Original Research ARTICLE
An Optimized Screen Reduces the Number of GA Transporters and provides Insights into NPF Substrate Determinants
- 1Copenhagen Plant Science Centre (CPSC), Denmark
- 2Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
- 3Department for Molecular Plant-Physiologie and Biophysics, University of Würzburg, Germany
- 4VILLUM Research Center for Plant Plasticity, University of Copenhagen, Denmark
Based on recent in vitro data, a relatively large number of the plant Nitrate transporter 1/Peptide transporter Family (NPF) proteins has been suggested to function as gibberellic acid (GA) transporters. Most GA transporting NPF proteins also appear to transport other structurally unrelated phytohormones or metabolites. Several of the GAs used in previous in vitro assays are membrane permeable weak organic acids whose movement across membranes are influenced by the pH-sensitive ion-trap mechanism. Moreover, a large proportion of in vitro GA transport activities have been demonstrated indirectly via long-term yeast-based GA-dependent growth assays that are limited to detecting transport of bioactive GAs. Thus, there is a need for an optimized transport assay for identifying and characterizing GA transport. Here, we develop an improved transport assay in Xenopus laevis oocytes wherein we directly measure movement of six different GAs across oocyte membranes over short time. We show that membrane permeability of GAs in oocytes can be predicted based on number of oxygen atoms and that several GAs do not diffuse over membranes regardless of changes in pH values. In addition, we show that small changes in internal cellular pH can result in strongly altered distribution of membrane permeable phytohormones. This prompts caution when interpreting heterologous transport activities. We use our transport assay to screen all Arabidopsis thaliana NPF proteins for transport activity towards six GAs (two membrane permeable and four non-permeable). The results presented here, significantly reduce the number of bona fide NPF GA transporters in Arabidopsis and narrow the activity to fewer subclades within the family. Furthermore, to gain first insight into the molecular determinants of substrate specificities towards organic molecules transported in the NPF, we charted all surface exposed amino acid residues in the substrate-binding cavity and correlated them to GA transport. This analysis identified distinct residues within the substrate-binding cavity that are shared between GA transporting NPF proteins; the potential roles of these residues in determining substrate specificity are discussed.
Keywords: gibberellic acid, transport, NPF, membrane permeable, phytohormones, in vitro, pH
Received: 21 May 2019;
Accepted: 13 Aug 2019.
Copyright: © 2019 Wulff, Ernst, Jørgensen, Lambertz, Maierhofer, Belew, Crocoll, Motawia, Geiger, Jørgensen, Mirza and Nour-Eldin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Prof. Hussam H. Nour-Eldin, Copenhagen Plant Science Centre (CPSC), Copenhagen, Denmark, firstname.lastname@example.org