Whole-genome, transcriptome, and methylome analyses provide insights into the evolution of platycoside biosynthesis in Platycodon grandiflorus, a medicinal plant.

Triterpenoid saponins (TSs) are common plant defense phytochemicals with potential pharmaceutical properties. Platycodon grandiflorus (Campanulaceae) has been traditionally used to treat bronchitis and asthma in East Asia. The oleanane-type TSs, platycosides, are a major component of the P. grandiflorus root extract. Recent studies show that platycosides exhibit anti-inflammatory, antiobesity, anticancer, antiviral, and antiallergy properties. However, the evolutionary history of platycoside biosynthesis genes remains unknown. In this study, we sequenced the genome of P. grandiflorus and investigated the genes involved in platycoside biosynthesis. The draft genome of P. grandiflorus is 680.1 Mb long and contains 40,017 protein-coding genes. Genomic analysis revealed that the CYP716 family genes play a major role in platycoside oxidation. The CYP716 gene family of P. grandiflorus was much larger than that of other Asterid species. Orthologous gene annotation also revealed the expansion of ß-amyrin synthases (bASs) in P. grandiflorus, which was confirmed by tissue-specific gene expression. In these expanded gene families, we identified key genes showing preferential expression in roots and association with platycoside biosynthesis. In addition, whole-genome bisulfite sequencing showed that CYP716 and bAS genes are hypomethylated in P. grandiflorus, suggesting that epigenetic modification of these two gene families affects platycoside biosynthesis. Thus whole-genome, transcriptome, and methylome data of P. grandiflorus provide novel insights into the regulation of platycoside biosynthesis by CYP716 and bAS gene families.

A Novel Multifunctional C-23 Oxidase, CYP714E19, is Involved in Asiaticoside Biosynthesis.

Centella asiatica is widely used as a medicinal plant due to accumulation of the ursane-type triterpene saponins asiaticoside and madecassoside. The molecular structure of both compounds suggests that they are biosynthesized from α-amyrin via three hydroxylations, and the respective Cyt P450-dependent monooxygenases (P450 enzymes) oxidizing the C-28 and C-2α positions have been reported. However, a third enzyme hydroxylating C-23 remained elusive. We previously identified 40,064 unique sequences in the transcriptome of C. asiatica elicited by methyl jasmonate, and among them we have now found 149 unigenes encoding putative P450 enzymes. In this set, 23 full-length cDNAs were recognized, 13 of which belonged to P450 subfamilies previously implicated in secondary metabolism. Four of these genes were highly expressed in response to jasmonate treatment, especially in leaves, in accordance with the accumulation patterns of asiaticoside. The functions of these candidate genes were tested using heterologous expression in yeast cells. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that yeast expressing only the oxidosqualene synthase CaDDS produced the asiaticoside precursor α-amyrin (along with its isomer ß-amyrin), while yeast co-expressing CaDDS and CYP716A83 also contained ursolic acid along with oleanolic acid. This P450 enzyme thus acts as a multifunctional triterpenoid C-28 oxidase converting amyrins into corresponding triterpenoid acids. Finally, yeast strains co-expressing CaDDS, CYP716A83 and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, showing that CYP714E19 is a multifunctional triterpenoid oxidase catalyzing the C-23 hydroxylation of oleanolic acid and ursolic acid. Overall, our results demonstrate that CaDDS, CYP716A83 and CYP714E19 are C. asiatica enzymes catalyzing consecutive steps in asiaticoside biosynthesis.

Characterization of the Asiatic Acid Glucosyltransferase, UGT73AH1, Involved in Asiaticoside Biosynthesis in Centella asiatica (L.) Urban.

