title: Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).
abstract: We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan. anakamur@nias.affrc.go.jp
authors.forename: Atsuko
authors.initials: A
authors.lastname: Nakamura
authors.forename: Atsunori
authors.initials: A
authors.lastname: Fukuda
authors.forename: Shingo
authors.initials: S
authors.lastname: Sakai
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D005800:Genes, Fungal; D017343:Genes, Plant; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012275:Oryza / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013045:Species Specificity; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D018118:Chloride Channels; D018076:DNA, Complementary; D018744:DNA, Plant; C089713:GEF1 protein, S cerevisiae; D010940:Plant Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride