Geissoschizine synthase controls flux in the formation of monoterpenoid indole alkaloids in a Catharanthus roseus mutant
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A Catharanthus roseus mutant accumulates high levels of ajmalicine at the expense of catharanthine and vindoline. The altered chemistry depends on increased expression and biochemical activities of strictosidine β-glucosidase and ajmalicine synthase activities and reduced expression and biochemical activity of geissoschizine synthase.
The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is a valuable source for several monoterpenoid indole alkaloids (MIAs), such as the powerful antihypertensive drug ajmalicine and the antineoplastic agents, vinblastine and vincristine. While biosynthesis of the common MIA precursor strictosidine and its reactive aglycones has been elucidated, the branch point steps leading to the formation of different classes of MIAs remain poorly characterized. Screening of 3600 ethyl methyl sulfonate mutagenized C. roseus plants using a simple thin-layer chromatography screen yielded a mutant (M2-0754) accumulating high levels of ajmalicine together with significantly lower levels of catharanthine and vindoline. Comparative bioinformatic analyses, virus-induced gene silencing, and biochemical characterization identified geissoschizine synthase, the gateway enzyme that controls flux for the formation of iboga and aspidosperma MIAs. The reduction of geissoschizine synthase transcripts in this high ajmalicine mutant, together with increased transcripts and enzyme activities of strictosidine β-glucosidase and of heteroyohimbine synthase, explains the preferential formation of ajmalicine in the mutant instead of catharanthine and vindoline that accumulates in the wild-type parent. Reciprocal crosses established that that the high ajmalicine phenotype is inherited as a Mendelian recessive trait.
KeywordsGeissoschizine synthase Heteroyohimbine synthase Mutant screen Strictosidine β-glucosidase Tetrahydroalstonine synthase Virus-induced gene silencing
Ethyl methyl sulfonate
Monoterpenoid indole alkaloid
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (V. D. L.), Canada Research Chairs (V. D. L.), and the Advanced Biomanufacturing Center (Brock University). We recognize the skilled technical work of next-generation sequencing personnel at the McGill University-Genome Québec-Innovation Centre. We are grateful to Christoph Sensen, Mei Xiao, and Ye Zhang for their dedicated bioinformatic support and large-scale gene annotation efforts that helped in the identification of differentially expressed genes from the Phytometasyn website. The computational part of this work was made possible by the facilities of the Shared Hierarchical Academic Research Computing Network (SHARCNET:http://www.sharcnet.ca) and Compute/Calcul Canada.
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