Overexpression of a chrysanthemum transcription factor gene <Emphasis Type="Italic">DgNAC1</Emphasis> improves drought tolerance in chrysanthemum

Phenolic acids and tanshinones are two groups of pharmaceutical components present in Salvia miltiorrhiza Bunge. Methyl jasmonate (MeJA) has been reported to influence the accumulation of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. However, there is currently a lack of information regarding the comparison of how these two groups of bioactive compounds in S. miltiorrhiza respond to MeJA under the same conditions. In the present study, the effect of 100 µM MeJA on the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza hairy roots was investigated. The results showed that MeJA dramatically induced the accumulation of five different phenolic acids, especially rosmarinic acid and salvianolic acid B, which reached their highest contents at day 3 (20.3 mg/g DW, 1.5-fold of control) and day 6 (47.49 mg/g DW, 2.5-fold of control), respectively. The total production of phenolic acids was induced by as much as 3.3-fold of the control (day 9 after treatment), reaching 357.5 mg/L at day 6. However, tanshinone I was almost unaffected by MeJA treatment, and the accumulation of tanshinone IIA was inhibited. Furthermore, cryptotanshinone and dihydrotanshinone I were moderately induced by MeJA. The gene expression results indicated that MeJA probably induced the whole pathways, especially the tyrosine-derived pathway and the methylerythritol phosphate pathway, and finally resulted in the increased production of these metabolites. This study will help us to further understand how the different biosynthetic mechanisms of phenolic acids and tanshinones respond to MeJA and provide a reference for the future selection of elicitors for application to improving the production of targeted compounds.     

Candidate genes involved in tanshinone biosynthesis in hairy roots of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> revealed by cDNA microarray

Salvia miltiorrhiza is a valuable Chinese herb (Danshen) that is widely used in traditional Chinese medicine. Diterpene quinones, known as tanshinones, are the main bioactive components of S. miltiorrhiza; however, there is only limited information regarding the molecular mechanisms underlying secondary metabolism in this plant. We used cDNA microarray analysis to identify changes in the gene expression profile at different stages of hairy root development in S. miltiorrhiza. A total of 203 genes were singled out from 4,354 cDNA clones on the microarray, and 114 unique differentially expressed cDNA clones were identified: six genes differentially expressed in 45-day hairy root compared with 30-day hairy root; 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root; and 12 genes unstably expressed at different stages. Among the 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root, a total of 57 genes were up-regulated, and 26 genes represent 29 metabolism-related enzymes. Copalyl diphosphate synthase, which catalyzes the conversion of the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate to copalyl diphosphate, was up-regulated 6.63 fold, and another six genes involved in tanshinone biosynthesis and eight candidate P450 genes were also differentially expressed. These data provide new insights for further identification of the enzymes involved in tanshinone biosynthesis.     

Promotion of phenolic compounds production in Salvia miltiorrhiza hairy roots by six strains of rhizosphere bacteria

Salvia miltiorrhiza Bunge is an important herb for the treatment of cerebrovascular and cardiovascular diseases with bioactive compounds (phenolic acids and tanshinones). Abundant studies showed that tanshinones could be stimulated by biotic and abiotic stresses, but limited information is available on biosynthesis of phenolic acids promoted by biotic stresses. The aim of the present work was to isolate and identify rhizosphere bacteria which stimulated phenolic compound in Salvia miltiorrhiza hairy roots and investigated the internal mechanism, providing a potential means to enhance content of pharmaceuticals in S. miltiorrhiza. The results showed that six bacteria, namely, HYR1, HYR26, SCR22, 14DSR23, DS6, and LNHR13, belonging to the genus Pseudomonas and Pantoea, significantly promoted the growth and content of major phenolic acids, RA and SAB. Bacteria LNHR13 was the most effective one, with the contents of RA and SAB reaching ～2.5‐fold (30.1 mg/g DW) and ～2.3‐fold (48.3 mg/g DW) as those of the control, respectively. Phytohormones and polysaccharides produced by bacteria showed potential responsibility for the growth and biosynthesis of secondary metabolites of S. miltiorrhiza. Meanwhile, we found that the more abundant the types and contents of phytohormones, the stronger their stimulating effect on the content of salvianolic acids.     

