Tanshinone Biosynthesis Research Articles

SmKSL overexpression combined with elicitor treatment enhances tanshinone production from Salvia miltiorrhiza hairy roots

Tanshinones are compounds of diterpenoid, which are used in the treatment of cardiovascular diseases; however there is an urgent need to improve their production using biotechnological methods. In the present study, Agrobacterium-mediated transformation was used to produce hairy roots of Salvia miltiorrhiza expressing kaurene synthase-like (SmKSL), a tanshinone biosynthesis gene. The results revealed that the yield of total tanshinone increased by a maximum of 2.7-fold in the transgenic Salvia miltiorrhiza hairy roots overexpressing SmKSL as compared with wild-type control lines. Treatment with elicitors further increased the total tanshinone production in the SmKSL-overexpressing lines. Yeast extract treatment of line p35S::SmKSL-15 produced the highest total tanshinone content (4.2 mg/g dry weight), representing an increase of 3.5-fold compared with that in the wild-type control lines. At the same time, qRT-PCR analysis indicated that tanshinone accumulation correlated positively with the transcriptional level of the two pathway genes.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

Promoting tanshinone synthesis of Salvia miltiorrhiza root by a seed endophytic fungus, Phoma herbarum D603

The interaction of endophytes and host plant is an effective mean to regulate the growth and secondary metabolism of medicinal plants. Here we want to elucidate the effects and mechanism of Phoma herbarum D603 on the root development and tanshinone synthesis in root of Salvia miltiorrhiza by endophyte-plant coculture system. The mycelium of P. herbarum D603 was colonized in the root tissue space, and formed a stable symbiotic relationship with host plant. The in vitro activities analysis showed that the concentration of IAA produced by D603 can reach(6.45±0.23) μg·mL~(-1), and this strain had some abilities of phosphorus solubilization and siderophore production activities. The coculture experiment showed that strain D603 can significantly promote the synthesis and accumulation of tanshinones in the root of S. miltiorrhiza, in which after 8 weeks of treatment with D603, the content of tanshinone Ⅱ_A in the roots reached up to(1.42±0.59) mg·g~(-1). By the qRT-PCR analysis results, we found that D603 could improve the expression levels of some key genes(DXR, DXS, GGPP, HMGR, CPS) of tanshinone biosynthesis pathway in host plant S. miltiorrhiza, but the promoting effect mainly occurred in the early stage of the interaction, and the enzyme activity level decreased in varying degrees of the later stage. In summary, seed-associated endophyte P. herbarum D603 can promote the growth and root development of S. miltiorrhiza by producing hormones, promoting nutrient absorption and siderophore production, and promote the synthesis and accumulation of tanshinones by regulating the expression level of key genes in the synthetic pathway in S. miltiorrhiza.

Genome-wide characterisation and expression profiling of the LBD family in Salvia miltiorrhiza reveals the function of LBD50 in jasmonate signaling and phenolic biosynthesis

Jasmonates (JAs) regulate growth, development, and secondary metabolism in many economically significant crops. The lateral organ boundaries domain (LBD) protein family is a class of plant-specific transcription factors involved in the regulation of lateral organ development and jasmonate signaling. However, the functions of LBD genes in Salvia miltiorrhiza Bunge, an emerging model medicinal plant, are unclear. In this study, a total of 51 LBD transcription factor genes were identified in the genome of S. miltiorrhiza, and were classified by phylogenetic analysis into seven subfamilies. The expression profile revealed that 10 of the LBD genes are tissue-specific and JA-inducible. Of the 51 LBD transcription factor genes, 11 % (six out of 51) were potentially involved in the biosynthesis of tanshinones or phenolic acids. Correlation analysis showed that the expression of SmLBD9, SmLBD13, SmLBD21, and SmLBD50 were observably regulated in JA zim domain (JAZ) proteins transgenic S. miltiorrhiza, indicating their critical roles in the JA signaling pathway. Further functional analysis indicated that the overexpression of SmLBD50 inhibited the synthesis of total phenolics, total flavonoids, and anthocyanins in S. miltiorrhiza. Consistent with these phytochemical changes, the expression of phenolic biosynthetic genes was also stimulated. Yeast two-hybrid (Y2H) assays indicated that the SmLBD50 protein interacts with SmJAZ1, SmMYB36/97, SmbHLH37 and SmMYC2a/2b, four important transcription factors involved in JA signaling. In conclusion, our study off ;ers the first genome-wide overview and annotation of the LBD transcription factor family in S. miltiorrhiza and provides a foundation for understanding the molecular basis and regulatory mechanisms of SmLBD50 in relation to the JA response and phenolic metabolism in S. miltiorrhiza.

