methyl-jasmonate and raubasine

The medicinal plant Catharanthus roseus accumulates large numbers of terpenoid indole alkaloids (TIAs), including the pharmaceutically important vinblastine, vincristine, ajmalicine, and serpentine. The phytohormone ethylene or methyl jasmonate (MeJA) can markedly enhance alkaloid accumulation. The interaction between ethylene or MeJA in the regulation of TIA biosynthesis in C. roseus is unknown. Here, a metabolomics platform is reported that is based on liquid chromatography (LC) coupled with time-of-flight mass spectrometry to study candidate components for TIA biosynthesis, which is controlled by ethylene or MeJA in C. roseus. Multivariate analysis identified 16 potential metabolites mostly associated with TIA metabolic pathways and seven targeted metabolites, outlining the TIA biosynthesis metabolic networks controlled by ethylene or MeJA. Interestingly, ethylene and MeJA regulate the 2-C-methyl-d-erythritol 4-phosphate (MEP) and acetate-mevalonate (MVA) pathways through AACT and HMGS and through DXS, respectively, to induce TIA biosynthesis in C. roseus. Overall, both nontargeted and targeted metabolomics, as well as transcript analysis, were used to reveal that MeJA and ethylene control different metabolic networks to induce TIA biosynthesis.

Topics: Acetates; Catharanthus; Chromatography, Liquid; Cyclopentanes; Ethylenes; Mass Spectrometry; Metabolic Networks and Pathways; Metabolomics; Mevalonic Acid; Oxylipins; Secologanin Tryptamine Alkaloids; Vinblastine; Vincristine

Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM β-CD and 150 μM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that β-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that β-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that β-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus, and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids.

Topics: Acetates; beta-Cyclodextrins; Catharanthus; Cells, Cultured; Cyclopentanes; Oxylipins; Plant Cells; Secologanin Tryptamine Alkaloids; Vinblastine; Vinca Alkaloids

Catharanthus roseus (L.) G. Don (Apocynaceae) is a medicinal plant that produces more than 130 alkaloids, with special attention given to the production of the anti-hypertensive monomeric indole alkaloids, serpentine and ajmalicine, and the antitumor dimeric alkaloids, vinblastine and vincristine.. This study evaluated the cytotoxic activity of the indole alkaloid-enriched bioactive extract obtained from suspension cultured-cells of C. roseus elicited with methyl jasmonate (MJ) and cyclodextrins (CDs) in three cell lines: JURKAT E.6 human lymphocytic leukemia, THP-1 human monocytic leukemia and BL 1395 non-tumor human B-cell line.. An indole alkaloid-enriched bioactive extract was obtained from C. roseus cell cultures elicited with MJ and CDs. The indole alkaloids were identified using an HPLC-diode array system coupled to a time-of-flight mass spectrometer using electrospray ionization (ESI) source. The cytotoxic assays were made using the colorimetric assay 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-S-[(phenylamino)carbonyl]-2 tetrazolium hydroxide (XTT).. Four indole alkaloids were identified (catharanthine, ajmalicine, tabersonine and lochnericine) but only catharanthine and ajmalicine were quantified. The concentration of the indole alkaloid-enriched bioactive extract that inhibited cell growth by 50% was 211 and 210 ng/mL for the JURKAT E.6 and THP-1 cell lines, respectively.. The results confirm that the powerful antitumor activity of this indole alkaloid-enriched bioactive extract is not due to the effect of a single compound but depends on the synergistic action of the four compounds identified.

Topics: Acetates; Antineoplastic Agents, Phytogenic; Catharanthus; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Cyclodextrins; Cyclopentanes; Drug Discovery; Humans; Indole Alkaloids; Inhibitory Concentration 50; Oxylipins; Plant Extracts; Plant Leaves; Quinolines; Secologanin Tryptamine Alkaloids; Spectrometry, Mass, Electrospray Ionization; Vinca Alkaloids

The joint use of cyclodextrins and methyljasmonate, when accompanied by a short exposure to UV, enhanced extracellular ajmalicine accumulation to 1040 ± 26.6 mg/l in suspension cultured cells of Catharanthus roseus. The success of this strategy is due to the use of cyclodextrins, which not only induce ajmalicine biosynthesis but also promote adduct formation. This removes ajmalicine from the medium, reduces feedback inhibition and ajmalicine degradation, and allows its accumulation in the culture medium at elevated concentrations.

