Molecular characterization of rice GTG/GPHR reveals a conserved role as an anion channel that regulates endomembrane pH

Abstract

Membrane proteins mediate processes that are fundamental for the survival of the organism. Expanding the knowledge of how those membrane proteins function in the cell will allow us to improve crop survival and seed yield that is critical for future food security. G protein-coupled receptor type-G proteins (GTGs)/Golgi pH regulator proteins (GPHRs) are a conserved family of membrane proteins in eukaryotes, which are yet to be fully characterized. In mammalian cells they are described as anion channels regulating Golgi pH (Maeda et al., 2008), whereas in plants GTGs have been shown to interact with the Gα subunit of G proteins and have been described as plasma membrane abscisic acid receptors (in Arabidopsis) or as cold receptors (in rice) (Pandey et al., 2009; Ma et al., 2015). This study determines whether there is a conserved physiological function for GTGs in plants through the analysis of rice and Arabidopsis GTGs.

Arabidopsis contains two GTGs, and the deletion of both genes generates defects in growth, development and fertility (Jaffé et al., 2012). A functional complementation approach demonstrated that rice GTG rescued the defects seen in root length, biomass, hypocotyl growth, cell shape and ER body number in the Arabidopsis gtg1gtg2 double mutant. This and the observation that rice GTG (OsGTG), like Arabidopsis GTG1, localises to the endoplasmic reticulum (ER) and Golgi, supports a conserved function for these proteins. OsGTG restored the increased pH observed in the ER and Golgi of the gtg1gtg2 Arabidopsis mutant to wild-type levels, indicating that OsGTG is also able to regulate pH at the endomembrane system. Similar to the Arabidopsis gtg1gtg2 mutant, knocking out OsGTG in rice led to several developmental defects, including increased tiller number, delayed senescence and reduced grain production. Recombinant OsGTG synthesised using a cell-free expression system and incorporated into a planar lipid bilayer, displayed single-channel events that were inhibited by the anion channel blocker, DIDS.

The interaction between GTGs with the Gα subunit was also explored. Although no evidence for protein-protein interaction was found using BiFC, a gtg1gtg2gpa1.4 triple mutant showed reduced hypocotyl growth and open cotyledons in the dark, and a reduced plant height in normal conditions, in comparison with WT, gtg1gtg2 and gpa1.4 mutants, suggesting a genetic interaction under particular conditions. The results of this thesis indicate that GTG/GPHRs have a conserved function across plant species as anion channels that regulate endomembrane pH. This thesis contributes to extend the knowledge of plant physiology by the characterization of this family of membrane proteins from its fundamental to its physiological role in the cell.

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ORCID for Matthew Terry: orcid.org/0000-0001-5002-2708

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