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An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole

Nature Plants volume 3, Article number: 16208 (2017) Cite this article

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Abstract

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

In the medicinal plant Catharanthus roseus, it has been shown that MIA synthesis takes place in at least three distinct cell types, the internal phloem associated parenchyma, laticifers/idioblasts and epidermal cells, with the epidermis hosting the final steps of strictosidine formation and many of the downstream reactions3. Notably, the epidermis-located steps also display compartmentalization at the intracellular level. The direct precursors of strictosidine, tryptamine and the monoterpene secologanin are synthesized in the cytoplasm and must be transported into the vacuole, where biosynthesis of the central intermediate strictosidine by the enzyme strictosidine synthase takes place3,4. The next enzyme in the MIA pathway, strictosidine β-glucosidase, is sequestered in the nucleus5. The separation of strictosidine synthase and strictosidine β-glucosidase has been hypothesized to serve as a firewall, preventing the inappropriate accumulation of strictosidine aglycone, which is a highly reactive dialdehyde that induces protein crosslinking5. Although the reasons for specific localization to the cytoplasm, vacuole and nucleus remain unproven, this organellar separation implicates the need for transport of MIA intermediates into and out of the vacuole.

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Figure 1: Localization of the MIA pathway in C. roseus.
Figure 2: Self-organizing map of C. roseus transcriptomic data.
Figure 3: In planta silencing of CrNPF2.9.
Figure 4: Overexpression of CrNPF2.9–YFP in C. roseus and onion cells.
Figure 5: In vitro characterization of CrNPF2.9.

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Acknowledgements

This work was supported by grants from the European Research Council (311363), BBSRC (BB/J004561/1) (S.E.O.), Danish National Research Foundation (DNRF99 for D.X., V.N., M.B., H.H.N., B.A.H. and 10-082858 for F.G.-F.), the Innovation Fund Denmark (j.nr. 76-2014-3) (H.H.N.) and from the Région Centre, France (ABISAL grant; E.F., A.O., T.D.D.B., V.C.). R.M.E.P. was supported by a BBSRC Studentship.

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Authors and Affiliations

  1. Department of Biological Chemistry, The John Innes Centre, Norwich Research Park, Norwich, NR4 7UK, UK

    Richard M. E. Payne, Evangelos C. Tatsis & Sarah E. O'Connor

  2. Department of Plant and Environmental Sciences, DynaMo Centre, Faculty of Science, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark

    Deyang Xu, Vlastimil Novak, Meike Burow & Hussam Hassan Nour-Eldin

  3. Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark

    Deyang Xu, Vlastimil Novak, Meike Burow, Carl-Erik Olsen, Barbara Ann Halkier, Fernando Geu-Flores & Hussam Hassan Nour-Eldin

  4. Département de Biologie et Physiologie Végétales, Université François-Rabelais de Tours, EA2106 Biomolécules et Biotechnologies Végétales, UFR Sciences et Techniques, Parc de Grandmont 37200 Tours, France

    Emilien Foureau, Marta Ines Soares Teto Carqueijeiro, Audrey Oudin, Thomas Dugé de Bernonville & Vincent Courdavault

  5. Department of Computational and Systems Biology, The John Innes Centre, Norwich Research Park, Norwich, NR4 7UK, UK

    D. Marc Jones

  6. Department of Cell and Developmental Biology, The John Innes Centre, Norwich Research Park, Norwich, NR4 7UK, UK

    Ali Pendle

  7. Department of Plant and Environmental Sciences, Section for Plant Biochemistry, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark

    Fernando Geu-Flores

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Contributions

R.M.E.P. made the initial discovery of CrNPF2.9 and designed and carried out all silencing experiments; D.X. and H.H.N-E designed and carried out kinetic experiments; E.F., M.I.S.T.C., T.D.B. and V.C. designed and carried out localization experiments; F.G-F., S.E.O., H.H.N-E, V.C. and B.A.H. contributed to the conception and design of the experiments; R.M.E.P., D.X., E.F., M.I.S.T.C., A.O., T.D.B, V.N., M.B., C-E.O, D.M.J., E.C.T. and A.P. contributed to the acquisition of data; R.M.E.P., D.X., E.F., M.I.S.T.C., F.G-F., V.C., H.H.N-E. and S.E.O. contributed to data analysis and interpretation; R.M.E.P. and S.E.O. drafted the manuscript and all authors critically revised and approved the final version of the manuscript for publication.

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Correspondence to Sarah E. O'Connor.

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Payne, R., Xu, D., Foureau, E. et al. An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole. Nature Plants 3, 16208 (2017). https://doi.org/10.1038/nplants.2016.208

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