Cold-induced CaCYP links protein degradation and antioxidant defense via CabHLH-79 in Capsicum annuum

Cold stress severely limits pepper (Capsicum annuum) production worldwide. We identified CaCYP (Capsicum annuum cysteine protease), a cold-inducible papain-like cysteine protease that enhances cold tolerance through its interaction with the bHLH transcription factor CabHLH-79, as confirmed by yeast two-hybrid screening, BiFC, luciferase complementation, and GUS transactivation assays. CaCYP expression increased 11-fold after 48 h of cold stress (4°C), and the protein localized predominantly to the nucleus, where it co-localized with CabHLH-79. Functional analysis using VIGS and transient overexpression showed that CaCYP promotes antioxidant gene expression through CabHLH-79. Overexpression plants exhibited 22-28-fold increases in CaCAT, CaPOD, and CaSOD transcript levels with corresponding rises in enzyme activities, whereas VIGS plants showed 70-80% reductions, collectively lowering ROS accumulation as revealed by NBT and DAB staining. Physiologically, CaCYP overexpression conferred marked cold protection, with 2.4-fold lower electrolyte leakage, 3.1-fold higher chlorophyll retention, 2.3-fold reduced MDA content, and maintained stomatal integrity relative to controls. These results indicate that CaCYP links protein degradation with antioxidant defense through CabHLH-79, although the precise downstream mechanism by which CabHLH-79 mediates antioxidant gene expression remains to be fully elucidated. Overall, CaCYP represents a promising target for improving cold tolerance in pepper and other crops.