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

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.

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin

“…GA-related DEGs also included the AtHB23 homeobox protein that is involved in light-regulated hypocotyl growth and cotyledon expansion [ 21 ] and the GA catabolic enzyme GA2ox6 [ 22 ] ( Table 2 and Supplementary Table S3 ). GA transport is considered to be mediated by NPF (NRT1/PTR FAMILY) transporters [ 23 , 24 , 25 ]. The NPF family has 53 members in Arabidopsis [ 26 ], but the expression of only 6 of them was altered in the rlck vi_a2 mutant.…”

The Arabidopsis NRT1/PTR FAMILY protein NPF7.3/NRT1.5 is an indole-3-butyric acid transporter involved in root gravitropism

“…3 ). Interestingly, it was shown recently that Xenopus oocytes expressing NPF7.3 took up significantly lower amounts of plant hormones including ABA, GA, and JA-Ile compared to control oocytes injected with water ( 46 ). This is possibly because NPF7.3 affected proton uptake and hence pH inside the oocytes, which in turn influenced membrane permeabilities of the chemicals indirectly.…”

Mechanistic insight into substrate specificity of plant glucosinolate transporters

“…Positon 70 of NPF2.9 was chosen and included as a 12 th position as a control for sample mix-up. It was identified as a central cavity residue (Wulff et al 2019) and identified in the HOLE and docking analysis, but it is not stringently differentially conserved (Ser in all 26 NPF2.10 and NPF2.11 orthologous but among the 13 NPF2.9 sequences it is Ala one time, Gly eight times and Ser four times).…”