Chromosome-level genome assembly of Salvia miltiorrhiza with orange roots uncovers the role of Sm2OGD3 in catalyzing 15,16-dehydrogenation of tanshinones.

Salvia miltiorrhiza is well known for its clinical practice in treating heart and cardiovascular diseases. Its roots, used for traditional Chinese medicine materials, are usually brick-red due to accumulation of red pigments, such as tanshinone IIA and tanshinone I. Here we report a S. miltiorrhiza line (shh) with orange roots. Compared with the red roots of normal S. miltiorrhiza plants, the contents of tanshinones with a single bond at C-15,16 were increased, whereas those with a double bond at C-15,16 were significantly decreased in shh. We assembled a high-quality chromosome-level genome of shh. Phylogenomic analysis showed that the relationship between two S. miltiorrhiza lines with red roots was closer than the relationship with shh. It indicates that shh could not be the mutant of an extant S. miltiorrhiza line with red roots. Comparative genomic and transcriptomic analyses showed that a 1.0 kb DNA fragment was deleted in shh Sm2OGD3m. Complementation assay showed that overexpression of intact Sm2OGD3 in shh hairy roots recovered furan D-ring tanshinone accumulation. Consistently, in vitro protein assay showed that Sm2OGD3 catalyzed the conversion of cyptotanshinone, 15,16-dihydrotanshinone I and 1,2,15,16-tetrahydrotanshinone I into tanshinone IIA, tanshinone I and 1,2-dihydrotanshinone I, respectively. Thus, Sm2OGD3 functions as tanshinone 15,16-dehydrogenase and is a key enzyme in tanshinone biosynthesis. The results provide novel insights into the metabolic network of medicinally important tanshinone compounds.

Comparative transcriptomic analysis reveals genes regulating the germination of morphophysiologically dormant Paris polyphylla seeds during a warm stratification.

We previously analyzed the expression of genes associated with Paris polyphylla var. yunnanensis seed maturation and dormancy release; however, we were unable to clarify the relationship between gene expression levels and these processes. To reveal the molecular mechanisms underlying P. polyphylla var. yunnanensis seed dormancy release during a warm stratification, the transcriptomes of dormant and germinating P. polyphylla var. yunnanensis seeds were separately analyzed by RNA sequencing and were also compared with the transcriptomes of stem-leaf and root tissues harvested during the seed maturation stage. The RNA sequencing of five tissues generated 234,331 unigenes, of which 10,137 (4.33%) were differentially expressed among the analyzed tissues. The 6,619 unigenes whose expression varied among mature dormant, sprouted, and germinated seeds included 95 metabolic and 62 signaling genes related to abscisic acid, gibberellin, auxin, brassinosteroid, cytokinin, ethylene, jasmonic acid and salicylic acid. Additionally, 243 differentially expressed genes were annotated as known seed dormancy/germination-related genes. Among these genes, 109 were regulated by hormones or involved in hormone signal transduction. Finally, 310 transcription factor unigenes, including 71 homologs of known seed dormancy/ germination-related genes, were observed to be differentially expressed during a warm stratification. These results confirm that multiple hormones and transcription factors influence P. polyphylla var. yunnanensis seed dormancy release and germination during a warm stratification. This study identified candidate genes (e.g., ABI5) that should be cloned and functionally characterized regarding their effects on the release of P. polyphylla var. yunnanensis seed morphophysiological dormancy.

Quality evaluation of Panax quinquefolium from different cultivation regions based on their ginsenoside content and radioprotective effects on irradiated mice.