SmMYB98b positive regulation to tanshinones in Salvia miltiorrhiza Bunge hairy roots

Tanshinones are major secondary metabolites in Salvia miltiorrhiza Bunge, the traditional Chinese medicinal plant Danshen. Increasing the production of tanshinones is important because of their significant economic value in human medicine, for the treatment of cardiovascular and cerebrovascular diseases and for their anti-tumor properties. Phosphate (Pi) starvation can change secondary metabolism in plants. MYB transcription factors (TFs) have been reported to be involved in both Pi starvation signaling and secondary metabolism regulation, enabling plants to build a relationship between the Pi starvation signal and secondary metabolism. In the present study, a nucleus-located R2R3 MYB TF, SmMYB98b, was found in S. militorrhiza. SmMYB98b was shown to belong to subgroup 20 of R2R3-MYB TFs, and expression could be induced by low-Pi conditions. The overexpression of SmMYB98b in S. militorrhiza hairy roots stimulated tanshinone accumulation. Plants overexpressing SmMYB98b exhibited Pi-starvation responses, such as decreased Pi concentration and activation of genes inducible by phosphate starvation, whereas transgenic plants with the SmMYB98b-RNA interference (RNAi) construct showed the opposite phenotype. In summary, the present study highlighted the role of SmMYB98b as a regulator of tanshinone biosynthesis and its potential function in Pi-starvation signaling. These results might have wider implications for studying the crosstalk between Pi stress responses and secondary metabolism pathways in plants. This study highlights the role of SmMYB98b as a positive regulator of tanshinone biosynthesis and its potential function in Pi-starvation signaling in Salvia miltiorrhiza.

SmGRAS3 negatively responds to GA signaling while promotes tanshinones biosynthesis in Salvia miltiorrhiza

Salvia miltiorrhiza Bunge is the origin plant of traditional Chinese medicine, Danshen. Demand of high-quality S. miltiorrhiza is increasing because of its excellent pharmaceutical value. Tanshinones and salvianolic acids (SAs) are the main active ingredients of S. miltiorrhiza. GA was reported to induce tanshinones and SAs accumulation, however, its mechanism was unclear. Here, we got a GA responsive protein, SmGRAS3. SmGRAS3 expressed highest in the periderm where tanshinones produced and accumulated. Results of SmGRAS3 over and antisense expression indicated that SmGRAS3 is a positive regulator of tanshinones biosynthesis in S. miltiorrhiza hairy roots. But overexpression of SmGRAS3 depressed SAs and GA biosynthesis as well as hairy roots growth, and application of GA could partially recover these depressions. Pathway enzyme genes expressions of tanshinones, SAs and GA biosynthesis were comprehensively changed in SmGRAS3 transgenic hairy roots. SmGRAS3 acted as a transcription factor that located in nucleus and has the transcription activation activity. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that SmGRAS3 directly bound to GARE-motif in promoter of KSL1 and activated KSL1 transcription. Our study suggested that the SmGRAS3 may be a good potential target for further metabolic engineering of bioactive components biosynthesis in S. miltiorrhiza.

