Triterpene Saponin Biosynthesis Research Articles

Investigation of the response of Platycodongrandiflorus (Jacq.) A. DC to salt stress using combined transcriptomics and metabolomics

BackgroundPlatycodon grandiflorus (Jacq.) A. DC is a famous traditional Chinese medicine in China and an authentic medicine in Inner Mongolia. It has been traditionally used as an expectorant in cough and also has anti-inflammatory and other pharmacological effects. As a homologous plant of medicine and food, P. grandiflorus is widely planted in Northeast China. Soil salinity isa limiting factor for its cultivation. In this study, we comprehensively described the physiological characteristics of P. grandiflorus and combined transcriptomics and metabolomics to study the response of roots of P. grandiflorus to salt stress.ResultsOverall, 8,988 differentially expressed genes were activated and significantly altered the metabolic processes. In total, 428 differentially abundant metabolites were affected by salt stress. After moderate and severe salt stress, most of the differentially abundant metabolites were enriched in the L-phenylalanine metabolic pathway. Through the comprehensive analysis of the interaction between key genes and metabolites, the main pathways such as lignin compound biosynthesis and triterpene saponin biosynthesis were completed. The relative content of compounds related to lignin biosynthesis, such as caffeic acid, coniferin, and syringing, increased under salt stress, and the related genes such as PAL, C4H, and the key enzyme gene UGT72E2 were activated to adapt to the salt stress. Platycodon saponin is one of the major triterpene saponins in P. grandiflorus, and Platycodin D is its most abundant major bioactive component. Under severe salt stress, Platycodin D level increased by nearly 1.77-fold compared with the control group. Most of the genes involved insynthetic pathway of Platycodin D, such as HMGCR, GGPS, SE, and LUP, were upregulated under salt stress.ConclusionSalt stress led to a decrease in the biomass and affected the activities of antioxidant enzymes and contents of osmotic regulators in the plant. These results provided not only novel insights into the underlying mechanisms of response of P. grandiflorus to salt stress but also a foundation for future studies on the function of genes related to salt tolerance in the triterpenoid saponin biosynthesis pathway.

Effect of Developmental Stages on the Biosynthesis of Triterpene Saponins and Related Gene Expression in Seedlings of Platycodon grandiflorus

Objective: To investigate the impact of developmental stages on triterpene saponin content and expression of key genes in their biosynthetic pathway in Platycodon grandiflorus seedlings. Method: The total saponins were extracted using ultrasonic extraction with methanol as the solvent. The quantification of Platycodin D in 6-month-old and 1-year-old Platycodon grandiflorus seedlings was performed using ultra-performance liquid chromatography tandem mass spectrometry, while the expression of key genes in the biosynthesis of saponins was determined by real-time quantitative polymerase chain reaction. Result: The content of Platycodin D and total saponins in 1-year-old Platycodon grandiflorus roots was significantly higher than that in of 6-month-old. The expression of key genes in saponin biosynthesis, such as PGMVK , PGMVD , PGDXR , PGMCT , PGMDS , PGHDS , PGHDR , PGFPS, and PGSS, was higher in 1-year-old roots compared to 6-month-old seedlings. Conclusion: The medicinal value of 1-year-old Platycodon grandiflorus root was higher than that of 6-month-old seedlings. The difference in medicinal value can be attributed to the variation in the expression of key genes involved in the saponin biosynthesis pathway.

