[Show abstract][Hide abstract] ABSTRACT: Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs, and the root of this plant contains pharmacologically active components,
called ginsenosides. Ginsenosides, a class of tetracyclic triterpene saponins, are synthesized from dammarenediol-II after
hydroxylation by cytochrome P450 (CYP) and then glycosylation by a glycosyltransferase. Protopanaxadiol synthase, which is
a CYP enzyme (CYP716A47) that catalyzes the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol, was recently characterized.
Here, we isolated two additional CYP716A subfamily genes (CYP716A52v2 and CYP716A53v2) and determined that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside
biosynthesis in P. ginseng. Both CYP716A47 and CYP716A53v2 mRNAs accumulated ubiquitously in all organs of ginseng plants. In contrast, CYP716A52v2 mRNA accumulated only in the rhizome. Methyl jasmonate (MeJA) treatment resulted in the obvious accumulation of CYP716A47 mRNA in adventitious roots. However, neither CYP716A52v2 nor CYP716A53v2 mRNA was affected by MeJA treatment during the entire culture period. The ectopic expression of CYP716A53v2 in recombinant WAT21 yeast resulted in protopanaxatriol production after protopanaxadiol was added to the culture medium.
In vitro enzymatic activity assays revealed that CYP716A53v2 catalyzed the oxidation of protopanaxadiol to produce protopanaxatriol. The chemical structures of the protopanaxatriol products
were confirmed using liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Our results
indicate that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that produces protopanaxatriol from protopanaxadiol, which is an important step in the
formation of dammarane-type triterpene aglycones in ginseng saponin biosynthesis.
Preview · Article · Aug 2012 · Plant and Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: Methyl jasmonate (MeJA) triggers the production of secondary metabolites in plants and participates in a diverse range of plant developmental processes. MeJA is derived from jasmonic acid (JA) via the octadecanoid pathway and the reaction is catalyzed by jasmonic acid carboxyl methyltransferase (JMT). In this study, transgenic Panax ginseng roots were constructed to express an Arabidopsis jasmonic acid carboxyl methyltransferase (AtJMT). The transgenic lines exhibited high expression of genes (PgSS1, PgSE1, and PgDDS) involved in ginsenoside biosynthetic pathways as well as a MeJA-responsive gene (PgPR10-2). These alterations of the gene expressions led to 3-fold increase in the growth of transgenic roots. Overexpression of AtJMT gene strongly affected the ginsenoside heterogeneity (protopanaxadiol/protopanaxatriol ratio) although total ginsenoside accumulation was slightly increased in transgenic roots. Protopanaxadiol group of ginsenosides (Rb1, Rc, and Rb2) increased about 2-fold in transgenic roots compared to those in wild-type. The results suggest that overexpression of AtJMT stimulate not only the growth of roots but also the production of protopanaxadiol-group in transgenic roots.
[Show abstract][Hide abstract] ABSTRACT: Glandular trichomes are the phytochemical factories of plants, and they secrete a wide range of commercially important natural products such as lipids, terpenes and flavonoids. Herein, we report that the Nicotiana tabacum LTP1 (NtLTP1) gene, which is specifically expressed in long glandular trichomes, plays a role in lipid secretion from trichome heads. NtLTP1 mRNA is abundantly transcribed in trichomes, but NtLTP3, NtLTP4 and NtLTP5 are not. In situ hybridization revealed that NtLTP1 mRNAs accumulate specifically in long trichomes and not in short trichomes or epidermal cells. X-gluc staining of leaves from a transgenic plant expressing the NtLTP1 promoter fused to a GUS gene revealed that NtLTP1 protein accumulated preferentially on the tops of long glandular trichomes. GFP fluorescence from transgenic tobacco plants expressing an NtLTP1-GFP fusion protein was localized at the periphery of cells and in the excreted liquid droplets from the glandular trichome heads. In vitro assays using a fluorescent 2-p-toluidinonaphthalene-6-sulfonate probe indicated that recombinant NtLTP1 had lipid-binding activity. The overexpression of NtLTP1 in transgenic tobacco plants resulted in the increased secretion of trichome exudates, including epicuticular wax. In transgenic NtLTP1-RNAi lines, liquid secretion from trichomes was strongly reduced, but epicuticular wax secretion was not altered. Moreover, transgenic tobacco plants overexpressing NtLTP1 showed increased protection against aphids. Taken together, these data suggest that NtLTP1 is abundantly expressed in long glandular trichomes, and may play a role in lipid secretion from long glandular trichomes.
