Resolving diurnal dynamics of the chloroplastic glutathione redox state in Arabidopsis reveals its photosynthetically derived oxidation

Plants are subjected to fluctuations in light intensity, and this might cause unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). Electrons needed for ROS detoxification are drawn, at least partially, from the cellular glutathione (GSH) pool via the ascorbate–glutathione cycle. Here, we explore the dynamics of the chloroplastic glutathione redox potential (chl-E_GSH) using high-temporal-resolution monitoring of Arabidopsis (Arabidopsis thaliana) lines expressing the reduction–oxidation sensitive green fluorescent protein 2 (roGFP2) in chloroplasts. This was carried out over several days under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-E_GSH oxidation during dark-to-light and light-to-dark transitions were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, suggesting two regulated oxidative states of the chl-E_GSH. These patterns were not affected in npq1 plants, which are impaired in non-photochemical quenching. Oscillations between the two oxidation states were observed under fluctuating light in WT and npq1 plants, but not in pgr5 plants, suggesting a role for PSI photoinhibition in regulating the chl-E_GSH dynamics. Remarkably, pgr5 plants showed an increase in chl-E_GSH oxidation during the nights following light stresses, linking daytime photoinhibition and nighttime GSH metabolism. This work provides a systematic view of the dynamics of the in vivo chloroplastic glutathione redox state during varying light conditions.

Monitoring the daily in vivo dynamics of the chloroplastic GSH redox state in light-stressed wild-type plants versus photoprotective mutants provides insight into the photosynthesis-dependent production of oxidants.     

Distinct Redox Regulation in Sub-Cellular Compartments in Response to Various Stress Conditions in Saccharomyces cerevisiae

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (E _GSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. E _GSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (−340 to −350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H⁺/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H⁺/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions.     

Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite Plasmodium falciparum

In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (E _GSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects E _GSH changes induced by oxidative and nitrosative stress. The cytosolic basal E _GSH of 3D7 and Dd2 were estimated to be −314.2±3.1 mV and −313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in E _GSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites'' E _GSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular E _GSH in P. falciparum. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in Plasmodium and might also stimulate redox research in other pathogens.     

The Response of Antioxidant Enzymes in Cellular Organelles in Cucumber (Cucumis sativus L.) Leaves to Methyl Viologen-induced Photo-oxidative Stress

In order to clarify the response of antioxidant systems in various cellular organelles to photo-oxidative stress, the activities of superoxide dismutase (SOD) and enzymes of the ascorbate–glutathione (AsA-GSH) cycle were investigated in chloroplasts, mitochondria and cytosol of cucumber leaves subjected to methyl viologen (MV) treatment. Photo-oxidation by MV resulted in significant reductions in net photosynthetic rate (Pn) and increases in the ratio of the quantum efficiency of photosystem II (PSII), Φ_PSII to that of the quantum efficiency of CO₂ fixation (ΦCO₂), followed by increased activities of SOD, and a general increase of AsA-GSH cycle enzymes in chloroplasts, mitochondria and cytosol. These increases were however, most significant in chloroplasts. There were also significant increases in dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) except that the content of ascorbate (AsA) in chloroplasts and cytosol was slightly decreased and little effected, respectively. However, GSSG in mitochondria and GSH in cytosol were little influenced by the MV treatment. The activity of ascorbate oxidase (AO) in these organelles was independent of the MV treatment while the activity of l-galactono-1,4- lactone dehydrogenase (GLDH) in mitochondria was slightly inhibited by MV treatment. These results indicate that disturbance of electron transport in chloroplasts by MV influenced the metabolism of whole cell by a crosstalk signaling system and that the AsA-GSH cycle played a primary role in sustaining the levels of AsA.     

Sensing stress responses in potato with whole-plant redox imaging

Environmental stresses are among the major factors that limit crop productivity and plant growth. Various nondestructive approaches for monitoring plant stress states have been developed. However, early sensing of the initial biochemical events during stress responses remains a significant challenge. In this work, we established whole-plant redox imaging using potato (Solanum tuberosum) plants expressing a chloroplast-targeted redox-sensitive green fluorescence protein 2 (roGFP2), which reports the glutathione redox potential (E_GSH). Ratiometric imaging analysis demonstrated the probe response to redox perturbations induced by H₂O₂, DTT, or a GSH biosynthesis inhibitor. We mapped alterations in the chloroplast E_GSH under several stress conditions including, high-light (HL), cold, and drought. An extremely high increase in chloroplast E_GSH was observed under the combination of HL and low temperatures, conditions that specifically induce PSI photoinhibition. Intriguingly, we noted a higher reduced state in newly developed compared with mature leaves under steady-state and stress conditions, suggesting a graded stress sensitivity as part of the plant strategies for coping with stress. The presented observations suggest that whole-plant redox imaging can serve as a powerful tool for the basic understanding of plant stress responses and applied agricultural research, such as toward improving phenotyping capabilities in breeding programs and early detection of stress responses in the field.

Whole-plant imaging of potato plants expressing a genetically encoded biosensor allows for spatially resolved and nondestructive mapping of stress-induced redox perturbations.     