A metabolic reprogramming in Bacopa monnieri plants induced by methyl-jasmonate and enhanced biosynthesis of triterpene saponins

The bacoside-A is the main bioactive metabolite present in Bacopa monnieri L. The yield of bacoside-A from plant sources is too less to meet industrial demand. This study investigated the effect of methyl jasmonate (MeJA) on metabolic profiles and bacoside-A biosynthesis in greenhouse-grown plants of B. monnieri. The greenhouse-grown plants were kept inside a closed plastic container and then elicited with MeJA vapour. It was found that MeJA treatment (200 µM for 2 h on day zero) followed by harvesting leaves on day three was the optimum treatment, resulting in an increase of 1.8-fold, 2.2-fold, 3.1-fold, and 2.5-fold in bacoside A3, bacopaside II, bacopaside X, and bacopasaponin C in comparison with control plants, respectively. The transcript levels of squalene synthase (SQS), a key gene involved in bacoside-A biosynthesis was transiently induced by MeJA and reached a peak of up to 3.7-fold at 24 h. The relationships between bacosides content and primary metabolites in plants treated with MeJA were analyzed by gas chromatography-mass spectrometry. MeJA enhances glycolysis and TCA cycles, generating acetyl-CoA, which acts as a precursor for bacosides synthesis, as revealed by untargeted metabolomics. Therefore, in planta application of MeJA appears to have a great impact on enhanced bacoside-A accumulation, in the greenhouse-grown plants of B. monnieri. In this study, farmers and the pharmaceutical industry will have access to a simple and time-saving elicitation strategy for improving bacosides production from greenhouse-grown plants without the need for expensive and time-consuming in vitro culture techniques.

Biosynthesis and Pharmacological Activities of Flavonoids, Triterpene Saponins and Polysaccharides Derived from Astragalus membranaceus.

Astragalus membranaceus (A. membranaceus), a well-known traditional herbal medicine, has been widely used in ailments for more than 2000 years. The main bioactive compounds including flavonoids, triterpene saponins and polysaccharides obtained from A. membranaceus have shown a wide range of biological activities and pharmacological effects. These bioactive compounds have a significant role in protecting the liver, immunomodulation, anticancer, antidiabetic, antiviral, antiinflammatory, antioxidant and anti-cardiovascular activities. The flavonoids are initially synthesized through the phenylpropanoid pathway, followed by catalysis with corresponding enzymes, while the triterpenoid saponins, especially astragalosides, are synthesized through the universal upstream pathways of mevalonate (MVA) and methylerythritol phosphate (MEP), and the downstream pathway of triterpenoid skeleton formation and modification. Moreover, the Astragalus polysaccharide (APS) possesses multiple pharmacological activities. In this review, we comprehensively discussed the biosynthesis pathway of flavonoids and triterpenoid saponins, and the structural features of polysaccharides in A. membranaceus. We further systematically summarized the pharmacological effects of bioactive ingredients in A. membranaceus, which laid the foundation for the development of clinical candidate agents. Finally, we proposed potential strategies of heterologous biosynthesis to improve the industrialized production and sustainable supply of natural products with pharmacological activities from A. membranaceus, thereby providing an important guide for their future development trend.

Interference between ER stress-related bZIP-type and jasmonate-inducible bHLH-type transcription factors in the regulation of triterpene saponin biosynthesis in Medicago truncatula.

Triterpene saponins (TS) are a structurally diverse group of metabolites that are widely distributed in plants. They primarily serve as defense compounds and their production is often triggered by biotic stresses through signaling cascades that are modulated by phytohormones such as the jasmonates (JA). Two JA-modulated basic helix-loop-helix (bHLH) transcription factors (TFs), triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2, have previously been identified as direct activators of TS biosynthesis in the model legume Medicago truncatula. Here, we report on the involvement of the core endoplasmic reticulum (ER) stress-related basic leucine zipper (bZIP) TFs bZIP17 and bZIP60 in the regulation of TS biosynthesis. Expression and processing of M. truncatula bZIP17 and bZIP60 proteins were altered in roots with perturbed TS biosynthesis or treated with JA. Accordingly, such roots displayed an altered ER network structure. M. truncatula bZIP17 and bZIP60 proteins were shown to localize in the nucleus and appeared to be capable of interfering with the TSAR-mediated transactivation of TS biosynthesis genes. Furthermore, interference between ER stress-related bZIP and JA-modulated bHLH TFs in the regulation of JA-dependent terpene biosynthetic pathways may be widespread in the plant kingdom, as we demonstrate that it also occurs in the regulation of monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

Comparative transcriptomic analysis of genes in the triterpene saponin biosynthesis pathway in leaves and roots of Ardisia kteniophylla A. DC., a plant used in traditional Chinese medicine.

