Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean

The anionic toxicity of plants under salt stress is mainly caused by chloride (Cl ). Thus Cl influx, transport and their regulatory mechanisms should be one of the most important aspects of plant salt tolerance studies, but are often sidelined by the focus on sodium (Na ) toxicity and its associated adaptations. Plant chloride channels (CLCs) are transport proteins for anions including Cl and nitrate (NO ), and are critical for nutrition uptake and transport, adjustment of cellular turgor, stomatal movement, signal transduction, and Cl and NO homeostasis under salt stress. Among the eight soybean CLC genes, the tonoplast-localized c2 has uniquely different transcriptional patterns between cultivated soybean N23674 and wild soybean BB52. Using soybean hairy root transformation, we found that GsCLC-c2 over-expression contributed to Cl and NO homeostasis, and therefore conferred salt tolerance, through increasing the accumulation of Cl in the roots, thereby reducing their transportation to the shoots where most of the cellular damages occur. Also, by keeping relatively high levels of NO in the aerial part of the plant, GsCLC-c2 could reduce the Cl /NO ratio. Wild type GsCLC-c2, but not its mutants (S184P, E227V and E294G) with mutations in the conserved domains, is able to complement Saccharomyces cerevisiae △gef1 Cl sensitive phenotype. Using two-electrode voltage clamp on Xenopus laevis oocytes injected with GsCLC-c2 cRNA, we found that GsCLC-c2 transports both Cl and NO with slightly different affinity, and the affinity toward Cl was pH-independent. This study revealed that the expression of GsCLC-c2 is induced by NaCl-stress in the root of wild soybean. The tonoplast localized GsCLC-c2 transports Cl with a higher affinity than NO in a pH-independent fashion. GsCLC-c2 probably alleviates salt stress in planta through the sequestration of excess Cl into the vacuoles of root cells and thus preventing Cl from entering the shoots where it could result in cellular damages.