The transcription factor GmbHLH3 confers Cl−/salt tolerance to soybean by upregulating GmCLC1 expression for maintenance of anion homeostasis

Chloride channels (CLCs)-mediated anion homeostasis plays a crucial role for plants to adapt to Cl−/salt stress; however, the transcriptional regulation of CLCs gene expression in response to Cl−/salt stress is not understood to date. Here, a novel bHLH transcription factor, GmbHLH3, upstream of the GmCLC1 gene, was identified by gene co-expression network analysis. GmbHLH3 localizes to the nucleus and can bind to the G-box at position − 550 of GmCLC1 promoter to up-regulate its expression. GmbHLH3-overexpressed soybean hairy root-composite plants and transgenic Arabidopsis exhibited enhanced tolerance to Cl−/salt stress, which was reflected by plant growth phenotype, leaf dead cells, cell membrane damage and lipid peroxidation, and Arabidopsis seed germination. NaCl-treated GmbHLH3-overexpressed soybean plants displayed up-regulated GmCLC1 expression, higher accumulation of Cl− and NO3− in roots for restricting the transport to shoots, and thus maintaining lower Cl−/NO3− ratio in whole plants. Similar effects of GmbHLH3 on the tissue distribution of Cl− and NO3−, and maintenance of anion homeostasis were further verified in transgenic Arabidopsis plants. These findings provide a valuable theoretical basis for further exploration of the transcriptional regulatory molecular networks of plant CLC family members in response to Cl− toxicity under salt stress, as well as seeking molecular genetic improvement approaches for Cl−/salt tolerance in soybean and other crops.