Subcellular glutathione homeostasis and characterisation of glutathione transport across the plasma membrane in Arabidopsis thaliana

Abstract

Challenging environmental conditions are known to alter glutathione homeostasis, notably by inducing the accumulation of oxidized glutathione, an effect that may be influential in the perception or transduction of stress signals. The tripeptide glutathione (reduced form: GSH; oxidized form: GSSG) is a key player in maintaining cellular thiol-redox homeostasis. In Arabidopsis, two proteins are responsible for GSH biosynthesis, GSH1 and GSH2. GSH1 is exclusively localized in plastids, while GSH2 is localized in both plastids and cytosol. Thus, GSH synthesis is restricted to plastids and cytosol but there is a requirement for glutathione also in other subcellular compartments with the need for intracellular GSH transport. In addition, there is good indication pointing at long-distance transport of glutathione between different organs. The latter implies the need for transport of glutathione across the plasma membrane which is poorly understood. Null mutants of GSH1 are embryo lethal, while disruption of GSH2 is seedling lethal. In several genetic screens, different gsh1 mutants with defects in GSH biosynthesis have been identified. However, the effect of these mutations at the subcellular level was still elusive. In this study, the effect of GSH1 mutations on subcellular glutathione distribution was analysed in an allelic series of gsh1 mutants and wild-type plants. Fluorescent labelling of GSH with monochlorobimane (MCB) and HPLC measurements showed that the total amount of glutathione was affected by the respective mutations. Furthermore, the relative glutathione redox potential (EGSH) in different subcellular compartments measured with redox-sensitive fluorescent protein2 (roGFP2), showed that these mutations differentially affect the subcellular glutathione pool. The data indicate that mutations in GSH1 have a stronger effect on cytosolic and plastidial glutathione homeostasis than on mitochondrial glutathione homeostasis. Furthermore, crossing of bir6 (a mutant with diminished glutathione turnover) with the severely GSH deficient mutants, zir1 (a mutant <20 % GSH, and restricted growth phenotype) and rml1 (a mutant with <5 % GSH and seedling lethal) partially rescued the growth and lethal phenotype respectively, which strongly suggests that the absolute amount of glutathione is responsible for the growth phenotype GSH deficient mutants. <br /> The glutathione concentration is limited by the γ-glutamyl cycle, which is based on glutathione synthesis, degradation and transport. While the γ-glutamyl cycle was suggested as a classical pathway involved in glutathione transport across the plasma membrane there is also evidence for cytosolic glutathione degradation. Under sulfur deficiency, intense MCB fluorescence in γ-glutamyl-cyclotransferase (GGCT) null mutants and diminished MCB fluorescence in wild-type suggests that GGCTs specifically degrade GSH in the cytosol. <br /> While glutathione transport studies in yeast (Saccharomyces cerevisiae) have led to identification of Hgt1p as a high affinity glutathione transporter, genes encoding glutathione-specific transporters in the plasma membrane of plants remain largely unknown. To investigate the transport of glutathione across the plasma membrane, the severely glutathione-deficient Arabidopsis mutant rml1 was analysed using the roGFP2, which are able to monitor the local EGSH. Changes in the fluorescence ratio of roGFP2 expressed in the cytosol of rml1 with external supply of GSH, in combination with inhibitor studies revealed a highly efficient secondary active uptake of GSH across the plasma membrane. Furthermore, reduction of roGFP2 was only seen with GSH, but not with individual amino acids or GSSG. Additionally, a generated opt4rml1 double mutant further proved that the oligopeptide transporter 4 (OPT4) reported earlier is not the only GSH transporter in Arabidopsis but is rather complemented by a yet unknown high affinity transporter. These results have major implications for our understanding of the glutathione homeostasis in plants, with a particular focus on subcellular compartmentation, degradation, functionality and transport.