Measuring Glutathione Redox Potential of HIV-1-infected Macrophages

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E_GSH; Grx1-roGFP2) and measured subcellular changes in E_GSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E_GSH (approximately −310 mV), active viral replication induces substantial oxidative stress (E_GSH more than −240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H₂O₂), induces significant deviations in subcellular E_GSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E_GSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E_GSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.     

Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis

Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (E_GSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal E_GSH dynamics, we show that stromal E_GSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.     

Early perturbation in mitochondria redox homeostasis in response to environmental stress predicts cell fate in diatoms

Diatoms are ubiquitous marine photosynthetic eukaryotes that are responsible for about 20% of global photosynthesis. Nevertheless, little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a redox-sensitive green fluorescent protein sensor targeted to various subcellular organelles in the marine diatom Phaeodactylum tricornutum, to map the spatial and temporal oxidation patterns in response to environmental stresses. Specific organelle oxidation patterns were found in response to various stress conditions such as oxidative stress, nutrient limitation and exposure to diatom-derived infochemicals. We found a strong correlation between the mitochondrial glutathione (GSH) redox potential (E_GSH) and subsequent induction of cell death in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), and a volatile halocarbon (BrCN) that mediate trophic-level interactions in marine diatoms. Induction of cell death in response to DD was mediated by oxidation of mitochondrial E_GSH and was reversible by application of GSH only within a narrow time frame. We found that cell fate can be accurately predicted by a distinct life-death threshold of mitochondrial E_GSH (−335 mV). We propose that compartmentalized redox-based signaling can integrate the input of diverse environmental cues and will determine cell fate decisions as part of algal acclimation to stress conditions.     

Monitoring the in vivo redox state of plant mitochondria: Effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge

In animals, the impact of ROS production by mitochondria on cell physiology, death, disease and ageing is well recognised. In photosynthetic organisms such as higher plants, however, the chloroplast and peroxisomes are the major sources of ROS during normal metabolism and the importance of mitochondria in oxidative stress and redox signalling is less well established. To address this, the in vivo oxidation state of a mitochondrially-targeted redox-sensitive GFP (mt-roGFP2) was investigated in Arabidopsis leaves. Classical ROS-generating inhibitors of mitochondrial electron transport (rotenone, antimycin A and SHAM) had no effect on mt-roGFP oxidation when used singly, but combined inhibition of complex III and alternative oxidase by antimycin A and SHAM did cause significant oxidation. Inhibitors of complex IV and aconitase also caused oxidation of mt-roGFP2. This oxidation was not apparent in the cytosol whereas antimycin A + SHAM also caused oxidation of cytosolic roGFP2. Menadione had a much greater effect than the inhibitors, causing nearly complete oxidation of roGFP2 in both mitochondria and cytosol. A range of severe abiotic stress treatments (heat, salt, and heavy metal stress) led to oxidation of mt-roGFP2 while hyperosmotic stress had no effect and low temperature caused a slight but significant decrease in oxidation. Similar changes were observed for cytosolic roGFP2. Finally, the recovery of oxidation state of roGFP in mitochondria after oxidation by H₂O₂ treatment was dramatically slower than that of either the cytosol or chloroplast. Together, the results highlight the sensitivity of the mitochondrion to redox perturbation and suggest a potential role in sensing and signalling cellular redox challenge.     

High Resolution Imaging of Temporal and Spatial Changes of Subcellular Ascorbate,Glutathione and H2O2 Distribution during Botrytis cinerea Infection in Arabidopsis

In order to study the mechanisms behind the infection process of the necrotrophic fungus Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H₂O₂) was monitored over a time frame of 96 h post inoculation (hpi) in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS) and the area around the IS which was defined as area adjacent to the inoculation site (AIS). H₂O₂ accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi) correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS) which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H₂O₂ in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts (e.g. decrease of chloroplast number, decrease of starch and thylakoid contents, increase of plastoglobuli size), chlorosis and necrosis of the leaves.     

Diurnal fluctuations in chloroplast GSH redox state regulate susceptibility to oxidative stress and cell fate in a bloom‐forming diatom

Diatoms are one of the key phytoplankton groups in the ocean, forming vast oceanic blooms and playing a significant part in global primary production. To shed light on the role of redox metabolism in diatom's acclimation to light–dark transition and its interplay with cell fate regulation, we generated transgenic lines of the diatom Thalassiosira pseudonana that express the redox‐sensitive green fluorescent protein targeted to various subcellular organelles. We detected organelle‐specific redox patterns in response to oxidative stress, indicating compartmentalized antioxidant capacities. Monitoring the GSH redox potential (E_GSH) in the chloroplast over diurnal cycles revealed distinct rhythmic patterns. Intriguingly, in the dark, cells exhibited reduced basal chloroplast E_GSH but higher sensitivity to oxidative stress than cells in the light. This dark‐dependent sensitivity to oxidative stress was a result of a depleted pool of reduced glutathione which accumulated during the light period. Interestingly, reduction in the chloroplast E_GSH was observed in the light phase prior to the transition to darkness, suggesting an anticipatory phase. Rapid chloroplast E_GSH re‐oxidation was observed upon re‐illumination, signifying an induction of an oxidative signaling during transition to light that may regulate downstream metabolic processes. Since light–dark transitions can dictate metabolic capabilities and susceptibility to a range of environmental stress conditions, deepening our understanding of the molecular components mediating the light‐dependent redox signals may provide novel insights into cell fate regulation and its impact on oceanic bloom successions.     

