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 (NO3-), and are critical for nutrition uptake and transport, adjustment of cellular turgor, stomatal movement, signal transduction, and Cl- and NO3- 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 NO3- 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 NO3- in the aerial part of the plant, GsCLC-c2 could reduce the Cl-/NO3- 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 NO3- 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 NO3- 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.