Structural insights revealed by crystal structures of CYP76AH1 and CYP76AH1 in complex with its natural substrate.

CYP76AH1 is the key enzyme in the biosynthesis pathway of tanshinones in Salvia miltiorrhiza, which are famous natural products with activities against various heart diseases and others. CYP76AH1 is a membrane-associated typical plant class II cytochrome P450 enzyme and its catalytic mechanism has not to be clearly elucidated. Structural determination of eukaryotic P450 enzymes is extremely challenging. Recently, we solved the crystal structures of CYP76AH1 and CYP76AH1 in complex with its natural substrate miltiradiene. The structure of CYP76AH1 complexed with miltiradiene is the first plant cytochrome P450 structure in complex with natural substrate. The studies revealed a unique array pattern of amino acid residues, which may play an important role in orienting and stabilizing the substrate for catalysis. This work would provide structural insights into CYP76AH1 and related P450s and the basis to efficiently improve tanshinone production by synthetic biology techniques.

Targeted mutagenesis of CYP76AK2 and CYP76AK3 in Salvia miltiorrhiza reveals their roles in tanshinones biosynthetic pathway.

Salvia miltiorrhiza Bunge, belonging to Lamiaceae family, is one of the most important Chinese medicinal herbs. The dried roots, also called Danshen in Chinese, are usually used in the formula of Chinese traditional medicine due to the bioactive constituents known as phenolic acids and tanshinones, which are a group of abietane nor-diterpenoid quinone natural products. Cytochrome P450 enzymes (CYPs) usually play crucial roles in terpenoids synthesis, especially in hydroxylation processes. Up to now, several important P450 enzymes, such as CYP76AH1, CYP76AH3, CYP76AK1, CYP71D373, and CYP71D375, have been functionally characterized in the tanshinones biosynthetic pathway. Nevertheless, the tanshinones biosynthesis is a so complex network that more P450 enzymes should be identified and characterized. Here, we report two novel P450 enzymes CYP76AK2 and CYP76AK3 that are involved in tanshinones biosynthetic pathway. These two P450 enzymes were highly homologous to previously reported CYP76AK1 and showed the same expression profile as CYP76AK1. Also, CYP76AK2 and CYP76AK3 could be stimulated by MeJA and SA, resulting in increased expression. We used a triple-target CRISPR/Cas9 system to generate targeted mutagenesis of CYP76AK2 and CYP76AK3 in S. miltiorrhiza. The content of five major tanshinones was significantly reduced in both cyp76ak2 and cyp76ak3 mutants, indicating that the two enzymes might be involved in the biosynthesis of tanshinones. This study would provide a foundation for the catalytic function identification of CYP76AK2 and CYP76AK3, and further enrich the understanding of the network of tanshinones secondary metabolism synthesis as well.

Endophytic fungus Mucor circinelloides DF20 promote tanshinone biosynthesis and accumulation in Salvia miltiorrhiza root.

As a traditional Chinese medicine, Salvia miltiorrhiza rhizome is mainly used to treat cardiovascular diseases. Symbiosis of endophytic fungi with their host plants, is an effectively regulatory means to promote the growth and secondary metabolism of medicinal plants. Here, an endophytic fungus Mucor circinelloides DF20 was co-cultivated with the sterile seedlings of S. miltiorrhiza, to clarify the promoting mechanism on tanshinone biosynthesis and accumulation in S. miltiorrhiza root. The assay of promoting-growth activities in vitro showed that DF20 have the ability to produce IAA and siderophores. DF20 could significantly promote the biosynthesis and accumulation of tanshinones in the root of S. miltiorrhiza, especially the content of tanshinone ⅡA, reaching 4.630 ± 0.342 mg/g after 56 days of DF20 treatment, which is 22-fold of the control group. The result also showed that the hyphae of M. circunelloides DF20 mainly colonized in the root tissue interspace of S. miltiorrhiza, and a small amount of hyphae were located inside the cells. The results of florescent real-time quantitative RT-PCR showed that DF20 colonization significantly increase the expression level of some key enzyme genes (DXS, DXR, HMGR, GGPPS) in tanshinone biosynthesis pathway, but the regulatory effect mainly occurred in the early stage of co-culture, while the expression level decreased in different degrees in the later stage. In conclusion, the endophytic fungus M. circunelloides DF20 can form an interaction relationship with its host, then to promote the biosynthesis and accumulation of tanshinones in root by upregulating the key enzyme genes expression levels of the biosynthesis pathway.

Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H+-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H+-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H+-ATPase isoforms were identified and named SmPHA1-SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H+-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H+-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza.

Expansion within the CYP71D subfamily drives the heterocyclization of tanshinones synthesis in Salvia miltiorrhiza.

Tanshinones are the bioactive nor-diterpenoid constituents of the Chinese medicinal herb Danshen (Salvia miltiorrhiza). These groups of chemicals have the characteristic furan D-ring, which differentiates them from the phenolic abietane-type diterpenoids frequently found in the Lamiaceae family. However, how the 14,16-epoxy is formed has not been elucidated. Here, we report an improved genome assembly of Danshen using a highly homozygous genotype. We identify a cytochrome P450 (CYP71D) tandem gene array through gene expansion analysis. We show that CYP71D373 and CYP71D375 catalyze hydroxylation at carbon-16 (C16) and 14,16-ether (hetero)cyclization to form the D-ring, whereas CYP71D411 catalyzes upstream hydroxylation at C20. In addition, we discover a large biosynthetic gene cluster associated with tanshinone production. Collinearity analysis indicates a more specific origin of tanshinones in Salvia genus. It illustrates the evolutionary origin of abietane-type diterpenoids and those with a furan D-ring in Lamiaceae.

Lipopolysaccharide Enhances Tanshinone Biosynthesis via a Ca2+-Dependent Manner in Salvia miltiorrhiza Hairy Roots.

Tanshinones, the major bioactive components in Salvia miltiorrhiza Bunge (Danshen), are synthesized via the mevalonic acid (MVA) pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and the downstream biosynthesis pathway. In this study, the bacterial component lipopolysaccharide (LPS) was utilized as a novel elicitor to induce the wild type hairy roots of S. miltiorrhiza. HPLC analysis revealed that LPS treatment resulted in a significant accumulation of cryptotanshinone (CT) and dihydrotanshinone I (DTI). qRT-PCR analysis confirmed that biosynthesis genes such as SmAACT and SmHMGS from the MVA pathway, SmDXS and SmHDR from the MEP pathway, and SmCPS, SmKSL and SmCYP76AH1 from the downstream pathway were markedly upregulated by LPS in a time-dependent manner. Furthermore, transcription factors SmWRKY1 and SmWRKY2, which can activate the expression of SmDXR, SmDXS and SmCPS, were also increased by LPS. Since Ca2+ signaling is essential for the LPS-triggered immune response, Ca2+ channel blocker LaCl3 and CaM antagonist W-7 were used to investigate the role of Ca2+ signaling in tanshinone biosynthesis. HPLC analysis demonstrated that both LaCl3 and W-7 diminished LPS-induced tanshinone accumulation. The downstream biosynthesis genes including SmCPS and SmCYP76AH1 were especially regulated by Ca2+ signaling. To summarize, LPS enhances tanshinone biosynthesis through SmWRKY1- and SmWRKY2-regulated pathways relying on Ca2+ signaling. Ca2+ signal transduction plays a key role in regulating tanshinone biosynthesis in S. miltiorrhiza.

JA-Responsive Transcription Factor SmMYB97 Promotes Phenolic Acid and Tanshinone Accumulation in Salvia miltiorrhiza.

