CaSNF1 interacts with and phosphorylates CaHD-ZIP to enhance cold tolerance in pepper

Cold stress severely restricts pepper (Capsicum annuum L.) growth and productivity by disrupting cellular homeostasis and inducing oxidative damage. Although HD-ZIP transcription factors play important roles in plant abiotic stress responses, the molecular mechanisms regulating their function during cold stress remain largely unknown. Here, we characterized the pepper HD-ZIP transcription factor CaHD-ZIP and demonstrated that it is a nuclear-localized, cold-responsive protein that positively contributes to cold stress tolerance. To further investigate the regulatory mechanism underlying CaHD-ZIP function, a yeast two-hybrid (Y2H) screen identified the sucrose non-fermenting 1-related protein kinase CaSNF1 as a previously unrecognized interacting partner of CaHD-ZIP. Their interaction was confirmed by yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation assays, with BiFC indicating that the interaction occurs predominantly in the nucleus. In vitro phosphorylation analysis further demonstrated that CaSNF1 directly phosphorylates CaHD-ZIP, providing biochemical evidence for a functional regulatory relationship between these proteins. Complementary loss- and gain-of-function analyses, including virus-induced gene silencing and transient overexpression in pepper together with stable overexpression in Arabidopsis, demonstrated that CaSNF1 positively regulates cold tolerance. Silencing of CaSNF1 increased oxidative damage, impaired antioxidant enzyme activities, and suppressed the expression of antioxidant and cold-responsive genes, whereas overexpression produced the opposite effects and significantly enhanced cold tolerance. Together, these findings identify CaSNF1 as a CaHD-ZIP-interacting kinase and reveal a potential CaSNF1-CaHD-ZIP regulatory module involved in pepper cold stress adaptation. This study advances our understanding of the molecular mechanisms underlying pepper cold tolerance and provides potential genetic targets for improving cold resilience in pepper and other Solanaceae crops.