Ginsenosides are one of major types of bioactive compounds in American ginseng (AG) and utilized to assess the quality of various AG samples. The contents of ginsenosides showed cultivation region-related variation, which is possibly associated with AG's pharmacological effect difference. Therefore, to reveal the quality difference of AGs in different cultivation regions, AG samples from seven cultivation regions were evaluated via analyzing their contents of nine ginsenosides and the biochemical parameters in AG-treated irradiated mice. Pre-administration of AG decoctions could reversely modulate the irradiation-induced changes of antioxidant enzymatic activity, cytokine level and hormone level in irradiated mice, which demonstrated that AG had the radioprotective effects due to its antioxidative, immunomodulatory and anti-inflammatory properties. However, this radioprotection effect varied among different cultivation regions of AGs. Collectively, Beijing and Canada-cultivated AGs had the best radioprotection. Heilongjiang and Jilin-originated AGs had the similar pharmacological effects while USA, Shandong and Shaanxi-grown AGs had closer pharmacological effects. This biochemical measurements-based PCA and heatmap clustering of AGs from seven cultivation regions was nearly consistent with ginsencoside content- and the previous serum metabolome-based analyses. However, the pearson correlation analysis revealed that only Rb3 and Rd were significantly correlated with some of assayed biochemical parameters in irradiated mice pretreated with different cultivation regions of AG extracts.

A Widely Metabolomic Analysis Revealed Metabolic Alterations of Epimedium Pubescens Leaves at Different Growth Stages.

Epimedium folium is the major medicinally-used organ of Epimedium species and its metabolic changes during the leaf growth have not been studied at the metabolomic level. E. pubescens is one of five recorded species in the Pharmacopoeia of the People's Republic of China and widely grows in China. A UPLC-ESI-MS/MS-based targeted metabolomic analysis was implemented to explore the metabolite composition in E. pubescens leaves under the cultivation condition and further to investigate their temporal variations among four representative growth stages. A total of 403 metabolites, including 32 hitherto known in Epimedium species, were identified in E. pubescens leaf, of which 302 metabolites showed the growth/development-dependent alterations. Flavonoid-type compounds were the major composition of the metabolites identified in this study. Most flavonoids, together with tannin-type and lignans and coumarin-type compounds, were up-regulated with E. pubescens leaf growth and maturation after the full flowering stage. Our results not only greatly enriched the existing Epimedium phytochemical composition database and also, for the first time, provided the metabolomics-wide information on metabolic changes during E. pubescens leaf growth and development, which would facilitate in the choice of an optimum harvest time to balance a higher biomass yield of Epimedium folium with its better medicinal quality.

UPLC-Q/TOF-MS-Based Serum Metabolomics Reveals Hypoglycemic Effects of Rehmannia glutinosa, Coptis chinensis and Their Combination on High-Fat-Diet-Induced Diabetes in KK-Ay Mice.

Diabetes is a worldwide severe health issue which causes various complications. This study aimed to evaluate the hypoglycemic effects of Rehmannia glutinosa (RG), Coptis chinensis (CC) alone and their combination on high-fat-diet-induced diabetes in mice via biochemical assays and UPLC-Q/TOF-MS-based serum metabolomic analysis. Diabetic KK-Ay mice were induced by high-fat diet and treated for eight weeks, separately with RG, CC and their combination and the positive control drug metformin. Administration of RG and CC alone, and their combination could decrease the fasting blood glucose level, ameliorate the tolerance of glucose, and recover the levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in sera of diabetic mice. Orthogonal partial least squares discriminant analysis (OPLS-DA) on serum metabolomes revealed that 79 ESI⁺ and 76 ESI- metabolites were changed by diabetes mellitus (DM) compared to the normal control. Heatmaps on these diabetes-related metabolites showed that CC and RG/CC were clustered closer with the normal control, indicating that they had the better antidiabetic effects at the metabolite level. Fifteen of the differential metabolites in DM serum were annotated and their related metabolic pathways were lipid metabolism. These data suggested that RG and CC alone and in combination treatment had the antidiabetic activity in lowering glycemia and improving lipid metabolism. UPLC-Q/TOF-MS-based metabolomics shed light on the differential metabolite effects of RG and CC in DM treatment. However, it should be noted that some differential metabolites were possibly generated or not detected due to our groupwise run order, which possibly contributed to or covered the group difference in our experiment. They need to be further discriminated in the future work.

[Changes of transport sugar content in different organs of Rehmannia glutinosa].

