Lipopolysaccharide Enhances Tanshinone Biosynthesis via a Ca²⁺-Dependent Manner in <i>Salvia miltiorrhiza</i> Hairy Roots

• Main Authors: Bin Zhang, Xueying Li, Xiuhong Li, Zhigang Lu, Xiaona Cai, Qing Ou Yang, Pengda Ma, Juane Dong
• Format: Article
• Language: English
• Published: MDPI AG 2020-12-01
• Series: International Journal of Molecular Sciences
• Subjects: lipopolysaccharide; tanshinones; Ca²⁺ signaling; <i>Sm</i>WRKY
• Online Access: https://www.mdpi.com/1422-0067/21/24/9576
• ISSN: 1661-6596; 1422-0067

Tanshinones, the major bioactive components in <i>Salvia miltiorrhiza</i> 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 <i>S. miltiorrhiza.</i> 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 <i>SmAACT</i> and <i>SmHMGS</i> from the MVA pathway, <i>SmDXS</i> and <i>SmHDR</i> from the MEP pathway, and <i>SmCPS</i>, <i>SmKSL</i> and <i>SmCYP76AH1</i> from the downstream pathway were markedly upregulated by LPS in a time-dependent manner. Furthermore, transcription factors <i>Sm</i>WRKY1 and <i>Sm</i>WRKY2, which can activate the expression of <i>SmDXR</i>, <i>SmDXS</i> and <i>SmCPS</i>, were also increased by LPS. Since Ca²⁺ signaling is essential for the LPS-triggered immune response, Ca²⁺ channel blocker LaCl₃ and CaM antagonist W-7 were used to investigate the role of Ca²⁺ signaling in tanshinone biosynthesis. HPLC analysis demonstrated that both LaCl₃ and W-7 diminished LPS-induced tanshinone accumulation. The downstream biosynthesis genes including <i>SmCPS</i> and <i>SmCYP76AH1</i> were especially regulated by Ca²⁺ signaling. To summarize, LPS enhances tanshinone biosynthesis through <i>Sm</i>WRKY1- and <i>Sm</i>WRKY2-regulated pathways relying on Ca²⁺ signaling. Ca²⁺ signal transduction plays a key role in regulating tanshinone biosynthesis in <i>S. miltiorrhiza</i>.