An Optimized Screen Reduces the Number of GA Transporters and Provides Insights Into Nitrate Transporter 1/Peptide Transporter Family Substrate Determinants.

Nikolai Wulff,Heidi Asschenfeldt Ernst,Dietmar Geiger,Morten Egevang Jørgensen,Christoph Crocoll,Tobias Maierhofer,Zeinu Mussa Belew,Mohammed Saddik Motawia,Osman Mirza,Hussam Hassan Nour-Eldin,Flemming Steen Jørgensen,Sophie Lambertz

doi:10.3389/fpls.2019.01106

Abstract

Based on recent in vitro data, a relatively large number of the plant nitrate transporter 1/peptide transporter family (NPF) proteins have 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 toward 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 suggests 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.

Highlights

Gibberellic acids (GAs) were detected in phloem sap more than 50 years ago suggesting that gibberellic acid (GA) are mobile phytohormones (Hoad and Bowen, 1968)
One of the most intriguing features of the nitrate transporter 1/peptide transporter family (NPF) phytohormone transporters is the apparent multi-specificity toward phytohormones and other metabolites with distinct chemical structure (Corratge-Faillie and Lacombe, 2017; Wang et al, 2018)
Our characterization of NPF7.3 provides an alternative explanation to what we first perceived as multispecificity towards different phytohormones

Summary

Introduction

Gibberellic acids (GAs) were detected in phloem sap more than 50 years ago suggesting that GAs are mobile phytohormones (Hoad and Bowen, 1968). Novel in vitro and in vivo approaches led to the identification of a large number of putative GA transporters (>25 different genes in Arabidopsis summarized in (Supplementary Table 1). The majority of these genes belong to the nitrate transporter 1/peptide transporter family (NPF) (Kanno et al, 2012; Chiba et al, 2015; Saito et al, 2015; Tal et al, 2016). Experiments in which expression of multiple GA transporting NPF members are inhibited or knocked-out simultaneously may be needed to unveil their distinct roles (Binenbaum et al, 2018)

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