs released and that have a negative impact on carbon balance is one of the objectives of precision agriculture. One way to reduce N fertilizers without dramatically affecting grain yields is to improve the nitrogen recycling and remobilization performances of plants. Mechanisms involved in nitrogen recycling, such as autophagy, are essential for nutrient remobilization at the whole-plant level and for seed quality. Studies on leaf senescence and nutrient recycling provide new perspectives for improvement. The aim of this review is to give an overview of the mechanisms involved in nitrogen recycling and remobilization during leaf senescence and to present the different approaches undertaken to improve nitrogen remobilization efficiency using both model plants and crop species.

Autophagy is a universal mechanism in eukaryotes that promotes cell longevity and nutrient recycling through the degradation of unwanted organelles, proteins and damaged cytoplasmic compounds. Autophagy is important in plant resistance to stresses and starvations and in remobilization. Autophagy facilitates bulk and selective degradations, through the delivery of cell material to the vacuole where hydrolases and proteases reside. Large metabolite modifications are observed in autophagy mutants showing the important role of autophagy in cell homeostasis. The control of autophagic activity by nutrients and energy status is supported by several studies in plant and animal. We review how autophagy contributes to nutrient management in plants and how nutrient status control this degradation pathway for adaptation to the environment.