The expression of BrMDHAR gene in chloroplasts and mitochondria enhances tolerance to freezing stress in Arabidopsis thaliana

In chloroplasts and mitochondria, antioxidant mechanisms include the ascorbate-glutathione cycle, and monodehydroascorbate reductase (MDHAR) is important for regeneration of ascorbate (AsA) from monodehydroascorbate (MDHA). To improve detoxification of reactive oxygen species (ROS), we established a construct of the MDHAR gene from Brassica rapa fused to the targeting signal peptides of Pisum sativum glutathione reductase (GR), which was controlled by a stress-inducible SWPA2 promoter, and introduced this expression system into Arabidopsis thaliana. Transgenic (TG) plants overexpressing BrMDHAR targeted to chloroplasts and mitochondria through signal peptides showed an elevated MDHAR activity and an increased ratio of AsA to dehydroascorbate (DHA) when compared to wild-type (WT) plants under a freezing stress. These led to increased photosynthetic parameters, redox homeostasis, and biomass in TG plants when compared to the WT plants. Our results suggest that the overexpression of the BrMDHAR gene targeted to chloroplasts and mitochondria conferred an enhanced tolerance against the freezing stress, and a stress adaptation of dual-targeted BrMDHAR was better than that of single BrMDHAR.     

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H₂O₂ in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H₂O₂ levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H₂O₂ is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).     

Higher sensitivity of pad2-1 and vtc2-1 mutants to cadmium is related to lower subcellular glutathione rather than ascorbate contents

Cadmium (Cd) interferes with ascorbate and glutathione metabolism as it induces the production of reactive oxygen species (ROS), binds to glutathione due to its high affinity to thiol groups, and induces the production of phytochelatins (PCs) which use glutathione as a precursor. In this study, changes in the compartment specific distribution of ascorbate and glutathione were monitored over a time period of 14 days in Cd-treated (50 and 100 μM) Arabidopsis Col-0 plants, and two mutant lines deficient in glutathione (pad2-1) and ascorbate (vtc2-1). Both mutants showed higher sensitivity to Cd than Col-0 plants. Strongly reduced compartment specific glutathione, rather than decreased ascorbate contents, could be correlated with the development of symptoms in these mutants suggesting that higher sensitivity to Cd is related to low glutathione contents rather than low ascorbate contents. On the subcellular level it became obvious that long-term treatment of wildtype plants with Cd induced the depletion of glutathione and ascorbate contents in all cell compartments except chloroplasts indicating an important protective role for antioxidants in chloroplasts against Cd. Additionally, we could observe an immediate decrease of glutathione and ascorbate in all cell compartments 12 h after Cd treatment indicating that glutathione and ascorbate are either withdrawn from or not redistributed into other organelles after their production in chloroplasts, cytosol (production centers for glutathione) and mitochondria (production center for ascorbate). The obtained data is discussed in respect to recently proposed stress models involving antioxidants in the protection of plants against environmental stress conditions.     

In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis

The glutathione redox couple (GSH/GSSG) and hydrogen peroxide (H₂O₂) are central to redox homeostasis and redox signaling, yet their distribution within an organism is difficult to measure. Using genetically encoded redox probes in Drosophila, we establish quantitative in vivo mapping of the glutathione redox potential (E_GSH) and H₂O₂ in defined subcellular compartments (cytosol and mitochondria) across the whole animal during development and aging. A chemical strategy to trap the in vivo redox state of the transgenic biosensor during specimen dissection and fixation expands the scope of fluorescence redox imaging to include the deep tissues of the adult fly. We find that development and aging are associated with redox changes that are distinctly redox couple-, subcellular compartment-, and tissue-specific. Midgut enterocytes are identified as prominent sites of age-dependent cytosolic H₂O₂ accumulation. A longer life span correlated with increased formation of oxidants in the gut, rather than a decrease.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L.

Abstract

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E_{GSSG/2 GSH}) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E_{CySS/2 Cys}). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a ‘thiol–disulphide redox environment’ (E_{thiol–disulphide}), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E_{CySS/2 Cys} to E_{thiol–disulphide} in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous nonprotein antioxidant defense molecule is the tripeptide glutathione (γ-glutamylcysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days postfertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione using HPLC and calculated the whole embryo total glutathione (GSH_T) concentrations and redox potentials (E_h) over 0–120 h of zebrafish development (including mature oocytes, fertilization, midblastula transition, gastrulation, somitogenesis, pharyngula, prehatch embryos, and hatched eleutheroembryos). GSH_T concentration doubled between 12 h postfertilization (hpf) and hatching. The GSH E_h increased, becoming more oxidizing during the first 12 h, and then oscillated around −190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) E_h (−220 mV). After hatching, E_h stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis.     

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X_L protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.