Dynamic transcriptomic profiling of Salix triandra under freezing stress identifies StWRKY31 as a positive regulator...

Salix triandra is a fast-growing shrub willow native to temperate and cold regions, possessing remarkable freezing tolerance. However, the molecular mechanisms underlying this natural cold hardiness remain largely unexplored. In this study, we performed a time-course transcriptomic analysis of S. triandra seedlings subjected to freezing stress at -4 °C. A total of 11,287 differentially expressed genes were identified and clustered into six distinct co-expression modules with dynamic temporal patterns, revealing a multi-phasic transcriptional response involving early stress perception, metabolic adjustment, and sustained cellular protection. Among the differentially expressed transcription factors, a WRKY family member designated StWRKY31 (EVM0008367) was identified as a cold-induced candidate gene, belonging to a mid-to-late adaptation module peaking at 24 h. Subcellular localization analysis showed that StWRKY31 localizes to the nucleus. Heterologous overexpression of StWRKY31 in Arabidopsis significantly enhanced freezing tolerance, as evidenced by improved survival rates under both non-acclimated and cold-acclimated freezing assays. In the StWRKY31-overexpressing Arabidopsis lines, reactive oxygen species (ROS) accumulation was reduced, superoxide dismutase (SOD) and peroxidase (POD) activities were increased, proline content was elevated, and malondialdehyde (MDA) levels were decreased. Moreover, StWRKY31 upregulated the expression of cold-responsive marker genes (AtGSTF8, AtZAT10, and AtHY5) in a time-dependent manner, with elevated basal expression prior to cold exposure, suggesting a potential defense priming state. These findings suggest that StWRKY31 functions as a positive regulator of freezing tolerance. This study contributes to understanding the regulatory dynamics of cold adaptation in woody plants and provides a valuable candidate gene for breeding cold-tolerant varieties.