Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences

Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolium), are thought to be representative plant of Panax species, have important commercial value and are used in worldwide. Panax species produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane-type and oleanane-type. Dammarane-type ginsenosides dominate over oleanane-type not only in amount but also in structural varieties. Researches shows that the saponins content in American ginseng is higher than that in Asian ginseng, the higher part of ginsenosides is from dammarane-type biosynthesis. It has been proposed that protopanaxadiol derived from dammarenediol-II, is a key hydroxylation by cytochrome P450 for the biosynthesis of ginsenosides, and the gene number of protopanaxadiol synthase has been published independent in Asian ginseng (PgCYP716A47). However, little is known about genes involved in hydroxylation and glycosylation in American ginseng ginsenoside biosynthesis. Here, we first cloned and identified a P450 gene named PqD12H encoding enzymes catalyzed dammarenediol-II to protopanaxadiol by RT-PCR using degenerate primers designed based on sequence homology. In vitro, the ectopic expression of PqD12H in recombinant WAT21 yeast resulted in protopanaxadiol production after dammarenediol-II was added to the culture medium. In vivo, we established both PgCYP716A47 and PqD12H RNAi transgenic. The RT-PCR and HPLC analysis of the final products of protopanaxadiol and protopanaxatriol showed a result that declined level of protopanaxadiol-type and protopanaxatriol-type ginsenosides. It suggested that the P450 synthase content or expression in American ginseng exceed than in Asian ginseng. The result elucidated the evolution relationship of P450s and the reason of different saponins content among Panax species.     

Both the mevalonate and the non-mevalonate pathways are involved in ginsenoside biosynthesis

Key Message

When one of them was inhibited, the two pathways could compensate with each other to guarantee normal growth. Moreover, the sterol biosynthesis inhibitor miconazole could enhance ginsenoside level.

Abstract

Ginsenosides, a kind of triterpenoid saponins derived from isopentenyl pyrophosphate (IPP), represent the main pharmacologically active constituents of ginseng. In plants, two pathways contribute to IPP biosynthesis, namely, the mevalonate pathway in cytosol and the non-mevalonate pathway in plastids. This motivates biologists to clarify the roles of the two pathways in biosynthesis of IPP-derived compounds. Here, we demonstrated that both pathways are involved in ginsenoside biosynthesis, based on the analysis of the effects from suppressing either or both of the pathways on ginsenoside accumulation in Panax ginseng hairy roots with mevinolin and fosmidomycin as specific inhibitors for the mevalonate and the non-mevalonate pathways, respectively. Furthermore, the sterol biosynthesis inhibitor miconazole could enhance ginsenoside levels in the hairy roots. These results shed some light on the way toward better understanding of ginsenoside biosynthesis.     

Global Identification of Multiple OsGH9 Family Members and Their Involvement in Cellulose Crystallinity Modification in Rice

Plant glycoside hydrolase family 9 (GH9) comprises typical endo-β-1,4-glucanase (EGases, EC3.2.1.4). Although GH9A (KORRIGAN) family genes have been reported to be involved in cellulose biosynthesis in plants, much remains unknown about other GH9 subclasses. In this study, we observed a global gene co-expression profiling and conducted a correlation analysis between OsGH9 and OsCESA among 66 tissues covering most periods of life cycles in 2 rice varieties. Our results showed that OsGH9A3 and B5 possessed an extremely high co-expression with OsCESA1, 3, and 8 typical for cellulose biosynthesis in rice. Using two distinct rice non-GH9 mutants and wild type, we performed integrative analysis of gene expression level by qRT-PCR, cellulase activities in situ and in vitro, and lignocellulose crystallinity index (CrI) in four internodes of stem tissues. For the first time, OsGH9B1, 3, and 16 were characterized with the potential role in lignocellulose crystallinity alteration in rice, whereas OsGH9A3 and B5 were suggested for cellulose biosynthesis. In addition, phylogenetic analysis and gene co-expression comparison revealed GH9 function similarity in Arabidopsis and rice. Hence, the data can provide insights into GH9 function in plants and offer the potential strategy for genetic manipulation of plant cell wall using the five aforementioned novel OsGH9 genes.     

Identification of candidate genes in Arabidopsis and Populus cell wall biosynthesis using text-mining, co-expression network analysis and comparative genomics

Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of biofuels from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidence supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database, and additional genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional characterization in relation to cell wall biosynthesis.     

