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.     

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.     

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.     

Molecular characterization and expression of 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose 5-phosphate reductoisomerase (DXR) gene from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plants. The present study describes the cloning and characterization of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255). Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with DXRs from other plant species. Phylogenetic tree analysis indicated that SmDXR belongs to the plant DXR superfamily and has the closest relationship with DXR from Lycopersicon esculentum. Tissue expression pattern analysis revealed that SmDXR expressed strongly in leaves, followed by roots and stems, implying that SmDXR was a constitutively expressed gene. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in tanshinone biosynthetic pathway in Salvia plants. The expression profiles revealed by RT-PCR under different elicitor treatments such as methyl jasmonate (MJ) and salicylic acid (SA) were compared for the first time, and the results revealed that SmDXR was an elicitor-responsive gene, which could be induced by SA in leaves and inhibited by exogenous MJ in three tested tissues. The functional color assay in Escherichia coli showed that SmDXR could accelerate the biosynthesis of lycopene, indicating that SmDXR encoded a functional protein. The characterization, expression profile and functional analysis of SmDXR gene will be helpful for further study in the role of SmDXR in tanshinones biosynthetic pathway and metabolic engineering to increase tanshinones production in S. miltiorrhiza.     

Deep Sequencing Identifies Tissue-Specific MicroRNAs and Their Target Genes Involving in the Biosynthesis of Tanshinones in Salvia miltiorrhiza

Salvia miltiorrhiza is one of the most popular traditional medicinal herbs in Asian nations. Its dried root contains a number of tanshinones, protocatechuic aldehyde, salvianolic acid B and rosmarinic, and is used for the treatment of various diseases. The finding of microRNAs (miRNAs) and their target genes will help understand their biological role on the biosynthesis of tanshinones in S. miltiorrhiza. In the present study, a total of 452 known miRNAs corresponding to 589 precursor miRNAs (pre-miRNAs), and 40 novel miRNAs corresponding to 24 pre-miRNAs were identified in different tissues of S. miltiorrhiza by high-throughput sequencing, respectively. Among them, 62 miRNAs express only in root, 95 miRNAs express only in stem, 19 miRNAs express only in leaf, and 71 miRNAs express only in flower, respectively. By the degradome analysis, 69 targets potentially cleaved by 25 miRNAs were identified. Among them, acetyl-CoA C-acetyltransferase was cleaved by miR5072, and involved in the biosynthesis of tanshinones. This study provided valuable information for understanding the tissue-specific expression patterns of miRNAs in S. miltiorrhiza, and offered a foundation for future studies of the miRNA-mediated biosynthesis of tanshinones.     

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.