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

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 (H 2 O 2 ), and the production rate of superoxide anion (O 2 -. ). 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. • We explored the mechanisms of plant adaptation to Cl - under salt stress. • We found that Malus hupehensis MhCLC-c1 protein enhance plant salt tolerance by promoting Cl - efflux under salt stress. • We searched for an upstream transcription factor MhbHLH122. MhbHLH122 activates MhCLC-c1 transcription.