Suppressor mutations in the Glutamine Dumper1 protein dissociate disturbance in amino acid transport from other characteristics of the Gdu1D phenotype.

Shi Yu,Cynthia Denbow,Guillaume Pilot,Réjane Pratelli

doi:10.3389/fpls.2015.00593

Abstract

Intracellular amino acid transport across plant membranes is critical for metabolic pathways which are often split between different organelles. In addition, transport of amino acids across the plasma membrane enables the distribution of organic nitrogen through the saps between leaves and developing organs. Amino acid importers have been studied for more than two decades, and their role in this process is well-documented. While equally important, amino acid exporters are not well-characterized. The over-expression of GDU1, encoding a small membrane protein with one transmembrane domain, leads to enhancement of amino acid export by Arabidopsis cells, glutamine secretion at the leaf margin, early senescence and size reduction of the plant, possibly caused by the stimulation of amino acid exporter(s). Previous work reported the identification of suppressor mutations of the GDU1 over-expression phenotype, which affected the GDU1 and LOG2 genes, the latter encoding a membrane-bound ubiquitin ligase interacting with GDU1. The present study focuses on the characterization of three additional suppressor mutations affecting GDU1. Size, phenotype, glutamine transport and amino acid tolerance were recorded for recapitulation plants and over-expressors of mutagenized GDU1 proteins. Unexpectedly, the over-expression of most mutated GDU1 led to plants with enhanced amino acid export, but failing to display secretion of glutamine and size reduction. The results show that the various effects triggered by GDU1 over-expression can be dissociated from one another by mutagenizing specific residues. The fact that these residues are not necessarily conserved suggests that the diverse biochemical properties of the GDU1 protein are not only born by the characterized transmembrane and VIMAG domains. These data provide a better understanding of the structure/function relationships of GDU1 and may enable modifying amino acid export in plants without detrimental effects on plant fitness.

Highlights

Amino acids are critical metabolites in plants which fulfill several roles, in addition to being the constituting blocks of proteins
Out of a total of 110,000 M2 seeds screened, three additional suppressor mutations were isolated that nearly restored the wild type size of the plants (Supplementary Figure S1), and suppressed the early senescence and Gln secretion
Sequencing of the GDU1 CDS in the T-DNA construct leading to its over-expression revealed that the mutations in log1-2, log1-3, and log1-4 corresponded to G202A, G119A, and C95T mutations, respectively, in the GDU1 DNA sequence. These mutations led to E68K, G40D, and S32L substitutions, respectively, in the GDU1 protein sequence (Figure 1). Because these mutations suppressed the Gdu1D visual phenotype, it was hypothesized that they affected GDU1 protein function

Summary

Introduction

Amino acids are critical metabolites in plants which fulfill several roles, in addition to being the constituting blocks of proteins. Importers utilize energy of the proton gradient across membranes to import amino acids against their concentration gradient, and are involved in many roles, like uptake from the soil, import into the phloem, phloem–xylem exchange, and transport into the embryo (Tegeder, 2014). Two APC members have been described as possible amino acid exporters: CAT8 (Yang et al, 2010) and BAT1/GABP1 (Dundar and Bush, 2009; Michaeli et al, 2011). One gene belonging to the Drug and Metabolite Transporter superfamily (Jack et al, 2001), SIAR1, has been shown to unequivocally mediate amino acid export from plant cells (Ladwig et al, 2012). The family SIAR1 belongs to contains 47 members with only one other gene characterized, the auxin transporter WAT1 (Ranocha et al, 2013)

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Discussion

Conclusion