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

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Highlights

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SmMYB98 was predominantly expressed in the lateral roots of Salvia miltiorrhiza.
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Tanshinones and salvianolic acids were improved in the SmMYB98-OE hairy roots.
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Tanshinone and salvianolic acid contents in SmMYB98-KO lines were reduced.
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SmMYB98 regulates the expression of tanshinones and salvianolic acids biosynthetic genes.
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SmMYB98 negatively regulates the biosynthesis of gibberellins in S. miltiorrhiza.

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.

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Most cited references 40

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Full-length transcriptome sequences and splice variants obtained by a combination of sequencing platforms applied to different root tissues of Salvia miltiorrhiza and tanshinone biosynthesis.

Zhichao Xu, Reuben J. Peters, Jason Weirather … (2015)

Danshen, Salvia miltiorrhiza Bunge, is one of the most widely used herbs in traditional Chinese medicine, wherein its rhizome/roots are particularly valued. The corresponding bioactive components include the tanshinone diterpenoids, the biosynthesis of which is a subject of considerable interest. Previous investigations of the S. miltiorrhiza transcriptome have relied on short-read next-generation sequencing (NGS) technology, and the vast majority of the resulting isotigs do not represent full-length cDNA sequences. Moreover, these efforts have been targeted at either whole plants or hairy root cultures. Here, we demonstrate that the tanshinone pigments are produced and accumulate in the root periderm, and apply a combination of NGS and single-molecule real-time (SMRT) sequencing to various root tissues, particularly including the periderm, to provide a more complete view of the S. miltiorrhiza transcriptome, with further insight into tanshinone biosynthesis as well. In addition, the use of SMRT long-read sequencing offered the ability to examine alternative splicing, which was found to occur in approximately 40% of the detected gene loci, including several involved in isoprenoid/terpenoid metabolism.

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Della proteins and gibberellin-regulated seed germination and floral development in Arabidopsis.

Joseph R. Ecker, Wladmir J. Alonso, Tai Sun … (2004)

RGA (repressor of ga1-3) and GAI (gibberellin insensitive) are negative regulators of plant hormone gibberellin (GA) signaling in Arabidopsis. The GA-deficient mutant ga1-3 is a nongerminating, extreme dwarf that flowers late and produces male-sterile flowers. The rga and gai null alleles interact synergistically to rescue vegetative growth and floral initiation in ga1-3, indicating that RGA and GAI are major repressors for these processes. However, rga and gai in combination cannot rescue seed germination or floral development in ga1-3. RGA and GAI belong to the DELLA subfamily within the GRAS family of plant regulatory proteins. Three additional DELLA proteins RGL1, RGL2, and RGL3 are present in Arabidopsis. Previous studies provided evidence that RGL2 and possibly RGL1 control seed germination. To investigate further the function of the RGL genes, we examined the expression profiles of all 5 DELLA protein genes by real-time PCR. RGA and, to a lesser extent, GAI mRNAs were expressed ubiquitously in all tissues, whereas RGL1, 2, and 3 transcripts were present at high levels only in germinating seeds and/or flowers and siliques. Using the newly isolated rgl1, rgl2, and rgl3 T-DNA insertion mutants, we demonstrated that RGL2 is the major repressor in seed germination. We further provided evidence that RGA, RGL1, and RGL2 are all involved in modulating floral development. Interestingly, RGL2 expression is regulated not only at the transcript level. We showed that RGL2 protein in imbibed seeds is rapidly degraded by GA treatment and that the F-box protein SLY1 is required for RGL2 degradation to occur.

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CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.

Juan Guo, Yongjin J. Zhou, Matthew Hillwig … (2013)

Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.

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Author and article information

Contributors

Guoyin Kai

Journal

Journal ID (nlm-ta): J Adv Res

Journal ID (iso-abbrev): J Adv Res

Title: Journal of Advanced Research

Publisher: Elsevier

ISSN (Print): 2090-1232

ISSN (Electronic): 2090-1224

Publication date PMC-release: 25 January 2020

Publication date Collection: May 2020

Publication date (Electronic): 25 January 2020

Volume: 23

Pages: 1-12

Affiliations

[a ]Laboratory of Medicinal Plant Biotechnology, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, PR China

[b ]Institute of Plant Biotechnology, School of Life Sciences, Shanghai Normal University, Shanghai 200234, PR China

Author notes

[* ]Corresponding author at: Laboratory of Medicinal Plant Biotechnology, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, PR China. kaiguoyin@ 123456zcmu.edu.cn

[1]

These authors contributed equally to this work.

