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Metal-tolerance proteins (MTPs) are divalent cation transporters that have been shown to be essential for metal homeostasis and tolerance in model plants and hyperaccumulators. Due to the lack of genomic resources, studies on MTPs in cultivated crops are lacking. Here, we present the first functional characterization of genes encoding cucumber proteins homologous to MTP1 and MTP4 transporters. CsMTP1 expression was ubiquitous in cucumber plants, whereas CsMTP4 mRNA was less abundant and was not detected in the generative parts of the flowers. When expressed in yeast, CsMTP1 and CsMTP4 were able to complement the hypersensitivity of mutant strains to Zn and Cd through the increased sequestration of metals within vacuoles using the transmembrane electrochemical gradient. Both proteins formed oligomers at the vacuolar membranes of yeast and cucumber cells and localized in Arabidopsis protoplasts, consistent with their function in vacuolar Zn and Cd sequestration. Changes in the abundance of CsMTP1 and CsMTP4 transcripts and proteins in response to elevated Zn and Cd, or to Zn deprivation, suggested metal-induced transcriptional, translational, and post-translational modifications of protein activities. The differences in the organ expression and affinity of both proteins to Zn and Cd suggested that CsMTP1 and CsMTP4 may not be functionally redundant in cucumber cells.

Cation diffusion facilitator (CDF) proteins are ubiquitous divalent cation transporters that have been proved to be essential for metal homeostasis and tolerance in Archaebacteria, Bacteria, and Eukaryota. In plants, CDFs are designated as metal tolerance proteins (MTPs). Due to the lack of genomic resources, studies on MTPs in other plants, including cultivated crops, are lacking. Here, the identification and organization of genes encoding members of the MTP family in cucumber are described. The first functional characterization of a cucumber gene encoding a member of the Mn-CDF subgroup of CDF proteins, designated as CsMTP8 based on the highest homology to plant MTP8, is also presented. The expression of CsMTP8 in Saccharomyces cerevisiae led to increased Mn accumulation in yeast cells and fully restored the growth of mutants hypersensitive to Mn in Mn excess. Similarly, the overexpression of CsMTP8 in Arabidopsis thaliana enhanced plant tolerance to high Mn in nutrition media as well as the accumulation of Mn in plant tissues. When fused to green fluorescent protein (GFP), CsMTP8 localized to the vacuolar membranes in yeast cells and to Arabidopsis protoplasts. In cucumber, CsMTP8 was expressed almost exclusively in roots, and the level of gene transcript was markedly up-regulated or reduced under elevated Mn or Mn deficiency, respectively. Taken together, the results suggest that CsMTP8 is an Mn transporter localized in the vacuolar membrane, which participates in the maintenance of Mn homeostasis in cucumber root cells.

International audience; Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

International audience; In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wildtype plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.

International audience; Anion channels/transporters are key to a wide spectrum of physiological functions in plants, such as osmoregulation, cell signaling, plant nutrition and compartmentalization of metabolites, and metal tolerance. The recent identification of gene families encoding some of these transport systems opened the way for gene expression studies, structure-function analyses of the corresponding proteins, and functional genomics approaches toward further understanding of their integrated roles in planta. This review, based on a few selected examples, illustrates that the members of a given gene family exhibit a diversity of substrate specificity, regulation, and intracellular localization, and are involved in a wide range of physiological functions. It also shows that post-translational modifications of transport proteins play a key role in the regulation of anion transport activity. Key questions arising from the increasing complexity of networks controlling anion transport in plant cells (the existence of redundancy, cross talk, and coordination between various pathways and compartments) are also addressed.

International audience; Nitrate, the major nitrogen source for plants, can be accumulated in the vacuole. Its transport across the vacuolar membrane is mediated by AtCLCa, an antiporter of the chloride channel (CLC) protein family. In contrast to other CLC family members, AtCLCa transports nitrate coupled to protons. Recently, the different behaviour towards nitrate of CLC proteins has been linked to the presence of a serine or proline in the selectivity filter motif GXGIP. By monitoring AtCLCa activity in its native environment, we show that if proline 160 in AtCLCa is changed to a serine (AtCLCa(P160S) ), the transporter loses its nitrate selectivity, but the anion proton exchange mechanism is unaffected. We also performed in vivo analyses in yeast and Arabidopsis. In contrast to native AtCLCa, expression of AtCLCa(P160S) does not complement either the ΔScCLC yeast mutant grown on nitrate or the nitrate under-accumulation phenotype of clca knockout plants. Our results confirm the significance of this amino acid in the conserved selectivity filter of CLC proteins and highlight the importance of the proline in AtCLCa for nitrate metabolism in Arabidopsis.

International audience; Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.

Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological level and, more recently, at the molecular level. Two classes of genes, NRT1 and NRT2, have been found to be potentially involved in the high and low affinity nitrate transport systems (HATS and LATS, respectively). The complexity of the molecular basis of nitrate uptake has been enhanced by the finding that in many plants both NRT1 and NRT2 classes are represented by multigene families. Furthermore, recent studies demonstrate that the control mechanisms that lead to an active protein at the plasma membrane act on gene transcription, modulating the steady-state levels of mRNA, and on the activation of the protein, possibly by a phosphorylation/dephosphorylation process. This is a review of recent progress in the characterization of the NRT2 nitrate transporters, the composition of this family in Arabidopsis, their possible role in nitrate acquisition, and some aspects of their regulation in plants.

We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 microM KNO3). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation.

The nitrate assimilation pathway has been the matter of intensive research during the past decade. Many genes involved in low and high affinity nitrate uptake have been identified in fungi, algae and, more recently, in plants. The plant genes so far isolated are transcriptionally regulated; their inducibility by nitrate seems to be a common feature, shared by their homologs in fungi and algae. A number of questions remain to be elucidated regarding the physiological roles of these transporters and the regulation of their expression.

Six monoclonal antibodies (MAbs) against a French isolate of barley yellow mosaic virus (BaYMV) pathotype 2 were produced and their isotypes determined. These MAbs were compared in ELISA for their reactivity with different isolates of BaYMV, wheat yellow mosaic virus (WYMV), wheat spindle streak mosaic virus (WSSMV) and oat mosaic virus (OMV).The six MAbs detected BaYMV in TAS ELISA and western blot, whereas in ACP ELISA no reaction was observed with isolates of BaYMV and WYMV. These MAbs could recognize the sequential motifs situated at the surface of viral particles. The six MAbs detected all the European isolates of BaYMV pathotype 1 and 2 and the Japanese isolate of this viral pathotype 1-1. In contrast to other MAbs, MAb IV did not react with the Japanese isolate of BaYMV pathotype II-1. In TAS ELISA. MAbs I, II, III, and IV detected the Japanese isolate of WYMV and American isolates of WSSMV only when they were captured by anti-WYMV polyclonal antibodies. A French isolate of OMV was detected only by the MAbs I and II in TAS ELISA with Polyclonal anti-BaYMV.

International audience; Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high-affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock-out mutant where the T-DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3 ' region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low-affinity system. Moreover, the deregulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild-type. These results demonstrate that plant NRT2 proteins do have a role in HATS.