SmGRAS1 and SmGRAS2 Regulate the Biosynthesis of Tanshinones and Phenolic Acids in Salvia miltiorrhiza.

Wenrui Li,Bingxue Zhang,Dongfeng Yang,Tianlin Pei,Zongsuo Liang,Zhenqing Bai,Renjun Mao,Chuangfeng Liu

doi:10.3389/fpls.2019.01367

Abstract

Salvia miltiorrhiza is one of the most widely used traditional Chinese medicinal plants because of its excellent performance in treating heart diseases. Tanshinones and phenolic acids are two important classes of effective metabolites, and their biosynthesis has attracted widespread interest. Here, we functionally characterized SmGRAS1 and SmGRAS2, two GRAS family transcription factors from S. miltiorrhiza. SmGRAS1/2 were highly expressed in the root periderm, where tanshinones mainly accumulated in S. miltiorrhiza. Overexpression of SmGRAS1/2 upregulated tanshinones accumulation and downregulated GA, phenolic acids contents, and root biomass. However, antisense expression of SmGRAS1/2 reduced the tanshinones accumulation and increased the GA, phenolic acids contents, and root biomass. The expression patterns of biosynthesis genes were consistent with the changes in compounds accumulation. GA treatment increased tanshinones, phenolic acids, and GA contents in the overexpression lines, and restored the root growth inhibited by overexpressing SmGRAS1/2. Subsequently, yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays (EMSA) showed SmGRAS1 promoted tanshinones biosynthesis by directly binding to the GARE motif in the SmKSL1 promoter and activating its expression. Yeast two-hybrid assays showed SmGRAS1 interacted physically with SmGRAS2. Taken together, the results revealed that SmGRAS1/2 acted as repressors in root growth and phenolic acids biosynthesis but as positive regulators in tanshinones biosynthesis. Overall, our findings revealed the potential value of SmGRAS1/2 in genetically engineering changes in secondary metabolism.

Highlights

Danshen, the dried roots of Salvia miltiorrhiza Bunge, is a traditional Chinese medicine in treatment of cardiovascular and cerebrovascular diseases (Dong et al, 2011)
Phylogenetic analysis indicated that SmGRAS1 clustered with Arabidopsis AtSHR, while SmGRAS2 clustered with AtPAT1 (Figure S1)
Improving S. miltiorrhiza root biomass and the accumulation of the two major bioactive compounds, tanshinones and phenolic acids, in S. miltiorrhiza roots has a crucial influence on the quality of medicinal materials

Summary

Introduction

The dried roots of Salvia miltiorrhiza Bunge, is a traditional Chinese medicine in treatment of cardiovascular and cerebrovascular diseases (Dong et al, 2011). It has many pharmaceutical activities, including anti-inflammatory, antibacterial, and antiancer properties (Jiang et al, 2013). The contents of tanshinones and phenolic acids are the major quality markers of S. miltiorrhiza medicinal materials, according to the Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2015). Phenolic acids are produced in phenylpropanoid and tyrosine-derived pathways (Pei et al, 2018), which included C4H, 4CL, TAT, and CYP98A14 biosynthetic genes (Xu et al, 2016). Relatively less is known about the regulatory mechanisms of transcriptional factors in the biosynthesis of tanshinones and phenolic acids in S. miltiorrhiza

Methods

Results

Discussion

Conclusion