We examined the function and intracellular localization of the product of the Na(+)/H(+) antiporter gene (OsNHX1) cloned from rice (Oryza sativa). OsNHX1 has the ability to suppress Na(+), Li(+) and hygromycin sensitivity of yeast nhx1 mutants and sensitivity to a high K(+) concentration, a novel phenotype of the nhx1 mutants. Analysis using rice cells expressing a fusion protein of OsNHX1 and green fluorescent protein and Western blot analysis using antibodies specific for OsNHX1 confirmed the localization of OsNHX1 on the tonoplasts. These results indicate that the OsNHX1 gene encodes a vacuolar (Na(+), K(+))/H(+) antiporter. Treatment with high concentrations of NaCl and KCl increased the transcript levels of OsNHX1 in rice roots and shoots. In addition, overexpression of OsNHX1 improved the salt tolerance of transgenic rice cells and plants. These results suggest that OsNHX1 on the tonoplasts plays important roles in the compartmentation of Na(+) and K(+) highly accumulated in the cytoplasm into the vacuoles, and the amount of the antiporter is one of the most important factors determining salt tolerance in rice.

We previously cloned a vacuolar Na+/H+ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon-intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na+, Li+, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K+ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter-GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter-GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na+ and K+ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.

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Two cDNA clones encoding vacuolar H+-inorganic pyrophosphatase (HVP1 and HVP10), one clone encoding the catalytic subunit (68 kDa) of vacuolar H+-ATPase (HvVHA-A), and one clone encoding vacuolar Na+/H+ antiporter (HvNHX1) were isolated from barley (Hordeum vulgare), a salt-tolerant crop. Salt stress increased the transcript levels of HVP1, HVP10, HvVHA-A, and HvNHX1, and osmotic stress also increased the transcript levels of HVP1 and HvNHX1 in barley roots. The transcription of HVP1 in response to salt stress was regulated differently from that of HVP10. In addition, the HVP1 expression changed in a pattern similar to that of HvNHX1 expression. These results indicate that the expression of HVP1 is co-ordinated with that of HvNHX1 in barley roots in response to salt and osmotic stresses.

We screened a rice (Oryza sativa L. 'Nipponbare') full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K 1 channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Dena1-4), which lacks a major component of Na 1 efflux. It also suppressed a K 1 -transport-defective phenotype of yeast strain CY162 (Dtrk1Dtrk2), suggesting the enhancement of K 1 uptake by OsKAT1. By the expression of OsKAT1, the K 1 contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na 1 than nonexpressing cells, but almost the same K 1 . The cellular Na 1 to K 1 ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K 1 content. These functions of OsKAT1 are likely to be common among Shaker K 1 channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K 1 channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na 1 .