The PhNH10-PhDREB1C-PhPOD/Ph3GT module positively regulates cold stress tolerance by scavenging reactive oxygen species in Petunia

Cold stress represents a primary abiotic constraint limiting the productivity and ornamental quality of floricultural crops. Although DREB/CBF transcription factors are established master regulators of cold acclimation, whether they can directly transcriptionally activate antioxidant enzyme-encoding genes-rather than merely correlating with enhanced antioxidant capacity-remains mechanistically unresolved. This study identified PhDREB1C, a DREB transcription factor that positively regulates cold tolerance in Petunia hybrida. Overexpression of PhDREB1C significantly enhanced cold tolerance, as evidenced by a 27.88% higher survival rate than that of wild-type plants after 2 wk of treatment at 0 °C. In addition, after 3 d of treatment at 4 °C, the transgenic plants showed a 28.3% reduction in superoxide anion accumulation, a 53.1% decrease in hydrogen peroxide levels, a 37.4% decrease in malondialdehyde content, and a 72.40% increase in anthocyanin content relative to wild-type plants. In contrast, phdreb1c knockout mutants exhibited hypersensitivity to cold stress. ChIP-seq combined with Y1H and EMSA analyses indicated that PhDREB1C directly interacts with the ACCGAC motif in the PhPOD promoter (encoding peroxidase) and the GTCGGC motif the Ph3GT promoter (encoding anthocyanidin 3-O-glucosyltransferase), activating their transcription. VIGS-mediated silencing of either PhPOD or Ph3GT compromised cold tolerance, confirming their functional requirement downstream of PhDREB1C. Furthermore, we established that the NAC transcription factor PhNH10 directly represses PhDREB1C transcription under ambient temperature by binding to TTATCC motifs in its promoter. Our findings establish a PhNH10-PhDREB1C-PhPOD/Ph3GT regulatory module that coordinates enzymatic and nonenzymatic antioxidant defenses, providing candidate genes for molecular breeding of cold-tolerant ornamental cultivars.