MhbHLH122-MhCLC-c1 regulates Malus hupehensis chloride salt tolerance by promoting Cl- efflux

Abstract

Chloride channels (CLCs) are crucial for adapting plants to salt stress. However, the mechanism by which CLCs regulate plants’ responses to chloride salt stress remains unclear. This study identified a basic helix-loop-helix (bHLH) transcription factor, MhbHLH122, which positively regulated MhCLC-c1 transcription. The transcription of MhbHLH122 was induced by chloride salt stress. Under conditions of chloride salt stress, M. hupehensis [Malus hupehensis (Pamp.) Rehd. var. pingyiensis Jiang] hairy root-composite plants with MhbHLH122 overexpressed alone, MhCLC-c1 overexpressed alone, and both of them overexpressed together, as well as Arabidopsis thaliana, all showed a decrease in the relative cell death rate, the content of malondialdehyde (MDA), the content of hydrogen peroxide (H₂O₂), and the production rate of superoxide anion (O₂^-.). Furthermore, in the ‘Orin’ apple calli with suppression of MhbHLH122, the expression level of MhCLC-c1 was down-regulated and it was more sensitive to chloride salt stress. Under conditions of chloride salt stress, overexpression of MhCLC-c1 promoted the efflux of Cl^- from M. hupehensis hairy root-composite plants. Moreover, the Cl^- efflux rate was further increased after MhbHLH122 was over-expressed in the MhCLC-c1-overexpression plants. Therefore, MhbHLH122 can regulate Cl^- efflux in the root system by upregulating MhCLC-c1 expression, thereby enhancing the tolerance of plants to chloride salt stress.