NtWHY1 negatively regulates tobacco cold tolerance via interfering with flavonoid biosynthesis.

Our fi ndings identify NtWHY1 as a potential genetic target for improving cold tolerance in crops. WHIRLY1 is a plant-specific transcription factor localized to both the nucleus and plastids, playing important roles in plant growth and stress responses. However, its function in tobacco under cold stress remains unclear. In this study, we found that NtWHY1 expression was strongly induced by cold (-4 °C). Surprisingly, overexpression of NtWHY1 enhanced cold sensitivity, causing more severe leaf wilting. Physiological analysis revealed higher accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), along with reduced antioxidant enzyme activity, compared with wild-type (WT) plants. Transcriptome profiling showed that NtWHY1 overexpression enriched differentially expressed genes in secondary metabolism, particularly suppressing the flavonoid biosynthesis pathway. Both qPCR and LC-MS/MS metabolomics confirmed downregulation of key flavonoid genes (NtCHS2, NtANS, NtCCOMT1/2/3) and decreased levels of multiple flavonoids, including morin, rutin, quercetin, narcissin, kaempferol, and isorhamnetin, with reductions ranging from 22.23% to 100%, and apigenin 7-O-(6-O-malonylglucoside) was completely abolished. Dual-luciferase and yeast one-hybrid assays further demonstrated that NtWHY1 protein transcriptionally represses NtCHS2 and NtANS promoter activity. Collectively, our results indicate that NtWHY1 protein acts as a cold-induced negative regulator that attenuates cold tolerance in tobacco by repressing flavonoid biosynthesis and compromising antioxidant capacity, providing new insights into transcription factor-mediated modulation of secondary metabolism under abiotic stress.