The phenylalanine ammonia-lyase gene family in Salvia miltiorrhiza: genome-wide characterization, molecular cloning and expression analysis

Salvia miltiorrhiza Bunge is a well-known material of traditional Chinese medicine. Hydrophilic phenolic acids, such as rosmarinic acid and salvianolic acid B, are a group of pharmaceutically important compounds in S. miltiorrhiza. The biosynthesis of rosmarinic acid requires the coordination of the phenylpropanoid pathway and the tyrosine-derived pathway. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. Systematic analysis of the SmPAL gene family has not been carried out. We report here the identification of three SmPALs through searching the recently obtained working draft of the S. miltiorrhiza genome and full-length cDNA cloning. Bioinformatic and phylogenetic analyses showed that SmPAL1 and SmPAL3 clustered in a sub-clade of dicot PALs, whereas SmPAL2 fell into the other one. Some important cis-elements were conserved in three SmPAL promoters, whereas the others were not. SmPAL1 and SmPAL3 were highly expressed in roots and leaves of S. miltiorrhiza, but SmPAL2 were predominately expressed in stems and flowers. It indicates that SmPAL1 and SmPAL3 function redundantly in rosmarinic acid biosynthesis. All SmPALs were induced in roots treated with PEG and MeJA, but the time and degree of responses were different, suggesting the complexity of SmPAL-associated metabolic network in S. miltiorrhiza. This is the first comprehensive study dedicated to SmPAL gene family characterization. The results provide a basis for elucidating the role of SmPAL genes in the biosynthesis of bioactive compounds.     

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.     

PEG and ABA Trigger the Burst of Reactive Oxygen Species to Increase Tanshinone Production in Salvia miltiorrhiza Hairy Roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Tanshinones, a group of active compounds in S. miltiorrhiza, are derived from two biosynthetic pathways: the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastids. Water stress is well known to stimulate the accumulation of secondary metabolites in plants. Reactive oxygen species (ROS) serve as important secondary messengers in water stress-induced signal transduction pathways. In this study, the effects of polyethylene glycol (PEG) and abscisic acid (ABA) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of ROS in PEG- and ABA-induced tanshinone production were further elucidated. The results showed that contents and yields of four tanshinones in S. miltiorrhiza hairy roots were significantly enhanced by 2 % PEG and 200?μM ABA. Simultaneously, the mRNA levels and activities of two key enzymes (3-hydroxy-3-methylglutaryl coenzyme A reductase and 1-deoxy-D-xylulose 5-phosphate synthase) involved in tanshinone biosynthesis were upregulated. Both PEG and ABA were able to trigger the burst of H₂O₂ and O₂ ^?. The PEG- and ABA-induced increases of tanshinone production, gene expression, and enzyme activity were all dramatically suppressed by two ROS scavengers, catalase and superoxide dismutase. In addition, ROS treatments resulted in a significant increase in tanshinone production. These results demonstrated that the MVA and MEP pathways were activated by PEG and ABA to stimulate tanshinone biosynthesis, and the increase of tanshinone production was probably via ROS signaling.     