Phenolic acids and tanshinones are active principles in Salvia miltiorrhiza Bunge administered for cardiovascular and cerebrovascular diseases. Jasmonic acid (JA) promotes secondary metabolite accumulation, but the regulatory mechanism is unknown in S. miltiorrhiza. We identified and characterized the JA-responsive gene SmMYB97. Multiple sequence alignment and phylogenetic tree analyses showed that SmMYB97 was clustered with AtMYB11, AtMYB12, and ZmP1 in the subgroup S7 regulating flavonol biosynthesis. SmMYB97 was highly expressed in S. miltiorrhiza leaves and induced by methyl jasmonate (MeJA). SmMYB97 was localized in the nucleus and had strong transcriptional activation activity. SmMYB97 overexpression increased phenolic acid and tanshinone biosynthesis and upregulated the genes implicated in these processes. Yeast one-hybrid and transient transcriptional activity assays disclosed that SmMYB97 binds the PAL1, TAT1, CPS1, and KSL1 promoter regions. SmJAZ8 interacts with SmMYB97 and downregulates the genes that it controls. This study partially clarified the regulatory network of MeJA-mediated secondary metabolite biosynthesis in S. miltiorrhiza.

Increased phenolic acid and tanshinone production and transcriptional responses of biosynthetic genes in hairy root cultures of Salvia przewalskii Maxim. treated with methyl jasmonate and salicylic acid.

The purpose of this study is to reveal the impact of the plant hormone salicylic acid (SA) and methyl jasmonate (MeJA) on the growth, effective components accumulation, and related gene expression of the hairy root of Salvia przewalskii Maxim. Various concentrations of SA (0, 25, 50, 100, 200 µM) or MeJA (0, 50, 100, 200, 400, 600 µM) were added to the culture medium of Salvia przewalskii Maxim. Low concentrations of SA promoted the growth of hairy root, while a high concentration inhibited it. 0 to 400 µM MeJA promoted the growth of hairy root, but 600 µM MeJA starts to inhibit its growth. 50 µM SA and 400 µM MeJA significantly enhanced the production of caffeic acid, rosmarinic acid, salvianolic acid B, cryptotanshinone, and tanshinone IIA. In general, 50 µM SA can be used to accumulate of tanshinone in hairy roots of S. przewalskii with 6 days. 400 µM MeJA can be used to accumulate of phenolic acids in hairy roots of S. przewalskii with 3 days. The selected genes in the tanshinone and phenolic acid biosynthetic pathway were upregulated with elicitation. To obtain a higher yield and content of secondary metabolites, it is advisable to use 50 µM SA or 400 µM MeJA as the optimal doses to cultivate the hairy root of S. przewalskii. This study provides, for the first time, an efficient tanshinone and phenolic acid production method for S. przewalskii.

Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK3 and SmMAPK1 in secondary metabolism.

BACKGROUND: The contribution of mitogen-activated protein kinase (MAPK) cascades to plant growth and development has been widely studied, but this knowledge has not yet been extended to the medicinal plant Salvia miltiorrhiza, which produces a number of pharmacologically active secondary metabolites. RESULTS: In this study, we performed a genome-wide survey and identified six MAPKKK kinases (MAPKKKKs), 83 MAPKK kinases (MAPKKKs), nine MAPK kinases (MAPKKs) and 18 MAPKs in the S. miltiorrhiza genome. Within each class of genes, a small number of subfamilies were recognized. A transcriptional analysis revealed differences in the genes' behaviour with respect to both their site of transcription and their inducibility by elicitors and phytohormones. Two genes were identified as strong candidates for playing roles in phytohormone signalling. A gene-to-metabolite network was constructed based on correlation analysis, highlighting the likely involvement of two of the cascades in the synthesis of two key groups of pharmacologically active secondary metabolites: phenolic acids and tanshinones. CONCLUSION: The data provide insight into the functional diversification and conservation of MAPK cascades in S. miltiorrhiza.

Functional Integration of Two CYP450 Genes Involved in Biosynthesis of Tanshinones for Improved Diterpenoid Production by Synthetic Biology.