Raffinose series oligosaccharides are the transport and storage sugars of many plants, Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, medicinal parts ist he roots. Root and tuber of R. glutinosa contains stachyose, raffinose and other oligosaccharides, but the study about the process of growth and development of other organs in the non-structural changes in sugar content is rare.In this study, leaves, stems and roots of R. glutinosa were used as materials to analyze the diurnal variation and the changes of sugar content of sucrose, raffinose and stachyose in different organs of R. glutinosa. The results showed that the content of sucrose in R. glutinosa leaves gradually increased from seedling stage.However, the content of stachyose did not change much at the early stage of growth, and the stachyose rapidly increased at the later stage of growth. The raffinose content gradually decreased throughout the growing season, young leaves of R. glutinosa have higher ability to sucrose synthesis than mature leaves, while mature leaf has higher raffinose and stachyose synthesis ability than young leaves. Sucrose and stachyose content in stem gradually increased, while there was little change in raffinose content. The content of raffinose and stachyose in root increased rapidly from the beginning of fast growing period, while the content of sucrose did not change much. The content of sucrose in leaves of R. glutinosa did not change much at day and night, while the daily changes of raffinose and stachyose contents were very obvious. The contents of raffinose and stachyose in daytime were higher than those at night. The content of raffinose in root and stem was not changed much, but the change of stachyose in root, stem and leaf was very obvious, especially in stem and leaf. In summary, the leaf is the main synthetic organ of raffinose, leaves, stems and roots are stachyose synthesis organ. Sucrose, raffinose and stachyose are the major transport forms of carbohydrates in R. glutinosa.

Comparison of Serum Metabolite Changes of Radiated Mice Administered with Panax quinquefolium from Different Cultivation Regions Using UPLC-Q/TOF-MS Based Metabolomic Approach.

Chemometric analysis of bioactive compounds revealed that American ginsengs (AGs) from different cultivation regions of China had a difference in quality, which indicates their possible pharmacological difference. A UPLC-Q/TOF-MS-based untargeted metabolomic approach was used to uncover serum metabolite changes in radiated mice pre-administered with AG root decoctions from seven cultivation regions and to further assess their quality difference. OPLS-DA revealed that 51 metabolites (ESI−) and 110 (ESI⁺) were differentially expressed in sera between the control and the radiated model mice. Heatmap analysis further revealed that AG could not reverse most of these radiation-altered metabolites, which indicates dietary supplement of AG before cobalt radiation had the weak potential to mediate serum metabolites that were altered by the sub-lethal high dose radiation. In addition, 83 (ESI−) and 244 (ESI⁺) AG altered metabolites were detected in radiated mice under radiation exposure. Both OPLS-DA on serum metabolomes and heatmap analysis on discriminant metabolites showed that AGs from different cultivation regions differentially influenced metabolic alterations in radiated mice, which indicates AGs from different cultivation regions showed the pharmacological difference in modulation of metabolite changes. AGs from Shandong, Shanxi, and Beijing provinces had more similar pharmacological effects than AGs from USA, Canada, Jilin, and Heilongjiang. Finally, 28 important potential biomarkers were annotated and assigned onto three metabolic pathways including lipid, amino acid, and energy metabolisms.

Simultaneous Determination of Catalpol, Aucubin, and Geniposidic Acid in Different Developmental Stages of Rehmannia glutinosa Leaves by High Performance Liquid Chromatography.

Although R. glutinosa roots are currently the only organ source in clinics, its leaves are a potential supplement for the roots especially in extraction of some important bioactive compounds. Our early work found that the contents of catalpol and total iridoid glycosides varied among different developmental stages of R. glutinosa leaves. Aucubin and geniposidic acid, the abundant major bioactive compounds in Eucommia ulmoides and Gardenia jasminoides, respectively, were found present in R. glutinosa roots, however, and have not been analyzed in its leaves. In this paper, we aimed to determine contents of these three iridoid glycosides in different developmental stages of R. glutinosa leaves using the optimized HPLC-UV conditions. Our results showed that aucubin and GPA in R. glutinosa leaves were much lower than catalpol and showed the increasing trend with the leaf development, which was different from catalpol. This work provided the important information for future exploitation of R. glutinosa leaves as a potential supplement for its roots in extraction of some important bioactive compounds and studying the relationship of aucubin and catalpol metabolism.

Determination of Saponin Content in Hang Maidong and Chuan Maidong via HPLC-ELSD Analysis.