Ardisia kteniophylla (Primulaceae) is highly valued in traditional medicine due to its production of the pharmacologically active secondary metabolites, especially triterpenoid saponins in its roots. Although A. kteniophylla is very important in traditional medicine, the genetic basis for its production of triterpenoid saponins remains largely unknown. Therefore, we sequenced transcriptomes of A. kteniophylla to identify putative genes involved in production of triterpenoid saponins in both leaves and roots, and we used the transcriptomes to compare expression levels of these genes between the two organ systems. The production of triterpenoid saponins in plants is usually induced through hormonal signaling on account of the presence of pests. Thus, we treated plants with the hormones salicylic acid (SA) and methyl jasmonate (MeJA) and used quantitative real‐time PCR (qRT‐PCR) to investigate expression levels of genes involved in triterpenoid saponin biosynthesis. In total, we obtained transcriptomes for leaf and root tissues representing 52,454 unigenes. Compared with the leaf transcriptome, we found that 6092 unigenes were upregulated in the root, especially enzymes involved in the direct synthesis of triterpenoid saponins, while 6001 genes appeared downregulated, including those involved in precursory steps in the triterpenoid saponin biosynthesis pathway. Our results from qRT‐PCR indicate that genes within the upstream parts of the triterpenoid saponin biosynthesis pathway may be upregulated under exposure to the applied hormones, but downstream genes are downregulated. This suggests possible conflicting effects of SA and MeJA in promoting the production of secondary metabolites on the one hand, and, on the other, limiting plant growth processes to devote energy to combating pests. We also performed an analysis of transcription factors (TFs) and found 997 unique transcripts belonging to 16 TF families. Our data may help to facilitate future work on triterpene saponins biosynthesis in A. kteniophylla with potential pharmacological and molecular breeding applications.

Comparative Transcriptome and Phytochemical Analysis Provides Insight into Triterpene Saponin Biosynthesis in Seeds and Flowers of the Tea Plant (Camellia sinensis).

Triterpene saponins exhibit various biological and pharmacological activities. However, the knowledge on saponin biosynthesis in tea plants (Camellia sinensis L.) is still limited. In this work, tea flower and seed samples at different developmental stages and leaves were collected and analyzed with UPLC-PDA-MS and RNA sequencing for saponin determination and transcriptome comparison. The saponin content reached around 19% in the freshly mature seeds and 7% in the green flower buds, and decreased with the fruit ripeness and flower blooming. Almost no saponins were detected in leaf samples. PCA and KEGG analysis suggested that the gene expression pattern and secondary metabolism in TF1 and TS2 vs. leaf samples were significantly different. Weighted gene coexpression network analysis (WGCNA) uncovered two modules related to saponin content. The mevalonate (MVA) instead of 2-C-methyl-d-erythritol-4-phospate (MEP) pathway was responsible for saponin accumulation in tea plants, and 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), diphosphomevalonate decarboxylase (MVD) and isopentenyl diphosphate isomerase (IDI) may be the key enzymes involved in saponin biosynthesis in tea seeds and flowers. Moreover, ten transcription factors (TFs) were predicted to regulate saponin biosynthesis in the tea plant. Taken together, our study provides a global insight into the saponin biosynthesis and accumulation in the tea plant.

Content changes of triterpene saponins in crude and sweated Dipsacus asper:an iTRAQ-based analysis

The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D. asper, and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ) combined with LC-MS/MS. After sweating, the content of total triterpene saponins decreased manifestly, while that of asperosaponin Ⅵ increased significantly. As revealed by the iTRAQ-LC-MS/MS analysis, 140 proteins with significant differential expression were figured out, with 50 up-regulated and 90 down-regulated. GO analysis indicated a variety of hydrolases, oxido-reductases, and transferases in the differential proteins. The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis. As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway, two enzymes(from CYP450 and UGT families, respectively, which are involved in the structural modification of triterpene saponins) were significantly down-regulated after sweating. The findings suggested that sweating of D. asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.

