Saponin Biosynthesis Research Articles

Colonization of endophyte Acremonium sp. D212 in Panax notoginseng and rice mediated by auxin and jasmonic acid.

Endophytic fungi can be beneficial to plant growth. However, the molecular mechanisms underlying colonization of Acremonium spp. remain unclear. In this study, a novel endophytic Acremonium strain was isolated from the buds of Panax notoginseng and named Acremonium sp. D212. The Acremonium sp. D212 could colonize the roots of P. notoginseng, enhance the resistance of P. notoginseng to root rot disease, and promote root growth and saponin biosynthesis in P. notoginseng. Acremonium sp. D212 could secrete indole-3-acetic acid (IAA) and jasmonic acid (JA), and inoculation with the fungus increased the endogenous levels of IAA and JA in P. notoginseng. Colonization of the Acremonium sp. D212 in the roots of the rice line Nipponbare was dependent on the concentration of methyl jasmonate (MeJA) (2-15 μmol/L) and 1-naphthalenacetic acid (NAA) (10-20 μmol/L). Moreover, the roots of the JA signaling-defective coi1-18 mutant were colonized by Acremonium sp. D212 to a lesser degree than those of the wild-type Nipponbare and miR393b-overexpressing lines, and the colonization was rescued by MeJA but not by NAA. It suggests that the cross-talk between JA signaling and the auxin biosynthetic pathway plays a crucial role in the colonization of Acremonium sp. D212 in host plants.

Transcriptome analysis of Clinopodium gracile (Benth.) Matsum and identification of genes related to Triterpenoid Saponin biosynthesis

BackgroundClinopodium gracile (Benth.) Matsum (C. gracile) is an annual herb with pharmacological properties effective in the treatment of various diseases, including hepatic carcinoma. Triterpenoid saponins are crucial bioactive compounds in C. gracile. However, the molecular understanding of the triterpenoid saponin biosynthesis pathway remains unclear.ResultsIn this study, we performed RNA sequencing (RNA-Seq) analysis of the flowers, leaves, roots, and stems of C. gracile plants using the BGISEQ-500 platform. The assembly of transcripts from all four types of tissues generated 128,856 unigenes, of which 99,020 were mapped to several public databases for functional annotation. Differentially expressed genes (DEGs) were identified via the comparison of gene expression levels between leaves and other tissues (flowers, roots, and stems). Multiple genes encoding pivotal enzymes, such as squalene synthase (SS), or transcription factors (TFs) related to triterpenoid saponin biosynthesis were identified and further analyzed. The expression levels of unigenes encoding important enzymes were verified by quantitative real-time PCR (qRT-PCR). Different chemical constituents of triterpenoid saponins were identified by Ultra-Performance Liquid Chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS).ConclusionsOur results greatly extend the public transcriptome dataset of C. gracile and provide valuable information for the identification of candidate genes involved in the biosynthesis of triterpenoid saponins and other important secondary metabolites.

Salinity effects on physiological and phytochemical characteristics and gene expression of two Glycyrrhiza glabra L. populations

Glycyrrhiza glabra (licorice) is a medicinal plant with valuable specialised metabolites such as triterpene sweetener glycyrrhizin. Salinity stress is the main environmental stress limiting plant growth and development. The effects of six levels of NaCl (0, 100, 200, 400, 600, and 800 mM) on growth, osmolyte content, oxidative stress markers, antioxidant enzyme activities, K+/Na+ ratio, glycyrrhizin content, and gene expression of glycyrrhizin biosynthesis (bAS, CYP88D6, and CYP72A154) were investigated in licorice rhizomes of two populations. The results showed that the salt stress progressively reduced the growth parameters and increased the proline concentrations in the rhizomes. K+/Na+ ratio showed a significant decrease under salinity as compared to the controls. Salt stress resulted in oxidative stress on the rhizomes, as indicated by increased lipid peroxidation and hydrogen peroxide concentrations and elevated the activities of antioxidant enzymes (i.e., ascorbate peroxidase and superoxide dismutase). The glycyrrhizin content increased only under 100 and 200 mM NaCl treatments. The same trend was observed in the expression of bAS, CYP88D6, and CYP72A154 genes in Fars population. Fars population was found to have more glycyrrhizin content than Khorasan population. But, growth, glycyrrhizin content, and biosynthesis genes of glycyrrhizin showed more reduction in Khorasan population as compared to those of Fars population. The results indicate that the application of 100 mM NaCl up-regulated the expression of key genes involved in the biosynthesis of triterpenoid saponins and directly enhanced the production of glycyrrhizin. Accordingly, G. glabra can be introduced as a halophyte plant.

