Glutathione Redox Status Research Articles

Optogenetic Monitoring of the Glutathione Redox State in Engineered Human Myocardium.

Redox signaling affects all aspects of cardiac function and homeostasis. With the development of genetically encoded fluorescent redox sensors, novel tools for the optogenetic investigation of redox signaling have emerged. Here, we sought to develop a human heart muscle model for in-tissue imaging of redox alterations. For this, we made use of (1) the genetically-encoded Grx1-roGFP2 sensor, which reports changes in cellular glutathione redox status (GSH/GSSG), (2) human embryonic stem cells (HES2), and (3) the engineered heart muscle (EHM) technology. We first generated HES2 lines expressing Grx1-roGFP2 in cytosol or mitochondria compartments by TALEN-guided genomic integration. Grx1-roGFP2 sensor localization and function was verified by fluorescence imaging. Grx1-roGFP2 HES2 were then subjected to directed differentiation to obtain high purity cardiomyocyte populations. Despite being able to report glutathione redox potential from cytosol and mitochondria, we observed dysfunctional sarcomerogenesis in Grx1-roGFP2 expressing cardiomyocytes. Conversely, lentiviral transduction of Grx1-roGFP2 in already differentiated HES2-cardiomyocytes and human foreskin fibroblast was possible, without compromising cell function as determined in EHM from defined Grx1-roGFP2-expressing cardiomyocyte and fibroblast populations. Finally, cell-type specific GSH/GSSG imaging was demonstrated in EHM. Collectively, our observations suggests a crucial role for redox signaling in cardiomyocyte differentiation and provide a solution as to how this apparent limitation can be overcome to enable cell-type specific GSH/GSSG imaging in a human heart muscle context.

H1/H2 Histamine Receptor Blockade Alters the Glutathione Redox Status in Skeletal Muscle Following a Bout of Prolonged Exercise

The effects of an acute bout of prolonged exercise on muscle include increases in oxidative metabolism and altered cellular redox environment. Histamine mediates many acute exercise responses, including sustained vasodilation and associated delivery of substrates to muscle. Previously, we demonstrated that H1 and H2 histamine receptor blockade with oral antihistamines resulted in a diminished capacity for mitochondrial H2O2 emission in skeletal muscle following an acute bout of prolonged exercise. Here, we tested the hypothesis that along with diminishing mitochondrial H2O2 emission, H1 and H2 histamine receptor blockade would attenuate exercise‐induced changes in skeletal muscle glutathione redox status. Adult male Wistar rats (83 ± 5.5 days old; 440.04 ± 40.70 g) were assigned to one of four groups (n = 12/group): exercise, in the form of one hour of continuous treadmill running at 20 cm/sec (Group 1); mass‐specific oral gavage with H1 antagonist fexofenadine (7.11 mg/kg) and H2 antagonist ranitidine (3.95 mg/kg) one hour prior to the same exercise protocol (Group 2); a group receiving oral H1 and H2 antagonists, but not exercised (Group 3); and a control group, which received neither antihistamines nor exercise (Group 4). An hour post‐exercise, total, reduced (GSH) and oxidized glutathione (GSSG) were assessed in red gastrocnemius (mixed fiber types) and soleus (oxidative) skeletal muscles. Redox status was expressed as GSH/GSSG. While no changes were observed in total glutathione for any treatment in either muscle, in soleus, antihistamine treatment, but not exercise decreased GSH/GSSG (main effect; p < 0.05). In red gastrocnemius, exercise, but not antihistamine treatment decreased GSH/GSSH (main effect; p < 0.05). These observations trend with those of a blunted capacity for mitochondrial H2O2 emission in skeletal muscle with antihistamine treatment and suggest an altered redox response to exercise following H1/H2 histamine receptor blockade in soleus. Given the widespread and routine use of antihistamines, further research into the effects of these antagonists on redox status in muscle is warranted.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada, Nova Scotia Health Research Foundation, Canada Foundation for Innovation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abscisic acid-determined seed vigour differences do not influence redox regulation during ageing.

