Publications

• A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus.

  Liscombe D.K., O'Connor S.E.

  The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies ... >> More

  The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003-0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro. << Less

  Phytochemistry 72:1969-1977(2011) [PubMed] [EuropePMC]

• A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus.

  Besseau S., Kellner F., Lanoue A., Thamm A.M., Salim V., Schneider B., Geu-Flores F., Hoefer R., Guirimand G., Guihur A., Oudin A., Glevarec G., Foureau E., Papon N., Clastre M., Giglioli-Guivarc'h N., St-Pierre B., Werck-Reichhart D., Burlat V., De Luca V., O'Connor S.E., Courdavault V.

  Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from ... >> More

  Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis. << Less

  Plant Physiol. 163:1792-1803(2013) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Homolog of tocopherol C methyltransferases catalyzes N methylation in anticancer alkaloid biosynthesis.

  Liscombe D.K., Usera A.R., O'Connor S.E.

  Madagascar periwinkle (Catharanthus roseus) is the sole source of the anticancer drugs vinblastine and vincristine, bisindole alkaloids derived from the dimerization of the terpenoid indole alkaloids vindoline and catharanthine. Full elucidation of the biosynthetic pathways of these compounds is a ... >> More

  Madagascar periwinkle (Catharanthus roseus) is the sole source of the anticancer drugs vinblastine and vincristine, bisindole alkaloids derived from the dimerization of the terpenoid indole alkaloids vindoline and catharanthine. Full elucidation of the biosynthetic pathways of these compounds is a prerequisite for metabolic engineering efforts that will improve production of these costly molecules. However, despite the medical and commercial importance of these natural products, the biosynthetic pathways remain poorly understood. Here we report the identification and characterization of a C. roseus cDNA encoding an S-adenosyl-L-methionine-dependent N methyltransferase that catalyzes a nitrogen methylation involved in vindoline biosynthesis. Recombinant enzyme produced in Escherichia coli is highly substrate specific, displaying a strict requirement for a 2,3-dihydro bond in the aspidosperma skeleton. The corresponding gene transcript is induced in methyl jasmonate-elicited seedlings, along with the other known vindoline biosynthetic transcripts. Intriguingly, this unique N methyltransferase is most similar at the amino acid level to the plastidic γ-tocopherol C methyltransferases of vitamin E biosynthesis, suggesting an evolutionary link between these two functionally disparate methyltransferases. << Less

  Proc. Natl. Acad. Sci. U.S.A. 107:18793-18798(2010) [PubMed] [EuropePMC]