DgWRKY6 Mediates Cold Tolerance by Activating DgGST for ROS Scavenging in Chrysanthemum

Cold stress is a major limiting factor for growth and quality in chrysanthemum. Enhancing cold tolerance helps plants better cope with low-temperature stress, increase antioxidant enzyme activity, and effectively inhibit excessive accumulation of reactive oxygen species (ROS). This study identifies the transcription factor DgWRKY6 as a key positive regulator in chrysanthemum’s cold response. DgWRKY6 is localized in the nucleus and shows high expression levels in leaf tissue, which is strongly induced by cold stress. Cold treatment also activates its promoter region. Physiological assays demonstrate that overexpression of DgWRKY6 enhances ROS scavenging, reduces membrane damage, and improves cold tolerance by increasing the activities of glutathione S-transferase (GST) and peroxidase (POD), whereas DgWRKY6 knockout lines exhibit the opposite phenotype. Real-time quantitative PCR (RT-qPCR), yeast one-hybrid (Y1H), dual-luciferase reporter assays (Dual-LUC), and chromatin immunoprecipitation-qPCR (ChIP-qPCR) confirmed that DgWRKY6 directly binds to the W-box element and activates DgGST transcription. In conclusion, DgWRKY6 plays a positive regulatory role in enhancing cold tolerance in chrysanthemum by activating DgGST transcription in response to cold stress, ultimately increasing GST activity, reducing ROS accumulation, and enhancing antioxidant responses under low temperatures. This finding provides a valuable molecular target for cold tolerance breeding in chrysanthemum and other related horticultural crops.