An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress

Cereal seed cells contain different mechanisms for protection against the oxidative stress that occurs during maturation and germination. One such mechanism is based on the antioxidant activity of a 1-Cys peroxiredoxin (1-Cys Prx) localized in the nuclei of aleurone and scutellum cells. However, nothing is known about the mechanism of activation of this enzyme. Here, we describe the pattern of localization of NADPH thioredoxin reductase (NTR) in developing and germinating wheat seeds using an immunocytochemical analysis. The presence of NTR in transfer cells, vascular tissue, developing embryo and root meristematic cells, agrees with the localization of thioredoxin h (Trx h ), and supports the important function of the NTR/Trx system in cell proliferation and communication. Interestingly, NTR is found in the nuclei of seed cells suffering oxidative stress, thus showing co-localization with Trx h and 1-Cys Prx. To test whether the NTR/Trx system serves as a reductant of the 1-Cys Prx, we cloned a full-length cDNA encoding 1-Cys Prx from wheat, and expressed the recombinant protein in Escherichia coli . Using the purified components, we show NTR-dependent activity of the 1-Cys Prx. Mutants of the 1-Cys Prx allowed us to demonstrate that the peroxidatic residue of the wheat enzyme is Cys46, which is overoxidized in vitro under oxidant conditions. Analysis of extracts from developing and germinating seeds confirmed 1-Cys Prx overoxidation in vivo . Based on these results, we propose that NADPH is the source of the reducing power to regenerate 1-Cys Prx in the nuclei of seed cells suffering oxidative stress, in a process that is catalyzed by NTR.     

AtPER1 enhances primary seed dormancy and reduces seed germination by suppressing the ABA catabolism and GA biosynthesis in Arabidopsis seeds

Seed is vital to the conservation of germplasm and plant biodiversity. Seed dormancy is an adaptive trait in numerous seed‐plant species, enabling plants to survive under stressful conditions. Seed dormancy is mainly controlled by abscisic acid (ABA) and gibberellin (GA) and can be classified as primary and secondary seed dormancy. The primary seed dormancy is induced by maternal ABA. Here we found that AtPER1, a seed‐specific peroxiredoxin, is involved in enhancing primary seed dormancy. Two loss‐of‐function atper1 mutants, atper1‐1 and atper1‐2, displayed suppressed primary seed dormancy accompanied with reduced ABA and increased GA contents in seeds. Furthermore, atper1 mutant seeds were insensitive to abiotic stresses during seed germination. The expression of several ABA catabolism genes (CYP707A1, CYP707A2, and CYP707A3) and GA biosynthesis genes (GA20ox1, GA20ox3, and KAO3) in atper1 mutant seeds was increased compared to wild‐type seeds. The suppressed primary seed dormancy of atper1‐1 was completely reduced by deletion of CYP707A genes. Furthermore, loss‐of‐function of AtPER1 cannot enhance the seed germination ratio of aba2‐1 or ga1‐t, suggesting that AtPER1‐enhanced primary seed dormancy is dependent on ABA and GA. Additionally, the level of reactive oxygen species (ROS) in atper1 mutant seeds was significantly higher than that in wild‐type seeds. Taken together, our results demonstrate that AtPER1 eliminates ROS to suppress ABA catabolism and GA biosynthesis, and thus improves the primary seed dormancy and make the seeds less sensitive to adverse environmental conditions.     

Nonredundant antioxidant defense by multiple two-cysteine peroxiredoxins in human prostate cancer cells

BACKGROUND: Peroxiredoxins (Prxs) are antioxidant enzymes expressed by most free-living organisms, often in multiple isoforms. Because mammalian Prxs have not been experimentally deleted or inhibited, it is not known how much they contribute to antioxidant defense, nor whether the multiple isoforms afford redundant or additive protection. MATERIALS AND METHODS: Expression of the four members of the 2-Cys family of human Prxs was tested in human tumor cell lines. Monospecific antibodies were developed and used to monitor the extent and specificity of inhibition of expression of each isoform in prostate cancer cells stably transfected with antisense constructs. RESULTS: Seventeen tumor lines transcribed genes for all four human Prxs. Prostate cancer cells coexpressed each isoform at the protein level. Stable transfection with antisense allowed partial, selective suppression of Prx 1, 2, 3, or 4. Prostate cancer cells were rendered more sensitive to hydrogen peroxide or an organic hydroperoxide when Prx 1, 2, or 3 but not 4 was partially suppressed, bringing them into the range of sensitivity of mouse cells. The effect of partially suppressing a single Prx was comparable to that of depleting glutathione. In contrast, sensitization to adriamycin, an antitumor agent with a redox-active quinone, followed the partial suppression of Prxs 1, 2, or 4 but not 3. Individual suppression of Prxs 1-4 had no effect on sensitivity of the cells to reactive nitrogen intermediates, tumor necrosis factor (TNF), paclitaxel (Taxol), or etoposide. CONCLUSIONS: The 2-Cys Prxs act in a mutually nonredundant and sometimes stress-specific fashion to protect human cells from oxidant injury. The substantial resistance of human cells to hydroperoxides may result in part from the additive action of multiple Prxs.     

Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity

Peroxiredoxins are ubiquitously expressed proteins that reduce hydroperoxides using disulfur-reducing compounds as electron donors. Peroxiredoxins (Prxs) have been classified in two groups dependent on the presence of either one (1-Cys Prx) or two (2-Cys Prx) conserved cysteine residues. Moreover, 2-Cys Prxs, also named thioredoxin peroxidases, have peroxide reductase activity with the use of thioredoxin as biological electron donor. However, the biological reducing agent for the 1-Cys Prx has not yet been identified. We report here the characterization of a 1-Cys Prx from yeast Saccharomyces cerevisiae that we have named Prx1p. Prx1p is located in mitochondria, and it is overexpressed when cells use the respiratory pathway, as well as in response to oxidative stress conditions. We show also that Prx1p has peroxide reductase activity in vitro using the yeast mitochondrial thioredoxin system as electron donor. In addition, a mutated form of Prx1p containing the absolutely conserved cysteine as the only cysteine residue also shows thioredoxin-dependent peroxide reductase activity. This is the first example of 1-Cys Prx that has thioredoxin peroxidase activity. Finally, exposure of null Prx1p mutant cells to oxidant conditions reveals an important role of the mitochondrial 1-Cys Prx in protection against oxidative stress.     

Repeated Superovulation via PMSG/hCG Administration Induces 2-Cys Peroxiredoxins Expression and Overoxidation in the Reproductive Tracts of Female Mice

Superovulation induced by exogenous gonadotropin treatment (PMSG/hCG) increases the number of available oocytes in humans and animals. However, Superovulatory PMSG/hCG treatment is known to affect maternal environment, and these effects may result from PMSG/hCG treatment-induced oxidative stress. 2-Cys peroxiredoxins (2-Cys Prxs) act as antioxidant enzymes that protect cells from oxidative stress induced by various exogenous stimuli. Therefore, the objective of this study was to test the hypothesis that repeated PMSG/hCG treatment induces 2-Cys Prx expression and overoxidation in the reproductive tracts of female mice. Immunohistochemistry and western blotting analyses further demonstrated that, after PMSG/hCG treatment, the protein expression levels of 2-Cys Prxs increased most significantly in the ovaries, while that of Prx1 was most affected by PMSG/hCG stimulation in all tissues of the female reproductive tract. Repeated PMSG/hCG treatment eventually leads to 2-Cys Prxs overoxidation in all reproductive organs of female mice, and the abundance of the 2-Cys Prxs-SO_2/3 proteins reported here supports the hypothesis that repeated superovulation induces strong oxidative stress and damage to the female reproductive tract. Our data suggest that excessive oxidative stress caused by repeated PMSG/hCG stimulation increases 2-Cys Prxs expression and overoxidation in the female reproductive organs. Intracellular 2-Cys Prx therefore plays an important role in maintaining the reproductive organ environment of female mice upon exogenous gonadotropin treatment.     

The Time Required for Dormancy Release in Arabidopsis Is Determined by DELAY OF GERMINATION1 Protein Levels in Freshly Harvested Seeds

Seed dormancy controls the start of a plant's life cycle by preventing germination of a viable seed in an unfavorable season. Freshly harvested seeds usually show a high level of dormancy, which is gradually released during dry storage (after-ripening). Abscisic acid (ABA) has been identified as an essential factor for the induction of dormancy, whereas gibberellins (GAs) are required for germination. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION1 (DOG1) was recently identified as a major regulator of dormancy in Arabidopsis thaliana. Here, we show that the DOG1 protein accumulates during seed maturation and remains stable throughout seed storage and imbibition. The levels of DOG1 protein in freshly harvested seeds highly correlate with dormancy. The DOG1 protein becomes modified during after-ripening, and its levels in stored seeds do not correlate with germination potential. Although ABA levels in dog1 mutants are reduced and GA levels enhanced, we show that DOG1 does not regulate dormancy primarily via changes in hormone levels. We propose that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed dormancy release, which functions largely independent from ABA.     

