The transcription factor SmWRKY20 positively regulates abscisic acid-induced salvianolic acids biosynthesis by activating SmC4H1 in Salvia miltiorrhiza

Salvianolic acids represent the predominant phenolic compounds in Salvia miltiorrhiza Bunge, a medicinal herb widely applied in cardiovascular and cerebrovascular therapies. Despite their pharmacological relevance, the transcriptional mechanisms controlling salvianolic acids production remain incompletely understood. Among potential regulators, WRKY is well known in modulating plant development, secondary metabolism, and stress adaptation, yet to be systematically characterized in relation to SmWRKY gene involvement in salvianolic acids biosynthesis. Here, abscisic acid (ABA) was found to enhance salvianolic acids accumulation in leaves and tanshinone ⅡA in roots of S. miltiorrhiza. Transcriptome analysis of ABA- and PEG6000-treated plants identified SmWRKY20 as a candidate transcription factor (TF) associated with salvianolic acids biosynthesis, as determined through co-expression analysis. SmWRKY20 was predominantly observed and strong responsiveness to ABA in the roots. In transgenic hairy roots, SmWRKY20-OE dramatically enhanced rosmarinic acid and salvianolic acid B levels, whereas RNAi suppression led to their marked reduction. Correspondingly, upregulated and downregulated SmC4H1 expression occurred in OE and RNAi lines, respectively. Dual-LUC, EMSA, together with Y1H experiments showed that SmWRKY20 directly binds to W-box1/2 elements in SmC4H1 promoter, activating its expression. SmWRKY20 promotes the biosynthesis of salvianolic acids by activating SmC4H1. This work elucidates ABA-dependent regulatory metabolic mechanisms in S. miltiorrhiza, establishing a conceptual framework for optimizing growth strategies to improve medicinal root materials.