Journal Article

Molecular Cloning, Functional Expression and Subcellular Localization of Two Putative Vacuolar Voltage-gated Chloride Channels in Rice (<i>Oryza sativa</i> L.)

Atsuko Nakamura, Atsunori Fukuda, Shingo Sakai and Yoshiyuki Tanaka

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 47, issue 1, pages 32-42
Published in print January 2006 | ISSN: 0032-0781
Published online January 2006 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci220
Molecular Cloning, Functional Expression and Subcellular Localization of Two Putative Vacuolar Voltage-gated Chloride Channels in Rice (Oryza sativa L.)

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  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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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.

Keywords: Chloride channel; Ion homeostasis; Rice; Salt stress; Vacuole; ORF, open reading frame; P-ATPase, plasma membrane ATPase; RFLP, restriction fragment length polymorphism; RT–PCR, reverse transcription–PCR; sGFP, synthetic green fluorescent protein. V-ATPase, vacuolar ATPase; V-PPase, vacuolar pyrophosphatase

Journal Article.  7018 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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