Fungal sensitivity to and enzymatic deglycosylation of ginsenosides

A ginseng pathogen, Cylindrocarpon destructans, and five nonpathogens were tested for their sensitivity to a total ginsenoside fraction (T-GF), a protopanaxadiol-type ginsenoside fraction (PPD-GF) and a protopanaxatriol-type ginsenoside fraction (PPT-GF) from the roots of Panax ginseng C.A. Meyer. The results showed that T-GF inhibited growth of the five ginseng nonpathogens, while it promoted growth of the ginseng pathogen C. destructans. PPT-GF and PPD-GF both inhibited the growth of the five ginseng nonpathogens, although the activity of PPT-GF was higher than that of PPD-GF. PPT-GF and PPD-GF exhibited different activities on C. destructans: PPT-GF inhibited its growth, whereas PPD-GF significantly enhanced its growth. The subsequent analysis of enzymatic degradation of ginsenosides by the test fungi showed that C. destructans can consecutively hydrolyze the terminal monosaccharide units from the sugar chains attached at C3 and C20 in PPD-type ginsenosides by extracellular glycosidase activity to yield four major products, gypenoside XVII (G-XVII), compound O, compound Mb and the ginsenoside F₂. By contrast, the ginseng nonpathogens Aspergillus nidulans and Cladosporium fulvum have no extracellular glycosidase activity toward sugar chains attached to C3 in PPD-type ginsenosides. These results indicated that ginsenosides might act as host chemical defenses, while the ginseng root pathogenic fungi might counter their toxicity by converting PPD-type ginsenosides into growth or host recognition factors. The ability of ginseng root pathogens to deglycosylate PPD-type ginsenosides may be a pathogenicity factor.     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis

To develop an experimental system for studying ginsenoside biosynthesis, we generated thousands of ginseng (Panax ginseng C.A. Meyer) hairy roots, genetically transformed roots induced by Agrobacterium rhizogenes, and analyzed the ginsenosides in the samples. 27 putative ginsenosides were detected in ginseng hairy roots. Quantitative and qualitative variations in the seven major ginsenosides were profiled in 993 ginseng hairy root lines using LC/MS and HPLC-UV. Cluster analysis of metabolic profiling data enabled us to select hairy root lines, which varied significantly in ginsenoside production. We selected hairy root lines producing total ginsenoside contents 4-5 times higher than that of a common hairy root population, as well as lines that varied in the ratio of the protopanaxadiol to protopanaxatriol type ginsenoside. Some of the hairy root lines produce only a single ginsenoside in relatively high amounts. These metabolites represent the end product of gene expression, thus metabolic profiling can give a broad view of the biochemical status or biochemical phenotype of a hairy root line that can be directly linked to gene function.     

Advances in study of ginsenoside biosynthesis pathway in <Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

Panax ginseng C. A. Meyer is one of the important nutraceutical and medicinal plants, which is used worldwide. Until now, ginseng has been reported to contain saponins, antioxidants, peptides, polysaccharides, fatty acids, vitamins, alkaloids, lignans, and flavonoids. The saponins, known as ginsenosides, are widely believed to be the major bioactive compounds of ginseng. In this article, ginsenoside biosynthesis pathway and key enzymes regulation are also described. This review provides a reference for improving ginsenoside contents through regulation of ginsenoside biosynthesis pathway.     

Genome-wide analysis reveals widespread roles for RcREM genes in floral organ development in Rosa chinensis

Members of the REM (Reproductive Meristem) gene family are expressed primarily in reproductive meristems and floral organs. However, their evolution and their functional profiles in flower development remain poorly understood. Here, we performed genome-wide identification and evolutionary analysis of the REM gene family in Rosaceae. This family has been greatly expanded in rose (Rosa chinensis) compared to other species, primarily through tandem duplication. Expression analysis revealed that most RcREM genes are specifically expressed in reproductive organs and that their specific expression patterns are dramatically altered in rose plants with mutations affecting floral organs. Protein-protein interaction analysis indicated that RcREM14 interact with RcAP1 (one of the homology of A class genes in ABCDE model), highlighting the roles of RcREM genes in floral organ identity. Finally, co-expression network analysis indicated that RcREM genes are co-expressed with a high proportion of key genes that regulate flowering time, floral organ development, and cell proliferation and expansion in R. chinensis.     

