Molecular Cloning and Characterization of SaCLCd, SaCLCf, and SaCLCg, Novel Proteins of the Chloride Channel Family (CLC) from the Halophyte Suaeda altissima (L.) Pall

Abstract

Coding sequences of the CLC family genes <i>SaCLCd</i>, <i>SaCLCf</i>, and <i>SaCLCg</i>, the putative orthologs of <i>Arabidopsis thaliana AtCLCd</i>, <i>AtCLCf</i>, and <i>AtCLCg</i> genes, were cloned from the euhalophyte <i>Suaeda altissima</i> (L.) Pall. The key conserved motifs and glutamates inherent in proteins of the CLC family were identified in SaCLCd, SaCLCf, and SaCLCg amino acid sequences. SaCLCd and SaCLCg were characterized by higher homology to eukaryotic (human) CLCs, while SaCLCf was closer to prokaryotic CLCs. Ion specificities of the SaCLC proteins were studied in complementation assays by heterologous expression of the <i>SaCLC</i> genes in the <i>Saccharomyces cerevisiae GEF1</i> disrupted strain <i>Δgef1</i>. <i>GEF1</i> encoded the only CLC family protein, the Cl^- transporter Gef1p, in undisrupted strains of this organism. Expression of <i>SaCLCd</i> in <i>Δgef1</i> cells restored their ability to grow on selective media. The complementation test and the presence of both the "gating" and "proton" conservative glutamates in SaCLCd amino acid sequence and serine specific for Cl^- in its selectivity filter suggest that this protein operates as a Cl^-/H⁺ antiporter. By contrast, expression of <i>SaCLCf</i> and <i>SaCLCg</i> did not complement the growth defect phenotype of <i>Δgef1</i> cells. The selectivity filters of SaCLCf and SaCLCg also contained serine. However, SaCLCf included only the "gating" glutamate, while SaCLCg contained the "proton" glutamate, suggesting that SaCLCf and SaCLCg proteins act as Cl^- channels. The <i>SaCLCd</i>, <i>SaCLCf</i>, and <i>SaCLCg</i> genes were shown to be expressed in the roots and leaves of <i>S. altissima</i>. In response to addition of NaCl to the growth medium, the relative transcript abundances of all three genes of <i>S. altissima</i> increased in the leaves but did not change significantly in the roots. The increase in expression of <i>SaCLCd</i>, <i>SaCLCf,</i> and <i>SaCLCg</i> in the leaves in response to increasing salinity was in line with Cl^- accumulation in the leaf cells, indicating the possible participation of SaCLCd, SaCLCf, and SaCLCg proteins in Cl^- sequestration in cell organelles. Generally, these results suggest the involvement of SaCLC proteins in the response of <i>S. altissima</i> plants to increasing salinity and possible participation in mechanisms underlying salt tolerance.