Constitutive activation of genes involved in triterpene saponins enhances the accumulation of saponins in three-year-old Panax notoginseng growing under moderate light intensity

Light is an important physical factor that regulates plant developmental patterns and induces the synthesis of secondary metabolites. The optimal light intensity for saponin accumulation in roots of medicinal plants with different ages is relatively poorly understood. After that, the correlation of light-driven biosynthesis of triterpene saponins with the expression of genes incorporated into the biosynthesis of triterpene saponins is still unknown. The expression levels of genes, the total saponin contents, and the biomass accumulation and allocation were examined in different age Panax notoginseng grown under different light intensities. A significant elevation in total saponin content was found in three-year-old plants grown under 9.6 % of full sunlight (FL) condition compared with the others. The expression level of dammarenediol-II synthase (DS) rapidly increased from one-year-old plants to three-year-old plants, which was consistent with the change in total saponins. Furthermore, there is a positive relationship between the expression level of DS and the content of saponins. Additionally, 9.6 % FL-driven biosynthesis of saponin has more activity in the three-year-old P. notoginseng, and the genes expression level of CYP716A47 was elevated. At the same time, the expression of geranylgeranyl pyrophosphate synthase (GGPS) and phosphomevalonate kinase (PMK) had no positive contribution to the accumulation of saponins in roots. Overall, DS and CYP716A47 might attribute to the accumulation of saponins in three-year-old P. notoginseng under a moderate light regime (about 10 % FL).

A hairy-root transformation protocol for Trigonella foenum-graecum L. as a tool for metabolic engineering and specialised metabolite pathway elucidation

The development of genetic transformation methods is critical for enabling the thorough characterization of an organism and is a key step in exploiting any species as a platform for synthetic biology and metabolic engineering approaches. In this work we describe the development of an Agrobacterium rhizogenes-mediated hairy root transformation protocol for the crop and medicinal legume fenugreek (Trigonella foenum-graecum). Fenugreek has a rich and diverse content in bioactive specialised metabolites, notably diosgenin, which is a common precursor for synthetic human hormone production. This makes fenugreek a prime target for identification and engineering of specific biosynthetic pathways for the production of triterpene and steroidal saponins, phenolics, and galactomanans. Through this transformation protocol, we identified a suitable promoter for robust transgene expression in fenugreek. Finally, we establish the proof of principle for the utility of the fenugreek system for metabolic engineering programs, by heterologous expression of known triterpene saponin biosynthesis regulators from the related legume Medicago truncatula in fenugreek hairy roots.

Comparative transcriptome analysis of rhizome nodes and internodes in Panax. japonicus var. major reveals candidate genes involved in the biosynthesis of triterpenoid saponins

The rhizome of P. japonicus var. major, one of the important herbs in Traditional Chinese medicine (TCM), has been used as tonic and hemostatic drugs in Tujia and Miao ethnic groups of China for thousand years. In this study, comparative metabolite and transcriptome analysis of rhizome nodes and internodes of wild P. japonicus var. major was performed to reveal their different roles in the biosynthesis of triterpene saponins. The results showed that the node was the crucial section for the synthesis of ginsenosides in the rhizome. The content of oleanane-type ginsenosides in the node was much higher than those in the internode. Most isoprenoid biosynthesis-related genes were highly expressed in the node. And, candidate UDP-glycosyltransferase (UGT) genes were also found to be differentially expressed between node and internode. Our study will provide a better understanding of the metabolism of ginsenosides in the rhizome of P. japonicus var. major.