SmbHLH3 acts as a transcription repressor for both phenolic acids and tanshinone biosynthesis in Salvia miltiorrhiza hairy roots

Phenolic acids and tanshinones are the two groups of pharmaceutically active metabolites in Salvia miltiorrhiza Bunge. Their contents are the key quality indicator to evaluate S. miltiorrhiza. bHLH transcription factors have important roles in regulation of plant specialised metabolism. In this study, an endogenous bHLH transcription factor, SmbHLH3, was identified and functionally analyzed. SmbHLH3 was presented in all the six tissues and mostly expressed in fibrous roots and flowers. It was localized to the nucleus. Overexpression of SmbHLH3 decreased both phenolic acids and tanshinones contents. Contents of caffeic acid and rosmarinic acid were both decreased to 50% of the control. And accumulation of salvianolic acid B was decreased as much as 62%. Content of cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA in SmbHLH3-overexpression lines were reduced 97%, 62%, 86% and 91%, respectively. In the transgenic lines, expression of C4H1, TAT and HPPR in phenolic acids pathways were reduced to about 43%, 66% and 77% of the control, respectively. For tanshinone biosynthetic pathways, transcripts of DXS3, DXR, HMGR1, KSL1, CPS1 and CYP76AH1 were reduced to 46%, 65%, 78%, 57%, 27% and 62% of the control, respectively. There was an E/G-box specific binding site in SmbHLH3, which may bind the E/G-box present in promoter region of these biosynthetic pathway genes. Y1H results indicated that SmbHLH3 could bind the promoter of TAT, HPPR, KSL1 and CYP76AH1. These findings indicated that SmbHLH3 downregulate both phenolic acids and tanshinone accumulation through directly suppressing the transcription of key enzyme genes.

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

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.

Transcriptomic analysis reveals potential genes involved in tanshinone biosynthesis in Salvia miltiorrhiza

Tanshinones are important bioactive components in Salvia miltiorrhiza and mainly accumulate in the periderms of mature roots. Tanshinone biosynthesis is a complicated process, and little is known about the third stage of the pathway. To investigate potential genes that are responsible for tanshinone biosynthesis, we conducted transcriptome profiling analysis of two S. miltiorrhiza cultivars. Differential expression analysis provided 2,149 differentially expressed genes (DEGs) for further analysis. GO and KEGG analysis showed that the DEGs were mainly associated with the biosynthesis of secondary metabolites. Weighted gene coexpression network analysis (WGCNA) was further performed to identify a “cyan” module associated with tanshinone biosynthesis. In this module, 25 cytochromes P450 (CYPs), three 2-oxoglutarate-dependent dioxygenases (2OGDs), one short-chain alcohol dehydrogenases (SDRs) and eight transcription factors were found to be likely involved in tanshinone biosynthesis. Among these CYPs, 14 CYPs have been reported previously, and 11 CYPs were identified in this study. Expression analysis showed that four newly identified CYPs were upregulated upon application of MeJA, suggesting their possible roles in tanshinone biosynthesis. Overall, this study not only identified candidate genes involved in tanshinone biosynthesis but also provided a basis for characterization of genes involved in important active ingredients of other traditional Chinese medicinal plants.

UHPLC-HRMSn Analysis Reveals the Dynamic Metabonomic Responses of Salvia miltiorrhiza Hairy Roots to Polysaccharide Fraction from Trichoderma atroviride

We have previously reported that Trichoderma atroviride, an endophytic fungus isolated from S. miltiorrhiza, promotes S. miltiorrhiza hairy root growth and significantly stimulates the biosynthesis of tanshinones specifically the polysaccharide fraction (PSF). However, this study only focused exclusively on six metabolites whilst ignoring changes to the whole metabolite composition of the S. miltiorrhiza hairy roots. In the present study, the dynamic metabonomic responses of S. miltiorrhiza hairy roots were investigated using ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMSn). UHPLC-HRMS typical total ions chromatograms (TICs) of PSF-treated hairy root samples were different from the control. Moreover, the results of principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and hierarchical clustering analysis (HCA) indicated that PSF-treated samples were significantly different from the control. Through the analysis of PLS-DA, a total of 114 and 99 differential metabolites were found from the positive and negative models respectively and a total of 33 differential metabolites were identified. Thus, S. miltiorrhiza hairy roots had been induced to regulate the metabolic profiling in response to PSF and the changes of the metabolic profiling contributed to promoting the biosynthesis of tanshinones notably whilst the biosynthesis of phenolic acids were slightly inhibited.

