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

Abstract

CLC family genes, comprising anion channels and anion/H+ antiporters, are widely represented in nearly all prokaryotes and eukaryotes. CLC proteins carry out a plethora of functions at the cellular level. Here the coding sequences of the SaCLCa2 and SaCLCc2 genes, homologous to Arabidopsis thaliana CLCa and CLCc, were cloned from the euhalophyte Suaeda altissima (L.) Pall. Both the genes cloned belong to the CLC family as supported by the presence of the key conserved motifs and glutamates inherent for CLC proteins. SaCLCa2 and SaCLCc2 were heterologously expressed in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encodes the only CLC family protein, the Cl– transporter Gef1p, in undisrupted strains of yeast. The Δgef1 strain is characterized by inability to grow on YPD yeast medium containing Mn2+ ions. Expression of SaCLCa2 in Δgef1 cells growing on this medium did not rescue the growth defect phenotype of the mutant. However, a partial growth restoration occurred when the Δgef1 strain was transformed by SaCLCa2(C544T), the gene encoding protein in which proline, specific for nitrate, was replaced with serine, specific for chloride, in the selectivity filter. Unlike SaCLCa2, expression of SaCLCc2 in Δgef1 resulted in a partial growth restoration under these conditions. Analysis of SaCLCa2 and SaCLCc2 expression in the euhalophyte Suaeda altissima (L.) Pall by quantitative real-time PCR (qRT-PCR) under different growth conditions demonstrated stimulation of SaCLCa2 expression by nitrate and stimulation of SaCLCc2 expression by chloride. The results of yeast complementation assay, the presence of both the “gating” and “proton” glutamates in aa sequences of both the proteins, as well results of the gene expression in euhalophyte Suaeda altissima (L.) Pall suggest that SaCLCa2 and SaCLCc2 function as anion/H+ antiporters with nitrate and chloride specificities, respectively. The general bioinformatic overview of seven CLC genes cloned from euhalophyte Suaeda altissima is given, together with results on their expression in roots and leaves under different levels of salinity.

Novel Proteins of the High-Affinity Nitrate Transporter Family NRT2, SaNRT2.1 and SaNRT2.5, from the Euhalophyte Suaeda altissima: Molecular Cloning and Expression Analysis

“…Total RNA from S. altissima plant organs was isolated by the hot phenolic method [ 56 ] and used as a template for the total first-strand cDNA synthesis. For amplification of the 3′- and 5′-ends of the SaNRT2.1 and SaNRT2.5 transcripts by the Step-Out RACE method, the first strand of cDNA was synthesized on the total RNA template isolated from Suaeda roots using MINT revertase (Evrogen, Moscow, Russia).…”

Identification and selection of reference genes for analysis of gene expression by quantitative real-time PCR in the euhalophyte Suaeda altissima (L.) Pall.

“…S. altissima (L.) Pall. was cultivated in hydroponics under controlled conditions, as it was described previously [ 26 ]. Briefly, the plants were illuminated with high pressure sodium lamps DNaZ_400 (Re- flux, Russia) with a photoperiod of 12 h/12 h (day/night) and a light intensity of 300 µmol photons/(m −2 s −1 ) and grown in Robinson-Downton medium [ 44 ].…”

Yeast Heterologous Expression Systems for the Study of Plant Membrane Proteins

“…Recently, the genes of anionic transporters of the CLC family from a salt-accumulating euhalophyte Suaeda altissima have been cloned [98][99][100][101]: SaCLCa1/a2 (the putative orthologs of AtCLCa encoding NO 3…”

“…Recently, the genes of anionic transporters of the CLC family from a salt-accumulating euhalophyte Suaeda altissima have been cloned [ 98 , 99 , 100 , 101 ]: SaCLCa1/a2 (the putative orthologs of AtCLCa encoding NO 3 − /H + -antiporter of A. thaliana [ 102 ]), SaCLCc1/c2 and SaCLCd (the putative orthologs, correspondingly, of AtCLCc and AtCLCd encoding Cl − /H + - antiporters of A. thaliana [ 103 , 104 ]), and SaCLCf and SaCLCg (the putative orthologs of AtCLCf and AtCLCg encoding Cl − channels [ 94 , 105 ]). The CLC genes of S. altissima were expressed in the mutant strain Δgef1 .…”