Pepper bHLH transcription factor CabHLH035 contributes to salt tolerance by modulating ion homeostasis and proline biosynthesis

Members of the bHLH family of transcription factors play important roles in multiple aspects of plant biological processes, for instance, abiotic stress responses. Previously, we characterized CaNAC035, a gene that positively regulates stress tolerance and identified CabHLH035, a CaNAC035-interacting protein in pepper (Capsicum annuum L.). In this study, we describe the role of CabHLH035 in the response to salt stress. Our results show that the expression of CabHLH035 increased following salt treatment. Transient expression of CabHLH035 (CabHLH035-To) in pepper enhanced salt tolerance, ectopic expression of CabHLH035 in Arabidopsis increased the salt stress tolerance, whereas knocking down the expression of CabHLH035 in pepper plants resulted in decreased salt tolerance. Homologs of the Salt Overly Sensitive 1 (SOS1) and pyrroline-5-carboxylate acid synthetase (P5CS) genes showed drastically increased expression in transgenic Arabidopsis plants expressing CabHLH035 and CabHLH035-To plants, but expression decreased in CabHLH035-silenced plants. Our results also showed that CabHLH035 can directly bind to the CaSOS1 and CaP5CS gene promoters and positively activate their expression. We found that transgenic Arabidopsis plants, ectopic expression of CabHLH035 and pepper plants transiently overexpressing CabHLH035 (CabHLH035-To) showed lower Na+ and higher proline contents in response to NaCl treatment, while CabHLH035-silenced plants had higher Na+ and lower proline concentrations. Overall, CabHLH035 plays important roles in salt tolerance through its effects on the intracellular Na+ : K+ ratio and proline biosynthesis.