Abstract
Salvia miltiorrhiza Bunge is an herb rich in bioactive tanshinone and salvianolic acid compounds. It is primarily used as an effective medicine for treating cardiovascular and cerebrovascular diseases. Liposoluble tanshinones and water-soluble phenolic acids are a series of terpenoids and phenolic compounds, respectively. However, the regulation mechanism for the simultaneous promotion of tanshinone and salvianolic acid biosynthesis remains unclear. This study identified a R2R3-MYB subgroup 20 transcription factor (TF), SmMYB98, which was predominantly expressed in S. miltiorrhiza lateral roots. The accumulation of major bioactive metabolites, tanshinones, and salvianolic acids, was improved in SmMYB98 overexpression (OE) hairy root lines, but reduced in SmMYB98 knockout (KO) lines. The qRT-PCR analysis revealed that the transcriptional expression levels of tanshinone and salvianolic acid biosynthesis genes were upregulated by SmMYB98-OE and downregulated by SmMYB98-KO. Dual-Luciferase (Dual-LUC) assays demonstrated that SmMYB98 significantly activated the transcription of SmGGPPS1, SmPAL1, and SmRAS1. These results suggest that SmMYB98-OE can promote tanshinone and salvianolic acid production. The present findings illustrate the exploitation of R2R3-MYB in terpenoid and phenolic biosynthesis, as well as provide a feasible strategy for improving tanshinone and salvianolic acid contents by MYB proteins in S. miltiorrhiza.
Keywords:
4CL, 4-coumarate-CoA ligase; AACT, acetoacetyl-CoA thiolase; C4H, cinnamate 4-hydroxylase; CDP-ME, 4-diphosphocytidyl-2-C-methyl-D-erythritol; CDP-MEP, 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; CMK, 4-(cytidine5-diphospho)-2-C-methylerythritol kinase; CPP, copalyldiphesphate; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; G3P, glyceraldehyde-3-phosphate; GGPP, geranylgeranyl diphosphate; HDR, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase; HDS, hydroxy-methybutenyl-4-diphosphate synthase; HMB-PP, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; HMGS, hydroxymethylglutaryl-CoA synthase; HPPR, 4-hydroxyphenylpyruvate reductase; IPP, isopentenyl diphosphate; IPPI, isopentenyl diphosphate isomerase; MCT, MEP cytidyl-transferase; MDC, mevalonate diphosphate decarboxylase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MEP, 2-C-methyl-D-erythritol 4-phosphate; MEcPP, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; MK, mevalonate kinase; MVA, mevalonate; MVAP, mevalonate-5-phosphate; MVAPP, mevalonate-5-pyrophosphate; Metabolic engineering; PAL, phenylalanine ammonia-lyase; PMK, phosphomevalonate kinase; Plant secondary metabolism; R2R3-MYB transcription factor; RAS, rosmarinic acid synthase; TAT, tyrosine aminotransferase; Traditional Chinese Medicine; Transcriptional regulation; ent-CPP, ent-Copalyldiphesphate.