Transcriptome-based identification of the WRKY gene family in Polygala tenuifolia Willd. and their response to cold stress

Polygala tenuifolia Willd. is an important traditional medicinal plant widely used in prescriptions for cognitive enhancement. Cold stress severely affects seedling growth and medicinal quality in P. tenuifolia; however, the molecular mechanisms underlying cold tolerance remain poorly understood. WRKY transcription factors (TFs) are key regulators of abiotic stress responses in plants, but their role in cold stress adaptation of P. tenuifolia has not been systematically explored. Cold stress caused pronounced but reversible growth inhibition in P. tenuifolia seedlings and was accompanied by dynamic physiological changes. During early cold exposure, proline content, chlorophyll a and b levels, and antioxidant enzyme activities increased, while membrane lipid peroxidation was effectively suppressed, indicating an active stress mitigation response. Transcriptome analysis revealed significant enrichment of genes involved in photosynthesis, oxidative stress response, secondary metabolite biosynthesis, and plant hormone signaling pathways. A total of 104 cold-responsive WRKY TF genes (PtWRKYs) were identified from transcriptome data. Promoter analysis showed that many PtWRKY genes contained low-temperature and abscisic acid-responsive cis-acting elements. Expression profiling further demonstrated marked diversity in cold responsiveness among PtWRKYs. Notably, Pt13G01037 and Pt17G01891 showed strong induction under cold stress and distinct tissue-specific expression patterns, suggesting their potential roles in regulating cold tolerance. This study reveals coordinated physiological adaptation and WRKY-mediated transcriptional regulation in P. tenuifolia under cold stress. The identification of cold-responsive PtWRKY genes, particularly Pt13G01037 and Pt17G01891, provides promising candidate regulators for future functional studies aimed at improving cold tolerance and stabilizing medicinal traits in P. tenuifolia.