DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark; Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark
DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark; Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark
Christoph Crocoll
DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark; Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark
Heidi Asschenfeldt Ernst
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
David Ramírez
Centro de Bioinformática y Simulación Molecular (CBSM), Universidad de Talca, Talca, Chile; Instituto de Innovación Basada en Ciencia, Universidad de Talca, Talca, Chile
Mohammed Saddik Motawia
Center for Plant Plasticity, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark
Carl Erik Olsen
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark
Osman Mirza
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark; Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark
DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark; Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark
Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.