IDENTIFICACIÓN DE cDNAs DE Agave tequilana Weber Var. azul SIMILARES A FUROSTANOL GLICÓSIDO 26-O-β-GLUCOSIDASAS

Furostanol glycoside 26-O-β-glucosidase (F26G) participates in the last step of steroidal saponin biosynthesis, cleaving the glucose bound to the C26 of the hydroxylin furostanol glucoside to allow the formation of spirostane. Despite the existence of numerous studies showing the structural diversity and biological activity of plant saponins, as well as their recognized biotechnological and pharmacological potential, few enzymes involved in the biosynthetic pathway of steroidal saponins have been studied and only one native F26G enzyme has been isolated and functionally characterized. In this study, by searching a database of Agave tequilana Weber var. azul (Agave DB), 46 ESTs (Expressed Sequence Tags) of complementary DNA (cDNAs) similar to the known F26G were identified through BLASTX, most come from cDNA from floral tissues (anthers and ovaries) or roots; both organs are saponins producers. Among them, six ESTs were identified whose alignment indicates that they represent three cDNAs different from each other. These ESTs were submitted to the dbEST database of NCBI (National Center for Biotechnology Information), being this the first record of native cDNA sequences putatively encoding F26G-like in Agave. The size of the cloned cDNAs and their alignment to the 5’ end of the known F26G suggest they are full-length cDNAs. Specific differential primers were designed for each type of cDNA and their expression in vegetative tissues of A. tequilana was analyzed by RT-PCR. Quantitative PCR protocols (qPCR) were established for future studies on gene expression regulation. The information reported here will serve as a basis for studying the function, regulation and enzymatic activity of F26G-like cloned from Agave.

Structure and Location Studies on Key Enzymes in Saponins Biosynthesis of Panax notoginseng.

Panax notoginseng is one of the most widely used traditional herbs for the treatment of various diseases, in which saponins were the main active components. At present, the research of P. notoginseng mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites of P. notoginseng. In our study, four coding sequences (CDS) encoding the key enzymes involved in saponin biosynthesis were cloned, namely farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), and dammarenediol-II synthase (DS), which contained open reading frame (ORF) of 1029 bp, 1248 bp, 1614 bp, and 2310 bp, and coded 342, 415, 537, and 769 amino acids, respectively. At the same time, their domains, secondary structures, three-dimensional structures, and phylogenetics trees were analyzed by kinds of bioinformatics tools. Their phylogenetics relationships were also analyzed. In addition, GFP (Green fluorescent protein) fusion genes were constructed by the plasmid transformation system to determine the subcellular localization. The results of subcellular localization showed that FPS, SE, and DS were mainly located in cytomembrane and its surrounding, while SS was located both in cytoplasm and cytomembrane. Our findings provided data demonstrating the expression patterns of genes involved in saponin biosynthesis and would facilitate efforts to further elucidate the biosynthesis of the bioactive components in P. notoginseng.

Molecular Analysis of Two Novel Missense Mutations in the Sg‐1 Gene Associated with Group A Saponin Biosynthesis in Soybean