High seed quality is a key trait to achieve successful crop establishment required for optimum yield and sustainable production. Seed storage conditions greatly impact two key seed quality traits; seed viability (ability to germinate and produce normal seedlings) and vigour (germination performance). Accumulated oxidative damage accompanies the loss of seed vigour and viability during ageing, indicating that redox control is key to longevity. Here, we studied the effects of controlled deterioration at 40°C and 75% relative humidity (RH) ('ageing') under two different O2 concentrations (21 and 78% O2) in Brassica oleracea Two B. oleracea genotypes with allelic differences at two QTLs that result in differences in abscisic acid (ABA) signalling and seed vigour were compared. Ageing led to a similar loss in germination speed in both genotypes that was lost faster under elevated O2 In both genotypes, an equal oxidative shift in the glutathione redox state and a minor loss of α-tocopherol progressively occurred before seed viability was lost. In contrast, ABA levels were not affected by ageing. In conclusion, both ABA signalling and seed ageing impact seed vigour but not necessarily through the same biochemical mechanisms.

Rooibos (Aspalathus linearis) and honeybush (Cyclopia species) modulate the oxidative stress associated injury of diesel exhaust particles in human umbilical vein endothelial cells

BackgroundPrevious evidence show foods and beverages rich in polyphenolic compounds to have favourable effects on the cardiovascular system. HypothesisThe current study assessed the modulation of oxidative stress and associated inflammation induced by diesel exhaust particles (DEP - SRM 2975) by pre-treatment of human umbilical vein endothelial cells (HUVECs) with aqueous extracts of rooibos [fermented (FR) as well as green form (GR)] and honeybush [fermented form (FH)]. Study designHUVEC are either exposed to DEP (10 µg/ml) for 4 h or pre-treated with 40 and 60 µg/ml of FR or GH or FR, or 50 µg/ml orientin (OR) for 6 h prior to DEP exposure. MethodsIn vitro antioxidant capacity of the extracts was assessed and the polyphenol contents were also assessed by HPLC. ROS, cell viability, lactate dehydrogenase leakage, lipid peroxidation, GSH:GSSG ratios, conjugated diene and protein carbonyl levels were determined as indices of oxidative stress and cytotoxicity. RT-qPCR and western blot were used to assess inflammatory cytokines and antioxidant genes expression. ResultsDEP caused a dose and time-dependent increase in ROS production, significant (p < 0.001) increase in protein carbonyl (PC) formation, thiobarbituric acid reactive substances and conjugated dienes levels (p < 0.01) and a significant reduction in glutathione (GSH) redox status. Pre-incubation with either the herbal extracts or orientin attenuated these effects. The significant increase in IL-1α, IL-6, IL-8, VCAM-1 and ATF4 gene expression caused by DEP (10 µg/ml) were also attenuated by the presence of the FR, GR and FH extracts, and OR . Pre-treatment with the rooibos extracts or flavone orientin enhanced cell viability, reduced LDH leakage, enhanced mRNA expression of NQO1 and Nrf2, but repressed CYP1B1 mRNA induced by DEP. Western blot showed both the herbal tea extracts and orientin to enhance NQO1 and γGSC protein induction by DEP. ConclusionTaken together, the herbal extracts offer protection against DEP-induced oxidative stress and inflammatory response.

Генетически кодируемый биосенсор roKate для регистрации редокс-состояния пула глутатиона

Genetically encoded fluorescent sensors are exploited to study a variety of biological processes in living organisms in real time. In recent years, a whole family of biosensors has been developed, serving to visualize changes in the glutathione redox state. The aim of our experiment was to design a biosensor based on the red fluorescent protein mKate2 for measuring the 2GSH/GSSG ratio. A pair of cysteine amino acid residues were introduced into the structure of the fluorescent protein using site-directed mutagenesis. These residues form a disulfide bridge when the surrounding glutathione pool is oxidized, affecting the spectral characteristics of the protein. Our biosensor, which we called roKate, was tested in vitro on an isolated protein. Specifically, we examined the spectral characteristics, pH and the redox potential of the sensor. Additionally, the performance of roKate was evaluated using the culture of living mammalian cells. The fluorescent signal emitted by the sensor was very bright and remarkably stable under pH conditions varying in the physiological range. Irreversibly oxidized in mammalian cells, roKate stands out from other members of this biosensor family. This biosensor should be preferred in the experiments when the time between the manipulations with the biological object and the subsequent analysis of the induced effect is substantial, as is the case with long sample preparation.

