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Background: ATP-binding cassette (ABC) transporters have been found to play important roles in metabolic transport in plant cells, influencing subcellular compartmentalisation and tissue distribution of these metabolic compounds. Salvia miltiorrhiza Bunge, known as Danshen in traditional Chinese medicine, is a highly valued medicinal plant used to treat cardiovascular and cerebrovascular diseases. The dry roots and rhizomes of S. miltiorrhiza contain biologically active secondary metabolites of tanshinone and salvianolic acid. Given an assembled and annotated genome and a set of transcriptome data of S. miltiorrhiza, we analysed and identified the candidate genes that likely involved in the bioactive metabolite transportation of this medicinal plant, starting with the members of the ABC transporter family., Results: A total of 114 genes encoding ABC transporters were identified in the genome of S. miltiorrhiza. All of these ABC genes were divided into eight subfamilies: 3ABCA, 31ABCB, 14ABCC, 2ABCD, 1ABCE, 7ABCF, 46ABCG, and 10 ABCI. Gene expression analysis revealed tissue-specific expression profiles of these ABC transporters. In particular, we found 18 highly expressed transporters in the roots of S. miltiorrhiza, which might be involved in transporting the bioactive compounds of this medicinal plant. We further investigated the co-expression profiling of these 18 genes with key enzyme genes involved in tanshinone and salvianolic acid biosynthetic pathways using quantitative reverse transcription polymerase chain reaction (RT-qPCR). From this RT-qPCR validation, we found that three ABC genes (SmABCG46, SmABCG40, and SmABCG4) and another gene (SmABCC1) co-expressed with the key biosynthetic enzymes of these two compounds, respectively, and thus might be involved in tanshinone and salvianolic acid transport in root cells. In addition, we predicted the biological functions of S. miltiorrhiza ABC transporters using phylogenetic relationships and analysis of the transcriptome to find biological functions., Conclusions: Here, we present the first systematic analysis of ABC transporters in S. miltiorrhiza and predict candidate transporters involved in bioactive compound transportation in this important medicinal plant. Using genome-wide identification, transcriptome profile analysis, and phylogenetic relationships, this research provides a new perspective on the critical functions of ABC transporters in S. miltiorrhiza.

Key Message: The novel AP2/ERF transcription factor SmERF128 positively regulates diterpenoid tanshinone biosynthesis by activating the expression of SmCPS1, SmKSL1, and SmCYP76AH1 in Salvia miltiorrhiza. Certain members of the APETALA2/ethylene-responsive factor (AP2/ERF) family regulate plant secondary metabolism. Although it is clearly documented that AP2/ERF transcription factors (TFs) are involved in sesquiterpenoid biosynthesis, the regulation of diterpenoid biosynthesis by AP2/ERF TFs remains elusive. Here, we report that the novel AP2/ERF TF SmERF128 positively regulates diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza. Overexpression of SmERF128 increased the expression levels of copalyl diphosphate synthase 1 (SmCPS1), kaurene synthase-like 1 (SmKSL1) and cytochrome P450 monooxygenase 76AH1 (SmCYP76AH1), whereas their expression levels were decreased when SmERF128 was silenced. Accordingly, the content of tanshinone was reduced in SmERF128 RNA interference (RNAi) hairy roots and dramatically increased in SmERF128 overexpression hairy roots, as demonstrated through Ultra Performance Liquid Chromatography (UPLC) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. Furthermore, SmERF128 activated the expression of SmCPS1, SmKSL1, and SmCYP76AH1 by binding to the GCC box, and to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs within their promoters during in vivo and in vitro assays. Our findings not only reveal the molecular basis of how the AP2/ERF transcription factor SmERF128 regulates diterpenoid biosynthesis, but also provide useful information for improving tanshinone production through genetic engineering.

Salvia miltiorrhiza Bunge (Labiatae) is an emerging model plant for traditional medicine, and tanshinones are among the pharmacologically active constituents of this plant. Although extensive chemical and pharmaceutical studies of these compounds have been performed, studies on the basic helix-loop-helix (bHLH) transcription factors that regulate tanshinone biosynthesis are limited. In our study, 127 bHLH transcription factor genes were identified in the genome of S. miltiorrhiza, and phylogenetic analysis indicated that these SmbHLHs could be classified into 25 subfamilies. A total of 19 sequencing libraries were constructed for expression pattern analyses using RNA-Seq. Based on gene-specific expression patterns and up-regulated expression patterns in response to MeJA treatment, 7 bHLH genes were revealed as potentially involved in the regulation of tanshinone biosynthesis. Among them, the gene expression of SmbHLH37, SmbHLH74 and SmbHLH92 perfectly matches the accumulation pattern of tanshinone biosynthesis in S. miltiorrhiza. Our results provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factors in S. miltiorrhiza.

