Identification and analysis of WRKY transcription factors related to triterpenoid biosynthesis in Panax japonicus

Panax japonicus represents a pharmacologically important species characterized by abundant triterpenoid saponins with demonstrated bioactivity. However, the transcriptional regulatory mechanisms underlying their biosynthesis remain largely unexplored. This study systematically investigated WRKY transcription factor functions in triterpenoid biosynthesis through a combination of genome-wide identification, metabolomics analysis, and transcriptional analysis. A total of 167 PjWRKY and 22 PjOSC genes were identified in the P. japonicus genome. Metabolomic analysis of five organs revealed that oleanane-type triterpenoids accumulate predominantly in roots, whereas key PjOSC and PjWRKY genes show elevated expression in aerial tissues. Utilizing co-expression network analysis, 13 PjWRKY candidate genes were identified demonstrating significant correlations with both triterpenoid accumulation and PjOSC expression profiles. Through yeast one-hybrid and dual-luciferase assays, PjWRKY2 , 21 , and 97 were validated to directly bind to the PjOSC20 promoter and to enhance its transcriptional activity. Subcellular localization confirmed their nuclear residence, supporting their role as transcription factors. Collectively, these results define a coherent regulatory module in which specific PjWRKYs target and regulate key OSC genes associated with triterpene skeleton biosynthesis. This study provides fundamental insights into the transcriptional regulation of specialized metabolism in Panax species, while simultaneously proposing genetic engineering targets for metabolic enhancement through molecular breeding strategies. • Triterpenoids were the most abundant metabolites in of Panax japonicus . • PjWRKY2 , 21 , and 97 were positively correlated with squalene cyclase. • PjWRKY2, 21, and 97 can specifically bind to W-box cis -elements. • PjWRKY2 , 21 , and 97 activating PjOSC20 expression by binding to promoter regions.