The clinical significance of serum oxidative stress biomarkers in breast cancer females

Background: This study was conducted to evaluate the role of some serum oxidative stress biomarkers for breast cancer diagnosis, incidence and monitoring the effects of surgery and chemotherapy. Methods: A total of 35 breast cancer patients (before surgery, after two weeks of surgery and after 6 cy­cles of chemotherapy) and 35 normal healthy controls were analyzed for serum oxidative stress markers including total antioxidant capacity (TAC), malondialdehyde (MDA), total, reduced (GSH) and oxidized (GSSG) glutathione and glutathione redox status (GSH/GSSG). Results: The serum levels of MDA and GSSG were significantly higher in breast cancer patients than controls. The serum levels of GSH, TAC and GSH/GSSG ratio were significantly lower in breast cancer patients than controls. After surgery, the serum levels of MDA and GSSG were significantly decreased, while the serum levels of GSH were significantly increased, compared with their levels before surgery. Six cycles of chemotherapy showed the non-significant effect on the serum levels of the assayed biomarkers. ROC curve analysis demonstrated that MDA and GSH were superior to the GSH/GSSG ratio, TAC and GSSG. Increased levels of MDA and GSSG and reduced levels of GSH, TAC and GSH/GSSG ratio were found to significantly increase the risk of breast cancer. Conclusions: All of the assayed biomarkers can be used for prediction of breast cancer with MDA and GSH being superior to the others. MDA, GSH and GSSG were able to monitor the effect of surgery. All of the assayed biomarkers were found to be associated with breast cancer risk. None of the assayed biomarkers was able to predict the effect of chemotherapy.

Redox Systemic Signaling and Induced Tolerance Responses During Soybean-Bradyrhizobium japonicum Interaction: Involvement of Nod Factor Receptor and Autoregulation of Nodulation.

The symbiotic relationship between legumes and nitrogen-fixing rhizobia induces local and systemic responses, which ultimately lead to nodule formation. The autoregulation of nodulation (AON) is a systemic mechanism related to innate immunity that controls nodule development and involves different components ranging from hormones, peptides, receptors to small RNAs. Here, we characterized a rapid systemic redox changes induced during soybean–Bradyrhizobium japonicum symbiotic interaction. A transient peak of reactive oxygen species (ROS) generation was found in soybean leaves after 30 min of root inoculation with B. japonicum. The ROS response was accompanied by changes in the redox state of glutathione and by activation of antioxidant enzymes. Moreover, the ROS peak and antioxidant enzyme activation were abolished in leaves by the addition, in either root or leaf, of DPI, an NADPH oxidase inhibitor. Likewise, these systemic redox changes primed the plant increasing its tolerance to photooxidative stress. With the use of non-nodulating nfr5-mutant and hyper-nodulating nark-mutant soybean plants, we subsequently studied the systemic redox changes. The nfr5-mutant lacked the systemic redox changes after inoculation, whereas the nark-mutant showed a similar redox systemic signaling than the wild type plants. However, neither nfr5- nor nark-mutant exhibited tolerance to photooxidative stress condition. Altogether, these results demonstrated that (i) the early redox systemic signaling during symbiotic interaction depends on a Nod factor receptor, and that (ii) the induced tolerance response depends on the AON mechanisms.

Plasma Albumin Redox State Is Responsive to the Amino Acid Balance of Dietary Proteins in Rats Fed a Low Protein Diet.

We recently reported that plasma albumin redox state, which correlates with albumin synthesis rate, could be associated with the quality of dietary protein. Aiming to elucidate the association between them, plasma albumin redox state was investigated in rats fed various kinds of AIN-93G-based low protein diets. Plasma albumin redox state was shifted to a more oxidized state in rats fed 3% casein (CN) diet than those fed 3% whey protein or 3% wheat gluten diet, while supplementing 3% CN diet with cystine reversed it to a more reduced state, indicating that cystine would complement the shortage of cysteine in CN, thereby increasing albumin synthesis rate. Supplementation with glutathione, a cysteine-containing antioxidative tripeptide, normalized hepatic glutathione redox state modulated by ingestion of 3% CN diet, but it only reversed the oxidized shift of plasma albumin redox state to an extent similar to cystine alone or the constituting amino acid mixture of glutathione (i.e., glutamic acid, cystine, and glycine), indicating that glutathione would primarily serve as a source of cysteine rather than exert its antioxidative activity. Plasma albumin would thus be influenced by amino acid balance in dietary proteins, and it could be useful as a biomarker that contributes to prevention of protein under-nutriton, caused by not only insufficient protein intake but also ingestion of poor-quality protein.