Topics: Acetates; Biotechnology; Catharanthus; Cell Culture Techniques; Culture Media; Cyclodextrins; Cyclopentanes; Oxylipins; Secologanin Tryptamine Alkaloids; Ultraviolet Rays

In Catharanthus roseus, the first step of monoterpenoid indole alkaloids (MIA) biosynthesis results from the condensation of the indole precursor tryptamine with the terpenoid precursor secologanin. Secologanin biosynthesis requires two successive biosynthetic pathways, the plastidial methyl-D: -erythritol 4-phosphate (MEP) pathway and the monoterpene secoiridoid pathway. In C. roseus cell culture, the expression of several genes encoding enzymes of these two pathways is dramatically down-regulated by auxin, while strongly enhanced by cytokinin and methyl-jasmonate. Furthermore, our previous studies have shown that protein prenylation events are also involved in the transcriptional activation of some of these genes. In the present work, we investigate the involvement of protein prenylation in the jasmonate signalling pathway leading to MIA biosynthesis. Inhibition of protein prenyltransferase down-regulates the methyl-jasmonate-induced expression of MEP and monoterpene secoiridoid pathway genes and thus abolishes MIA biosynthesis. Jointly, it also inhibits the methyl-jasmonate-induced expression of the AP2/ERF transcription factor ORCA3 that acts as a central regulator of MIA biosynthesis. Finally, a specific silencing of protein prenyltransferases mediated by RNA interference in C. roseus cells shows that inhibition of type I protein geranylgeranyltransferase (PGGT-I) down-regulates the methyl-jasmonate-induced expression of ORCA3, suggesting that PGGT-I prenylated proteins are part of the early steps of jasmonate signalling leading to MIA biosynthesis.

Topics: Acetates; Alkyl and Aryl Transferases; Catharanthus; Cells, Cultured; Cyclopentanes; Gene Expression Regulation, Plant; Oxylipins; Plant Proteins; Protein Prenylation; RNA Interference; RNA, Plant; Secologanin Tryptamine Alkaloids; Signal Transduction; Transcription Factors

The effects of methyl jasmonate, salicylic acid and ethylene on alkaloid accumulation in in vitro cell suspension, hairy roots and rootless shoot cultures of Catharanthus roseus were analyzed. Ajmalicine, but not catharanthine, accumulation was promoted by jasmonate and ethylene treatments in cell suspensions. In hairy roots, jasmonate induced the accumulation of both alkaloids, whereas ethylene only induced catharanthine accumulation. In shoot cultures, positive effects of jasmonate and ethylene were recorded only in vindoline accumulation. Ethylene diminished catharanthine accumulation in these cultures. No effect of salicylic acid was observed in any of the studied in vitro culture systems.

Topics: Acetates; Catharanthus; Cells, Cultured; Cyclopentanes; Ethylenes; Oxylipins; Plant Roots; Plant Shoots; Salicylic Acid; Secologanin Tryptamine Alkaloids; Vinca Alkaloids

Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the precursor availability for TIA biosynthesis. C. roseus suspensions were induced with MJ (100 microM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM), loganin plus tryptamine, or geraniol (0.1-1.0 mM) on day 7. While MJ increased ajmalicine production by 3-fold, induced cultures were still limited by terpenoid precursors. However, both induced and non-induced cultures became tryptamine-limited with excess loganin. Geraniol feeding also increased ajmalicine production in non-induced cultures. But MJ appeared to increase geraniol availability in induced cultures, due presumably to the increased expression of Dxs with MJ addition.

Topics: Acetates; Acyclic Monoterpenes; Catharanthus; Cells, Cultured; Cyclopentanes; Iridoids; Oxylipins; Secologanin Tryptamine Alkaloids; Terpenes; Tryptamines

Cytosolic Ca(2+) and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca(2+) and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl(2) (3, 23, and 43 mM) and MJ (0, 10, and 100 microM) on day 6 of growth. Increased Ca(2+) influx through the addition of extracellular CaCl(2) suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 microM). In the presence of 3 mM CaCl(2) in the medium, the addition of Ca(2+) chelator EGTA (1, 2.5, and 5 mM) or Ca(2+) channel blocker verapamil (1, 10, and 50 muM) to MJ-induced (100 microM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca(2+) inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca(2+) concentration and a low extracellular Ca(2+) concentration (3 mM) enhanced MJ-induced ajmalicine production.

Topics: Acetates; Calcium; Calcium Signaling; Catharanthus; Cells, Cultured; Cyclopentanes; Egtazic Acid; Oxylipins; Secologanin Tryptamine Alkaloids; Verapamil

The optimum growth stage for enhancing ajmalicine production in Catharanthus roseus cultures with methyl jasmonate (MJ) was after 6 d growth. MJ added at 10 or 100 microm on day 6 gave a maximum ajmalicine production of 10.2 mg l(-1), a 300% increase over that of non-elicited cultures.

Topics: Acetates; Catharanthus; Cell Proliferation; Cells, Cultured; Cyclopentanes; Dose-Response Relationship, Drug; Oxylipins; Secologanin Tryptamine Alkaloids; Time Factors