Computer-Aided Saturation Mutagenesis of Arabidopsis thaliana Ent-Copalyl Diphosphate Synthase

Ent-copalyl diphosphate synthase controls the biosynthesis of gibberellin plant hormones, which in turn coordinate the expression of numerous enzymes. Some gibberellin-dependent genes encode enzymes coordinating the biosynthesis of tanshinones: diterpene derivatives with broad medical applications. New biotechnological approaches, such as metabolic engineering using naturally occurring or mutated enzymes, have been proposed to meet the growing demand for tanshinones which is currently met by the Chinese medicinal plant Salvia miltiorrhiza Bunge. These mutants may be prepared by directed evolution, saturation mutagenesis or rational enzyme design. In the presented paper, 15,257 non-synonymous variants of Arabidopsis thaliana ent-copalyl diphosphate synthase were obtained using the SNAP2 tool. The obtained forms were screened to isolate variants with potentially improved biological functions. A group of 455 mutants with potentially improved stability was isolated and subjected to further screening on the basis of ligand–substrate affinity, and both secondary structure and active site structure stability. Finally, a group of six single mutants was obtained, which were used to construct double mutants with potentially improved stability and ligand affinity. The potential influence of single mutations on protein stability and ligand affinity was evaluated by double mutant cycle analysis. Finally, the procedure was validated by in silico assessment of the experimentally verified enzyme mutants with reduced enzymatic activity.

The ethylene response factor SmERF8 regulates the expression of SmKSL1 and is involved in tanshinone biosynthesis in Saliva miltiorrhiza hairy roots

Saliva miltiorrhiza ethylene response factor (SmERF), predicted to be expressed genome-wide, is the potential regulator of tanshinone biosynthesis. However, few studies have investigated its transcriptional regulation pathways in tanshinone biosynthesis. Here, we report an ethylene response factor (SmERF8), which was screened by the SmKSL1 (a key gene in tanshinone biosynthesis) promoter from the S. miltiorrhiza cDNA library. The SmERF8, highly expressed in S. miltiorrhiza root head, is sensitive to Eth stress, and its protein was enriched in the nucleus. The SmERF8 recognizes the GCC-box in the SmKSL1 promoter. Overexpression and RNAi of SmERF8 in S. miltiorrhiza transgenic hairy roots showed that the tanshinone contents were significantly increased in the overexpression transgenic lines and decreased in RNAi lines. These results suggest that the SmERF8 may be a central activator that regulates the expression of SmKSL1 by binding the GCC-box and then promoting tanshinone biosynthesis. Thus, the SmERF8 may functionally accelerate tanshinone biosynthesis by the transcriptional regulation of its key gene.

Screening of long non-coding RNA related to CYP450s involved in biosynthesis of tanshinones

Tanshinones are abietane-type norditerpenoid quinones that make up the main bioactive ingredients of traditional Chinese medicine Salvia miltiorrhiza. Cytochrome CYP450 plays an important role in the post-structural modification of tanshinone biosynthesis pathway. Long non-coding RNA( lncRNA) have been defined as transcripts longer than 200 nucleotides,which have been functionally characterized in regulating the growth and development,secondary metabolism and stress of medicinal plants. In this study,we perform a comprehensive identification of lncRNAs in response to tanshinone metabolism induced by yeast extract( YE) and Ag~+ S. miltiorrhiza hairy roots. Deep RNA sequencing was used to identify a set of different 8 942 lncRNAs,of which 6 755 were intergenic lncRNAs. We predicted a total of 1 115 814 lncRNA-coding gene pairs,including 122 lncRNA-coding gene as cis pairs. The correlation analysis between lncRNA and CYP450 related to tanshinone biosynthesis was carried out and a total of 16 249 lncRNA-CYP450 target gene pairs were identified. Further analysis with functional known CYP76 AH1,CYP76 AH3 and CYP76 AK1 involved in tanshinone biosynthesis,we also identified a set of 216 target genes. These candidate genes will be the important target in the downstream regulation mechanism analysis of the tanshinone biosynthesis pathway.