Comprehensive metabolic and transcriptomic profiling of various tissues provide insights for saponin biosynthesis in the medicinally important Asparagus racemosus

Asparagus racemosus (Shatavari), belongs to the family Asparagaceae and is known as a “curer of hundred diseases” since ancient time. This plant has been exploited as a food supplement to enhance immune system and regarded as a highly valued medicinal plant in Ayurvedic medicine system for the treatment of various ailments such as gastric ulcers, dyspepsia, cardiovascular diseases, neurodegenerative diseases, cancer, as a galactogogue and against several other diseases. In depth metabolic fingerprinting of various parts of the plant led to the identification of 13 monoterpenoids exclusively present in roots. LC-MS profiling led to the identification of a significant number of steroidal saponins (33). However, we have also identified 16 triterpene saponins for the first time in A. racemosus. In order to understand the molecular basis of biosynthesis of major components, transcriptome sequencing from three different tissues (root, leaf and fruit) was carried out. Functional annotation of A. racemosus transcriptome resulted in the identification of 153 transcripts involved in steroidal saponin biosynthesis, 45 transcripts in triterpene saponin biosynthesis, 44 transcripts in monoterpenoid biosynthesis and 79 transcripts in flavonoid biosynthesis. These findings will pave the way for better understanding of the molecular basis of steroidal saponin, triterpene saponin, monoterpenoids and flavonoid biosynthesis in A. racemosus.

Cloning and analyzing a squalene synthase gene in Pseudostellaria heterophylla

Triterpene saponins are primary bioactive compounds in the tuberous roots of Pseudostellaria heterophylla, which is one of well-known traditional Chinese medicines. Squalene synthase (SQS) is the key speed-limiting enzyme of saponin biosynthesis pathway. Here, we cloned and analyzed a SQS gene in P. heterophylla. The full length cDNA of SQS was cloned and designated PhSQS (GenBank accession no. KY436585). The open reading frame (ORF) of PhSQS contains 1245 bp and encodes a putative protein of 414 amino acids. In-silico analysis of amino acid sequence of PhSQS showed a conserved isoprenoid family domain and catalytic sites and the three-dimensional (3D) structure of PhSQS was not deviated from that of the SQSs from other species. The phylogenetic relationship of PhSQS was more closely related to that from Beta vulgaris. The semi-quantitative RT-PCR revealed the expression level of PhSQS was significantly higher in root tissue than that of other tissues. Our results suggest that PhSQS is a candidate of functional gene of squalene synthase and may be involved in triterpene saponin biosynthesis.

Functional characterization of the β-amyrin synthase gene involved in platycoside biosynthesis in Platycodon grandiflorum

Platycodon grandiflorum A. DC. contains a variety of triterpene saponins induced from the base structure of oleanane-type in roots. The β-amyrin synthase (bAS) plays an important role in controlling the biosynthesis of triterpene saponins and we obtained the full-length cDNA of the gene, which is named PlgOSC1 in P. grandiflorum. To identify the PlgOSC1 products, we expressed it in heterologous yeast cells. Results of GC-MS analysis showed a unique peak that was consistent with that of the authentic β-amyrin standard with exactly the same retention time. Furthermore, the pattern of MS fragments of the peak was also exactly the same as that of the β-amyrin. In qRT-PCR analysis of four organs of P. grandiflorum, no difference in PlgOSC1 expression level was observed; therefore, platycosides derived from β-amyrin might be present in all P. grandiflorum tissues. In conclusion, PlgOSC1 encodes a bAS enzyme that catalyzes the first committed step of platycosides biosynthesis in P. grandiflorum.

Two Cycloartenol Synthases for Phytosterol Biosynthesis in Polygala tenuifolia Willd.

