Lipopolysaccharide Enhances Tanshinone Biosynthesis via a Ca(2+)-Dependent Manner in Salvia miltiorrhiza Hairy Roots

Tanshinones, the major bioactive components in Salvia miltiorrhiza Bunge (Danshen), are synthesized via the mevalonic acid (MVA) pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and the downstream biosynthesis pathway. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. HPLC analysis revealed that LPS treatment resulted in a significant accumulation of cryptotanshinone (CT) and dihydrotanshinone I (DTI). qRT-PCR analysis confirmed that biosynthesis genes such as SmAACT and SmHMGS from the MVA pathway, SmDXS and SmHDR from the MEP pathway, and SmCPS, SmKSL and SmCYP76AH1 from the downstream pathway were markedly upregulated by LPS in a time-dependent manner. Furthermore, transcription factors SmWRKY1 and SmWRKY2, which can activate the expression of SmDXR, SmDXS and SmCPS, were also increased by LPS. Since Ca(2+) signaling is essential for the LPS-triggered immune response, Ca(2+) channel blocker LaCl(3) and CaM antagonist W-7 were used to investigate the role of Ca(2+) signaling in tanshinone biosynthesis. HPLC analysis demonstrated that both LaCl(3) and W-7 diminished LPS-induced tanshinone accumulation. The downstream biosynthesis genes including SmCPS and SmCYP76AH1 were especially regulated by Ca(2+) signaling. To summarize, LPS enhances tanshinone biosynthesis through SmWRKY1- and SmWRKY2-regulated pathways relying on Ca(2+) signaling. Ca(2+) signal transduction plays a key role in regulating tanshinone biosynthesis in S. miltiorrhiza.