Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate

It has been recognized that ginsenoside Rg3 is not naturally produced in ginseng although this ginsenoside can accumulate in red ginseng as the result of a thermal process. In order to determine whether or not Rg3 is synthesized in ginseng, hairy roots were treated with methyl jasmonate (MJ). From HPLC analysis, no peak for Rg3 was observed in the controls. However, Rg3 did accumulate in hairy roots that were MJ-treated for 7?days. Rg3 content was 0.42?mg/g (dry weight). To gain more insight into the effects of MJ on UDP-glucosyltransferase (UGT) activity, we attempted to evaluate ginsenoside Rg3 biosynthesis by UGT. A new peak for putative Rg3 was observed, which was confirmed by LC-MS/MS analysis. Our findings indicate that the proteins extracted from our hairy root lines can catalyze Rg3 from Rh2. This suggests that our ginseng hairy root lines possess Rg3 biosynthesis capacity.     

Production of some benzylisoquinoline alkaloids in Papaver armeniacum L. hairy root cultures elicited with salicylic acid and methyl jasmonate

Papaver armeniacum hairy roots were induced by four Rhizobium rhizogenes strains on three explants (shoot, root, and hypocotyl). Also, the effects of two concentrations (100 and 200 μM) of methyl jasmonate (MJ) and salicylic acid (SA) were assessed on productions of papaverine, noscapine, thebaine, morphine, and codeine and expression of some related genes (TYDC, DBOX, BBE, SalAT, T6ODM, and COR) in P. armeniacum L. hairy root culture at 24 and 48 h after elicitation. R. rhizogenes strain C58C1 induced the highest hairy root rate on hypocotyl explant. Application of 100 μM MJ resulted in the highest contents of thebaine, codeine, and morphine by enhancing the expression of SalAT, COR, and T6ODM genes, respectively, while application of 100 μM SA resulted in the highest contents of papaverine and noscapine by upregulating DBOX and BBE genes, respectively. 100 μM MJ can be used as an effective elicitor in P. armeniacum hairy root culture to increase studied morphinan alkaloids. Also, SA can be suggested for enhancing papaverine and noscapine contents in P. armeniacum hairy root culture. It may be due to that there is a SA- and MJ-signaling crosstalk, which results in reciprocal antagonism between SA and MJ signaling pathways. The effects of MJ and SA elicitors on benzylisoquinoline alkaloids (BIAs) production were level-dependent.

     

Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate

We isolated a gene encoding for farnesyl diphosphate synthase (FPS) from Panax ginseng, a species that produces a large quantity of triterpene saponins such as ginsenosides. The deduced amino acid sequence of PgFPS was 77, 84 and 95 % identical to those of Arabidopsis, Hevea, and Centella. Southern blot analysis indicated that P. ginseng contained more than two genes encoding for FPS. When the cDNA of PgFPS was expressed in Escherichia coli, the recombinant enzyme, purified with a His-tag column, was found to possess FPS activity. When cultures of ginseng hairy root were treated with 0.1 mM methyl jasmonate (MJ), PgFPS mRNA was detected within 12 h of the treatment, and achieved maximum after 24 h. Also FPS activity in the hairy root cultures after 12 h of MJ treatment was higher than that of the control.     

Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors

The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated. Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50–400 M); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration. The highest ginsenoside yield was obtained at 200 M MJ. In the second experiment, 200 M MJ was added on day 15 during the cultivation. The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment. Rb group gisnsenosides accumulated more than Rg group ginsenosides. Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly. Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides. These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.     

A squalene epoxidase from Nigella sativa participates in saponin biosynthesis and mediates terbinafine resistance in yeast