Glutathione-redox status on hydro alcoholic root bark extract of Premna integrifolia Linn in high fat diet induced atherosclerosis model

BackgroundPremna integrifolia Linn. is a medicinal plant of an Ayurvedic importance and proved to have an anti-inflammatory, anti-diabetic, anti-microbial and hypo-lipidemic activity. Glutathione (GSH) redox status is an important parameter to assess the antioxidant activity of any neutraceuticals. ObjectiveIn order to assess the anti-oxidant potential of hydro alcoholic extract (HAE) of P. integrifolia, this study was aimed to evaluate the GSH redox status in high fat diet induced experimental atherosclerosis. Materials and methodsThe present study comprises sixty Wistar rats and they were divided into six groups: the first group served as control, the second group was fed with high fat diet and the third, fourth and fifth groups were fed with high fat diet along with various concentrations of HAE of 200, 400 and 500 g/kg.b.wt respectively and the sixth group was administered high fat diet along with 10 mg/kg b.wt of atorvastatin for 30 days. GSH-dependent enzymes like GSH-peroxidase (GPx), GSH-reductase (GR) and glucose 6-phosphate dehydrogenase (G6PD) were estimated in hemolysate, kidney, heart and liver of experimental rats. ResultsAnalysis of GSH levels showed a significant decrease in hemolysate, heart and kidney (p < 0.05) and liver (p < 0.01) in high fat-fed rats when compared to control. Activities of GPx, GR and G6PD in hemolysate and heart (p < 0.001), liver and kidney (p < 0.05) in high fat-fed rats when compared to control. Dose-dependent increase was observed in rats treated with various concentrations of HAE. ConclusionThe HAE of root bark of P. integrifolia is proved to have a protective role on antioxidant defense in high fat diet induced atherosclerosis model. As a whole P. integrifolia increases the GSH content in a dose-dependent manner and in turn altered the redox cycle.

ANTIHYPERGLYCEMIC, ANTIOXIDANT AND ANTI-INFLAMMATORY EFFECTS OF AQUEOUS EXTRACT OF MISTLETOE (Cladocolea loniceroides) IN STZ-INDUCED DIABETIC MICE

Background: Inhibition of carbohydrate hydrolyzing enzymes, such as α-amylase and α-glucosidase, is a key element in the regulation of diabetes mellitus (DM). The purpose of this work was to study the inhibition of carbohydrate hydrolyzing enzymes, and the antihyperglycemic activity of aqueous extract of Cladocolea (C. loniceroides) in streptozotocin (STZ)- induced diabetic mice. Materials and Methods: The inhibitory activities of C. loniceroides aqueous extract on α-amylase and α-glucosidase were investigated in vitro. Glucose tolerance test was performed in normoglycemic (NG) mice which were fed with starch or sucrose. The effect of mistletoe aqueous extract (ME) was measured in (STZ)-induced diabetic mice. On day 35 of the treatment, the effect of decreasing oxidative stress (lipid peroxidation, glutathione redox state, GPx and GR specific activities, cytokines and aminotransferases analysis) was assessed. Results: ME showed a competitive mode of inhibition for the carbohydrate hydrolyzing enzymes (CHE). The maximum antihyperglycemic activity in mice was observed for the unripe fruit aqueous extract (UFAE) for -amylase and stem aqueous extract (SAE); for -glucosidase due to the glycemic response reduction by 23% or 35%, respectively. UFAE decreased malondialdehyde (MDA) 1.76 times; GSH/GSSG ratio was mantained (3.08 ± 0.66); GPx activity was reduced (24%); IL-6 was reduced (18%) and the concentration of TNF- (37%) was leveled with respect to the (STZ)-induced diabetic mice; ALT and AST (liver transaminases) levels were nearly the same compared with those found in the NG mice. Conclusion: UFAE of C. loniceroides exhibited the highest antidiabetic activity in (STZ)-induced diabetic mice.

Effects of N-Acetyl-Cysteine Supplementation through Drinking Water on the Glutathione Redox Status during the Weaning Transition of Piglets.