A network pharmacology approach to explore the potential role of Panax ginseng on exercise performance

[Purpose]As Panax ginseng C. A. Meyer (ginseng) exhibits various physiological activities and is associated with exercise, we investigated the potential active components of ginseng and related target genes through network pharmacological analysis. Additionally, we analyzed the association between ginseng-related genes, such as the G-protein-coupled receptors (GPCRs), and improved exercise capacity.[Methods]Active compounds in ginseng and the related target genes were searched in the Traditional Chinese Medicine Database and Analysis Platform (TCMSP). Gene ontology functional analysis was performed to identify biological processes related to the collected genes, and a compound-target network was visualized using Cytoscape 3.7.2.[Results]A total of 21 ginseng active compounds were detected, and 110 targets regulated by 17 active substances were identified. We found that the active compound protein was involved in the biological process of adrenergic receptor activity in 80%, G-protein-coupled neurotransmitter in 10%, and leucocyte adhesion to arteries in 10%. Additionally, the biological response centered on adrenergic receptor activity showed a close relationship with G protein through the beta-1 adrenergic receptor gene reactivity.[Conclusion]According to bioavailability analysis, ginseng comprises 21 active compounds. Furthermore, we investigated the ginseng-stimulated gene activation using ontology analysis. GPCR, a gene upregulated by ginseng, is positively correlated to exercise. Therefore, if a study on this factor is conducted, it will provide useful basic data for improving exercise performance and health.     

Comparative Analysis of the Gut Microbiota in People with Different Levels of Ginsenoside Rb1 Degradation to Compound K

Panax ginseng (family Araliaceae) which contains ginsenoside Rb1 as a main constituent is traditionally used as a remedy for cancer, inflammation, stress, and ageing. The ginsenoside Rb1 in orally administered ginseng is metabolized to bioactive compounds by gut microbiota before their absorptions to the blood. However, its metabolizing activities in individuals are significantly different as we previously demonstrated. Here, we selected 5 samples with fecal activity potently metabolizing ginsenoside Rb1 to compound K (FPG; metabolic activity, 0.058±0.029 pmol/min/mg) and 5 samples with fecal activity non-metabolizing ginsenoside Rb1 to compound K (FNG) from a pool of 100 subjects investigated in a previous study and analyzed fecal microbiota by 16S rRNA gene pyrosequencing. Taxonomy-based analysis showed that the population levels of Firmicutes and Proteobacteria in FPG were lower than in FNG, but those of Bacteroidetes and Tenericutes in FPG were higher than in FNG. At the genus level, the population levels of Clostridiales_uc_g, Oscillibacter, Ruminococcus, Holdemania, and Sutterella in FPG were significantly higher than in FNG, but that of Leuconostoc in FPG was lower than in FNG. The population levels of Bacteroides and Bifidobacterium, which potently metabolizes ginsenoside Rb1 to compound K were dramatically increased in FPG. The gut microbiota compositions of FPG and FNG were segregated on PCO2 by Principal Coordinate Analysis. Intestinal bacterial metabolism of ginseng, particularly ginsenoside Rb1, may be dependent on the composition of gut microbiota, such as Ruminococcus spp., Bacteroides spp. and Bifidobacterium spp.     

Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng

Panax ginseng is one of the most highly valued herbal medicines in the Orient, where it has gained an almost magical reputation for being able to maintain the quality of life. The root of ginseng contains noble tetracyclic triterpenenoid saponins, which are thought to be the major effective ingredients in P. ginseng. The first committed step in ginsenoside synthesis is the cyclization of 2,3-oxidosqualene to dammarenediol II by oxidosqualene cyclase, dammarenediol synthase (DDS). The gene encoding DDS has been characterized. Here, we investigated the expression of the DDS gene together with the genes involved in ginsenoside biosynthesis (SS, SE, PNX, PNY, PNY2 and PNZ). Expression of DDS mRNA was higher in flower buds compared with root, leaf and petiole of ginseng plants. Elicitor (methyl jasmonate) treatment up-regulated the expression of DDS mRNA. Ectopic expression of DDS in a yeast mutant (erg7) lacking lanosterol synthase resulted in the production of dammarenediol and hydroxydammarenone which were confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). RNA interference (RNAi) of DDS in transgenic P. ginseng resulted in silencing of DDS expression which leads to a reduction of ginsenoside production to 84.5% in roots. These results indicate that expression of DDS played a vital role in the biosynthesis of ginsenosides in P. ginseng.