Characterization,expression profiling,and functional identification of a gene encoding geranylgeranyl diphosphate synthase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes including tanshinone. In this study, a full-length cDNA encoding GGPPS was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmGGPPS (GenBank Accession No. FJ643617). The full-length cDNA of SmGGPPS was 1,234 bp containing a 1,092 bp open reading frame (ORF) encoding a polypeptide of 364 amino acids. Analysis of SmGGPPS genomic DNA revealed that it contained 2 exons and 1 intron. Bioinformatics analyses revealed that the deduced SmGGPPS had extensive homology with other plant GGPPSs contained all 5 conserved domains and functional aspartate-rich motifs of the prenyltransferases. Molecular modeling showed that SmGGPPS is a new GGPPS with a spatial structure similar to other plant GGPPSs. Phylogenetic tree analysis indicated that SmGGPPS belongs to the plant GGPPS super-family and has the closest relationship with GGPPS from Nicotiana attenuate. The functional identification in Escherichia coli showed that SmGGPPS could accelerate the biosynthesis of carotenoid, demonstrating that SmGGPPS encoded a functional protein. Expression pattern analysis implied that SmGGPPS expressed higher in leaves and roots, weaker in stems. The expression of SmGGPPS could be up-regulated by Salicylic acid (SA) in leaves and inhibited by methyl jasmonate (MeJA) in 3 tested tissues, suggesting that SmGGPPS was elicitor-responsive. This work will be helpful to understand more about the role of SmGGPPS involved in the tanshinones biosynthesis pathway and metabolic engineering to improve tanshiones production in S. miltiorrhiza.     

Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures

 Salvia miltiorrhiza contains two groups of biologically active secondary metabolites termed phenolic compounds (e.g. rosmarinic acid) and tanshinones (e.g. cryptotanshinone). Their roles in plant defense responses were examined using a simplified system consisting of a yeast elicitor and a Ti C58 transformed S. miltiorrhiza cell line. Both dosage and time course studies were carried out on the effects of yeast elicitor on the formation of rosmarinic acid and cryptotanshinone. It was found that the yeast elicitor reduced the constituent level of rosmarinic acid (from ca. 5% to ca. 3.0% of dry cell weight) whereas the level of cryptotanshinone was enhanced greatly (from a negligible amount to ca. 20 mg/l). These results suggest that in S. miltiorrhiza, rosmarinic acid and cryptotanshinone may take part in plant passive and active defense responses, respectively, against pathogen attack. Cryptotanshinone was identified as a phytoalexin in S. miltiorrhiza for the first time. Results of the treatment of cell cultures with 2-aminoindan-2-phosphonic acid, a highly specific and potent inhibitor of phenylalanine ammonia-lyase (EC 4.3.1.5), indicated that this compound did not inhibit yeast elicitor induced tanshinone formation, but did inhibit rosmarinic acid biosynthesis. Received: 22 May 1999 / Revision received: 27 August / Accepted: 1 September 1999     

Coding single nucleotide polymorphisms and SmCPS1 and SmKSL1 subcellular localization are associated with tanshinone biosynthesis in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> Bunge roots

Tanshinone is one of the major medicinal components of the roots of Salvia miltiorrhiza Bunge, and SmCPS1 and SmKSL1 are key enzymes in the tanshinone biosynthesis pathway. To increase our understanding of the coding single nucleotide polymorphisms (cSNPs) involved in tanshinone biosynthesis, seven S. miltiorrhiza landraces were examined. Our results revealed that the tanshinone content was significantly different among the seven landraces. In total, 48 cSNPs in SmCPS1 and 47 cSNPs in SmKSL1 were identified, and of these, 38 and 42 cSNPs, respectively, were associated with tanshinone content. The highest A/G and C/T base substitution rates were in SmCPS1 and SmKSL1, respectively. SmKSL1 expression was significantly, positively correlated with tanshinone IIA and tanshinone I contents, and SmCPS1 expression was significantly associated with tanshinone IIA content. Interestingly, subcellular SmCPS1 and SmKSL1 expression was enriched in the plastids. Therefore, cSNPs of SmCPS1 and SmKSL1 are involved in tanshinone biosynthesis in the plastids, where SmCPS1 and SmKSL1 enzymes catalyze tanshinone production in this species.     

Roles of reactive oxygen species in methyl jasmonate and nitric oxide-induced tanshinone production in Salvia miltiorrhiza hairy roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O₂ ⁻ and H₂O₂ were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.