Tanshinone and salvianolic acid biosynthesis are regulated by SmMYB98 in Salvia miltiorrhiza hairy roots

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.

Graphical abstract

Fig. 1

Biosynthetic pathway of tanshinones and GAs in S. miltiorrhiza. MVA pathway, mevalonate pathway; MEP pathway, 2-C-methyl-D-erythritol 4-phosphate pathway. Abbreviations for synthetases and compounds refer to the Abbreviations table and Introduction text.

Fig. 2

Salvianolic acid biosynthetic pathway in S. miltiorrhiza. Abbreviations for synthetases and compounds refer to the Abbreviations table and Introduction text.

Fig. 3

Comparative analysis of SmMYB98 and other related sequences. (A) Phylogenetic analysis of the SmMYB98 gene and 18 members of the R2R3-MYB S20 subgroup from different plants. (B) Protein sequence alignment of SmMYB98 and four R2R3-MYB S20 subgroup proteins from different plants. The conserved R2 and R3 domains are underlined in red; the WxPRL core sequence is underlined in blue.

Fig. 4

Tissue expression and subcellular localization patterns of SmMYB98. (A, B) The differential transcriptional expression levels of the SmMYB98, SmCPS1, SmKSL1, and SmCYP76AH1 genes from five different S. miltiorrhiza tissues (i.e., taproots, lateral roots, stems, leaves, and flowers) were detected by qRT-PCR. The transcriptional expression level of each gene in the taproots was set to 1. (C) Subcellular localization of 35S:SmMYB98-YFP (scale bars: 10 μm) and 35S:YFP (scale bars: 20 μm) in N. benthamiana leaf epidermal cells.

Fig. 5

Generation of the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines. (A, C) The transcriptional expression levels of SmMYB98 in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines were detected by qRT-PCR. The average transcriptional expression level of SmMYB98 in the two control hairy root lines was set to 1. The S. miltiorrhiza actin gene was used as the internal reference gene. Error bars represent the SD of three technical replicates. (B) Genomic SmMYB98 DNA sequences from different SmMYB98-KO transgenic hairy root lines were detected by DNA sequencing. The original sequence of SmMYB98 is displayed at the top; the PAM (GGG) area is highlighted in the blue box. Detailed DNA insertions and point mutations of the SmMYB98 sequence in different SmMYB98-KO transgenic hairy root lines are presented below the original sequence.

Fig. 6

Analysis of the tanshinones and salvianolic acids in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines. (A, D) The phenotype and tanshinone and salvianolic acid extracts of the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines (scale bars: 1 cm). (B, E) The contents of four tanshinones in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines were detected by HPLC. Error bars represent the SD of three technical replicates. (C, F) The contents of four salvianolic acids in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines were detected by HPLC. Error bars represent the SD of three technical replicates. TI, tanshinone I; TIIA, tanshinone IIA; CT, cryptotanshinone; DT, dihydrotanshinone; RA, rosmarinic acid; SAB, salvianolic acid B; SAA, salvianolic acid A; CA, caffeic acid.

Fig. 7

Transcriptional expression analysis of tanshinone biosynthetic genes in selected SmMYB98-OE and SmMYB98-KO transgenic hairy root lines. Transcriptional expression levels of MEP pathway genes (A), MVA pathway genes (B), and downstream tanshinones biosynthetic genes (C) in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines were detected by qRT-PCR. The average transcriptional expression level of each gene in the two control hairy root lines was set to 1. The S. miltiorrhiza actin gene was used as the internal reference gene. Error bars represent the SD of three technical replicates.

Fig. 8

Transcriptional expression analysis of salvianolic acid biosynthetic genes in selected SmMYB98-OE and SmMYB98-KO transgenic hairy root lines. (A, B) The transcriptional expression levels of salvianolic acid biosynthesis genes in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines were detected by qRT-PCR. The average transcriptional expression level of each gene in the two control hairy root lines was set to 1. The S. miltiorrhiza actin gene was used as the internal reference gene. Error bars represent the SD of three technical replicates.

Fig. 9

The effects of SmMYB98 on the promoter of tanshinone (A) and salvianolic acid (B) biosynthetic genes were detected by transient Dual-LUC analysis using N. benthamiana leaves. The relative folds of LUC/REN represent the activation level of SmMYB98 on the promoters. Error bars represent the SD of three biological replicates.

Fig. 10

Analysis of GA biosynthesis in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines. (A, C) The GA contents in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines were detected by HPLC. Error bars represent the SD of three technical replicates. (B, D) The transcriptional expression levels of GA biosynthetic genes in the SmMYB98-OE and SmMYB98-KO transgenic hairy root lines were detected by qRT-PCR. The average transcriptional expression level of each gene in the two control hairy root lines was set to 1. The S. miltiorrhiza actin gene was used as the internal reference gene. Error bars represent the SD of three technical replicates.