Zhejiang and Sichuan are regarded as two genuine producing areas of Ophiopogonis radix in China. To study the difference in the quality of Ophiopogonis radix from these two places, the contents of three reported bioactive saponins, that is, ophiopogonins B, D, and D', in tubers and fibrous roots of Ophiopogon japonicus from Cixi city of Zhejiang and Santai county of Sichuan were quantified using HPLC-ELSD method and compared. Ophiopogonin B and ophiopogonin D' content in tubers of HMD were higher than those in radix of CMD, whereas ophiopogonin D in HMD was about twice lower than that of CMD. Three ophiopogonins were all detected in fibrous roots of both HMD and CMD. Their averaged content in fibrous roots of CMD was about twice higher than that in tubers of CMD. Ophiopogonin D in fibrous roots of CMD was about five times higher than that of HMD. Our biochemical assay revealed that fibrous roots and tubers of CMD can be of an important saponin source, especially for ophiopogonin D.

The Effects of Weather Factors on Hand, Foot and Mouth Disease in Beijing.

The morbidity and mortality of hand, foot and mouth disease (HFMD) are increasing in Beijing, China. Previous studies have indicated an association between incidents of HFMD and weather factors. However, the seasonal influence of these factors on the disease is not yet understood, and their relationship with the enterovirus 71 (EV71) and Coxsackie virus A16 (CV-A16) viruses are not well documented. We analysed 84,502 HFMD cases from 2008 to 2011 in Beijing to explore the seasonal influence of weather factors (average temperature [AT], average relative humidity [ARH], total precipitation [TP] and average wind speed [AWS]) on incidents of HFMD by using a geographically weighted regression (GWR) model. The results indicated that weather factors differ significantly in their influence on HFMD depending on the season. AT had the greatest effect among the four weather factors, and while the influence of AT and AWS was greater in the summer than in the winter, the influence of TP was positive in the summer and negative in the winter. ARH was negatively correlated with HFMD. Also, we observed more EV71-associated cases than CV-A16 but there is no convincing evidence to show significant differences between the influences of the weather factors on EV71 and CV-A16.

Identification and developmental expression profiling of putative alkaloid biosynthetic genes in Corydalis yanhusuo bulbs.

Alkaloids in bulbs of Corydalis (C.) yanhusuo are the major pharmacologically active compounds in treatment of blood vessel diseases, tumors and various pains. However, due to the absence of gene sequences in C. yanhusuo, the genes involved in alkaloid biosynthesis and their expression during bulb development remain unknown. We therefore established the first transcriptome database of C. yanhusuo via Illumina mRNA-Sequencing of a RNA composite sample collected at Bulb initiation (Day 0), early enlargement (Day 10) and maturation (Day 30). 25,013,630 clean 90 bp paired-end reads were de novo assembled into 47,081 unigenes with an average length of 489 bp, among which 30,868 unigenes (65.56%) were annotated in four protein databases. Of 526 putative unigenes involved in biosynthesis o f various alkaloids, 187 were identified as the candidate genes involved in the biosynthesis of benzylisoquinoline alkaloids (BIAs), the only alkaloid type reported in C. yanhusuo untill now. BIAs biosynthetic genes were highly upregulated in the overall pathway during bulb development. Identification of alkaloid biosynthetic genes in C. yanhusuo provide insights on pathways and molecular regulation of alkaloid biosynthesis, to initiate metabolic engineering in order to improve the yield of interesting alkaloids and to identify potentially new alkaloids predicted from the transcriptomic information.

[Influence of plant growth regulater on yield and quality of Salvia miltiorrhiza].

The study is aimed to investigate the effect of plant growth regulators on yield and quality of the Salvia miltiorrhiza. The plant growth regulators was spraying on Salvia plants in July or August in field experiment, then the yield, ingredient content and the antioxidant activity were determined. The results showed that plant growth regulator 'Zhuanggenling' could increase the yield of Salvia with root-planting by 38.45%. Plant growth regulator 'Duoxiaozuo' could increase the yield of Salvia with seedling planting by 14.19%. Both plant growth regulator significantly reduced the antioxidant activity of Salvia in vitro, but they had no significant effect on active ingredient contents.