Centella asiatica (L.) Urban contains two ursane-type triterpene saponins, asiaticoside and madecassoside, as major secondary metabolites. In order to select candidate genes encoding UDP-glucosyltransferases (UGTs) involved in asiaticoside biosynthesis, we performed transcriptomic analysis of leaves elicited by methyl jasmonate (MeJA). Among the unigenes, 120 isotigs and 13 singletons of unique sequences were annotated as UGTs, including 37 putative full-length cDNAs, and 15 of the putative UGT genes were named according to the UGT committee nomenclature protocols. One of them, UGT73AH1, was characterized by heterologous expression in Escherichia coli BL21 (DE3) cells. After induction with IPTG, a total protein extract was assayed with UDP-glucose and asiatic acid. UPLC-QTOF/MS analysis showed that UGT73AH1 catalyzes the glycosylation of asiatic acid to its monoglucoside. It remains unclear whether glycosylation occurs on the triterpene C-2α, C-3ß, C-23, or C-28 position. However, it is very likely that UGT73AH1 glucosylates the C-28 position, because only C-28 bears a glucose moiety in the final pathway product of asiatic acid, while C-2α, C-3ß, and C-23 remain un-conjugated.

Development of Genome-Wide SSR Markers from Angelica gigas Nakai Using Next Generation Sequencing.

Angelica gigas Nakai is an important medicinal herb, widely utilized in Asian countries especially in Korea, Japan, and China. Although it is a vital medicinal herb, the lack of sequencing data and efficient molecular markers has limited the application of a genetic approach for horticultural improvements. Simple sequence repeats (SSRs) are universally accepted molecular markers for population structure study. In this study, we found over 130,000 SSRs, ranging from di- to deca-nucleotide motifs, using the genome sequence of Manchu variety (MV) of A. gigas, derived from next generation sequencing (NGS). From the putative SSR regions identified, a total of 16,496 primer sets were successfully designed. Among them, we selected 848 SSR markers that showed polymorphism from in silico analysis and contained tri- to hexa-nucleotide motifs. We tested 36 SSR primer sets for polymorphism in 16 A. gigas accessions. The average polymorphism information content (PIC) was 0.69; the average observed heterozygosity (HO) values, and the expected heterozygosity (HE) values were 0.53 and 0.73, respectively. These newly developed SSR markers would be useful tools for molecular genetics, genotype identification, genetic mapping, molecular breeding, and studying species relationships of the Angelica genus.

Global Profiling of Various Metabolites in Platycodon grandiflorum by UPLC-QTOF/MS.

In this study, a method of metabolite profiling based on UPLC-QTOF/MS was developed to analyze Platycodon grandiflorum. In the optimal UPLC, various metabolites, including major platycosides, were separated well in 15 min. The metabolite extraction protocols were also optimized by selecting a solvent for use in the study, the ratio of solvent to sample and sonication time. This method was used to profile two different parts of P. grandiflorum, i.e., the roots of P. grandiflorum (PR) and the stems and leaves of P. grandiflorum (PS), in the positive and negative ion modes. As a result, PR and PS showed qualitatively and quantitatively different metabolite profiles. Furthermore, their metabolite compositions differed according to individual plant samples. These results indicate that the UPLC-QTOF/MS-based profiling method is a good tool to analyze various metabolites in P. grandiflorum. This metabolomics approach can also be applied to evaluate the overall quality of P. grandiflorum, as well as to discriminate the cultivars for the medicinal plant industry.

Isolation and characterization of an oxidosqualene cyclase gene encoding a ß-amyrin synthase involved in Polygala tenuifolia Willd. saponin biosynthesis.

KEY MESSAGE: Expression of PtBS (Polygala tenuifolia ß-amyrin synthase) led to the production of ß-amyrin as sole product. ABSTRACT: Polygala tenuifolia Willdenow is a rich source of triterpene saponins, onjisaponins and polygalasaponins, used as herbal medicine to treat phlegms and for detumescence in traditional Asian healing. The Polygala saponins share the oleanane backbone structure and are, therefore, likely synthesized via ß-amyrin as a common precursor. We hypothesized that, in analogy to diverse other plant species, this central intermediate should be formed by a ß-amyrin synthase catalyzing the complex cyclization of oxidosqualene. This member of the oxidosqualene cyclase (OSC) family of enzymes is thus defining an important branch point between primary and secondary metabolisms, and playing a crucial role in the control of oleanane-type triterpene saponin biosynthesis. From P. tenuifolia roots, we isolated an OSC cDNA containing a reading frame of 2,289 bp nucleotides. The predicted protein of 763 amino acids (molecular weight 87.353 kDa) showed particularly high amino acid sequence identities to known ß-amyrin synthases (85-87 %) and was, therefore, named PtBS. Expression of PtBS in the triterpenoid synthase-deficient yeast mutant GIL77 led to the production of ß-amyrin as sole product. qRT-PCR analysis of various P. tenuifolia organs showed that PtBS transcript levels were highest in the roots, consistent with onjisaponin accumulation patterns. Therefore, we conclude that PtBS is the ß-amyrin synthase enzyme catalyzing the first committed step in the biosynthesis of onjisaponins and polygalasaponins in P. tenuifolia.

Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene.

Paclitaxel is produced by various species of yew trees and has been extensively used to treat tumors. In our research, a taxadiene synthase (TS) gene from Taxus brevifolia was used to transform the roots of cultured ginseng (Panax ginseng C.A. Meyer) to produce taxadiene, the unique skeletal precursor to taxol. The TS gene was successfully introduced into the ginseng genome, and the de novo formation of taxadiene was identified by mass spectroscopy profiling. Without any change in phenotypes or growth difference in a TS-transgenic ginseng line, the transgenic TSS3-2 line accumulated 9.1 µg taxadiene per gram of dry weight. In response to the treatment of methyl jasmonate for 3 or 6 days, the accumulation was 14.6 and 15.9 µg per g of dry weight, respectively. This is the first report of the production of taxadiene by engineering ginseng roots with a taxadiene synthase gene.

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer).

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.

Upregulation of phytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPS) plays an essential role in organ development in plants. However, FPS has not previously been identified as a key regulatory enzyme in triterpene biosynthesis. To elucidate the functions of FPS in triterpene biosynthesis, C. asiatica was transformed with a construct harboring Panax ginseng FPS (PgFPS)-encoding cDNA coupled to the cauliflower mosaic virus 35S promoter. Higher levels of CaDDS (C. asiatica dammarenediol synthase) and CaCYS (C. asiatica cycloartenol synthase) mRNA were detected in all hairy root lines overexpressing when compared with the controls. However, no differences were detected in any expression of the CaSQS (C. asiatica squalene synthase) gene. In particular, the upregulation of CaDDS transcripts suggests that FPS may result in alterations in triterpene biosynthesis capacity. Squalene contents in the T17, T24, and T27 lines were increased to 1.1-, 1.3- and 1.5-fold those in the controls, respectively. The total sterol contents in the T24 line were approximately three times higher than those of the controls. Therefore, these results indicated that FPS performs a regulatory function in phytosterol biosynthesis. To evaluate the contribution of FPS to triterpene biosynthesis, we applied methyl jasmonate as an elicitor of hairy roots expressing PgFPS. The results of HPLC analysis revealed that the content of madecassoside and asiaticoside in the T24 line was transiently increased by 1.15-fold after 14 days of MJ treatment. This result may indicate that FPS performs a role not only in phytosterol regulation, but also in triterpene biosynthesis.

Characterization of a dammarenediol synthase in Centella asiatica (L.) Urban.

To elucidate the exact function of CabAS in Centella asiatica, which was previously reported as a putative beta-amyrin synthase [Plant Cell Rep, 24:304-311, 2005], this gene was functionally expressed in the lanosterol synthase-deficient yeast mutant (erg7). After inducing the CabAS gene with galactose, a peak consistent with the dammarenediol standard was detected in LC/APCIMS analyses and the accumulated product was confirmed as dammarenediol. CabAS should therefore be renamed to C. asiatica dammarenediol synthase (CaDDS). The confirmation of this gene function may allow us to better understand the generation of numerous triterpene carbon skeletons.

Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate.

