NTR/NRX define a new thioredoxin system in the nucleus of Arabidopsis thaliana cells.

Thioredoxins (TRX) are key components of cellular redox balance, regulating many target proteins through thiol/disulfide exchange reactions. In higher plants, TRX constitute a complex multigenic family whose members have been found in almost all cellular compartments. Although chloroplastic and cytosolic TRX systems have been largely studied, the presence of a nuclear TRX system has been elusive for a long time. Nucleoredoxins (NRX) are potential nuclear TRX found in most eukaryotic organisms. In contrast to mammals, which harbor a unique NRX, angiosperms generally possess multiple NRX organized in three subfamilies. Here, we show that Arabidopsis thaliana has two NRX genes (AtNRX1 and AtNRX2), respectively, belonging to subgroups I and III. While NRX1 harbors typical TRX active sites (WCG/PPC), NRX2 has atypical active sites (WCRPC and WCPPF). Nevertheless, both NRX1 and NRX2 have disulfide reduction capacities, although NRX1 alone can be reduced by the thioredoxin reductase NTRA. We also show that both NRX1 and NRX2 have a dual nuclear/cytosolic localization. Interestingly, we found that NTRA, previously identified as a cytosolic protein, is also partially localized in the nucleus, suggesting that a complete TRX system is functional in the nucleus. We show that NRX1 is mainly found as a dimer in vivo. nrx1 and nrx2 knockout mutant plants exhibit no phenotypic perturbations under standard growth conditions. However, the nrx1 mutant shows a reduced pollen fertility phenotype, suggesting a specific role of NRX1 at the haploid phase.

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

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 approximately 65% of the total GR activity is attributed to GR1, whereas approximately 35% 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.