taxuyunnanine-c and jasmonic-acid

Taxus suspension cell culture has the potential to provide a sustainable source of anticancer drug paclitaxel (Taxol) and other taxoids. In the cell culture of Taxus chinensis, Taxuyunnanine C (Tc) is the primary taxoid. To design a rational strategy for redirecting the precursor fluxes from other taxoids into paclitaxel production, we employed Real-time Quantitative PCR (RQ-PCR) to understand the dynamic profiling of key biosynthetic pathway genes of palcitaxel and taxoids during the culture process. Six genes (TASY, TDAT, T5alphaH, TalphaH, T10betaH and T14betaH) were quantified under the process condition of double elicitation by 2,3-dihydroxylpropanyl jasmonate (DHPJA) (100 micromol/L on day 7 and day 12), and sucrose feeding (20 g/L) on day 7. This process treatment led to a high accumulation of Tc at (554.46 +/- 21.28) mg/L 8 days after the first elicitation. Then 9 days after the second elicitation, Tc production was as high as (997.72 +/- 1.51) mg/L. The early pathway genes TASY and TDAT were significantly up-regulated by 182-fold and 98-fold, respectively for the first DHPJA elicitation and by 208-fold and 131-fold, respectively for the second elicitation. The induction occurred after each elicitation lasted for about 24 h before their abundances decreased. Things are somewhat different in the case of the other four genes T5alphaH, TalphaH, T10betaH and T14betaH. For gene TalphaH, it was highly up-regulated by 3061-fold for the first DHPJA elicitation and by 1016-fold for the second elicitation. For the other three genes T5alphaH, T10betaH, T14betaH, they were up-regulated by 13-fold, 38-fold and 20-fold, respectively for the first DHPJA elicitation and by 7-fold, 16-fold and 6-fold, respectively for the second elicitation. The RQ-PCR results showed that there is tight correlation between gene expression and Tc accumulation. Gene expression was in accordance with Tc yield. Elicitation could improve expression of six genes. While along with culture course, high expression of the genes weakened. Elicitation for the second time would promote high expression of the genes again.

Topics: Cell Culture Techniques; Culture Media; Cyclopentanes; Gene Expression Regulation, Plant; Oxylipins; Plant Growth Regulators; Sucrose; Taxoids; Taxus; Transcriptome

Two novel 2,6-dichloroisonicotinic acid (INA) derivatives [trifluoroethyl 2,6-dichloroisonicotinate (TFINA) and 2-(2,6-dichloro-pyridine-4-carbonyloxy)-ethyl jasmonate (DPCEJ)] were chemically synthesized and evaluated by bioassay as potential elicitors for inducing the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which stably produces a high level of bioactive taxuyunnanine C (Tc), was taken as a model plant cell system. A significant increase in Tc accumulation was observed in the presence of TFINA or DPCEJ. For example, addition of 100 microM TFINA or DPCEJ on day 7 led to a high Tc content of 21.6 +/- 2.0 or 27.7 +/- 1.0 mg g(-1) (on day 21), while the Tc content was 13.7 +/- 1.0 and 17.1 +/- 0.9 mg g(-1) for the control and that with addition of 100 microM INA, respectively. To the best of our knowledge, this is the first report on the use of synthetic INA derivatives for inducing the biosynthesis of plant secondary metabolites. The results indicate that the newly synthesized INA analogues can act as promising elicitors for secondary metabolism induction in plant cell cultures.

Topics: Biological Assay; Biomass; Cyclopentanes; Isonicotinic Acids; Oxylipins; Plant Growth Regulators; Random Allocation; Taxoids; Taxus

Suspension cultures of Taxus chinensis were used as a model plant cell system to evaluate novel synthetic jasmonates as elicitors for stimulating the biosynthesis of secondary metabolites. Significant increases in accumulation of taxuyunnanine C (Tc) were observed in the presence of newly synthesized 2-hydroxyethyl jasmonate (HEJA) and trifluoroethyl jasmonate (TFEJA) without their inhibition on cell growth. Addition of 100 microM HEJA or TFEJA on day 7 led to a high Tc content of 44.3 +/- 1.1mg/g or 39.7 +/- 1.1 mg/g (at day 21), while the Tc content was 14.0 +/- 0.1 mg/g and 32.4 +/- 1.6 mg/g for the control and that with addition of 100 microM methyl jasmonate (MJA), respectively. The superior stimulating ability of HEJA and TFEJA over MJA, which was generally considered as the best chemical for eliciting taxoid biosynthesis, suggests that the novel jasmonate analogues may have great potential in application to other cell culture systems for effcient elicitation of plant secondary metabolites.

Topics: Cell Culture Techniques; Cell Division; Cells, Cultured; Cyclopentanes; Dose-Response Relationship, Drug; Oxylipins; Taxoids; Taxus

Novel hydroxyl-containing jasmonate derivatives were chemically synthesized and evaluated by bioassay as potential elicitors for stimulating the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which produces a bioactive taxoid, taxuyunnanine C (Tc), was taken as a model plant cell system. Experiments on the timing of addition of jasmonates and dose response indicated that day 7 and 100 microM was the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Tc accumulation was increased more in the presence of novel hydroxyl-containing jasmonates compared to that with methyljasmonate (MJA) addition. For example, addition of 100 microM 2,3-dihydroxypropyl jasmonate on day 7 led to a very high Tc content of 47.2 +/- 0.5 mg/g (at day 21), whereas the Tc content was 29.2 +/- 0.6 mg/g (on the same day) with addition of 100 microM MJA. Quantitative structure-activity analysis of various jasmonates suggests that the optimal lipophilicity and the number of hydroxyl groups may be two important factors affecting their elicitation activity. In addition, the jasmonate elicitors were found to induce plant defense responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL). Interestingly, a higher level of H(2)O(2) production and PAL activity was detected with elicitation by the synthesized jasmonates compared with that by MJA, which corresponded well to the superior stimulating activity in the former. This work indicates that the newly synthesized hydroxyl-containing jasmonates can act as powerful inducing signals for secondary metabolite biosynthesis in plant cell cultures.

Topics: Acetates; Cell Line; Cyclopentanes; Esters; Glucosides; Hydrogen Peroxide; Molecular Structure; Oxylipins; Phenylalanine Ammonia-Lyase; Plant Growth Regulators; Quantitative Structure-Activity Relationship; Taxoids; Taxus

To develop more effective abiotic elicitors for cell suspension cultures of T. chinensis to meet the needs for paclitaxel as anti-tumor drug, some fluoro- or hydroxyl-containing groups are introduced to the ester moiety of jasmonic acid by the esterification or acylation with bis(trichloromethyl) carbonate and corresponding alcohol. Some of them are found to be novel and effective elicitors, which can enhance the production of taxuyunnanine C (Tc) up to 60% more than that by methyl jasmonate (MJA) in T. chinensis cell cultures.

Topics: Acylation; Cells, Cultured; Cyclopentanes; Esterification; Esters; Hydrocarbons, Fluorinated; Oxylipins; Stereoisomerism; Structure-Activity Relationship; Taxoids; Taxus; Time Factors