Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells

Genes encoding ion transporters that regulate ion homeostasis in soybean have not been carefully investigated. Using degenerate primers, we cloned a putative chloride channel gene (GmCLC1) and a putative Na+/H+ antiporter gene (GmNHX1) from soybean. Confocal microscopic studies using yellow fluorescent fusion proteins revealed that GmCLC1 and GmNHX1 were both localized on tonoplast. The expressions of GmCLC1 and GmNHX1 were both induced by NaCl or dehydration stress imposed by polyethylene glycol (PEG). Using mitochondrial integrity and cell death as the damage indicators, a clear alleviation under NaCl stress (but not PEG stress) was observed in both GmCLC1 and GmNHX1 transgenic cells. Using fluorescent dye staining and quenching, respectively, a higher concentration of chloride ion (Cl-) or sodium ion (Na+) was observed in isolated vacuoles in the cells of GmCLC1 and of GmNHX1 transgenic lines. Our result suggested that these vacuolar-located ion transporters function to sequester ions from cytoplasm into vacuole to reduce its toxic effects.