The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis

Abstract

Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that Ϸ65% of the total GR activity is attributed to GR1, whereas Ϸ35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (E GSH) in the cytosol. Using this tool, it is shown that E GSH in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.redox homeostasis ͉ redox imaging ͉ redox-sensitive GFP ͉ thioredoxin reductase

RAC2-P38 MAPK-dependent NADPH oxidase activity is associated with the resistance of quiescent cells to ionizing radiation

“…25 NADPH-dependent glutathione reductase is a member of the FAD-binding disulfide reductase superfamily and the major enzyme responsible for reduction of GSSG in most organisms, which provides a defense system against oxidants. [26][27][28] GSH oxidation also causes stimulation of the hexose monophosphate shunt and increases the production of NADPH. 26 The low consumption of NADPH and low yields of ROS are associated with low expression levels of RACs in G0 cells, which result in a high antioxidant capacity and radioresistance.…”

“…For example, the reduction of GSSG by glutathione reductase also involves NADPH. 26 ROS damages DNA bases or the sugar backbone that lead to SSBs. 29 DSBs also result from ROS-induced lesions 30 ; however, indirect interactions mediated by ROS dominates during X-ray exposure, so yields of SSBs are higher than DSBs.…”

Glutathione – linking cell proliferation to oxidative stress

“…GR1 encodes the cytosolic/peroxisomal localized form of the enzyme, while GR2 encodes a dual-targeted chloroplast and mitochondrial GR form. Although the knockout mutants in GR1 give rise to a lower GSH: GSSG ratio they are able to undergo normal plant development (74). However, GR2 knockout mutants produce a lethal phenotype and show growth arrest at the stage of embryo development (74).…”

“…Although the knockout mutants in GR1 give rise to a lower GSH: GSSG ratio they are able to undergo normal plant development (74). However, GR2 knockout mutants produce a lethal phenotype and show growth arrest at the stage of embryo development (74). Hence, the activity of the cytosolic GR form is insufficient to compensate for loss of the chloroplast and mitochondrial enzymes.…”

“…While the knockout gr1 mutants do not show a growth or development phenotype even under stress conditions, and the glutathione redox potential of the cytosol is significantly shifted towards enhanced oxidation in the absence of cytosolic/peroxisomal GR activity. The NADPH-dependent TRX system can reduce GSSG in the cytosol and so act as a backup system for GR1 activity (74). Such a functional redundancy might have additional physiological important because the cytosolic glutathione pool is direct contact with the nuclear glutathione pool.…”