Squalene epoxidase catalyzes the formation of 2,3-oxidosqualene from squalene and in plants is the last enzyme common to all biosynthetic pathways leading to an array of triterpene derivatives like phytosterols, brassinosteroid phytohormones or saponins. In this work, we present a squalene epoxidase gene (NSSQE1) from the triterpene saponin producing plant Nigella sativa. The gene product showed a high degree of homology to functional squalene epoxidases (SQEs) from Arabidopsis thaliana and was able to complement SQE deficient yeast that harboured a knockout mutation in the underlying erg1 gene. Moreover, the expression of the NSSQE1 gene in ERG1 wild type yeast revealed that NSSQE1 conferred resistance towards terbinafine, an inhibitor of fungal SQEs. The latter suggested that a terbinafine-dependent NSSQE1 selection marker system can be developed for yeast. The gene NSSQE1 was ubiquitously expressed in all plant tissues analysed, including roots where no triterpene saponins are produced. Therefore, we argue that NSSQE1 is a housekeeping gene for triterpene metabolism in Nigella sativa. Similar to triterpene saponins, NSSQE1 was up-regulated by methyl jasmonate in leaves and should also be functionally involved in saponin biosynthesis in Nigella sativa.     

Saponin production by hairy root cultures ofPanax ginseng CA meyer: Influence of PGR and polyamines

A culture of hairy roots ofPanax ginseng C.A. Meyer was set up in order to investigate the possibility of producing ginseng saponin. Roots cultured in 1/2 MS medium in the presence of 2 mg/L IAA and 0.1 mM spermidine showed the maximal growth rate, whereas other polyamines increased the growth of hairy roots only slightly or not at all. High saponin root contents were obtained in culture media supplemented with 0.5 mg/L GA and 1 mM putrescine.     

Roles of reactive oxygen species in methyl jasmonate and nitric oxide-induced tanshinone production in Salvia miltiorrhiza hairy roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O₂ ⁻ and H₂O₂ were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.     

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150 microM and cultured for 40 days. Up to 100 microM MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 microM MJ peaked after 10 days at 48 mg g(-1) dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.     

Growth kinetics and ginsenosides production in transformed hairy roots of American ginseng—Panax quinquefolium L

A thin, profusely branched, fast growing hairy root line of Panax quinquefolium (American ginseng) was established by co-culturing epicotyl explants with a wild type strain of Agrobacterium rhizogenes. The transformed roots grew by over 10-fold from the initial inoculum within 8 weeks. The crude ginsenosides content in the roots was about 0.2 g/g dry wt level up to the 10th week of culture. Ginsenosides Rb2, Rd, Re, Rf and Rg1 constituted 47–49% of the crude saponin fraction between 6 and 8 weeks of growth whereas, Rc ginsenoside was accumulated only after 9th weeks when the biomass started receding. PCR amplification analysis of the hairy roots confirmed their transgenic nature by showing the presence of Ri-TL DNA with rolA, rolB and rolC genes in their genome.     

Cytostatic effects of structurally different ginsenosides on yeast cells with altered sterol biosynthesis and transport

Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.     

Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer)

Summary Hairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in the range 1.0−5.0 mg l⁻¹ (4.8–23.8 μM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone (300 mg l⁻¹) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g⁻¹, respectively. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.     

Temporal changes in the growth, saponin content and antioxidant defense in the adventitious roots of Panax ginseng subjected to nitric oxide elicitation

Nitric oxide (NO) is a diffusible, gaseous signaling molecule. In plants, NO influences growth and development, and it can also affect plant responses to various stresses. Because NO induces root differentiation and interacts with reactive oxygen species, we examined the temporal effect of NO elicitation on root growth, saponin accumulation and antioxidant defense responses in the adventitious roots of mountain ginseng (Panax ginseng). The observations revealed that NO is involved in root growth and saponin production. Elicitation with sodium nitroprusside (SNP) activated O₂ ⁻-generating NADPH oxidase (NOX) activity, which most probably subsequently enhanced growth of adventitious roots of mountain ginseng. A severe inhibition of NOX activity and decline in dry weight of SNP elicited adventitious roots in the presence of NOX inhibitor (diphenyl iodonium, DPI), which further supports involvement of NOX in root growth. Enhanced activities of antioxidant enzymes by SNP appear to be responsible for low H₂O₂, less lipid peroxidation, and modulation of ascorbate and non-protein thiol statuses in the adventitious roots of mountain ginseng. Dry mass, saponin content and NOX activity was related with NO content present in adventitious roots of mountain ginseng.     

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Valerenic acid (VA) is a pharmacologically-active sesquiterpene found in valerian (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L^?1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.     

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