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Plant and Cell Physiology Advance Access originally published online on October 25, 2005
Plant and Cell Physiology 2006 47(1):32-42; doi:10.1093/pcp/pci220
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Molecular Cloning, Functional Expression and Subcellular Localization of Two Putative Vacuolar Voltage-gated Chloride Channels in Rice (Oryza sativa L.)

Atsuko Nakamura1,2,*, Atsunori Fukuda2, Shingo Sakai1 and Yoshiyuki Tanaka2

1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, 305-8572 Japan
2 National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602 Japan

* Corresponding author: E-mail, anakamur{at}nias.affrc.go.jp; Fax, +81-298-38-8347.

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.

The nucleotide sequences reported in this paper have been submitted to the DDBJ/EMBL/GenBank database under the accession numbers AB069966 [GenBank] (OsCLC-1) and AB069967 [GenBank] (OsCLC-2).

(Received October 4, 2005; Accepted October 18, 2005)
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