Management of oxidative stress in plants: dissecting the dual role of glutathione in oxidant reduction and protein S-thiolation

Abstract

The regulation of enzyme activity by post-translational modifications enables a fast acclimation at the metabolic level under changing environmental conditions. The amino acid cysteine is very susceptible to redox modifications such as S-glutathionylation, a reversible mixed disulfide between a cysteine thiol group and the tripeptide glutathione (GSH, γ-Glu-Cys-Gly). Class I glutaredoxins (GRXs) facilitate the transfer of electrons from glutathione to target proteins and can (de)glutathionylate proteins. For their efficient reduction GRXs require GSH. A high concentration of reduced GSH is maintained by the glutathione reductase (GR), which reduces oxidized glutathione (GSSG) to two GSH using NADPH as electron donor. The absence of GR leads to a less reducing glutathione redox potential (<i>E</i>_GSH). Under oxidative stress conditions, increased levels of S-glutathionylation are observed, potentially resulting from an altered GSH:GSSG ratio and <i>E</i>_GSH. To what extend GRX function, the GSH:GSSG ratio or changes in <i>E</i>_GSH contribute to increasing levels of protein S-glutathionylation is largely unknown.<br> To explore the evolutionary conservation of the glutathione-dependent redox network including GRX and GR in plastids, I conducted a phylogenetic analysis showing evolutionary conservation of GR and GRX clades from streptophyte algae to flowering plants. I provide an overview of known plastidial glutathionylation target proteins and the respective S-glutathionylation sites based on available literature.<br> To investigate the influence of <i>E</i>_GSH and GSH:GSSG on protein S-glutathionylation, I used redox-sensitive GFP (roGFP2) as glutathionylation target and exposed it to various GSH:GSSG ratios at constant <i>E</i>_GSH. GRX-mediated roGFP2 oxidation kinetics increased with higher ratios of GSH:GSSG, indicating an important function of the GSH:GSSG ratio on the glutathionylation rate. To test the in vivo influence of <i>E</i>_GSH on protein glutathionylation, the cytosolic glutathionylation target GAPC1-YFP (glyceraldehyde-3-phosphate dehydrogenase C1) was introduced into the Arabidopsis GR mutant gr1-1, displaying a less reducing <i>E</i>_GSH. I observed higher expression of GAPC1-YFP in gr1-1, suggesting that changes in <i>E</i>_GSH affect GAPC1 promotor activity. However, aggregate formation triggered by S-glutathionylation of GAPC1 was not observed.<br> While class I GRX have been well characterized in vitro, in vivo studies provided only limited information due to functional redundancy. To elucidate the in planta role of class I GRX, I generated viable knock-outs of the single plastid class I GRX present in Physcomitrium patens (∆grxc5). To date, stress treatments did not reveal phenotypic differences between ∆grxc5 and WT, in contrast to null mutants of glutathione reductase (∆gr1). I conclude that stromal GRX malfunction is not the cause for the dwarfed and stress-sensitive ∆gr1 phenotype.<br> PpGRXC5 showed a high oxidoreductase activity in vitro. Plastid-targeted roGFP2 in ∆grxc5 revealed a complete loss of this activity observed by slower roGFP2 reduction kinetics after oxidative stress in vivo. I performed Western blot analyses on protein extracts of wild-type (WT) and ∆grxc5 before and after oxidative stress and observed increased total protein S-glutathionylation levels in both, suggesting that PpGRXC5 is not necessary for S-glutathionylation under these conditions.