Characterization of the role and regulation of the plant AtNHX cation /proton antiporter family

A family of genes from Arabidopsis (designated AtNHX) was cloned on the basis of its similarity to the yeast NHX1, a putative endosomal Na+/H+ antiporter. Expression of the AtNHX family of genes in the yeast ? nhx1 background complemented some of the mutant's observed phenotypes including sensitivity to the antibiotic hygromycin, and the pH and sodium-dependent growth inhibition. Unlike the rest of the family, AtNHX5 complemented the mutant's growth inhibition by both high K+ and Li+. AtNHX1 was immunologically detected in yeast vacuoles using antibodies raised against its hydrophilic C-terminus. Increased Na + tolerance in complemented yeast expressing AtNHX1 correlated with increased intracellular Na+. This suggests vacuolar Na + sequestration as the mechanism for increased Na+ tolerance upon the heterologous expression of the plant AtNHX-gene family. A cDNA clone with significant similarity to the AtNHX gene family, designated BvNHX1, was cloned from the halophyte Beta vulgaris by degenerate RT-PCR. Antibodies were raised against a central portion of the predicted ORF of BvNHX1 and used for the immunological detection of its gene product. BvNHX1 was detected in tonoplast-enriched membrane fractions and its abundance was increased when cells were exposed to 100 mM NaCl. This increase in protein abundance correlated with the measured increase in salt-induced vacuolar Na+/H + antiporter activity, suggesting a central role for BvNHX1 in salinity tolerance. AtNHX1 was immunologically detected in both tonoplast-enriched vesicles as well as in isolated vacuoles from Arabidopsis. Overexpression of AtNHX1 in Arabidopsis plants resulted in increased Na+-dependent H+ exchange in isolated vacuoles measured by fluorescence quenching. Furthermore, transgenic Arabidopsis plants showed significant increase in Na + tolerance exhibiting sustained growth under 200 mM NaCl as well as increased Na+ content when compared to wild type plants. These results demonstrated that the sequestration of Na+ through the action of vacuolar Na+/H+ exchange is central for salinity tolerance in plants. The C-terminus of AtNHX1 was observed to specifically interact with a calmodulin-like protein (designated as CaMX), isolated by a yeast interaction trap screening. The interaction domain was mapped within the C-terminus of AtNHX1 to a region that was observed to have the propensity to form an amphiphilic ?-helixâ€”the hallmark of calmodulin binding targets. Co-expression of AtNHX1 along with CaMX in the yeast ?nhx1 mutant background resulted in enhanced complementation of the hygromycin sensitivity and Na+-dependent yeast growth inhibition. In addition, K+ and Li+-dependent growth inhibition were complemented by co-expressing both proteins in yeast. Furthermore, overexpression of the hydrophilic C-terminus of AtNHX1 fused to GFP at its N-terminus, in Arabidopsis, resulted in reduced leaf size of transgenic plants when grown under high Na+ and high K+. These results taken together suggest the action of positive regulators of AtNHX1 vacuolar Na+/H+ antiporter activity as determinants of tolerance to ionic stress.