No abstract

Flavonoids are a class of important secondary metabolites with a broad spectrum of pharmacological functions. Salvia miltiorrhiza Bunge (Danshen) is a well-known traditional Chinese medicinal herb with a broad diversity of flavonoids. However, flavonoid biosynthetic enzyme genes have not been systematically and comprehensively analyzed in S. miltiorrhiza. Through genome-wide prediction and molecular cloning, twenty six flavonoid biosynthesis-related gene candidates were identified, of which twenty are novel. They belong to nine families potentially encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavone synthase (FNS), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), respectively. Analysis of intron/exon structures, features of deduced proteins and phylogenetic relationships revealed the conservation and divergence of S. miltiorrhiza flavonoid biosynthesis-related proteins and their homologs from other plant species. These genes showed tissue-specific expression patterns and differentially responded to MeJA treatment. Through comprehensive and systematic analysis, fourteen genes most likely to encode flavonoid biosynthetic enzymes were identified. The results provide valuable information for understanding the biosynthetic pathway of flavonoids in medicinal plants.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants and plays important roles in posttranscriptional regulation. In this study, we combined whole genome sequencing and transcriptomes to systematically investigate PPRs in Salvia miltiorrhiza, which is a well-known material of traditional Chinese medicine and an emerging model system for medicinal plant studies. Among 562 identified SmPPRs, 299 belong to the P subfamily while the others belong to the PLS subfamily. The majority of SmPPRs have only one exon and are localized in the mitochondrion or chloroplast. As many as 546 SmPPRs were expressed in at least one tissue and exhibited differential expression patterns, which indicates they likely play a variety of functions in S. miltiorrhiza. Up to 349 SmPPRs were salicylic acid-responsive and 183 SmPPRs were yeast extract and Ag+-responsive, which indicates these genes might be involved in S. miltiorrhiza defense stresses and secondary metabolism. Furthermore, 23 salicylic acid-responsive SmPPRs were co-expressed with phenolic acid biosynthetic enzyme genes only while 16 yeast extract and Ag+-responsive SmPPRs were co-expressed with tanshinone biosynthetic enzyme genes only. Two SmPPRs were co-expressed with both phenolic acid and tanshinone biosynthetic enzyme genes. The results provide a useful platform for further investigating the roles of PPRs in S. miltiorrhiza.

Small RNA-mediated gene silencing is a vital regulatory mechanism in eukaryotes that requires ARGONAUTE (AGO) proteins. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant. Therefore, it is important to characterize S. miltiorrhiza AGO family genes as they may be involved in multiple metabolic pathways. This chapter introduces the detailed protocol for SmAGO gene prediction and molecular cloning. In addition, an Agrobacterium-mediated genetic transformation method for S. miltiorrhiza is presented. These methodologies can be used to functionally study SmAGO genes as well as other genes of interest in S. miltiorrhiza.

Salvia miltiorrhiza is a model medicinal plant with significant economic and medicinal value. Its roots produce a group of diterpenoid lipophilic bioactive components, termed tanshinones. Biosynthesis and regulation of tanshinones has attracted widespread interest. However, the methylome of S. miltiorrhiza has not been analyzed and the regulatory mechanism of DNA methylation in tanshinone production is largely unknown. Here we report single-base resolution DNA methylomes from roots and leaves. Comparative analysis revealed differential methylation patterns for CG, CHG and CHH contexts and the association between DNA methylation and the expression of genes and small RNAs. Lowly methylated genes had always higher expression levels and 24-nucleotide sRNAs could be key players in the RdDM pathway in S. miltiorrhiza. DNA methylation variation analysis showed that CHH methylation contributed mostly to the difference. Go enrichment analysis showed that diterpenoid biosynthetic process was significantly enriched for genes with downstream overlapping with hypoCHHDMR in July_root when comparing with those in March_root. Tanshinone biosynthesis-related enzyme genes, such as DXS2, CMK, IDI1, HMGR2, DXR, MDS, CYP76AH1, 2OGD25 and CYP71D373, were less CHH methylated in gene promoters or downstream regions in roots collected in July than those collected in March. Consistently, gene expression was up-regulated in S. miltiorrhiza roots collected in July compared with March and the treatment of DNA methylation inhibitor 5-azacytidine significantly promoted tanshinone production. It suggests that DNA methylation plays a significant regulatory role in tanshinone biosynthesis in S. miltiorrhiza through changing the levels of CHH methylation in promoters or downstreams of key enzyme genes.