Enhancement of tanshinone production in Salvia miltiorrhiza hairy root cultures by metabolic engineering

BackgroundTanshinones are diterpenoid compounds that are used to treat cardiovascular diseases. As current extraction methods for tanshinones are inefficient, there is a pressing need to improve the production of these bioactive compounds to meet increasing demand.ResultsOverexpression of SmMDS (2-c-methyl-d-erythritol 2,4-cyclodiphosphate synthase, a tanshinone biosynthesis gene) in transgenic Salvia miltiorrhiza hairy roots significantly increased the tanshinone yield compared to the control, and total tanshinone content in SmMDS-overexpressing lines increased after elicitor treatment. Total tanshinones increased to 2.5, 2.3, and 3.2 mg/g DW (dry weight) following treatment with Ag+, YE (yeast extract), and MJ (methyl jasmonate), respectively, compared with the non-induced transgenic line (1.7 mg/g DW). Also, qRT-PCR analysis showed that the expression levels of two pathway genes was positively correlated with increased accumulation of tanshinone.ConclusionsOur study provides an effective strategy for increasing the content of tanshinones and other natural compounds using a combination of genetic engineering and elicitor treatment.

Proteomic analysis reveals novel insights into tanshinones biosynthesis in Salvia miltiorrhiza hairy roots

Salvia miltiorrhiza is a medicinal plant highly appreciated by its content of tanshinones and salvianolic acids. Tanshinones are of particular relevance for their anti-oxidant, anti-tumoral and anti-inflammatory properties. Abiotic and biotic agents as silver nitrate and yeast extract have shown efficiently to stimulate tanshinone accumulation, but the underlying molecular mechanism remains essentially unknown. By using hairy roots as experimental material and the elicitors mentioned, were obtained up to 22 mg of tanshinones per gram of dry weight. Differential label-free quantitative proteomic analysis was applied to study the proteins involved in tanshinone biosynthesis. A total of 2650 proteins were identified in roots extracts, of which 893 showed statistically (p < 0.05) significant change in relative abundance compared to control roots, 251 proteins were upregulated and 642 downregulated. Among the upregulated proteins the predominant functional categories were metabolism (47%), stress defense (18%) and redox homeostasis (10%). Within the metabolism category, isoprenoid metabolism enzymes, cytochromes P450 and FAD-binding berberine proteins showed abundance profile linked to tanshinone concentration. The results presented here allowed to propose 5 new cytochromes P450 and 5 berberine enzymes as candidates to be involved into tanshinone biosynthesis, a novel finding that opens new avenues to improve tanshinone production through biotechnological approaches.

Smoke-Isolated Karrikins Stimulated Tanshinones Biosynthesis in Salvia miltiorrhiza through Endogenous Nitric Oxide and Jasmonic Acid.

Although smoke-isolated karrikins (KAR1) could regulate secondary metabolism in medicinal plants, the signal transduction mechanism has not been reported. This study highlights the influence of KAR1 on tanshinone I (T-I) production in Salvia miltiorrhiza and the involved signal molecules. Results showed KAR1-induced generation of nitric oxide (NO), jasmonic acid (JA) and T-I in S. miltiorrhiza hairy root. KAR1-induced increase of T-I was suppressed by NO-specific scavenger (cPTIO) and NOS inhibitors (PBITU); JA synthesis inhibitor (SHAM) and JA synthesis inhibitor (PrGall), which indicated that NO and JA play essential roles in KAR1-induced T-I. NO inhibitors inhibited KAR1-induced generation of NO and JA, suggesting NO was located upstream of JA signal pathway. NO-induced T-I production was inhibited by SHAM and PrGall, implying JA participated in transmitting signal NO to T-I accumulation. In other words, NO mediated the KAR1-induced T-I production through a JA-dependent signaling pathway. The results helped us understand the signal transduction mechanism involved in KAR1-induced T-I production and provided helpful information for the production of S. miltiorrhiza hairy root.

