CaSnRK2.4‐mediated phosphorylation of CaNAC035 regulates abscisic acid synthesis in pepper (Capsicum annuum L.) responding to cold stress

Plant NAC transcription factors play a crucial role in enhancing cold stress tolerance, yet the precise molecular mechanisms underlying cold stress remain elusive. In this study, we identified and characterized CaNAC035, an NAC transcription factor isolated from pepper (Capsicum annuum) leaves. We observed that the expression of the CaNAC035 gene is induced by both cold and abscisic acid (ABA) treatments, and we elucidated its positive regulatory role in cold stress tolerance. Overexpression of CaNAC035 resulted in enhanced cold stress tolerance, while knockdown of CaNAC035 significantly reduced resistance to cold stress. Additionally, we discovered that CaSnRK2.4, a SnRK2 protein, plays an essential role in cold tolerance. In this study, we demonstrated that CaSnRK2.4 physically interacts with and phosphorylates CaNAC035 both in vitro and in vivo. Moreover, the expression of two ABA biosynthesis-related genes, CaAAO3 and CaNCED3, was significantly upregulated in the CaNAC035-overexpressing transgenic pepper lines. Yeast one-hybrid, Dual Luciferase, and electrophoretic mobility shift assays provided evidence that CaNAC035 binds to the promoter regions of both CaAAO3 and CaNCED3 in vivo and in vitro. Notably, treatment of transgenic pepper with 50 μm Fluridone (Flu) enhanced cold tolerance, while the exogenous application of ABA at a concentration of 10 μm noticeably reduced cold tolerance in the virus-induced gene silencing line. Overall, our findings highlight the involvement of CaNAC035 in the cold response of pepper and provide valuable insights into the molecular mechanisms underlying cold tolerance. These results offer promising prospects for molecular breeding strategies aimed at improving cold tolerance in pepper and other crops.