Oxidosqualene cyclases (OSCs) are enzymes that play a key role in control of the biosynthesis of phytosterols and triterpene saponins. In order to uncover OSC genes from Polygala tenuifolia seedlings induced by methyl jasmonate (MeJA), RNA-sequencing analysis was performed using the Illumina sequencing platform. A total of 148,488,632 high-quality reads from two samples (control and the MeJA treated) were generated. We screened genes related to phytosterol and triterpene saponin biosynthesis and analyzed the transcriptional changes of differentially expressed unigene (DEUG) values calculated by fragments per kilobase million (FPKM). In our datasets, two full-length cDNAs of putative OSC genes, PtCAS1, and PtCAS2, were found, in addition to the PtBS (β-amyrin synthase) gene reported in our previous studies and the two cycloartenol synthase genes of P. tenuifolia. All genes were isolated and characterized in yeast cells. The functional expression of the two PtCAS genes in yeast cells showed that the genes all produce a cycloartenol as the sole product. When qRT-PCR analysis from different tissues was performed, the expressions of PtCAS1 and PtCAS2 were highest in flowers and roots, respectively. After MeJA treatment, the transcripts of PtCAS1 and PtCAS2 genes increased by 1.5- and 2-fold, respectively. Given these results, we discuss the potential roles of the two PtCAS genes in relation to triterpenoid biosynthesis.

From Plant Extract to a cDNA Encoding a Glucosyltransferase Candidate: Proteomics and Transcriptomics as Tools to Help Elucidate Saponin Biosynthesis in Centella asiatica.

Centella asiatica (L.) Urban (Apiaceae), a small annual plant that grows in India, Sri Lanka, Malaysia, and other parts of Asia, is well-known as a medicinal herb with a long history of therapeutic uses. The bioactive compounds present in C. asiatica leaves include ursane-type triterpene sapogenins and saponins-asiatic acid, madecassic acid, asiaticoside, and madecassoside. Various bioactivities have been shown for these compounds, although most of the steps in the biosynthesis of triterpene saponins, including glycosylation, remain uncharacterized at the molecular level. This chapter describes an approach that integrates partial enzyme purification, proteomics methods, and transcriptomics, with the aim of reducing the number of cDNA candidates encoding for a glucosyltransferase involved in saponin biosynthesis and facilitating the elucidation of the pathway in this medicinal plant.

The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula.

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula.

Global transcriptome analysis profiles metabolic pathways in traditional herb Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao.

BackgroundAstragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao (A. mongolicus, family Leguminosae) is one of the most important traditional Chinese herbs. Among many secondary metabolites it produces, the effective bioactive constituents include isoflavonoids and triterpene saponins. The genomic resources regarding the biosynthesis of these metabolites in A. mongolicus are limited. Although roots are the primary material harvested for medical use, the biosynthesis of the bioactive compounds and its regulation in A. mongolicus are not well understood. Therefore, a global transcriptome analysis on A. mongolicus tissues was performed to identify the genes essential for the metabolism and to profile their expression patterns in greater details.ResultsRNA-sequencing was performed for three different A. mongolicus tissues: leaf, stem, and root, using the Illumina Hiseq2000 platform. A total of 159.5 million raw sequence reads were generated, and assembled into 186,324 unigenes with an N50 of 1,524bp. Among them, 129,966 unigenes (~69.7%) were annotated using four public databases (Swiss-Prot, TrEMBL, CDD, Pfam), and 90,202, 63,946, and 78,326 unigenes were found to express in leaves, roots, and stems, respectively. A total of 8,025 transcription factors (TFs) were identified, in which the four largest families, bHLH, MYB, C3H, and WRKY, were implicated in regulation of tissue development, metabolisms, stress response, etc. Unigenes associated with secondary metabolism, especially those with isolavonoids and triterpene saponins biosynthesis were characterized and profiled. Most genes involved in the isoflavonoids biosynthesis had the lowest expression in the leaves, and the highest in the stems. For triterpene saponin biosynthesis, we found the genes in MVA and non-MVA pathways were differentially expressed among three examined tissues, indicating the parallel but compartmentally separated biosynthesis pathways of IPP and DMAPP in A. mongolicus. The first committed enzyme in triterpene saponin biosynthesis from A. mongolicus, cycloartenol synthase (AmCAS), which belongs to the oxidosqualene cyclase family, was cloned by us to study the astragalosides biosynthesis. Further co-expression analysis indicated the candidate CYP450s and glycosyltransferases (GTs) in the cascade of triterpene saponins biosynthesis. The presence of the large CYP450 families in A. mongolicus was further compared with those from Medicago truncatula and Arabidopsis thaliana, and the diversity and phylegenetic relationships of the CYP450 families were established.ConclusionA transcriptome study was performed for A. mongolicus tissues to construct and profile their metabolic pathways, especially for the important bioactive molecules. The results revealed a comprehensive profile for metabolic activities among tissues, pointing to the equal importance of leaf, stem, and root in A. mongolicus for the production of bioactive compounds. This work provides valuable resources for bioengineering and in vitro synthesis of the natural compounds for medical research and for potential drug development.

