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Geissoschizine synthase controls flux in the formation of monoterpenoid indole alkaloids in a Catharanthus roseus mutant

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Abstract

Main conclusion

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.

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Abbreviations

EMS:

Ethyl methyl sulfonate

GS/Gs :

Geissoschizine synthase

HYS/Hys :

Heteroyohimbine synthase

MIA:

Monoterpenoid indole alkaloid

SGD/Sgd :

Strictosidine-β-glucosidase

THAS/Thas :

Tetrahydroalstonine synthase

TLC:

Thin-layer chromatography

References

  1. Asada K, Salim V, Masada-Atsumi S, Edmunds E, Nagatoshi M, Terasaka K, Mizukami H, De Luca V (2013) A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secologanin biosynthesis in Madagascar periwinkle. Plant Cell 25:4123–4134

  2. Blom TJM, Sierra M, van Vliet TB, Franke-van Dijk MEI, de Koning P, van Iren F, Verpoorte R, Libbenga KR (1991) Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.) G. Don. and its conversion into serpentine. Planta 183:170–177

  3. Brown S, Clastre M, Courdavault V, O’Connor SE (2015) De novo production of the plant-derived alkaloid strictosidine in yeast. Proc Natl Acad Sci USA 112:3205–3210

  4. Carqueijeiro I, Noronha H, Duarte P, Gerós H, Sottomayor M (2013) Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport. Plant Physiol 162:1486–1496

  5. Chaudhary S, Sharma V, Prasad M, Bhatia S, Tripathi BN, Yadav G, Kumar S (2011) Characterization and genetic linkage mapping of the horticulturally important mutation leafless inflorescence (lli) in periwinkle Catharanthus roseus. Sci Hortic 129:142–153

  6. De Luca V, Salim V, Masada-Atsumi S, Yu F (2012) Mining the biodiversity of plants: a revolution in the making. Science 336:1658–1661

  7. Deus Neumann B, Zenk MH (1984) A highly selective alkaloid uptake system in vacuoles of higher plants. Planta 162:250–260

  8. Edge A, Qu Y, Easson MLAE, Thamm AMK, Kim KH (2017) A tabersonine 3-reductase mutant accumulates vindoline pathway intermediates. Planta. https://doi.org/10.1007/s00425-017-2775-8

  9. Facchini PJ, Bohlmann J, Covello PS, De Luca V, Mahadevan R, Page JE, Ro DK, Sensen CW, Storms R, Martin VJ (2012) Synthetic biosystems for the production of high-value plant metabolites. Trends Biotechnol 30:127–131

  10. Geu-Flores F, Sherden NH, Courdavault V, Burlat V, Glenn WS, Wu C, Nims E, Cui Y, O’Connor SE (2012) An alternative route to cyclic terpene by reductive cyclization in iridoid biosynthesis. Nature 492:138–142

  11. Hemscheidt T, Zenk MH (1985) Partial purification and characterization of a NADPH dependent tetrahydroalstonine synthase from Catharanthus roseus cell suspension cultures. Plant Cell Rep 4(4):216–219

  12. Hong B, Cheng W, Wu J, Zhao C (2010) Screening and identification of many of the compounds present in Rauvolfia verticillata by use of high-pressure LC and quadrupole TOF MS. Chromatographia 72:841–847

  13. Kellner F, Geu-Flores F, Sherden NH, Brown S, Foureau E, Courdavault V, O’Connor SE (2015) Discovery of a P450-catalyzed step in vindoline biosynthesis: a link between the aspidosperma and eburnamine alkaloids. Chem Commun 51:7626–7628

  14. Kulkarni RN, Baskaran K (2015) Increasing total leaf alkaloid concentrations in periwinkle (Catharanthus roseus) by combining the macro-mutant traits of two induced leaf. J Hort Sci Biotech 316:513–518

  15. Kulkarni RN, Baskaran K, Chandrashekara RS, Kumar S (1999) Inheritance of morphological traits of periwinkle mutants with modified contents and yields of leaf and root alkaloids. Plant Breeding 118:71–74

  16. Liscombe DK, Usera AR, O’Connor SE (2010) Homolog of tocopherol C methyltransferases catalyzes N methylation in anticancer alkaloid biosynthesis. Proc Natl Acad Sci USA 107:18793–18798

