Rosmarinic Acid Synthase 1 Phosphorylation by SmMAPK3 Is Required for Salicylic Acid‐Induced Salvianolic Acid Accumulation in Salvia miltiorrhiza Hairy Roots

Salvianolic acid is the main active component of Salvia miltiorrhiza and holds significant value in the clinical treatment of myocardial ischemia and hypoxia. Previous studies have shown that salicylic acid (SA) can significantly promote the accumulation of salvianolic acid, but its molecular mechanism remains incompletely understood. To reveal the molecular mechanism by which SA regulates salvianolic acid, this study treated transgenic hairy roots overexpressing mitogen-activated protein kinase 3 (SmMAPK3) with SA and demonstrated that SmMAPK3 is essential for SA-induced salvianolic acid biosynthesis, with this function relying on the kinase activity of SmMAPK3. Through yeast two-hybrid screening, Rosmarinic Acid Synthase 1 (SmRAS1) was identified as a direct interacting protein of SmMAPK3. In vitro kinase assays showed that SmMAPK3 can catalyse phosphorylation of SmRAS1 at the Ser178 site, a process critical for salvianolic acid biosynthesis. Further studies confirmed that SA promotes the interaction between SmMAPK3 and SmRAS1, activates the kinase activity of SmMAPK3, and enhances the phosphorylation level and protein stability of SmRAS1. Additionally, SA treatment significantly promoted salvianolic acid accumulation in transgenic hairy roots coexpressing SmRAS1 and SmMAPK3. This study elucidates the mechanism by which the SA-SmMAPK3-SmRAS1 signalling module regulates salvianolic acid biosynthesis through post-translational modification, providing new theoretical targets for metabolic engineering and yield improvement of S. miltiorrhiza.