ABSTRACTSoybean [Glycine max (L.) Merr.] accumulates several different types of saponins, including the Aa and Ab series of saponins, which contribute to the bitter and astringent aftertaste in soy food products. Modification of the soybean genome is being applied to develop bitter‐free soybean cultivars. In this study, two new ethyl‐methanesulfonate (EMS)‐induced mutants, PE937 and PE1015, were characterized for their ability to accumulate the bitter‐free A0‐αg saponin and low level of the bitter Ab‐series saponins compared with the wild cultivar. These phenotypes resemble that of previously reported sg‐1° mutants that accumulate A0‐αg and no Ab‐series saponins, which are controlled by the Sg‐1b (Glyma.07G254600) gene. Sequence analysis of Sg‐1b in PE937 and PE1015 revealed that each mutant has a single‐nucleotide polymorphism that causes an amino acid change from Gly349 to Asp349 and from Gly241 to Asp241 in the Sg‐1b proteins. We therefore designated these mutations as alleles sg‐1b1 and sg‐1b2, respectively. Multiple alignment analysis showed that the Gly349 and Gly241 are highly conserved among the UDP‐dependent glycosyltransferase (UGT) proteins. In light of the above results, the fact that there are low levels of Ab‐series saponins in both of the mutants indicates that these mutations may reduce the UGT activity of Sg‐1b proteins in the biosynthesis of Ab‐series saponins. Cosegregation analysis revealed that the novel sg‐1b1 and sg‐1b2 variants are recessive to Sg‐1b and are tightly linked to A0‐αg saponin accumulation.

Characterization of a new sg-5 variant with reduced biosynthesis of group A saponins in soybean (Glycine max (L.) Merr.)

Triterpenoid saponins are major secondary metabolites in soybean; they are classified into group A and DDMP saponins. Group A saponins cause bitterness and an astringent aftertaste, whereas DDMP saponins are less bitter and more beneficial to human health. Therefore, a need exists for genetic improvement to develop bitter-free soybeans without losing other beneficial saponins. In this study, an ethyl methanesulfonate (EMS)–induced mutant, PE1327, was isolated and characterized as having a reduced level of group A saponins by chromatography. The PE1327 phenotype, which exhibits no accumulation of group A saponins, resembles that of a previously reported sg-5 wild soybean mutant whose biosynthesis is controlled by the Sg-5 (Glyma.15G243300) gene. The Sg-5 gene sequence in PE1327 revealed a single-nucleotide polymorphism (G1036A) that causes an amino acid change from Glu346 to Lys346 in the sg-5 protein. Multiple alignment analysis of cytochrome P450 enzymes from numerous organisms revealed that Glu346 is highly conserved in eukaryotes, including plants and animals, but not in prokaryotes. The predicted 3D model of the Sg-5 protein showed that the Glu346 residue is located in the J helix and is likely involved in the linkage between the J and K helices and the stabilization of the J-K loop, suggesting that the amino acid substitution from Glu346 to Lys346 in the PE1327 mutant results in hypofunction of Sg-5. Co-segregation analysis revealed that the Sg-5 locus is tightly linked to reduced group A saponin biosynthesis in PE1327 and that the novel sg-5 variant is recessive to Sg-5.

Large-Scale Profiling of Saponins in Different Ecotypes of Medicago truncatula.

A total of 1,622 samples representing 201 Medicago truncatula ecotypes were analyzed using ultrahigh pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS) to ascertain saponin profiles in different M. truncatula ecotypes and to provide data for a genome-wide association study and subsequent line selection for saponin biosynthesis. These ecotypes originated from 14 different Mediterranean countries, i.e., Algeria, Cyprus, France, Greece, Israel, Italy, Jordan, Libya, Morocco, Portugal, Spain, Syria, Tunisia, and Turkey. The results revealed significant differences in the saponin content among the ecotypes. European ecotypes generally contained higher saponin content than African ecotypes (p < 0.0001). This suggests that M. truncatula ecotypes modulate their secondary metabolism to adapt to their environments. Significant differences in saponin accumulation were also observed between the aerial and the root tissues of the same ecotypes (p < 0.0001). While some saponins were found to be present in both the aerial and root tissues, zanhic acid glycosides were found predominantly in the aerial tissues. Bayogenin and hederagenin glycosides were found mostly in roots. The differential spatially resolved accumulation of saponins suggests that saponins in the aerial and root tissues play different roles in plant fitness. Aerial saponins such as zanhic glycosides may act as animal feeding deterrent and root saponins may protect against soil microbes.

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.