Article

Publisher ID: S2090-1232(20)30012-6

DOI: 10.1016/j.jare.2020.01.012

PMC ID: 7016019

PubMed ID: 32071787

SO-VID: 51cc8796-a21e-4b73-b13a-cbb73c9fa756

License:

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

History

Date received : 24 September 2019

Date revision received : 4 January 2020

Date accepted : 22 January 2020

Categories

Subject: Article

Keywords: plant secondary metabolism,transcriptional regulation,r2r3-myb transcription factor,traditional chinese medicine,metabolic engineering,ipp, isopentenyl diphosphate,dmapp, dimethylallyl diphosphate,ggpp, geranylgeranyl diphosphate,mva, mevalonate,mep, 2-c-methyl-d-erythritol 4-phosphate,aact, acetoacetyl-coa thiolase,hmgs, hydroxymethylglutaryl-coa synthase,hmgr, 3-hydroxy-3-methylglutaryl-coenzyme a reductase,mk, mevalonate kinase,pmk, phosphomevalonate kinase,mdc, mevalonate diphosphate decarboxylase,ippi, isopentenyl diphosphate isomerase,dxs, 1-deoxy-d-xylulose-5-phosphate synthase,dxr, 1-deoxy-d-xylulose-5-phosphate reductoisomerase,mct, mep cytidyl-transferase,cmk, 4-(cytidine5-diphospho)-2-c-methylerythritol kinase,mds, 2-c-methyl-d-erythritol 2,4-cyclodiphosphate synthase,hds, hydroxy-methybutenyl-4-diphosphate synthase,hdr, 1-hydroxy-2-methyl-2-(e)-butenyl-4-diphosphate reductase,tat, tyrosine aminotransferase,hppr, 4-hydroxyphenylpyruvate reductase,pal, phenylalanine ammonia-lyase,c4h, cinnamate 4-hydroxylase,4cl, 4-coumarate-coa ligase,ras, rosmarinic acid synthase,mvap, mevalonate-5-phosphate,mvapp, mevalonate-5-pyrophosphate,g3p, glyceraldehyde-3-phosphate,dxp, 1-deoxy-d-xylulose-5-phosphate,cdp-me, 4-diphosphocytidyl-2-c-methyl-d-erythritol,cdp-mep, 4-diphosphocytidyl-2-c-methyl-d-erythritol 2-phosphate,mecpp, 2-c-methyl-d-erythritol 2,4-cyclodiphosphate,hmb-pp, (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate,cpp, copalyldiphesphate,ent-cpp, ent-copalyldiphesphate

Data availability:

Keywords: plant secondary metabolism, transcriptional regulation, r2r3-myb transcription factor, traditional chinese medicine, metabolic engineering, ipp, isopentenyl diphosphate, dmapp, dimethylallyl diphosphate, ggpp, geranylgeranyl diphosphate, mva, mevalonate, mep, 2-c-methyl-d-erythritol 4-phosphate, aact, acetoacetyl-coa thiolase, hmgs, hydroxymethylglutaryl-coa synthase, hmgr, 3-hydroxy-3-methylglutaryl-coenzyme a reductase, mk, mevalonate kinase, pmk, phosphomevalonate kinase, mdc, mevalonate diphosphate decarboxylase, ippi, isopentenyl diphosphate isomerase, dxs, 1-deoxy-d-xylulose-5-phosphate synthase, dxr, 1-deoxy-d-xylulose-5-phosphate reductoisomerase, mct, mep cytidyl-transferase, cmk, 4-(cytidine5-diphospho)-2-c-methylerythritol kinase, mds, 2-c-methyl-d-erythritol 2,4-cyclodiphosphate synthase, hds, hydroxy-methybutenyl-4-diphosphate synthase, hdr, 1-hydroxy-2-methyl-2-(e)-butenyl-4-diphosphate reductase, tat, tyrosine aminotransferase, hppr, 4-hydroxyphenylpyruvate reductase, pal, phenylalanine ammonia-lyase, c4h, cinnamate 4-hydroxylase, 4cl, 4-coumarate-coa ligase, ras, rosmarinic acid synthase, mvap, mevalonate-5-phosphate, mvapp, mevalonate-5-pyrophosphate, g3p, glyceraldehyde-3-phosphate, dxp, 1-deoxy-d-xylulose-5-phosphate, cdp-me, 4-diphosphocytidyl-2-c-methyl-d-erythritol, cdp-mep, 4-diphosphocytidyl-2-c-methyl-d-erythritol 2-phosphate, mecpp, 2-c-methyl-d-erythritol 2,4-cyclodiphosphate, hmb-pp, (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, cpp, copalyldiphesphate, ent-cpp, ent-copalyldiphesphate

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

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Point-Of-Care Testing for STIs

Most cited references 40

Full-length transcriptome sequences and splice variants obtained by a combination of sequencing platforms applied to different root tissues of Salvia miltiorrhiza and tanshinone biosynthesis.

Della proteins and gibberellin-regulated seed germination and floral development in Arabidopsis.

CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.

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