Transcriptome analysis of leaves, roots and flowers of Panax notoginseng identifies genes involved in ginsenoside and alkaloid biosynthesis.

BackgroundPanax notoginseng (Burk.) F.H. Chen is one of the most highly valued medicinal plants in the world. The major bioactive molecules are triterpene saponins, which are also known as ginsenosides. However, its large genome size has hindered the assembly of a draft genome by whole genome sequencing. Hence, genomic and transcriptomic details about P. notoginseng, especially its biosynthetic pathways and gene expression in different parts of the plant, have remained largely unknown until now.ResultsIn this study, RNA sequencing of three different P. notoginseng tissues was performed using next generation DNA sequencing. After assembling the high quality sequencing reads into 107,340 unigenes, biochemical pathways were predicted and 9,908 unigenes were assigned to 135 KEGG pathways. Among them, 270 unigenes were identified to be involved in triterpene saponin biosynthesis. In addition, 350 and 342 unigenes were predicted to encode cytochrome P450s and glycosyltransferases, respectively, based on the annotation results, some of which encode enzymes responsible for the conversion of the triterpene saponin backbone into different ginsenosides. In particular, one unigene predominately expressed in the root was annotated as CYP716A53v2, which probably participates in the formation of protopanaxatriol from protopanaxadiol in P. notoginseng. The differential expression of this gene was further confirmed by real-time PCR.ConclusionsWe have established a global transcriptome dataset for P. notoginseng and provided additional genetic information for further genome-wide research and analyses. Candidate genes involved in ginsenoside biosynthesis, including putative cytochrome P450s and glycosyltransferases were obtained. The transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of P. notoginseng.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1477-5) contains supplementary material, which is available to authorized users.

Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii.

Mogrosides, the major bioactive components isolated from the fruits of Siraitia grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins that are used worldwide as high-potency sweeteners and possess a variety of notable pharmacological activities. Mogrosides are synthesized from 2,3-oxidosqualene via a series of reactions catalyzed by cucurbitadienol synthase (CbQ), Cyt P450s (P450s) and UDP glycosyltransferases (UGTs) in vivo. However, the relevant genes have not been characterized to date. In this study, we report successful identification of SgCbQ and UGT74AC1, which were previously predicted via RNA-sequencing (RNA-seq) and digital gene expression (DGE) profile analysis of the fruits of S. grosvenorii. SgCbQ was functionally characterized by expression in the lanosterol synthase-deficient yeast strain GIL77 and was found to accumulate cucurbitadienol as the sole product. UGT74AC1 was heterologously expressed in Escherichia coli as a His-tag protein and it showed specificity for mogrol by transfer of a glucose moiety to the C-3 hydroxyl to form mogroside IE by in vitro enzymatic activity assays. This study reports the identification of CbQ and glycosyltransferase from S. grosvenorii for the first time. The results also suggest that RNA-seq, combined with DGE profile analysis, is a promising approach for discovery of candidate genes involved in biosynthesis of triterpene saponins.