Transcriptome Analysis of Clinopodium chinense (Benth.) O. Kuntze and Identification of Genes Involved in Triterpenoid Saponin Biosynthesis.

Clinopodium chinense (Benth.) O. Kuntze (C. chinense) is an important herb in traditional Chinese medicine. Triterpenoid saponins are a major class of active compounds in C. chinense with broad pharmacological activities and hemostatic, antitumor, and anti-hyperglycemic effects. To identify genes involved in triterpenoid saponin biosynthesis, transcriptomic analyses of leaves, stems, and roots from C. chinense were performed. A total of 135,968 unigenes were obtained by assembling the leaf, stem, and root transcripts, of which 102,154 were annotated in public databases. Differentially expressed genes were determined based on expression profile analysis and analyzed for differential expression of unique genes related to triterpenoid saponin biosynthesis. Multiple unigenes encoding crucial enzymes or transcription factors involved in triterpenoid saponin synthesis were identified and analyzed. The expression levels of unigenes encoding enzymes were experimentally validated using quantitative real-time PCR. This study greatly broadens the public transcriptome database for this species and provides a valuable resource for identifying candidate genes involved in the biosynthesis of triterpenoid saponins and other secondary metabolites.

Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins.

Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. UHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed. Correlation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of β-amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2β-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2β-hydroxyoleanolic acid, hederagenin and total saponin levels. The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2β-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis.

Enhanced patient care, reduced costs and measurable better healthcare performance through implementation of intelligent liver function testing (ILFT)

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica.

Screening and evaluation of adventitious root lines of Panax notoginseng by morphology, gene expression, and metabolite profiles.

Panax notoginseng is a commonly used Traditional Chinese Medicine (TCM) and has diverse pharmacological activities with triterpenoid saponins as its main active ingredient. In the cultivation of P. notoginseng, continuous cropping is a serious problem, which could induce reduced productivity, low tuber quality, and plant mortality. With unique advantages of easy control, relative stability, high yields, tissue culture is widely used in the protection of TCM resources. In this study, we screened one adventitious root line, multi-branched (MB) root induced from wild-type roots of P. notoginseng, with a high yield of total triterpenoid saponins (17.92mg/g). The morphology analysis showed that MB root had structure similar to that of wild-type roots, except for the highly branched phenotype. MB root also showed close gene expression levels and metabolite profiles, which were also similar to those of wild-type roots of Demonstration Park (S3Y). Pearson's correlation coefficient analysis confirmed the importance of key gene, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), and transcription factor, PnERF1, in regulating triterpenoid saponin biosynthesis in P. notoginseng. These results suggested that MB root possesses potential value in the large-scale cultivation of P. notoginseng.

Transcriptome analyses of Paris polyphylla var. chinensis, Ypsilandra thibetica, and Polygonatum kingianum characterize their steroidal saponin biosynthesis pathway

Steroidal saponins, one of the most diverse groups of plant-derived natural products, elicit biological and pharmacological activities; however, the genes involved in their biosynthesis and the corresponding biosynthetic pathway in monocotyledon plants remain unclear. This study aimed to identify genes involved in the biosynthesis of steroidal saponins by performing a comparative analysis among transcriptomes of Paris polyphylla var. chinensis (PPC), Ypsilandra thibetica (YT), and Polygonatum kingianum (PK). De novo transcriptome assemblies generated 57,537, 140,420, and 151,773 unigenes from PPC, YT, and PK, respectively, of which 56.54, 47.81, and 44.30% were successfully annotated, respectively. Among the transcriptomes for PPC, YT, and PK, we identified 194, 169, and 131; 17, 14, and 26; and, 80, 122, and 113 unigenes corresponding to terpenoid backbone biosynthesis; sesquiterpenoid and triterpenoid biosynthesis; and, steroid biosynthesis pathways, respectively. These genes are putatively involved in the biosynthesis of cholesterol that is the primary precursor of steroidal saponins. Phylogenetic analyses indicated that lanosterol synthase may be exclusive to dicotyledon plant species, and the cytochrome P450 unigenes were closely related to clusters CYP90B1 and CYP734A1, which are UDP-glycosyltransferases unigenes homologous with the UGT73 family. Thus, unigenes of β-glucosidase may be candidate genes for catalysis of later period modifications of the steroidal saponin skeleton. Our data provide evidence to support the hypothesis that monocotyledons biosynthesize steroidal saponins from cholesterol via the cycloartenol pathway.

