CaNAC76 enhances lignin content and cold resistance in pepper by regulating CaCAD1

Low temperature restricts the growth, development, and yield of peppers, significantly limiting the development of the pepper industry. NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) are implicated in plant responses to cold stress, but their specific mechanisms in peppers are unclear. In this study, we isolated a cold-induced NAC transcription factor, CaNAC76, from pepper (Capsicum annuum L.). CaNAC76 is localized in the nucleus and cytoplasm and exhibits transcriptional activation activity. Silencing CaNAC76 expression reduced the activities of superoxide dismutase, peroxidase, and catalase enzymes, resulting in decreased cold tolerance in peppers. Conversely, overexpressing CaNAC76 increased the activities of antioxidant enzymes and the expression of cold stress-responsive genes (ICE-CBF-COR) in Arabidopsis, enhancing the plant's freezing tolerance. Transcriptional regulation analysis showed that CaNAC76 directly binds to the promoter region of CaCAD1 and induces its expression. Similarly, low temperatures induced the expression of CaCAD1. Ectopic expression of CaCAD1 improved Arabidopsis freezing tolerance, whereas silencing CaCAD1 expression increased sensitivity to low temperatures. Furthermore, we observed that CaNAC76 overexpression enhanced CAD activity and lignin content in Arabidopsis, leading to lignin deposition in the xylem and interfascicular fibers. In summary, the results demonstrate that CaNAC76 can enhance cold tolerance in peppers by affecting both CBF-dependent (ICE-CBF-COR) and CBF-independent pathways (promoting CaCAD1 expression).