CsNAC25 negatively regulates cold tolerance in tea plants through modulation of CBF pathway and ethylene biosynthesis

The NAC (NAM-ATAF1/2-CUC) family proteins are crucial regulators of diverse biological processes and stress responses in plants. However, the specific function of CsNAC25 in the cold stress response of tea plants (Camellia sinensis L.) remains unclear. In this study, we systematically investigated CsNAC25 using a combination of approaches, from heterologous overexpression in tobacco and virus-induced gene silencing in tea plants to physiological, transcriptomic, and molecular analyses. Quantitative real-time PCR analysis revealed that cold stress significantly suppressed CsNAC25 expression. Overexpression of CsNAC25 in tobacco compromised cold tolerance, as indicated by impaired water retention, increased electrolyte leakage, decreased soluble sugar content, and enhanced reactive oxygen species accumulation. Transcriptome analysis and gene validation further revealed that CsNAC25 overexpression suppressed processes related to photosynthesis and cell wall remodeling, while activating ethylene biosynthesis and signaling pathways. In contrast, silencing CsNAC25 in tea plants enhanced cold tolerance, alleviating leaf wilting and electrolyte leakage, activating the CBF signaling pathway, promoting osmoprotectant accumulation, and suppressing cold-induced ethylene biosynthesis and signaling. Yeast one-hybrid and luciferase reporter assays confirmed that CsNAC25 directly binds to the promoter of the ethylene synthase gene CsACS2 and activates its expression. Overall, this study uncovering the negative regulatory role of CsNAC25 in tea plant cold tolerance, highlighting its multifaceted regulatory functions.