  17. Miettinen K, Dong L, Navrot N, Schneider T, Burlat V, Pollier J, Woittiez L, van der Krol S, Lugan R, Ilc T, Verpoorte R, Oksman-Caldentey K-M, Martinoia E, Bouwmeester H, Goossens A, Memelink J, Werck-Reichhard D (2014) The seco-iridoid pathway from Catharanthus roseus. Nat Commun 5:3606

  18. Murata J, Roepke J, Gordon H, De Luca V (2008) The leaf epidermome of Catharanthus roseus reveals its biochemical specialization. Plant Cell 20:524–542

  19. Qu Y, Easson MLAE, Froese J, Simionescu R, Hudlicky T, De Luca V (2015) Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast. Proc Natl Acad Sci USA 112:6224–6229

  20. Roepke J, Wu M, Salim V, Thamm A, Murata J, Ploss K, Boland W, De Luca V (2010) Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle. Proc Natl Acad Sci USA 107:15287–15292

  21. Salim V, Yu F, Altarejos J, De Luca V (2013) Virus induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis. Plant J 76:754–765

  22. Salim V, Wiens B, Masada-Atsumi S, Yu F, De Luca V (2014) 7-deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis. Phytochemistry 101:23–31

  23. Simkin AJ, Miettinen K, Claudel P, Burlat V, Guirimand G, Courdavault V, Papon N, Meyer S, Godet S, St-Pierre B, Giglioli-Guivarc’h N, Fischer MJC, Memelink J, Clastre M (2013) Characterization of the plastidial geraniol synthase from Madagascar periwinkle which initiates the monoterpenoid branch of the alkaloid pathway in internal phloem associated parenchyma. Phytochemistry 85:36–43

  24. Stravrinides A, Tatsis EC, Foureau E, Caputi L, Kellner F, Courdavault V, O’Connor SE (2015) Unlocking the diversity of alkaloids in Catharanthus roseus: nuclear localization suggests metabolic channelling in secondary metabolism. Chem Biol 22:336–341

  25. Stravrinides A, Tatsis EC, Caputi L, Foureau E, Stevenson CEM, Lawson DM, Courdavault V, O’Connor SE (2016) Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity. Nat Commun 7:12116

  26. Tatsis E, Carqueijeiro I, Dugé de Bernonville T, Frankel J, Dang TTT, Oudin A, Lanoue A, Lafonaine F, Stravinides AK, Clastre M, Courdavault V, O’Connor SE (2017) A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate. Nat Commun 8:316

  27. Thamm AMK, Qu Y, De Luca V (2016) Discovery and metabolic engineering of iridoid/secoiridoid and monoterpenoid indole alkaloid biosynthesis. Phytochem Rev 15:339–361

  28. Van der Heijden R, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R (2004) The Catharanthus alkaloids: pharmacognosy and biotechnology. Current Med Chem 11:607–628

  29. Xiao M, Zhang Y, Chen X, Lee EJ, Barber CJ, Chakrabarty R, Desgagné-Penix I, Haslam TM, Kim YB, Liu E, MacNevin G, Masada-Atsumi S, Reed DW, Stout JM, Zerbe P, Zhang Y, Bohlmann J, Covello PS, De Luca V, Page JE, Ro DK, Martin VJ, Facchini PJ, Sensen CW (2013) Transcriptome analysis based on next-generation sequencing of non-model plants producing specialized metabolites of biotechnological interest. J Biotechnol 166:122–134

  30. Yu F, De Luca V (2013) ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus. Proc Natl Acad Sci USA 110:15830–15835

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Acknowledgements

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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Correspondence to Vincenzo De Luca.

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Qu, Y., Thamm, A.M.K., Czerwinski, M. et al. Geissoschizine synthase controls flux in the formation of monoterpenoid indole alkaloids in a Catharanthus roseus mutant. Planta 247, 625–634 (2018). https://doi.org/10.1007/s00425-017-2812-7

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Keywords

  • Geissoschizine synthase
  • Heteroyohimbine synthase
  • Mutant screen
  • Strictosidine β-glucosidase
  • Tetrahydroalstonine synthase
  • Virus-induced gene silencing