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Figure 5. Phylogenetic relationship and transport specificity of GTR homologs from selected species.(A) Selected part of Bayesian inference (MrBayes) tree (s.d. < 0.01) of GTR homologs from selected species (Full phylogenetic tree of NPFs from selected species is found as Figure 5—figure supplement 1). Green circles at nodes represent a posterior probability of 1 (maximum is 1). Values at nodes separated by a backslash represent MrBayes values below 1 in red, followed by RAxML generated bootstrap values in black (only reported when Mrbayes value is below 1). Asterix indicates that Cp17.188 lacks the highly conserved EXXE[R/K] motif involved in proton-coupling (Jørgensen et al., 2015). Subclades with green, purple and pink background denote the GTR1 subclade, GTR3 subclade and GTR-like subclade (genes that cluster with GTR homologs from C.papaya), respectively. Genes in bold were tested for glucosinolate transport in B). (B) Uptake of 4MTB and I3M by X. laevis oocytes expressing selected GTR homologs (bold) from A. thaliana, B. rapa, C. papaya, T. cacao and M. esculenta from the colored subclades and Me15G176100, which clusters outside the GTR-like subclade). Genes were expressed individually in X. laevis oocytes and transport activity was measured in the presence of 0.2 mM 4MTB (black bars) or 0.2 mM I3M (green bars) at external pH 5. Dotted line represents substrate concentration in external media. Accumulated 4MTB or I3M was quantified by LC-MS in 5 × 1 oocytes for each gene (Error bars represent ± s.d. of mean, n = 5, experiment repeated two times). (C) 4MTB (black circles)- and I3M (green circles)-induced currents in oocytes expressing GTR homologs that showed glucosinolate uptake in B). Expressing oocytes were exposed to 0.2 mM 4MTB or I3M and induced currents were measured at membrane potentials clamped between 0 mV and −180 mV in 20 mV increments at pH 5 (Error bars represent ± s.d. of mean, n = 4, experiment repeated two times). (D) Normalized 4MTB-induced currents of CpGTRL2 (Cp17.190) measured at a membrane potential of −60 mV at pH 5 plotted against increasing 4MTB concentrations. The saturation curve was fitted with a Michaelis-Menten equation represented by a solid line (Error bars represent ± s.d. of mean for data obtained from four different oocytes per experiment). Each oocyte dataset was normalized to currents elicited at 1 mM 4MTB concentration at −60 mV. The insert shows the apparent Km as a function of membrane potential.DOI: http://dx.doi.org/10.7554/eLife.19466.017Figure 5—figure supplement 1. Phylogenetic relationships of NPF transporters from selected species.Bayesian inference (MrBayes) tree (s.d.< 0.01) of NPF transporters from selected species. Green circles at nodes represent a posterior probability of 1 (maximum is 1). Values at nodes separated by a backslash represent MrBayes values below 1 in red, followed by RAxML generated bootstrap values in black (only reported when Mrbayes value is below 1). Scale bar indicates number of substitutions per site.DOI: http://dx.doi.org/10.7554/eLife.19466.018 Image published in: Jørgensen ME et al. (2017) © 2017, Jørgensen et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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