Functional analysis and expression characteristics of chloroplastic Prx IIE

Peroxiredoxins (Prxs) are ubiquitous thiol-dependent peroxidases capable of eliminating a variety of peroxides through reactive catalytic cysteines, which are regenerated by reducing systems. Based on amino acid sequences and their mode of catalysis, five groups of thiol peroxidases have been distinguished in plants, and type II Prx is one of them with representatives in many sub-cellular compartments. The mature form of poplar chloroplastic Prx IIE was expressed as a recombinant protein in Escherichia coli . The protein is able to reduce H₂O₂ and tert-butyl hydroperoxide and is regenerated by both glutaredoxin (Grx) and thioredoxin (Trx) systems. Nevertheless, compared with Trxs, Grxs, and more especially chloroplastic Grx S12, are far more efficient reductants towards Prx IIE. The expression of Prx IIE at both the mRNA and protein levels as a function of organ type and abiotic stress conditions was investigated. Western blot analysis revealed that Prx IIE gene is constitutively expressed in Arabidopsis thaliana , mostly in young and mature leaves and in flowers. Under photo-oxidative treatment and water deficit, almost no change was observed in the abundance of Prx IIE in A.   thaliana , while the level of Prx Q (one of the two other chloroplastic Prxs with 2-Cys Prx) increased in response to both stresses, indicating that plastidic members of the Prx family exhibit specific patterns of expression under stress.     

Glutathionylation induces the dissociation of 1-Cys D-peroxiredoxin non-covalent homodimer

1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) pi via the formation of a GST-Prx heterodimer and Prx glutathionylation. In contrast, 1-Cys D-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys D-Prx induces the dissociation of the Prx non-covalent homodimer, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent dimer-monomer switch in the Prx family, similar to the decamer-dimer switch for the 2-Cys Prxs.     

The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.     

Sodium nitroprusside, cyanide, nitrite, and nitrate break Arabidopsis seed dormancy in a nitric oxide-dependent manner

The seeds of many plant species are dormant at maturity and dormancy loss is a prerequisite for germination. Numerous environmental and chemical treatments are known to lessen or remove seed dormancy, but the biochemical changes that occur during this change of state are poorly understood. Several lines of research have implicated nitric oxide (NO) as a participant in this process. Here, we show that dormant seeds of Arabidopsis thaliana (L.) Heynh. will germinate following treatment with the NO donor sodium nitroprusside (SNP), cyanide (CN), nitrite or nitrate. In all cases, the NO scavenger c-PTIO effectively promotes the maintenance of seed dormancy. c-PTIO does not, however, inhibit germination of fully after-ripened seeds, and c-PTIO does not interact directly with nitrite, nitrate or CN. We also show that volatile CN effectively breaks dormancy of Arabidopsis seeds, and that CN is the volatile compound in SNP that promotes dormancy loss. Our data support the hypothesis that NO is a signaling molecule that plays an important role in the loss of seed dormancy.     

Nitrate, a signal relieving seed dormancy in Arabidopsis

Nitrate is an important nitrogen source for plants, but also a signal molecule that controls various aspects of plant development. In the present study the role of nitrate on seed dormancy in Arabidopsis was investigated. The effects of either mutations affecting the Arabidopsis nitrate reductase genes or of different nitrate regimes of mother plants on the dormancy of the seeds produced were analysed. Altogether, data show that conditions favouring nitrate accumulation in mother plants and in seeds lead to a lower dormancy of seeds with little other morphological or biochemical differences. Analysis of germination during seed development indicated that nitrate does not prevent the onset of dormancy but rather its maintenance. The effect of an exogenous supply of nitrate on seed germination was tested: nitrate in contrast to glutamine or potassium chloride clearly stimulated the germination of dormant seeds. Data show, moreover, that the Arabidopsis dual affinity nitrate transporter NRT1.1 (CHL1) may be involved in conveying the nitrate signal into seeds. Thus, nitrate provided exogenously or by mother plants to the produced seeds, acts as a signal molecule favouring germination in Arabidopsis. This signalling may involve interaction with the abscisic acid or gibberellin pathway.     

