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One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl ions.

Coding sequences of the CLC family genes SaCLCd, SaCLCf, and SaCLCg, the putative orthologs of Arabidopsis thaliana AtCLCd, AtCLCf, and AtCLCg genes, were cloned from the euhalophyte Suaeda altissima (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 SaCLC genes in the Saccharomyces cerevisiae GEF1 disrupted strain Δgef1. GEF1 encoded the only CLC family protein, the Cl− transporter Gef1p, in undisrupted strains of this organism. Expression of SaCLCd in Δgef1 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 SaCLCf and SaCLCg did not complement the growth defect phenotype of Δgef1 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 SaCLCd, SaCLCf, and SaCLCg genes were shown to be expressed in the roots and leaves of S. altissima. In response to addition of NaCl to the growth medium, the relative transcript abundances of all three genes of S. altissima increased in the leaves but did not change significantly in the roots. The increase in expression of SaCLCd, SaCLCf, and SaCLCg 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 S. altissima plants to increasing salinity and possible participation in mechanisms underlying salt tolerance.

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