Cytochrome P450s (CYPs) are important enzymes in the secondary metabolism of plants and have been recognized as key players in bioengineering and synthetic biology. Previously reported CYP76AH1 and CYP76AH3, having greater than 80% sequence homology, played a continuous catalytic role in the biosynthesis of tanshinones in Salvia miltiorrhiza. Homology modeling indicates that four sites might be responsible for differences in catalytic activity between the two enzymes. A series of modeling-based mutational variants of CYP76AH1 were designed to integrate the functions of the two CYPs. The mutant CYP76AH1D301E,V479F, which integrated the functions of CYP76AH1 and CYP76AH3, was found to efficiently catalyze C11 and C12 hydroxylation and C7 oxidation of miltiradiene substrates. Integration and utilization of CYP76AH1D301E,V479F by synthetic biology methods allowed the robust production of 11-hydroxy ferruginol, sugiol, and 11-hydroxy sugiol in yeast. The functionally integrated CYP gene after active site modifications improves catalytic efficiency by reducing the transfer of intermediate metabolites between component proteins. This provides a synthetic biology reference for improving the catalytic efficiencies of systems that produce plant natural products in microorganisms.

[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.

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.

Transcriptome sequencing of Salvia miltiorrhiza after infection by its endophytic fungi and identification of genes related to tanshinone biosynthesis.

Context: Salvia miltiorrhiza Bunge (Labiatae) is a traditional Chinese herb. Endophytic fungi, which are biotic elicitors, can induce accumulation of secondary metabolites in their host plants.Objective: To analyze the interaction mechanism between S. miltiorrhiza and endophytic fungi.Materials and methods: Endophytic fungi U104 producing tanshinone IIA were isolated from the healthy disease-free tissue of root of S. miltiorrhiza by conventional methods. The endophytic fungus U104 of S. miltiorrhiza was co-cultured with the sterile seedlings of S. miltiorrhiza for 20 d (temp:day/night = 26 °C/18 °C, photoperiod:12/12 h, illuminance:2000 Lx). Transcriptome sequencing of S. miltiorrhiza seedlings after 20 d of co-cultivation was performed using the Illumina platform.Results: A total of 3713 differentially expressed genes (DEGs) were obtained. These different expression genes, such as STPII, LTP2, MYB transcription factors, CNGC, CDPK, Rboh, CaM, MAP2K1/MEK1, WRKY33, SGT1/SGT and Hsp90/htpG, showed that host S. miltiorrhiza had biological defence response in the initial stage of interaction. Under the induction of endophytic fungi, 14 key enzyme genes were up-regulated in the tanshinone biosynthesis pathway: DXS, DXS2, DXR, HMGR3, AACT, MK, PMK, GGPPS2, GPPS, KSL, IDI, IPII, FDPS and CPS.Discussion and conclusions: A total of 14 key genes were obtained from the tanshinone component synthesis and metabolic pathways, providing a reasonable explanation for the accumulation of tanshinone components, an accumulation induced by endophytic fungi, in the host plants. The large amounts of data generated in this study provide a strong and powerful platform for future functional and molecular studies of interactions between host plants and their endophytic fungi.

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.

Structure of a polysaccharide from Trichoderma atroviride and its promotion on tanshinones production in Salvia miltiorrhiza hairy roots.

This study evaluates the chemical structure of a heteropolysaccharide (PSF-W-1) from the endophytic fungus Trichoderma atroviride and its effects on the production of tanshinones in Salvia miltiorrhiza hairy roots. The total carbohydrate content of isolated PSF-W-1 was determined to be 97.72%. PSF-W-1 has a relative molecular weight of 36.13 kDa and contains mannose, glucose and galactose in molar ratios of 1.00:4.86:2.25. Through methylation analysis, IR and NMR, PSF-W-1 was determined to possess a backbone of →4)-ß-D-Glcp-(1→6)-α-D-Galp-(1→4)-ß-D-Manp-(1→6)-α-D-Galp-(1→ with two side chains ß-D-Glcp-(1→4)-ß-D-Glcp-(1→ attached to O3 of 1,6-α-D-Galp. Bioactivity tests suggested that PSF-W-1 was responsible for boosting the S. miltiorrhiza hairy root growth and the biosynthesis of dihydrotanshinone I, tanshinone I, tanshinone IIA and cryptotanshinone in hairy roots. According to this study, PSF-W-1 might be utilized as a potent stimulator of tanshinones synthesis.

[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.

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.

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.

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