Full-text · Article · Dec 2011 · The Plant Journal
[Show abstract][Hide abstract] ABSTRACT: Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs and contains pharmacologically active components, ginsenosides, in its roots. Ginsenosides, a class of tetracyclic triterpene saponins, are thought to be synthesized from dammarenediol-II after hydroxylation by the Cyt P450 (CYP) enzyme and then glycosylation by glycosyltransferase (GT). However, no genes encoding the hydroxylation and glycosylation in ginsenoside biosynthesis have been identified. Here, we identify protopanaxadiol synthase, which is a CYP enzyme (CYP716A47), to be involved in the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol. Nine putative full CYP sequences were isolated from the expressed sequence tags (ESTs) of methyl jasmonate (MeJA)-treated adventitious ginseng roots. The CYP716A47 gene product was selected as the putative protopanaxadiol synthase because this gene was transcriptionally activated not only by MeJA treatment but also in transgenic ginseng that overexpresses squalene synthase and overproduces ginsenosides. In vitro enzymatic activity assays revealed that CYP716A47 catalyzed the oxidation of dammarenediol-II to produce protopanaxadiol. Ectopic expression of CYP716A47 in recombinant WAT21 yeasts that were fed dammarenediol-II yielded protopanaxadiol. Furthermore, co-expression of the dammarenediol synthase gene (PgDDS) and CYP716A47 in yeast yielded protopanaxadiol without adding dammarenediol-II. The chemical structures of the protopanaxadiol products from dammarenediol-II were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Thus, CYP716A47 is a dammarenediol 12-hydroxylase that produces protopanaxadiol from dammarenediol-II.
Preview · Article · Dec 2011 · Plant and Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: Panax ginseng is one of the famous medicinal plants. Ginsenosides, a class of tetracyclic triterpene saponins, are mainly responsible for its pharmacological activity. Most ginsenosides are composed of dammarenediol-II aglycone with various sugar moieties. Dammarenediol-II synthase is the first enzyme in the biosynthesis of ginsenosides. Here, we report that transgenic tobacco expressing the P. ginseng dammarenediol-II synthase gene (PgDDS) produced dammarenediol-II, and conferred resistance to Tobacco mosaic virus (TMV). Upon infection with TMV, lesions developed more rapidly in transgenic tobacco plants, and their size was smaller than those of wild-type plants. Transgenic tobacco plants showed a low level of both the viral titer and mRNA accumulation of TMV coat protein (CP) compared with the wild type. The production of dammarenediol-II in transgenic tobacco stimulated the expression of tobacco pathogenesis-related genes (PR1 and PR2) under both virus-untreated and -treated conditions. When the leaves of wild-type plants were inoculated with a mixture of TMV and dammarenediol-II, the leaves exhibited a reduced viral concentration and TMV-CP expression than those receiving TMV treatment alone. When the leaves of P. ginseng were infected with TMV, transcription of PgDDS was significantly increased. Transgenic P. ginseng plants harboring a β-glucuronidase (GUS) gene driven by the PgDDS promoter were constructed. The GUS expression was activated when the transgenic ginseng plants were treated with TMV. These results indicate that the medicinally important dammarenediol-II can be ectopically produced in tobacco, and the production of dammarenediol-II in tobacco plants allows them to adopt a viral defense system.
Full-text · Article · Nov 2011 · Plant and Cell Physiology