SmWRKY33 coordinates tanshinone biosynthesis through concurrent activation of precursor supply and metabolic branch points in Salvia miltiorrhiza

Salvia miltiorrhiza is a well-established medicinal species in which the biosynthesis of bioactive tanshinone diterpenoids is governed by intricate transcriptional regulatory networks. While the jasmonate-responsive transcription factor SmWRKY33 was known to activate downstream biosynthetic genes, its full regulatory scope remained unclear. This study comprehensively elucidates the function and mechanism of SmWRKY33 as a master coordinator of tanshinone metabolism. SmWRKY33 exhibited root-predominant expression and was rapidly induced by both methyl jasmonate and gibberellin. Functional analyses in hairy roots demonstrated that overexpression of SmWRKY33 significantly increased tanshinone accumulation, whereas antisense suppression reduced it. The data demonstrate that SmWRKY33 directly activates not only the upstream 2-methyl- D -erythritol 4-phosphate (MEP) pathway genes SmDXS2 and SmDXR to boost precursor geranylgeranyl diphosphate (GGPP) production, but also the competitive branch-point gene SmCPS5 , thereby managing flux partitioning at a critical metabolic node. This coordinated transcriptional regulation was confirmed by yeast one-hybrid, dual-luciferase reporter, and electrophoretic mobility shift assays. These findings expand the role of SmWRKY33 from a downstream pathway activator to a systemic regulator that synchronizes precursor supply, competitive branch-point flux, and core pathway activation. This work provides a novel mechanistic understanding of growth-defense balance in specialized metabolism and presents an effective strategy for engineering diterpenoid production in medicinal plants.