methyl-jasmonate and triptolide

A novel GA13-oxidase ofTripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase. It specifically catalyzes the conversion of GA

Topics: Acetates; Biosynthetic Pathways; Cyclopentanes; Dioxygenases; Diterpenes; Epoxy Compounds; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gibberellins; Ketoglutaric Acids; Oxidoreductases; Oxylipins; Phenanthrenes; Phylogeny; Plant Proteins; Tripterygium

Tripterygium wilfordii Hook. f. is the traditional medicinal plants in China. Triptolide, wilforgine, and wilforine are the bioactive compounds in T. wilfordii. In this study, the contents of three metabolites and transcription levels of 21 genes involved in three metabolites biosynthesis in T. wilfordii were examined using high-performance liquid chromatography and reverse transcription PCR after application of methyl jasmonate (MeJA) on hairy roots in time course experiment (3-24 h). The results indicated that application of MeJA inhibited triptolide accumulation and promoted wilforgine and wilforine metabolites biosynthesis. In hairy roots, wilforgine content reached 693.36 μg/g at 6 h after adding MeJA, which was 2.23-fold higher than control. The accumulation of triptolide and wilforine in hairy roots increased the maximum at 9 h, which was 1.3- and 1.6-folds more than the control. Most of the triptolide secretes into the medium, but wilforgine and wilforine cannot secrete into the medium. The expression levels of unigenes which involved terpenoid backbone biosynthesis exist the correlation with marker metabolites (triptolide, wilforgine and wilforine) after induction by MeJA, and can be then used to infer flux bottlenecks in T. wilfordii secondary metabolites accumulation. These results showed that these genes may have potential applications in the metabolic engineering of T. wilfordii metabolites production.

Topics: Acetates; China; Chromatography, High Pressure Liquid; Cyclopentanes; Diterpenes; Drugs, Chinese Herbal; Epoxy Compounds; Lactones; Molecular Structure; Oxylipins; Phenanthrenes; Plants, Medicinal; Pyridines; Terpenes; Tripterygium

We found triptolide synthesis is correlated with the expressions of TwGGPPS1 and TwGGPPS4 . This lays the foundation for future studies of biosynthetic pathways for triptolide and other diterpenoids in T. wilfordii. Tripterygium wilfordii is a traditional Chinese medical plant commonly used to treat rheumatoid arthritis. One of its main bioactive compounds is triptolide, which is identified as an abietane-type diterpenoid natural product. Geranylgeranyl diphosphate synthase (GGPPS) catalyses the synthesis of GGPP (geranylgeranyl diphosphate), the common precursor of diterpenes, and is therefore a crucial enzyme in diterpene biosynthesis. A previous study showed that GGPP could be catalyzed by copalyl diphosphate synthase and kaurene synthase like of Salvia miltiorrhiza (SmCPS, SmKSL) to miltiradiene, a key intermediate in tanshinone biosynthesis. In this paper, five new full-length cDNAs (TwGGPPS) encoding GGPP synthases were cloned from T. wilfordii. Sequence comparisons revealed that all six TwGGPPSs (including TwGGPPS2 cloned previously) exhibit similarities to GGPPSs of other plants. Subsequent functional complement assays demonstrated that TwGGPPS1, TwGGPPS4 and TwGGPPS5 can participate in miltiradiene biosynthesis in the recombinant E. coli. Correlation analysis of gene expressions and secondary metabolite accumulation indicated that TwGGPPS1 and TwGGPPS4 are likely involved in the biosynthesis of triptolide. These findings lay the foundation for future studies of the biosynthetic pathways for triptolide and other diterpenoids in T. wilfordii.

Topics: Acetates; Amino Acid Sequence; Biosynthetic Pathways; Cloning, Molecular; Cyclopentanes; Diterpenes; Epoxy Compounds; Escherichia coli; Farnesyltranstransferase; Molecular Sequence Data; Oxylipins; Phenanthrenes; Phylogeny; Plant Growth Regulators; Plant Proteins; Polyisoprenyl Phosphates; Sequence Alignment; Tripterygium

To study the effects of the extract of fungal elicitor, AgNO3, MeJA and yeast on the growth and content of secondary metabolites of adventitious roots in Tripterygium wilfordii. The above elicitors were supplemented to the medium, the growth and the content of secondary metabolites were measured. When the medium was supplemented with the elicitor Glomerella cingulata or Collectotrichum gloeosporioides, the content of triptolide was increased by 2.24 and 1.93-fold, the alkaloids content was increased by 2.02 and 2.07-fold, respectively. The optimal concentration of G. cingulata was 50 μg/mL for accumulation of triptolide, alkaloids and for the growth of adventitious roots. AgNO3 inhibited the growth of adventitious roots and the accumulation of the alkaloids, whereas it (at 25 μmol/L) increased the accumulation of triptolide by 1.71-fold compared to the control. The growth of adventitious roots, the contents of triptolide and alkaloids were increased 1.04, 1.64 and 2.12-folds, respectively when MeJA was at 50 μmol/L. When the concentration of yeast reached 2 g/L, the content of triptolide increased 1.48-folds. This research demonstrated that supplementation of AgNO3 and yeast enhanced the biosynthesis of triptolide in adventitious roots and the synergism of G. cingulata and MeJA could promote the biosynthesis of both triptolide and alkaloids.

Topics: Acetates; Alkaloids; Colletotrichum; Cyclopentanes; Diterpenes; Drugs, Chinese Herbal; Epoxy Compounds; Oxylipins; Phenanthrenes; Phyllachorales; Plant Roots; Secondary Metabolism; Tripterygium