Cloning and characterization of a novel vacuolar Na+/H+ antiporter gene (VuNHX1) from drought hardy legume, cowpea for salt tolerance

Abstract

Soil salinity is a major abiotic stress that seriously affects crop productivity worldwide. One of the mechanisms that allow plants to withstand salt stress is vacuolar sequestration of Na+, through a Na+/H+ antiporter. We isolated a new vacuolar Na+/H+ antiporter gene (VuNHX1) from a drought hardy grain legume, cowpea (Vigna unguiculata L.). The cDNA is 1,981 bp, with an open reading frame of 1,629 bp encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. VuNHX1 displays a conserved amiloride binding domain (84LFFIYLLPPI93) in third transmembrane (TM3) region. Phylogenetic and bioinformatic analysis indicated VuNHX1 belonging to Class-I clade of plant NHX exchangers with high similarity with legume Na+/H+ antiporters. To assess its role in Na+ exchange, we performed complementation studies using the salt sensitive yeast mutant strain AXT3. The results showed that VuNHX1 complemented for the loss of yeast NHX1 under NaCl, KCl and LiCl stress in the salt sensitive phenotype of the yeast strain AXT3. The expression profiles revealed significant induction of VuNHX1 in cowpea seedlings under salt, cold and dehydration stress. Both expression analysis and ion estimation under salt stress indicated the VuNHX1 expression preferentially in roots than in leaves. Overexpression of VuNHX1 in transgenic Arabidopsis conferred enhanced salt tolerance in transgenic Arabidopsis lines while the wild type plants exhibited growth retardation. This study shows that VuNHX1 is a potential gene for salt tolerance, and can be used in future for developing cisgenic salt tolerant cowpea and transgenic salt tolerant crops.