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bioRxiv · 10.1101/2023.08.01.551495

In vivo characterization of a secologanin transporter from Catharanthus roseus

Li, F.·Shahsavarani, M.·Handy-Hart, C.-J.·Montgomery, V.·Beech, R. N.·Liu, L.·Bayen, S.·Qu, Y.·De Luca, V.·Dastmalchi, M.
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Abstract

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have fully characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1/SLTr. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. A full panel of in vivo biochemical characterization using the Xenopus laevis oocyte expression system was used to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, rapidly transporting 1 mM of secologanin within 25 min. Notably, the absence of CrMATE1 leads to a transport bottleneck, resulting in the conversion of secologanin to its reduced form, secologanol, both in planta and in the X. laevis system. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of metabolic flux, mediating the balance between anti-herbivory potency and cell homeostasis in planta. MIA and secoiridoid transporters could also be deployed in heterologous systems to guide biosynthetic pathways and improve titers of valuable and life-saving MIAs. SIGNIFICANCEWe have fully characterized CrMATE1, a multidrug and toxic compound extrusion (MATE) family transporter in Catharanthus roseus, as a vacuolar importer of secologanin. The translocation of secologanin into the vacuole is necessary for the first committed step of monoterpenoid indole alkaloid (MIA) biosynthesis.

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Li, F., Shahsavarani, M., Handy-Hart, C.-J., Montgomery, V., Beech, R. N., Liu, L., Bayen, S., Qu, Y., De Luca, V., Dastmalchi, M.. 2023-08-02. In vivo characterization of a secologanin transporter from Catharanthus roseus. https://doi.org/10.1101/2023.08.01.551495

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