The PtrC2H2.2-6-PtrCYP86A7/A8 Module Regulates Poplar Drought Tolerance Through Mediating Cutin and Wax Biosynthesis Pathways.

The plant cuticle, composed of cutin and wax, is crucial for reducing non-stomatal water loss and enhancing drought tolerance. CYP86A genes are key regulators of cutin and wax biosynthesis, yet the mechanisms controlling their expression remain unclear. Here, we identified PtrC2H2.2-6, a C2H2-type transcription factor that is downregulated at both transcriptional and protein levels under water stress in poplar. Through bioinformatics and biochemical analyses, we demonstrated that PtrC2H2.2-6 interacts with PtrPPK1 to regulate drought tolerance by modulating two critical cuticle biosynthesis genes, PtrCYP86A7 and PtrCYP86A8. PtrC2H2.2-6 negatively regulates cutin and wax biosynthesis by binding to the CACT motif in the promoters of PtrCYP86A7 and PtrCYP86A8, thereby suppressing their expression. Conversely, PtrPPK1 interacts with PtrC2H2.2-6, phosphorylates it, and promotes its degradation, thereby relieving its repression of PtrCYP86A7/8. Functional validation revealed that RNAi-mediated silencing of PtrC2H2.2-6 enhanced wax accumulation and drought tolerance, while overexpression of PtrC2H2.2-6 reduced wax accumulation and drought resistance. Furthermore, overexpression of PtrCYP86A7 or PtrCYP86A8 increased wax accumulation, enhanced the water retention capacity of the leaf surface, and improved drought resistance. Collectively, the PtrPPK1-PtrC2H2.2-6-PtrCYP86A7/A8 module is a critical regulatory mechanism for drought tolerance in poplar, offering potential targets for breeding drought-resistant forest trees.