The molecular mechanism of CsWRKY24-4 and CsWRKY24-5 in response to cold stress in tea plants (Camellia sinensis).

Cold stress has emerged as a major limiting factor for tea yield. In this study, we identified two low-temperature responsive WRKY transcription factors (CsWRKY24-4 and CsWRKY24-5) from tea plants (Camellia sinensis). Their expression was significantly upregulated under cold induction. Heterologous overexpression of CsWRKY24-4 and CsWRKY24-5 in Arabidopsis thaliana markedly enhanced cold tolerance in transgenic lines. Tea seedlings overexpressing CsWRKY24-4 or CsWRKY24-5 showed significantly stronger cold resistance under low-temperature stress, confirming their roles in enhancing cold tolerance. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays demonstrated that CsWRKY24-5 directly interacts with the W-box element within the CsERF061 promoter, whereas CsWRKY24-4 fails to bind to this motif. Moreover, overexpression of CsERF061 in tea plants also improved the cold tolerance. Yeast two-hybrid (Y2H) and luciferase complementation assays (LCA) illustrated that CsWRKY24-4 and CsWRKY24-5 independently interact with the Squamosa promoter binding protein-like 15 (CsSPL15) transcription factor, which is also upregulated under low-temperature treatment. Taken together, our findings indicate that both CsWRKY24-4 and CsWRKY24-5 positively regulate cold stress responses in tea plants, providing valuable insights into the molecular mechanisms involved in adapt to cold conditions for tea plants.