CaZAT5 negatively regulates cold tolerance in pepper by inhibiting lignin synthesis and antioxidant capacity

Low-temperature stress affects crop yield and economic value. Various transcription factors are crucial in plants' response to cold stress. However, the role and mechanism of ZAT subfamily proteins in the zinc finger protein family in regulating cold in pepper remain unclear. In this study, TF CaZAT5, a low-temperature-repressed ZFP that is localized in both the nucleus and cytoplasm, was isolated from the leaves of pepper ( Capsicum annuum L.) and was comprehensively analyzed to decipher its mechanism in regulating plant resistance to cold stress. A 24-hour low-temperature treatment at 4 °C reduced the transcriptional level of CaZAT5 in pepper. Pepper plants with silenced CaZAT5 exhibited stronger cold tolerance, while Arabidopsis and tomato plants overexpressing (OE) CaZAT5 were more sensitive to low temperatures. Transcriptome analysis revealed that the DEGs between the OE CaZAT5 tomato plants and wild-type plants under both normal and low temperatures were primarily enriched in phenylpropanoid biosynthesis and glutathione metabolism pathways. DAP-seq analysis revealed potential downstream target genes of CaZAT5 , including cinnamyl alcohol dehydrogenase 1 ( CaCAD1 ), glutamine S-transferase 1 ( CaGST1 ), and glutaredoxin S2 ( CaGRXS2 ). Y1H, EMSA, and Dual-LUC assays further revealed that CaZAT5 specifically binds to the promoter regions of CaCAD1 , CaGST1 , and CaGRXS2 , inhibiting their expression. Subsequent physiological and biochemical analyses revealed that OE CaZAT5 significantly reduced the lignin content, GST activity, and GRX content of transgenic plants while silencing CaZAT5 yielded opposite effects in pepper plants. The findings of this study postulate that CaZAT5 acts as a negative regulator of cold stress response in pepper and offers valuable theoretical insights into the role of ZAT TFs in controlling lignin synthesis and antioxidant activity during plant responses to cold stress.