Abstract
WRKY (WRKY domain-containing) transcription factors represent one of the largest families of plant-specific transcription regulators and play pivotal roles in plant growth, development, nutrient acquisition, and responses to biotic and abiotic stresses. In this study, the coding sequence of TaWRKY65-3B was cloned from the hexaploid wheat cultivar Hanxuan 10. Domain analysis revealed that TaWRKY65-3B contains a conserved WRKY domain and a typical C2H2-type zinc finger motif. The gene spans 1478 bp and comprises two exons and one intron. Promoter analysis identified multiple cis-acting regulatory elements, including those responsive to abscisic acid, methyl jasmonate, drought, and low temperature. Quantitative real-time PCR (qRT-PCR) demonstrated that TaWRKY65-3B was significantly upregulated under polyethylene glycol (PEG)-simulated-drought stress in wheat. Overexpression of TaWRKY65-3B in rice resulted in marked growth inhibition, characterized by reduced plant height, shortened grains, and decreased grain width, perimeter, and surface area. These morphological changes led to a significant reduction in thousand-grain weight and overall grain yield. Mechanistically, TaWRKY65-3B activated the expression of OsCPS2, OsCPS4, and OsKS4, which encode key enzymes in the diterpenoid phytoalexin biosynthetic pathway. Subsequent oxidative modifications catalyzed by the cytochrome P450 monooxygenase OsCYP76M7 facilitated the accumulation of the defensive diterpenoid neoabietic acid. Integrated transcriptomic and metabolomic analyses revealed that constitutive TaWRKY65-3B overexpression reprograms simulated-drought-responsive metabolism in rice, with a diterpenoid metabolite annotated as neoabietic acid as a key correlative feature—though definitive structural confirmation requires authentic standards. These findings identify a WRKY-mediated regulatory module connecting diterpenoid phytoalexin metabolism to simulated-drought stress responses, although the precise molecular cascades governing the growth-defence trade-off between stress adaptation and yield warrant further investigation.