Different Roles of the Mevalonate and Methylerythritol Phosphate Pathways in Cell Growth and Tanshinone Production of Salvia miltiorrhiza Hairy Roots

Dongfeng Yang,Zongsuo Liang,Ruilian Han,Xiao Liang,Fenghua Liu,Jianjun Zhao,Xuhong Du,Yan Liu,Miguel A Blazquez

doi:10.1371/journal.pone.0046797

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.

Highlights

It is well known that terpenoids are biosynthesized via two pathways in plants: the mevalonate (MVA) pathway in the cytosol [1] and the methylerythritol phosphate (MEP) pathway in the plastids [2] (Figure 1)
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

Summary

Introduction

It is well known that terpenoids are biosynthesized via two pathways in plants: the mevalonate (MVA) pathway in the cytosol [1] and the methylerythritol phosphate (MEP) pathway in the plastids [2] (Figure 1). The MEP pathway in most eubacteria is used to supply precursors for terpenoid biosynthesis, and only the MVA pathway in fungi and animals is used. In plants, both the MVA and MEP pathways are used for terpenoid production [3]. Some evidence indicated that IPP exchanges were probably in both direction, whereas, the delivery of precursor from the plastids to the cytosol seemed to operate more readily [5]

Methods

Results

Discussion

Conclusion