Molecular Cloning, Subcellular Localization and Functional Analysis of ThCLC-a from Thellungiella halophila

Often, nitrate is the major source of available nitrogen for plants. Nitrate can accumulate in central vacuoles via tonoplast transporters. In the present study, a gene termed ThCLC-a that encodes a chloride channel protein was isolated from Thellungiella halophila. Deduced amino acid sequence analysis revealed high identity with AtCLC-a. RT-PCR analysis showed that the ThCLC-a gene was expressed ubiquitously in all major organs and its expression was induced by nitrate treatment. Confocal microscopy using green fluorescent fusion proteins revealed that ThCLC-a was localized specifically to the tonoplast membrane. Furthermore, an RNAi construct expressing a ThCLC-a cDNA fragment was used to silence the endogenous ThCLC-a in T. halophila. HPLC analysis showed that the nitrate content in shoots or roots of silenced plants was 19-36 % lower than in wild-type plants. Transgenic Arabidopsis plants ectopically expressing the ThCLC-a gene could accumulate 15-21 % more nitrate content than wild type plants under limited nitrogen conditions. Finally, our results suggest ThCLC-a may play an important role in the transport of nitrate via the vacuolar membrane.