[Study on distribution and dynamic accumulation of catalpol and total iridoid in fresh Rehmannia glutinosa].

Iridoid glycosides were the main active ingredient of Rehmannia glutinosa, of which catalpol has the highest content. This work will provide theoretical basis for metabolic study and cultivation of iridoids on the basis of the dynamic accumulation of catalpol and total iridoids in the growth of R. glutinosa. The samples of rehmannia 85-5 were gathered in the same filed from July to October. The contents of catalpol and total iridoid glycosides were measured by HPLC and specteophotometric, respectively. The results showed that youngest leaves had the higher content of catalpol and total iridoid glyosides than that of the other two leaf ages in the same growth stage from July to September, while their content of catalpol and total iridoid glycosides were all decreased as the growth of leaves of R. glutinosa. The content of catalpol didn't differ significantly from July to September, whereas it has significantly increased in October in the three leaf stage. In the same stage, the wider the root diameter is, the higher content of the effective components are. In August and September, the total iridoid glycosides have the fastest accumulation. The content of catalpol was increased as the accumulation of total iridoid glycosides.

Transcriptional data mining of Salvia miltiorrhiza in response to methyl jasmonate to examine the mechanism of bioactive compound biosynthesis and regulation.

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.

[Pharmacodynamic differences between hangmaidong and chuanmaidong based on metabonomics].

To evaluate the differences of Ophiopogonjaponicus from different cultivations, the metabolomics based method was conducted to compare the effects of Hangmaidong and Chuanmaidong (Chinese name) on plasma endogenous metabolites of normal rats. Data were collected by ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS), and were analyzed by multivariate statistical method, such as Principal Component Analysis and Orthogonal Signal Correction Partial Least Square Discriminant Analysis. Results revealed that the plasma metabolites profiling of low and middle dose group of Chuanmaidong were similar to the control group, but different from the high dose group obviously. Meanwhile the high, middle and low dose groups of Hangmaidong were different from control group notably, and the difference is dose dependent. Lysophosphatidylcholines, the possible endogenous metabolites which contribute to the classification most significantly, are closely related to cardiovascular system diseases. Compared with the group of Chuanmaidong, Hangmaidong has greater impact on the plasma metabolic profiling of normal rats. Hangmaidong and Chuanmaidong showed significant differences pharmacodynamically.

The complete chloroplast genome sequence of the medicinal plant Salvia miltiorrhiza.

Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.

[Growth and development of fruit and seed of Panax quinquefolium].

OBJECTIVE: To understand the embryo after-ripening phenomenon of Panax quinquefolium, the growth and development process of fruit and seed was investigated in this study. METHOD: The growth and development characteristics of fruit and seed were obtained by field observation, the morphological changes were measured with a vernier caliper, paraffin section was used as well. RESULT AND CONCLUSION: The plant reached the most flowering numbers in 5-8 d after initial blooming time and the lag phase of embryo occurred in about 70 d. The size, fresh and dry weight of fruit and seed were all reached maximum in fruit maturation period. As the result showed, the development of seed was stopped at torpedo form of embryos, this conclusion can be applied to explore the morphological after-ripening mechanism of P. quinquefolium seed.

[Effects of seedling quality on growth of Rehmannia glutinosa and oligosaccharide content].

OBJECTIVE: To study the effect of seedling quality on growth, yield and quality of Rehmannia glutinosa at harvest and build a basis for its GAP. METHOD: The seedling quality of R. glutinosa in main producing regions was surveyed to understand the current status of seedling quality. Field experiments with different varieties and seedling quality were conducted to measure dry matter accumulation with different growth of R. glutinosa and oligosaccharide content, and economic yield at harvest. RESULT: The seedling was randomly selected by farmers in R. glutinosa producing regions. Seedling quality could significantly improve on seedling emergence rate, and promote seedling growth, especially with early stage R. glutinosa, finally increase yield at harvest. At harvest, 63% and 50% of yield with A and B seedling could be improved for variety of 85-5, and 50% and 47% of yield could be increased for variety of Beijing No. 1, compared to the C seeding. CONCLUSION: In cultivation, the seedlings with the diameter > 1.5 cm should be transplanted firstly.

Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza.

Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering.