Molecular biology and transport properties of grapevine Na⁺/H⁺ antiporter

Abstract

Na⁺/H⁺ antiporters are involved in the transport of sodium and hydrogen ions across membranes and contribute in pH regulation of actively metabolizing cells. They play a primary role in homeo-stasis and are found in every biological kingdom, from bacteria to humans to higher plants. In plants, vacuolar Na⁺/H⁺ antiporters use the proton electrochemical gradient generated by the vacuolar H⁺-translocating enzymes, H⁺-ATPase, and H⁺-PP_iase to couple the downhill movement of H⁺ with the uphill movement of Na⁺. Moreover, it has been shown that they compartmentalize Na⁺ into the vacuoles for detoxification and improve consequently the salt tolerance in yeasts and plants. Recently, genes encoding these Na⁺/H⁺ antiporters have been identified and studied using a molecular genetic approach in the model systemsArabidopsis or Saccharomyces cerevisiae. We describe here the identification, cloning, molecular characterization and functional properties in yeast heterologous system of a vacuolar Na⁺/H⁺antiporter from grapevine. To identify a Na⁺/H⁺ antiporter from grapevine we applied a candidate gene approach. A 1.83-kb genomic sequence adjacent to the VvNHX1 gene was isolated using the thermal asymmetric interlaced-PCR. Histochemical localization of β-glucuronidase gene (GUS) activity was directed by VvNHX1 promoter-GUS fusion in transgenic Arabidopsis. To determine the subcellular localization of the VvNHX1 protein by heterologous expression in yeast and transient expression in onion epidermal cells, chimera constructions were prepared using a modified green fluorescent protein mGFP6. An RT-PCR approach was used to examine the VvNHX1 mRNA levels in different organs and tissues of grapevine plants. To assess VvNHX1 transport properties, VvNHX1 was expressed in the nhx1 mutant TY001 (that lacks the endogenous Nhx1 Na⁺/H⁺ antiporter) and the rates of H⁺-coupled transport was measured by fluorescence quenching. Rates of cation-dependent proton movements in vacuoles isolated from yeast expressing VvNHX1 were measured.