The family of chloride channel proteins that mediate Cl- transportation play vital roles in plant nutrient supply, cellular action potential and turgor pressure adjustment, stomatal movement, hormone signal recognition and transduction, Cl- homeostasis, and abiotic and biotic stress tolerance. The anionic toxicity, mainly caused by chloride ions (Cl-), on plants under salt stress remains poorly understood. In this work, we investigated the function of soybean Cl-/H+ antiporter GmCLC1 under salt stress in transgenic Arabidopsis thaliana, soybean, and yeast. We found that GmCLC1 enhanced salt tolerance in transgenic A. thaliana by reducing the Cl- accumulation in shoots and hence released the negative impact of salt stress on plant growth. Overexpression of GmCLC1 in the hairy roots of soybean sequestered more Cl- in their roots and transferred less Cl- to their shoots, leading to lower relative electrolyte leakage values in the roots and leaves. When either the soybean GmCLC1 or the yeast chloride transporter gene, GEF1, was transformed into the yeast gef1 mutant, and then treated with different chloride salts (MnCl2, KCl, NaCl), enhanced survival rate was observed. The result indicates that GmCLC1 and GEF1 exerted similar effects on alleviating the stress of diverse chloride salts on the yeast gef1 mutant. Together, this work suggests a protective function of GmCLC1 under Cl- stress.

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.