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

Abstract

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