Molecular cloning and characterisation of SaCLCa1, a novel protein of the chloride channel (CLC) family from the halophyte Suaeda altissima (L.) Pall

Abstract

The SaCLCa1 gene, a putative orthologue of AtCLCa, the Arabidopsis thaliana gene encoding a NO3–/H+ antiporter, was cloned from the halophyte Suaeda altissima. It belonged to the CLC family, comprising anionic channels and anion/H+ antiporters. SaCLCa1 ion specificity was studied by heterologous expression of this gene in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encoded the only CLC family protein, the Cl– transporter Gef1p, in undisrupted strains of this organism. For comparison, the function of another recently identified S. altissima CLC family gene, SaCLCc1, was also characterised. Expression of SaCLCc1 in Δgef1 cells restored their ability to grow on selective media. This supported the chloride specificity of this transporter. By contrast, expression of SaCLCa1 did not complement the growth defect phenotype of Δgef1 cells. However, growth of the Δgef1 mutant on the selective media was partially restored when it was transformed with SaCLCa1(C562 T), encoding the modified protein SaCLCa1(P188S), in which proline responsible for NO3– selectivity in selective filter was replaced by serine providing chloride selectivity. Quantitative real-time polymerase chain reactions (qRT-PCR) showed that significant induction of SaCLCa1 occurred in the roots of S. altissima when plants were grown on nitrate-deficient medium, while SaCLCc1 activation was observed in S. altissima leaves of plants grown in increasing Cl– concentrations of nutrient solution. These results suggested that SaCLCa1 and SaCLCc1 function as anionic transporters with nitrate and chloride specificities, respectively.