DgbHLH2 positively regulates cold tolerance by activating DgGST expression to mediate ROS scavenging in chrysanthemum

Basic helix-loop-helix (bHLH) transcription factors play critical roles in plant growth, development and stress adaptation, yet their functions in chrysanthemum cold tolerance remain largely unclear. Here, we identified and functionally characterized DgbHLH2 , a cold-inducible bHLH transcription factor from Dendranthema grandiflorum var. Jinba. DgbHLH2 was localized to the nucleus, and exhibited predominant expression in leaves and strong induction by low temperature. Under cold stress, overexpression of DgbHLH2 improved ROS-scavenging ability and alleviated cell membrane injury, while RNAi-mediated silencing of DgbHLH2 weakened cold tolerance. Transcriptome and RT‑qPCR analyses showed that DgbHLH2 extensively modulated the expression of cold‑responsive genes, including DgGST, DgPOD, DgCBF, etc, with functional enrichment in oxidoreductase activity and the MAPK signaling pathway. We screened the WRKY71OS (TGAC) motif using TF-centered yeast one-hybrid (TF-Y1H) assays and identified it in the DgGST promoter. Further molecular assays, including Dual-luciferase reporter assays (Dual-LUC), electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-qPCR (ChIP-qPCR), demonstrated that DgbHLH2 directly binds to the WRKY71OS (TGAC) motif in the DgGST promoter and transcriptionally activates its expression. Our findings reveal that DgbHLH2 acts as a positive regulator of chrysanthemum cold tolerance by directly activating DgGST and orchestrating antioxidant and cold‑responsive networks. This study establishes the DgbHLH2–DgGST regulatory module and provides a promising target for molecular breeding of cold-tolerant chrysanthemum. • DgbHLH2 is a positive regulator in chrysanthemum and responds to cold. • DgbHLH2 binds the WRKY71OS (TGAC) element in DgGST promoter and activates its transcription. • DgbHLH2 boosts ROS scavenging, membrane stability via DgGST to enhance cold tolerance.