Comparative analysis of proteomic and metabolomic profiles of different species of Paris

An extract prepared from species of Paris is the most widely consumed herbal product in China. The genus Paris includes a variety of genotypes with different medicinal component contents but only two are defined as official sources. Closely related species have different medicinal properties because of differential expression of proteins and metabolites. To better understand the molecular basis of these differences, we examined proteomic and metabolomic changes in rhizomes of P. polyphylla var. chinensis, P. polyphylla var. yunnanensis, and P. fargesii var. fargesii using a technique known as sequential window acquisition of all theoretical mass spectra as well as gas chromatography–time-of-flight mass spectrometry. In total, 419 proteins showed significant abundance changes, and 33 metabolites could be used to discriminate Paris species. A complex analysis of proteomic and metabolomic data revealed a higher efficiency of sucrose utilization and an elevated protein abundance in the sugar metabolic pathway of P. polyphylla var. chinensis. The pyruvate content and efficiency of acetyl-CoA-utilization in saponin biosynthesis were also higher in P. polyphylla var. chinensis than in the other two species. The results expand our understanding of the proteome and metabolome of Paris and offer new insights into the species-specific traits of these herbaceous plants. SignificanceThe traditional Chinese medicine Paris is the most widely consumed herbal product for the treatment of joint pain, rheumatoid arthritis and antineoplastic. All Paris species have roughly the same morphological characteristics; however, different members have different medicinal compound contents. Efficient exploitation of genetic diversity is a key factor in the development of rare medicinal plants with improved agronomic traits and malleability to challenging environmental conditions. Nevertheless, only a partial understanding of physiological and molecular mechanisms of different plants of Paris can be achieved without proteomics. To better understand the molecular basis of these differences and facilitate the use of other Paris species, we examine proteomic metabolomic changes in rhizomes of Paris using the technique known as SWATH-MS and GC/TOF-MS. Our research has provided information that can be used in other studies to compare metabolic traits in different Paris species. Our findings can also serve as a theoretical basis for the selection and cultivation of other Paris species with a higher medicinal value.

The cytochrome P450 CYP72A552 is key to production of hederagenin-based saponins that mediate plant defense against herbivores.

Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins invitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin-based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C-23 to hederagenin. In vitro-produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin-based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

Oleanane-Type Saponins Biosynthesis in Panax notoginseng via Transformation of β-Amyrin Synthase Gene from Panax japonicus

Oleanane-type saponins considered as the main medicinal ingredients in Panax japonicus are not found in Panax notoginseng. β-Amyrin synthase (βAS) was recognized as the first key enzyme in the biosynthetic branch of oleanane-type saponins. In this study, βAS gene from P. japonicus ( PjβAS) was transferred into P. notoginseng cells. Along with PjβAS expression in the transgenic cells, the expression levels of several key enzyme genes related to triterpenoid saponins biosynthesis and the content of P. notoginseng saponins were also increased. Two oleanane-type saponins, chikusetsusaponin IV and chikusetsusaponin IVa, contained in P. japonicus were first discovered in transgenic P. notoginseng cells. This study successfully constructed a biosynthetic pathway of oleanane-type saponins in P. notoginseng by introducing just one gene into the species. On the basis of this discovery and previous studies, the common biosynthetic pathway of triterpenoid saponins in Panax genus may be unified to some extent.