Transformed root ("hairy root") cultures have been shown to be a good model for the study of many secondary metabolites. However, economically important compounds such as asiaticoside and madecassoside are produced in insignificant amounts in the root of Centella asiatica (L.) Urban. To overcome this problem, C. asiatica was transformed using Agrobacterium rhizogenes strain R1000 that harbors pCAMBIA1302 encoding the hygromycin phosphotransferase (hpt) and green fluorescence protein (mgfp5) genes and the hairy culture was coupled with elicitation technique. Hairy roots were obtained at a frequency of up to 14.1% from a tissue junction between the leaf and petiole. Abundant hairy roots were observed when co-cultivation of the plant with A. rhizogenes was done for 7 days (36.1%). Transformation was confirmed by PCR and Southern blot analyses. Five weeks after inoculation, no asiaticoside was detected in the hairy root samples. However, when 0.1 mM methyl jasmonate (MJ) was applied as an elicitor to the culture medium for 3 weeks, a large quantity of asiaticoside was generated (7.12 mg/g, dry wt). In the case of gene expression, 12 h after MJ treatment the expression of the CabAS (C. asiatica putative beta-amyrin synthase) gene in the hairy roots is significantly different from that of the control and this level of transcripts was maintained for 14 days. Our results showed that production of C. asiatica hairy roots could be optimized and the resulting cultures could be elicited with MJ treatment for enhanced production of asiaticoside.

Fingerprinting analysis of fresh ginseng roots of different ages using 1H-NMR spectroscopy and principal components analysis.

Fingerprinting analysis of fresh ginseng according to root age was performed using 1H-NMR spectroscopy and multivariate analysis techniques. Various peaks were detected in the aliphatic (0-3 ppm), sugar (3-6 ppm), and aromatic (6-9 ppm) regions of the 1H-NMR spectra of the water extracts of fresh ginseng root. The use of principal components (PCs) analysis (PCA) for metabolomic profiling allowed the large 1H-NMR data set obtained for various metabolites to be reduced to PC1, PC2, and PC3. Two dimensional score plots showed clear separations with these three components at different roots ages, and explained 89.6% of the total variance. Canonical discriminant analysis identified the ginseng roots at various ages from the NMR results with over 89.9% discrimination accuracy. These results indicate that the combination of 1H-NMR and PCA provides a very promising tool for the authentication and quality control of fresh ginseng roots at different ages.

Cloning and expression of a farnesyl diphosphate synthase in Centella asiatica (L.) Urban.

A cDNA encoding farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2.5.1.10) was isolated from Centella asiacita (L.) Urban, using degenerate primers based on two highly conserved domains. A full-length cDNA clone was subsequently isolated by rapid amplification of cDNA ends (RACE) PCR. The sequence of the CaFPS (C. asiatica farnesyl diphosphate synthase) cDNA contains an open reading frame of 1029 nucleotides encoding 343 amino acids with a molecular mass of 39.6 kDa. The deduced CaFPS amino acid sequence exhibits 84, 79, and 72%, identity to the FPSs of Artemisia annua, Arabidopsis thaliana, and Oryza sativa, respectively. Southern blot analysis suggested that the C. asiatica genome contains only one FPS gene. An artificially expressed soluble form of the CaFPS was identified by SDS-PAGE. It had high specific activity and produced farnesyl diphosphate as the major isoprenoid.

Cloning of a cDNA probably encoding oxidosqualene cyclase associated with asiaticoside biosynthesis from Centella asiatica (L.) Urban.

A homology-based PCR method was used to clone a cDNA encoding oxidosqualene cyclase from Centella asiatica, which produces a large quantity of triterpene saponins such as asiaticoside and madecassoside. Sequence analysis of one clone found sequences related to beta-amyrin synthase. An open reading frame in the full-length clone was named CabAS (Centella asiatica putative beta-amyrin synthase). On the basis of amino acid sequence, CabAS appears to be an enzyme (beta-amyrin synthase) that synthesizes beta-amyrin. Southern analysis showed that the C. asiatica genome contains one copy of the CabAS gene. Northern blot analysis demonstrated that the CabAS gene is expressed in leaves with no detectable transcript in other plant tissues, consistent with the organ-specific accumulation of the asiaticoside. Up-regulation of expression of CabAS by methyl jasmonate in leaves was also demonstrated.