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., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Main Conclusion: Twenty-nine genes related to phenolic acid biosynthesis were identified in the Salvia miltiorrhiza genome. Nineteen of these are described for the first time, with ten genes experimentally correlating to phenolic acid biosynthesis. Vast stores of secondary metabolites exist in plants, many of which possess biological activities related to human health. Phenolic acid derivatives are a class of valuable bioactive pharmaceuticals abundant in the widely used Chinese medicinal herb, Salvia miltiorrhiza. The biosynthetic pathway for phenolic acids differs in this species from that of other investigated plants. However, the molecular basis for this is unknown, with systematic analysis of the genes involved not yet performed. As the first step towards unraveling this complex biosynthetic pathway in S. miltiorrhiza, the current genome assembly was searched for putatively involved genes. Twenty-nine genes were revealed, 19 of which are described here for the first time. These include 15 genes predicted in the phenylpropanoid pathway; seven genes in the tyrosine-derived pathway; six genes encoding putative hydroxycinnamoyltransferases, and one CYP98A, namely CYP98A78. The promoter regions, gene structures and expression patterns of these genes were examined. Furthermore, conserved domains and phylogenetic relationships with homologous proteins in other species were revealed. Most of the key enzymes, including 4-coumarate: CoA ligase, 4-hydroxyphenylpyruvate reductase and hydroxycinnamoyltransferase, were found in multiple copies, each exhibiting different characteristics. Ten genes putatively involved in rosmarinic acid biosynthesis are also described. These findings provide a foundation for further analysis of this complex and diverse pathway, with potential to enhance the synthesis of water-soluble medicinal compounds in S. miltiorrhiza.

Salvia miltiorrhiza is a Chinese herb with significant pharmacologic effects because of the bioactive compounds of tanshinones and phenolic acids. Methyl jasmonate (MeJA) has been used as an effective elicitor to enhance the production of these compounds. However, the molecular mechanism of MeJA-mediated tanshinone and salvianolic acid biosynthesis remains unclear. The transcriptional profiles of S. miltiorrhiza leaves at 12 h (T12) after MeJA elicitation and mock-treated leaves (T0) were generated using the Illumina deep RNA sequencing (RNA-seq) strategy to detect the changes in gene expression in response to MeJA. In total, 37 647 unique sequences were obtained from about 21 million reads, and 25 641 (71.53%) of these sequences were annotated based on the blast searches against the public databases. A total of 5287 unique sequences were expressed differentially between the samples of T0 and T12, which covered almost all the known genes involved in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza. Many of the transcription factors (e.g. MYB, bHLH and WRKY) and genes involved in plant hormone biosynthesis and signal transduction were expressed differentially in response to the MeJA induction. Importantly, three and four candidate cytochrome P450s (P450s) that could be involved in the tanshinone and phenolic acid biosynthesis, respectively, were selected from the RNA-seq data based on co-expressed pattern analysis with SmCPS1/SmKSL1 and SmRAS, which are the key genes responsible for biosynthesis. This comprehensive investigation of MeJA-induced gene expression profiles can shed light on the molecular mechanisms of the MeJA-mediated bioactive compound biosynthesis and regulation in S. miltiorrhiza., (© 2014 Scandinavian Plant Physiology Society.)

Tanshinones are a class of bioactive components in the traditional Chinese medicine Salvia miltiorrhiza , and their biosynthesis and regulation have been widely studied. Current studies show that basic leucine zipper (bZIP) proteins regulate plant secondary metabolism, growth and developmental processes. However, the bZIP transcription factors involved in tanshinone biosynthesis are unknown. Here, we conducted the first genome-wide survey of the bZIP gene family and analyzed the phylogeny, gene structure, additional conserved motifs and alternative splicing events in S. miltiorrhiza. A total of 70 SmbZIP transcription factors were identified and categorized into 11 subgroups based on their phylogenetic relationships with those in Arabidopsis . Moreover, seventeen SmbZIP genes underwent alternative splicing events. According to the transcriptomic data, the SmbZIP genes that were highly expressed in the Danshen root and periderm were selected. Based on the prediction of bZIP binding sites in the promoters and the co-expression analysis and co-induction patterns in response to Ag + treatment via quantitative real-time polymerase chain reaction (qRT-PCR), we concluded that SmbZIP7 and SmbZIP20 potentially participate in the regulation of tanshinone biosynthesis. These results provide a foundation for further functional characterization of the candidate SmbZIP genes, which have the potential to increase tanshinone production. [ABSTRACT FROM AUTHOR]