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. 2012;7(11):e46797.
doi: 10.1371/journal.pone.0046797. Epub 2012 Nov 29.

Different roles of the mevalonate and methylerythritol phosphate pathways in cell growth and tanshinone production of Salvia miltiorrhiza hairy roots

Dongfeng Yang  1 , Xuhong Du, Xiao Liang, Ruilian Han, Zongsuo Liang, Yan Liu, Fenghua Liu, Jianjun Zhao
Affiliations

Different roles of the mevalonate and methylerythritol phosphate pathways in cell growth and tanshinone production of Salvia miltiorrhiza hairy roots

Dongfeng Yang et al. PLoS One. 2012.

Abstract

Salvia miltiorrhiza has been widely used in the treatment of coronary heart disease. Tanshinones, a group of diterpenoids are the main active ingredients in S. miltiorrhiza. Two biosynthetic pathways were involved in tanshinone biosynthesis in plants: the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme of the MVA pathway. The 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) are the key enzymes of the MEP pathway. In this study, to reveal roles of the MVA and the MEP pathways in cell growth and tanshinone production of S. miltiorrhiza hairy roots, specific inhibitors of the two pathways were used to perturb metabolic flux. The results showed that the MVA pathway inhibitor (mevinolin, MEV) was more powerful to inhibit the hairy root growth than the MEP pathway inhibitor (fosmidomycin, FOS). Both MEV and FOS could significantly inhibit tanshinone production, and FOS was more powerful than MEV. An inhibitor (D, L-glyceraldehyde, DLG) of IPP translocation strengthened the inhibitory effects of MEV and FOS on cell growth and tanshinone production. Application of MEV resulted in a significant increase of expression and activity of HMGR at 6 h, and a sharp decrease at 24 h. FOS treatment resulted in a significant increase of DXR and DXS expression and DXS activity at 6 h, and a sharp decrease at 24 h. Our results suggested that the MVA pathway played a major role in cell growth, while the MEP pathway was the main source of tanshinone biosynthesis. Both cell growth and tanshinone production could partially depend on the crosstalk between the two pathways. The inhibitor-mediated changes of tanshinone production were reflected in transcript and protein levels of genes of the MVA and MEP pathways.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The mevalonate (MVA) and methylerythritol phosphate (MEP) pathways in biosynthesis of terpenoid.
HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; DMAPP, dimethylallyl pyrophosphate; IPP, isopentenyl diphosphate; FPP, farnesyl diphosphate; DLG, D, L-glyceraldehyde; GA-3P, glyceraldehyde 3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; MEP, 2-C-methyl-d-erythritol-4-phosphate; GGPP, Geranylgeranyl diphosphate; GPP, Geranyl diphosphate; GGPPS, Geranylgeranyl diphosphate synthase.
Figure 2
Figure 2. Tanshinones and Salvia miltiorrhiza.
(A) Chemical structures of tanshinone I, cryptotanshinone, dihydrotanshinone I and tanshinone II A in Salvia miltiorrhiza; (B) S. miltiorrhiza hairy root culture; (C) The whole plant of S. miltiorrhiza.
Figure 3
Figure 3. Effects of mevinolin, fosmidomycin and D, L-glyceraldehyde on cell growth of S. miltiorrhiza hairy roots.
CK, the control; MEV, mevinolin; FOS, fosmidomycin; DLG, D, L-glyceraldehyde. Different letters indicate significant difference at p≤0.05 using Duncan's multiple range test. Means ± standard deviation (S.D.) (n = 3) are shown.
Figure 4
Figure 4. Effects of mevinolin, fosmidomycin and D, L-glyceraldehyde on tanshinone content in S. miltiorrhiza hairy roots.
CK, the control; MEV, mevinolin; FOS, fosmidomycin; DLG, D, L-glyceraldehyde. Different letters indicate significant difference at p≤0.05 using Duncan's multiple range test. Means ± standard deviation (S.D.) (n = 3) are shown.
Figure 5
Figure 5. Effects of mevinolin, fosmidomycin and D, L-glyceraldehyde on tanshinone release to the medium.
CK, the control; MEV, mevinolin; FOS, fosmidomycin; DLG, D, L-glyceraldehyde. Different letters indicate significant difference at p≤0.05 using Duncan's multiple range test. Means ± standard deviation (S.D (n = 3) are shown.
Figure 6
Figure 6. Effects of mevinolin, fosmidomycin and D, L-glyceraldehyde on tanshinone yield of S. miltiorrhiza hairy roots.
CK, the control; MEV, mevinolin; FOS, fosmidomycin; DLG, D, L-glyceraldehyde. Different letters indicate significant difference of the total tanshinone yields of four tanshinones at p≤0.05 using Duncan's multiple range test. Means ± standard deviation (S.D.) (n = 3) are shown.
Figure 7
Figure 7. Effects of mevinolin, fosmidomycin and D, L-glyceraldehyde on the expression of genes involved in the MVA and MEP pathways.
CK, the control; MEV, mevinolin; FOS, fosmidomycin; DLG, D, L-glyceraldehyde; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Different letters indicate significant difference at p≤0.05 using Duncan's multiple range test. Means ± standard deviation (S.D.) (n = 3) are shown.
Figure 8
Figure 8. Effects of mevinolin, fosmidomycin and D, L-glyceraldehyde on activities of HMGR and DXS.
CK, the control; MEV, mevinolin; FOS, fosmidomycin; DLG, D, L-glyceraldehyde; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase. Different letters indicate significant difference at p≤0.05 using Duncan's multiple range test. Means ± standard deviation (S.D.) (n = 3) are shown.
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This work was supported by Specialized Research Fund for the Doctoral Program of Higher Education (20110204110028) and Science Foundation of Zhejiang Sci-Tech University (ZSTU) under Grant No. 1204806-Y and No. 1204822-Y. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.