JrTTG2 confers drought tolerance in walnut via the JrGSTU8-mediated antioxidant pathway and interaction with JrEPSIN1

Drought significantly limits the productivity and quality of Juglans regia, yet the regulatory mechanisms governing drought tolerance in this economically vital nut tree remain largely elusive. Here, we report that the WRKY transcription factor JrTTG2 acts as a central hub in the walnut drought response by integrating transcriptional and protein-interaction networks. We identified JrTTG2 as a hypersensitive early-response gene, exhibiting a robust 333-fold induction in walnut roots under drought stress. Ectopic expression of JrTTG2 in Arabidopsis and poplar significantly enhanced drought tolerance, primarily by maintaining root architecture, increasing biomass, and reducing oxidative damage. Conversely, RNAi-mediated silencing of PagTTG2 in poplar led to stunted root systems and compromised antioxidant defense under water deficit. Transcriptomic and molecular analyses revealed that JrTTG2 directly binds to the W-box in the promoter of the JrGSTU8 gene to activate its transcription, thereby facilitating ROS scavenging. Furthermore, yeast two-hybrid and BiFC assays demonstrated that JrTTG2 physically interacts with the vesicle trafficking protein JrEPSIN1. Both JrGSTU8 and JrEPSIN1 showed highly synchronized induction with JrTTG2 in walnut roots during drought. Taken together, this study provides a novel dimension to the molecular mechanisms of woody plant adaptation and offers a promising candidate gene for breeding drought-tolerant walnut varieties.