English

Javanese ginseng (Talinum paniculatum) is an herb and one of the traditional medicinal plants in Indonesia which accumulate saponin in the root organ. However, slow root growth hampers the accumulation of the compound in this plant. Therefore, in vitro culture of adventitious roots offers an alternative way to increase the production of saponin. Furthermore, this study was performed to investigate the effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the growth and saponin content in adventitious root of Javanese ginseng. Adventitious root culture was induced on solid Murashige and Skoog (MS) medium supplemented by 10 µM IBA (indole-3-butyric acid). Subsequently, adventitious roots were subcultured into a half strength MS liquid medium with the addition of different concentrations of MeJA or SA and incubated for 5, 10 and 15 days. Despite inhibition on the root growth, saponin production increased by 1.5 and 1.3 fold upon elicitation with 0.2 mM MeJA and SA for 15 days, respectively. Thus, MeJA and SA elicitations regulate saponin biosynthesis in adventitious root culture of Javanese ginseng as a time and dose-dependent manner. Key words: Adventitious root, elicitation, Javanese ginseng, saponin, Talinum paniculatum.

Short-term water changes response of saponin biosynthesis process in Astragalus membranceus

The study is aimed to explore the effect of different water on the content of total saponins,astragaloside Ⅳ and gene expression in the growth of Astragalus membranceus. In this study, one-year-old A. membranaceus was used as the experimental material, by pot culture different water treatments were simulated at herbal garden in Jilin Agricultural University. The content of astragaloside Ⅳ was determined by HPLC and the total saponins by UV spectrophotometry. With 18 S RNA as a reference gene, fluorescent quantitative PCR was applied to analyze the eight key enzymes in astragalus saponin synthesis pathway AACT,HMGS,HMGR,IDI,FPS,SS,SE,CAS expression. With the decrease of soil water, the content of astragaloside Ⅳ in the root tissue of A. membranaceus showed an increasing trend, up to 1.46 mg·g~(-1). The total saponin content tended to increase, up to 6.80 mg·g~(-1). The results of relative expression of genes showed that the eight genes showed different effects at different water. With the change of soil water content, the amount of(AACT,IDI,SS) relative expression in drought stress group firstly increased and then decreased, then increased, and then decreased. The amount of(HMGS,HMGR,FPS) relative expression in drought stress group increased firstly and then decreased. The amount of(SE,CAS) relative expression in drought stress group increased firstly and then decreased, and continued to decrease after rehydration. The expression of key enzyme genes involved in the synthesis of astragaloside was influenced by each other, and the expression of key enzyme in roots showed a correlation with the content of astragaloside. Correlation analysis showed that there was a very significant positive correlation between HMGR gene and total saponins content in drought stress group and a significant negative correlation between content of CAS and total saponins. The contents of FPS,SE,CAS and astragaloside Ⅳ were very significantly and negative correlated. The relationship between other genes and quality was positive. Therefore, HMGR, FPS, SE and CAS genes have significant effects on the regulation of saponin content under water control. On the 15 th day after water regulation, the total amount of astragaloside and total saponins reached the highest value and could be harvested.

Transcriptomic analysis in Anemone flaccida rhizomes reveals ancillary pathway for triterpene saponins biosynthesis and differential responsiveness to phytohormones

Anemone flaccida Fr. Schmidt is a perennial medicinal herb that contains pentacyclic triterpenoid saponins as the major bioactive constituents. In China, the rhizomes are used as treatments for a variety of ailments including arthritis. However, yields of the saponins are low, and little is known about the plant's genetic background or phytohormonal responsiveness. Using one-quarter of the 454 pyrosequencing information from the Roche GS FLX Titanium platform, we performed a transcriptomic analysis to identify 157 genes putatively encoding 26 enzymes involved in the synthesis of the bioactive compounds. It was revealed that there are two biosynthetic pathways of triterpene saponins in A. flaccida. One pathway depends on β-amyrin synthase and is similar to that found in other plants. The second, subsidiary (“backburner”) pathway is catalyzed by camelliol C synthase and yields β-amyrin as minor byproduct. Both pathways used cytochrome P450-dependent monooxygenases (CYPs) and family 1 uridine diphosphate glycosyltransferases (UGTs) to modify the triterpenoid backbone. The expression of CYPs and UGTs were quite different in roots treated with the phytohormones methyl jasmonate, salicylic acid and indole-3-acetic acid. This study provides the first large-scale transcriptional dataset for the biosynthetic pathways of triterpene saponins and their phytohormonal responsiveness in the genus Anemone.