Two MscS Homologs Provide Mechanosensitive Channel Activities in the Arabidopsis Root

In bacterial and animal systems, mechanosensitive (MS) ion channels are thought to mediate the perception of pressure, touch, and sound 1, 2 and 3. Although plants respond to a wide variety of mechanical stimuli, and although many mechanosensitive channel activities have been characterized in plant membranes by the patch-clamp method, the molecular nature of mechanoperception in plant systems has remained elusive [4]. Likely candidates are relatives of MscS (Mechanosensitive channel of small conductance), a well-characterized MS channel that serves to protect E. coli from osmotic shock [5]. Ten MscS-Like (MSL) proteins are found in the genome of the model flowering plant Arabidopsis thaliana 4, 6 and 7. MSL2 and MSL3, along with MSC1, a MscS family member from green algae, are implicated in the control of organelle morphology 8 and 9. Here, we characterize MSL9 and MSL10, two MSL proteins found in the plasma membrane of root cells. We use a combined genetic and electrophysiological approach to show that MSL9 and MSL10, along with three other members of the MSL family, are required for MS channel activities detected in protoplasts derived from root cells. This is the first molecular identification and characterization of MS channels in plant membranes

Focus on plant proteomics.

Proteomics covers the systematic analysis of proteins expressed by a genome, from the identification of their primary amino-acid sequence to the determination of their relative amounts, their state of modification and association with other proteins or molecules of different types. Tremendous progress has been made in this field in the past few years, especially in plant biology, mostly due to major developments of mass spectrometry dedicated to protein analyses and advanced information technology. The aim of this special issue of Plant Physiology and Biochemistry devoted to Plant Proteomics is not to present a comprehensive coverage of this rapidly expanding field but to focus on the representation of some key aspects to illustrate the importance of proteomics in plant functional genomics

Caractérisation fonctionnelle du transporteur vacuolaire ClCa de Arabidopsis thaliana (activité d'échange de NO3-/H+ et régulation par les nucléotides)

Le nitrate représente pour la majeure partie des plantes terrestres une importante source d azote. Les plantes absorbent le nitrate du sol, l assimilent dans des composés azotés et en stockent le surplus dans la vacuole centrale. Les protéines responsables du transport intracellulaire du nitrate sont encore inconnues, mais il a été suggéré que des protéines de la famille des CLC (ChLoride Channel) pouvaient être impliquées dans ces fonctions. Ces travaux de thèse démontrent que la protéine AtClCa (Arabidopsis thaliana ClC) est localisée dans la membrane vacuolaire et transporte des anions au travers du tonoplaste. Ils démontrent également que AtClCa est un antiport NO3-/H+ avec une stoechiométrie de 2 NO3- transportés pour chaque H+ transféré. Sa propriété d antiport, ainsi que sa spécificité pour le nitrate, permettent à AtClCa d accumuler le nitrate dans la vacuole. Son activité de transport est inhibée par l ATP, alors que l ADP et l AMP n ont pas d effet sur le courant porté par AtClCa. Cependant, l AMP et l ATP entrent en compétition pour le site d interaction avec AtClCa. Ce site d interaction avec les nucléotides se trouve probablement dans le domaine C-terminal de la protéine. Le domaine C-terminal de AtClCa a été modélisé en utilisant la structure du C-terminal de la protéine humaine hClC-5. Les données de dynamique moléculaire obtenues via ce modèle s reproduisent les propriétés d interaction entre AtCLCa et les nucléotides déterminées expérimentalement. L ensemble de ces données montre que AtClCa est un élément clef de l homéostasie du nitrate intracellulaire, et que son activité de transport est régulée en fonction de l état métabolique de la cellule.Nitrate is the major nitrogen source for plants. Plants absorb nitrate from the soil, assimilate it in nitrogen compounds and stock the surplus of nitrate in the central vacuole. The proteins responsible for the intracellular transport of nitrate are unknown. It has been suggested that proteins that belong to the CLC family (ChLoride Channel) could be involved in nitrate intracellular homeostasis. In the present thesis we showed that AtClCa (Arabidopsis thaliana ClCa) is localized in the vacuolar membrane, and demonstrated its ability to mediate anions currents across the tonoplast. We could also demonstrate that AtClCa is a NO3-/H+ antiporter with a stoichiometry of 2NO3- transported for each H+ transferred. The antiporter property, together with nitrate specificity, enable AtClCa to mediate the accumulation of nitrate in the vacuole. We also showed that the current mediated by AtClCa is inhibited by ATP. ADP and AMP have no effect on AtClCa current, but AMP competes with ATP for the site of interaction with AtClCa. The interaction of nucleotides with AtClCa takes place presumably at its C-terminal. The C-terminal domain of AtClCa has been modelled by homology using the structure of the C-terminal of hClC-5. The data obtained with this model by molecular dynamics simulations can reproduce the experimental data on the interaction properties of AtClCa and the nucleotides. The set of data presented in this thesis shows that AtClCa is a key element for the homeostasis of intracellular nitrate, and that its transport activity is regulated in function of the metabolic state of the cell.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

AtMSL9 and AtMSL10: Sensors of plasma membrane tension in Arabidopsis roots

Plant cells, like those of animals and bacteria, are able to sense physical deformation of the plasma membrane. Mechanosensitive (MS) channels are proteins that transduce mechanical force into ion flux, providing a mechanism for the perception of mechanical stimuli such as sound, touch and osmotic pressure. We recently identified AtMSL9 and AtMSL10, two mechanosensitive channels in Arabidopsis thaliana, as molecular candidates for mechanosensing in higher plants.1 AtMSL9 and AtMSL10 are members of a family of proteins in Arabidopsis that are related to the bacterial MS channel MscS, termed MscS-Like (or MSL).2 MscS (Mechanosensitive channel of Small conductance) is one of the best-characterized MS channels, first identified as an electrophysiological activity in the plasma membrane (PM) of giant E. coli spheroplasts.3,4 Activation of MscS is voltage-independent, but responds directly to tension applied to the membrane and does not require other cellular proteins for this regulation.5,6 MscS family members are widely distributed throughout bacterial and archaeal genomes, are present in all plant genomes yet examined, and are found in selected fungal genomes.2,7,8 MscS homolgues have not yet been identified in animals

Nitrate efflux is an essential component of the cryptogein signaling pathway leading to defense responses and hypersensitive cell death in tobacco.

There is much interest in the transduction pathways by which avirulent pathogens or derived elicitors activate plant defense responses. However, little is known about anion channel functions in this process. The aim of this study was to reveal the contribution of anion channels in the defense response triggered in tobacco by the elicitor cryptogein. Cryptogein induced a fast nitrate (NO(3)(-)) efflux that was sensitive to anion channel blockers and regulated by phosphorylation events and Ca(2+) influx. Using a pharmacological approach, we provide evidence that NO(3)(-) efflux acts upstream of the cryptogein-induced oxidative burst and a 40-kD protein kinase whose activation seems to be controlled by the duration and intensity of anion efflux. Moreover, NO(3)(-) efflux inhibitors reduced and delayed the hypersensitive cell death triggered by cryptogein in tobacco plants. This was accompanied by a delay or a complete suppression of the induction of several defense-related genes, including hsr203J, a gene whose expression is correlated strongly with programmed cell death in plants. Our results indicate that anion channels are involved intimately in mediating defense responses and hypersensitive cell death