Structure, fonction et régulation du transporteur de nitrate/proton AtCLCa chez arabidopsis thaliana

AtCLCa chez Arabidopsis thaliana appartient à la famille de transporteurs d anions appelés ChLoride Channel (CLC). AtCLCa est plus sélectif pour le nitrate que pour le chlorure. C est un antiporteur NO3-/H+ à l origine de l accumulation du nitrate dans la vacuole. La différence de sélectivité est due à un simple changement d un acide aminé. Nous avons pu montrer que le changement de cet acide aminé transforme AtCLCa en un transporteur de chlorure et est incapable de réaliser sa fonction dans l accumulation de nitrate dans la vacuole in planta. De plus, nous avons démontré que AtCLCa est directement régulé par la liaison de l ATP et que cet effet est influencé par le rapport ATP/AMP, l AMP supprimant l effet de l ATP. Ceci suggère que la régulation de AtCLCa est dépendante du statut énergétique de la cellule. En supplément, nous avons aussi montré que cette protéine est régulée par phosphorylation dans sa partie N-terminale. Nous avons identifié une classe de protéines, les SnRKs, qui sont capables de phosphoryler AtCLCa in vitro et d interagir avec AtCLCa in vivo. SnRK1.1 et SnRK2.6. SnRK1.1 est exprimée de manière ubiquitaire dans la plante et peut inhiber l activité de la nitrate réductase. Des études d expression GUS montrent que AtCLCa est exprimé au travers de la plante (comme SnRK1.1) mais une forte expression observée dans les cellules de garde comme pour SnRK2.6. Des analyses phénotypiques des mutants nuls clca démontrent la fonction de AtCLCa non seulement dans l accumulation du nitrate mais aussi dans les mouvements stomatiques. Ces résultats suggèrent une possible interaction dans les cellules de garde de la kinase SnRK2.6 et ATCLCa in planta.The Arabidopsis thaliana CLCa belongs to the ChLoride Channel (CLC) family of anion transport proteins. AtCLCa is most selective for nitrate and not for chloride. It mediates the accumulation of nitrate into the vacuole by a NO3-/H+ exchanger mechanism. The difference in selectivity is accompanied by a single amino acid difference. We could show that an exchange of this amino acid turns AtCLCa into a chloride transporter and abolishes its function of nitrate accumulation in planta. Furthermore, we could show that AtCLCa is directly regulated by ATP and that this effect is influenced by the ATP/AMP ratio, as AMP abolished the effect of ATP, suggesting an energy-state dependent regulation of AtCLCa in the cell. Additionally to the regulation of AtCLCa by nucleotides, we could also show that it is regulated by phosphorylation on its N-terminus. We identified a specific class of kinases, the SnRKs, which are able to phosphorylate AtCLCa in vitro and interact with it in vivo. We focused on two candidate kinases within this family, SnRK1.1 and SnRK2.6. SnRK1.1 can inhibit the nitrate reductase. Therefore, the activity of SnRK1.1 is connected to nitrate metabolism, like the activity of AtCLCa. SnRK2.6 is strongly expressed in stomata guard cells and GUS expression studies showed that AtCLCa is expressed throughout the plant (like SnRK1.1), but shows a particular high expression in stomata guard cells like SnRK2.6. Subsequent phenotype analyses of clca knock-out mutants demonstrated a role of AtCLCa not only in nitrate accumulation but also in stomata movement, suggesting a possible interaction of the guard cell kinase SnRK2.6 and AtCLCa in planta.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display

International audienc

Nitrate transport : a key step in nitrate assimilation

International audienc

Nitrate transport in plants: which gene and which control ?

55 ref., special issue: inorganic nitrogen assimilationInternational audienc

Production and characterization of monoclonal antibodies to Barley Yellow Mosaic Virus and their use in detection of four Bymoviruses

International audienc

Signalling mechanisms integrating root and shoot responses to changes in the nitrogen supply.

During their life cycle, plants must be able to adapt to wide variations in the supply of soil nitrogen (N). Changes in N availability, and in the relative concentrations of NO3 −and NH4 +, are known to have profound regulatory effects on the N uptake systems in the root, on C and N metabolism throughout the plant, and on root and shoot morphology. Optimising the plant’s responses to fluctuations in the N supply requires co-ordination of the pathways of C and N assimilation, as well as establishment of the appropriate allocation of resources between root and shoot growth. Achieving this integration of responses at the whole plant level implies long-distance signaling mechanisms that can communicate information about the current availability of N from root-to-shoot, and information about the C/N status of the shoot in the reverse direction. In this review we will discuss recent advances which have contributed to our understanding of these long-range signaling pathways

Nitrate and glutamate sensing by root plants.

The architecture of a root system plays a major role in determining how efficiently a plant can capture water and nutrients from the soil. Growth occurs at the root tips and the process of exploring the soil volume depends on the behaviour of large numbers of individual root tips at different orders of branching. Each root tip is equipped with a battery of sensory mechanisms that enable it to respond to a range of environmental signals, including nutrients, water potential, light, gravity and touch. We have previously identified a MADS (MCM1, agamous, deficiens and SRF) box gene (ANR1) in Arabidopsis thaliana that is involved in modulating the rate of lateral root growth in response to changes in the external NO3- supply. Transgenic plants have been generated in which a constitutively expressed ANR1 protein can be post-translationally activated by treatment with dexamethasone (DEX). When roots of these lines are treated with DEX, lateral root growth is markedly stimulated but there is no effect on primary root growth, suggesting that one or more components of the regulatory pathway that operate in conjunction with ANR1 in lateral roots may be absent in the primary root tip. We have recently observed some very specific effects of low concentrations of glutamate on root growth, resulting in significant changes in root architecture. Experimental evidence suggests that this response involves the sensing of extracellular glutamate by root tip cells. We are currently investigating the possible role of plant ionotropic glutamate receptors in this sensory mechanism

Comparison and differentiation of Wheat Yellow Mosaic Virus (WYMV), Wheat Spindle Streak Mosaic Virus (WSSMV) and Barley Yellow Mosaic Virus (BaYMV) isolates using WYMV monoclonal antibodies

International audienc

Characterization of the Chloride Channel-Like, AtCLCg, Involved in Chloride Tolerance in Arabidopsis thaliana

International audienceIn plant cells, anion channels and transporters are essential for key functions such as nutrition, ion homeostasis and resistance to biotic or abiotic stresses. We characterized AtCLCg, a member of the chloride channel (CLC) family in Arabidopsis localized in the vacuolar membrane. When grown on NaCl or KCl, atclcg knock-out mutants showed a decrease in biomass. In the presence of NaCl, these mutants overaccumulate chloride in shoots. No difference in growth was detected in response to osmotic stress by mannitol. These results suggest a physiological function of AtCLCg in the chloride homeostasis during NaCl stress. AtCLCg shares a high degree of identity (62%) with AtCLCc, another vacuolar CLC essential for NaCl tolerance. However, the atclcc atclccg double mutant is not more sensitive to NaCl than single mutants. As the effects of both mutations are not additive, gene expression analyses were performed and revealed that: (i)AtCLCg is expressed in mesophyll cells, hydathodes and phloem while AtCLCc is expressed in stomata; and (ii)AtCLCg is repressed in the atclcc mutant background, and vice versa. Altogether these results demonstrate that both AtCLCc and AtCLCg are important for tolerance to excess chloride but not redundant, and form part of a regulatory network controlling chloride sensitivity