Tanshinone production could be increased by the expression of SmWRKY2 in Salvia miltiorrhiza hairy roots

Tanshinones are the main bioactive diterpenes in Salvia miltiorrhiza Bunge, are widely used for treating cardiovascular and cerebrovascular diseases. However, the biosynthetic mechanisms of these compounds have not yet been fully explained. In this study, a transcription factor named SmWRKY2 was isolated and functionally characterized. Multiple sequence analysis indicated it was classified into subgroup I of the WRKY family. Expression pattern showed that SmWRKY2 was mainly expressed in the stem and leaf and was inducible by methyl jasmonate (MeJA) treatment. Subcellular localization showed that SmWRKY2 was localized in the nucleus. Overexpression of SmWRKY2 in S. miltiorrhiza hairy roots significantly increased the expression of SmDXS2 and SmCPS, resulting in increased accumulation of tanshinones and the highest total tanshinone content was detected in OE-SmWRKY2-1 line, which was 1.83 times of the control. Meanwhile, tanshinone production was slightly reduced in the antisense-SmWRKY2 line. Dual-Luciferase assay showed that SmWRKY2 can positively regulate SmDXS2 and SmCPS expression, However, Y1H and EMSA experiments indicate that SmWRKY2 only binds to the W-box of the SmCPS promoter. Our study shows that SmWRKY2 is a positive regulator of tanshinone biosynthesis by mainly activating SmCPS. This study thus sheds new light on the regulatory role of SmWRKY2 in tanshinone biosynthesis.

Tanshinones, Critical Pharmacological Components in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge, a member of the Lamiaceae family, is valued in traditional Chinese Medicine. Its dried root (named Danshen) has been used for hundreds of years, primarily for the treatment of cardiovascular and cerebrovascular diseases. Tanshinones are the main active ingredients in S. miltiorrhiza and exhibit significant pharmacological activities, such as antioxidant activity, anti-inflammatory activity, cardiovascular effects, and antitumor activity. Danshen dripping pill of Tianshili is an effective drug widely used in the clinical treatment of cardiovascular diseases. With the increasing demand for clinical drugs, the traditional method for extracting and separating tanshinones from medicinal plants is insufficient. Therefore, in combination with synthetic biological methods and strategies, it is necessary to analyze the biosynthetic pathway of tanshinones and construct high-yield functional bacteria to obtain tanshinones. Moreover, the biosynthesis of tanshinones has been studied for more than two decades but remains to be completely elucidated. This review will systematically present the composition, extraction and separation, pharmacological activities and biosynthesis of tanshinones from S. miltiorrhiza, with the intent to provide references for studies on other terpenoid bioactive components of traditional Chinese medicines and to provide new research strategies for the sustainable development of traditional Chinese medicine resources.

The AP2/ERF transcription factor SmERF128 positively regulates diterpenoid biosynthesis in Salvia miltiorrhiza.

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.

Expression patterns of some genes involved in tanshinone biosynthesis in Salvia miltiorrhiza roots

Tanshinones are the major bioactive components of Salvia miltiorrhiza, and synthesized through the cytosol-localized mevalonic acid (MVA) and plastid-localized methylerythritol 4-phosphate (MEP) pathways. To reveal correlations between gene expression and tanshinone accumulation, transcript-level variations in five candidate genes involved in tanshinone biosynthesis in the roots of S. miltiorrhiza were investigated at different developmental stages. Additionally, the accumulation of tanshinone I, tanshinone IIA, and cryptotanshinone were analyzed by liquid chromatography tandem mass spectrometry. Compared with the other genes examined, SmCMK (encoding the enzyme 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase) expression was significantly positively correlated with tanshinone production, and SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) likely functions as a non-rate-limiting enzyme in the tanshinone-biosynthesis pathways of S. miltiorrhiza during different developmental stages, with significant correlations observed between SmDXR and SmCMK expression. Furthermore, no significant correlations were observed between the expression of SmAACT (encoding the enzyme acetyl-CoA acyltransferase), SmHMGR2 (encoding the enzyme 3-hydroxy-3-methylglutaryl-COA reductase), and SmFPPS (encoding the enzyme farnesyl diphosphate synthase), which are involved in the MVA pathway, and tanshinone production during the developmental stages examined, suggesting that the MVA pathway might contribute less to tanshinone accumulation as compared with the MEP pathway. The results in this study indicated that SmCMK might play an important role in their biosynthesis and accumulation. Overall, this study contributes to a better understanding of tanshinone biosynthesis during the different developmental stages of S. miltiorrhiza.