Crystal structure of Escherichia coli thiol peroxidase in the oxidized state: insights into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins

Thioredoxin-dependent thiol peroxidase (Tpx) from Escherichia coli represents a group of antioxidant enzymes that are widely distributed in pathogenic bacterial species and which belong to the peroxiredoxin (Prx) family. Bacterial Tpxs are unique in that the location of the resolving cysteine (CR) is different from those of other Prxs. E. coli Tpx (EcTpx) shows substrate specificity toward alkyl hydroperoxides over H2O2 and is the most potent reductant of alkyl hydroperoxides surpassing AhpC and BCP, the other E. coli Prx members. Here, we present the crystal structure of EcTpx in the oxidized state determined at 2.2-A resolution. The structure revealed that Tpxs are the second type of atypical 2-Cys Prxs with an intramolecular disulfide bond formed between the peroxidatic (CP, Cys61) and resolving (Cys95) cysteine residues. The extraordinarily long N-terminal chain of EcTpx folds into a beta-hairpin making the overall structure very compact. Modeling suggests that, in atypical 2-Cys Prxs, the CR-loop as well as the CP-loop may alternately assume the fully folded or locally unfolded conformation depending on redox states, as does the CP-loop in typical 2-Cys Prxs. EcTpx exists as a dimer stabilized by hydrogen bonds. Its substrate binding site extends to the dimer interface. A modeled structure of the reduced EcTpx in complex with 15-hydroperoxyeicosatetraenoic acid suggests that the size and shape of the binding site are particularly suited for long fatty acid hydroperoxides consistent with its greater reactivity.     

Isolation and characterization of high temperature-resistant germination mutants of Arabidopsis thaliana

Temperature is a primary environmental cue for seed germination of many weeds and vegetables. To investigate the mechanism of germination regulation by temperature, we selected five high temperature (thermoinhibition)-resistant germination mutants (TRW lines) from 20,000 T-DNA insertion lines of Arabidopsis. Segregation analyses indicated that each of the five lines had single locus recessive mutations. The seeds of TRW134-15 and TRW187 showed reduced sensitivity to ABA and also to the gibberrellin biosynthesis inhibitor, paclobutrazol. Genetic and nucleotide sequencing analyses indicated that TRW187 is a new allele of abi3 (abi3-14). TRW71-1 exhibited a maternal effect for both thermoinhibition-resistant and transparent testa phenotypes, and genetic analysis revealed that the mutation was allelic to tt7 (tt7-4 sib). Interestingly, the seeds of reduced dormancy mutants rdo1, rdo2, rdo3 and rdo4 were also thermoinhibition tolerant, and all the TRW seeds showed reduced dormancy. Like rdo3, TRW13-1 had shorter siliques and slightly shorter stems than the wild type. The mutation of TRW13-1 was mapped to the bottom arm of chromosome 1 where rdo3 has also been mapped, but the two mutants are not allelic. We designated TRW13-1 as thermoinhibition-resistant germination 1 (trg1). We also mapped the ABA-insensitive mutation of TRW134-15 to the bottom arm of chromosome 5 and named it trg2. These results show that both embryo/endosperm and maternal factors contribute to germination inhibition at supraoptimal temperatures in Arabidopsis. In addition, we confirm the role of ABA in thermoinhibition of seed germination and a link between seed physiological dormancy and response to high temperature.     

Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins

Cysteine residues of certain peroxiredoxins (Prxs) undergo reversible oxidation to sulfinic acid (Cys-SO2H) and the reduction reaction is catalyzed by sulfiredoxin (Srx). Specific Cys residues of various other proteins are also oxidized to sulfinic acid, suggesting that formation of Cys-SO2H might be a novel posttranslational modification that contributes to regulation of protein function. To examine the susceptibility of sulfinic forms of proteins to reduction by Srx, we prepared such forms of all six mammalian Prx isoforms and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Purified sulfiredoxin reduced the sulfinic forms of the four 2-Cys members (Prx I to Prx IV) of the Prx family in vitro, but it did not affect those of Prx V, Prx VI, or GAPDH. Furthermore, Srx bound specifically to the four 2-Cys Prxs in vitro and in cells. Sulfinic forms of Prx I and Prx II, but not of Prx VI or GAPDH, present in H2O2-treated A549 cells were gradually reduced after removal of H2O2; overexpression of Srx increased the rate of the reduction of Prx I and Prx II but did not induce that of Prx VI or GAPDH. These results suggest that reduction of Cys-SO2H by Srx is specific to 2-Cys Prx isoforms. For proteins such as Prx VI and GAPDH, sulfinic acid formation might be an irreversible process that causes protein damage.