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 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.

Chloride Channel Family in the Euhalophyte Suaeda altissima (L.) Pall: Cloning of Novel Members SaCLCa2 and SaCLCc2, General Characterization of the Family

“…OM994379, accordingly). This approach led earlier to cloning of 5 CLC genes from S. altissima [ 31 , 32 , 33 ].…”

“…Cloned cDNAs of the SaCLCa2 and SaCLCc2 genes contained open reading frames (ORFs) encoding polypeptides consisting of 800 and 708 amino acids, with calculated molecular masses of 88.2 and 77.5 kDa, respectively. Amino acid sequences of the SaCLCa2 and SaCLCc2 were aligned with those of other SaCLC proteins revealed by us previously [ 31 , 32 , 33 ]. The SaCLCa2 protein is comparable in size with SaCLCa1 (811 a.a.), SaCLCc1 (791 a.a.), SaCLCd (793 a.a.), and SaCLCg (776 a.a.) ( Figure 1 ), as well with AtCLCa (775 a.a.), AtCLCb (780 a.a.) [ 36 ], and a protein encoded by a sequence found in S. fruticosa transcriptome, SfCLCa2 (800 a.a.) ( Figure S1a ).…”

“…In previous studies, we have cloned 5 genes of the CLC family from the euhalophyte Suaeda altissima : SaCLCa1 , SaCLCc1 , SaCLCd , SaCLCf , and SaCLCg [ 31 , 32 , 33 ], homologs of the CLCs from A. thaliana : AtCLCa , AtCLCc , AtCLCd , AtCLCf , and AtCLCg , respectively. Effects of salinity on expression of these genes and ionic specificity of the encoded proteins have been also investigated.…”

Research Advances on Molecular Mechanism of Salt Tolerance in Suaeda

“…In Suaeda altissima (L.) Pall., the expression of chloride channel (CLC) family genes SaCLCc1 , SaCLCd , SaCLCf and SaCLCg in leaves increased with the increase of salt concentration, consistent with the accumulation of Cl − in leaf cells. The results indicated that SaCLCc1, SaCLCd, SaCLCf and SaCLCg proteins may be involved in the separation of Cl − in organelles, and may be involved in the mechanism of salt tolerance [ 42 , 43 ]. In addition, the complementation assay and bioinformatic analyses indicated that SaCLCc1 and SaCLCd proteins are Cl − /H + antiporters, while the SaCLCf and SaCLCg proteins are likely Cl − channels [ 42 , 43 ].…”

“…The results indicated that SaCLCc1, SaCLCd, SaCLCf and SaCLCg proteins may be involved in the separation of Cl − in organelles, and may be involved in the mechanism of salt tolerance [ 42 , 43 ]. In addition, the complementation assay and bioinformatic analyses indicated that SaCLCc1 and SaCLCd proteins are Cl − /H + antiporters, while the SaCLCf and SaCLCg proteins are likely Cl − channels [ 42 , 43 ].…”