This study investigated the effect of N-acetyl-cysteine (NAC) supplementation through drinking water on animal performance and the glutathione (GSH) redox system in weaned piglets, particularly in relation to the immediate post-weaning feed intake. To this end, 168 piglets were weaned and either fed ad libitum or fasted the first two days, and either or not administered 200 mg/L NAC via the drinking water until d14 post-weaning. Next to animal performance until day 42 (d42), the GSH redox system was measured in erythrocytes, small intestinal mucosa, liver, lung, and kidney tissue at d0, d2, and d14 post-weaning. Animal performance and GSH levels were not affected by NAC, nor by fasting. Irrespective of treatment, a significant drop in GSH at d2 post-weaning was found as compared to d0, in particular in liver (−69%), distal jejunal mucosa (−72%), and lung tissue (−80%). Post-weaning changes of the GSH redox status were strongly tissue-dependent. To conclude, this research indicates that GSH redox homeostasis was largely affected in multiple organs during the weaning transition. NAC supplementation did not increase GSH levels in any tissue, not even in fasted animals, questioning the fact if cysteine is the first or only limiting factor determining the rate of GSH synthesis in the early post-weaning phase.

Impact of a dietary challenge with peroxidized oil on the glutathione redox status and integrity of the small intestine in weaned piglets

Glutathione (GSH) is considered to play an important role in maintaining the integrity of the small intestine. In piglets, altered mucosal GSH levels might therefore be involved in weaning-induced changes of the small intestinal morphology and barrier function. To test this hypothesis, we aimed to challenge the mucosal GSH redox status during the first 28 days after weaning, by feeding diets containing 5% fresh linseed oil (CON), or 2.5% (OF1) or 5% (OF2) peroxidized linseed oil (peroxide value 225 mEq O2/kg oil) and exploring the effects on gut integrity. Piglets were pair-fed and had a total daily feed allowance of 32 g/kg BW. A fourth treatment included animals that were fed the control diet ad libitum (ACON). Animals were sampled at days 5 and 28 post-weaning. The malondialdehyde (MDA) concentration and GSH redox status (GSH/GSSG Eh) were determined in blood, liver and small intestinal mucosa. Histomorphology of the duodenal and jejunal mucosa was determined, and Ussing chambers were used to assess fluorescein isothiocyanate dextran (FD4) and horseradish peroxidase (HRP) fluxes across the mucosa. Results show that peroxidized linseed oil imposed an oxidative challenge at day 28, but not at day 5 post-weaning. At day 28, increasing levels of dietary peroxides to pair-fed pigs linearly increased MDA levels in duodenal and jejunal mucosa. Moreover, FD4 fluxes were significantly increased in OF1 (+75%) and OF2 (+64%) in the duodenum, and HRP fluxes tended (P=0.099) to show similar differences, as compared to CON. This co-occurred with a significant 11 mV increase of the hepatic GSH/GSSG Eh, potentiated by a significantly increased GSH peroxidase activity for treatments OF1 (+47%) and OF2 (+63%) in liver as compared to CON. Furthermore; duodenal HRP flux significantly correlated with the hepatic glutathione disulphide (GSSG) level (r=0.650), as also observed in the jejunum for hepatic GSSG (r=0.627) and GSH/GSSG Eh (r=0.547). The jejunal permeability was not affected, but FD4 and HRP fluxes significantly correlated with the local GSH (r=0.619; r=0.733) and GSSG (r=0.635; r=0586) levels. Small intestinal histomorphology was not affected by dietary lipid peroxides, nor were there any correlations found with the GSH redox system. To conclude, under oxidative stress conditions, jejunal barrier function is related to the local and hepatic GSH redox system. It is suggested that the hepatic GSH system participates in the elimination of luminal peroxides, and thereby impacts on duodenal barrier function.

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.

Redox Regulation of Monodehydroascorbate Reductase by Thioredoxin y in Plastids Revealed in the Context of Water Stress.

Thioredoxins (TRXs) are key players within the complex response network of plants to environmental constraints. Here, the physiological implication of the plastidial y-type TRXs in Arabidopsis drought tolerance was examined. We previously showed that TRXs y1 and y2 have antioxidant functions, and here, the corresponding single and double mutant plants were studied in the context of water deprivation. TRX y mutant plants showed reduced stress tolerance in comparison with wild-type (WT) plants that correlated with an increase in their global protein oxidation levels. Furthermore, at the level of the main antioxidant metabolites, while glutathione pool size and redox state were similarly affected by drought stress in WT and trxy1y2 plants, ascorbate (AsA) became more quickly and strongly oxidized in mutant leaves. Monodehydroascorbate (MDA) is the primary product of AsA oxidation and NAD(P)H-MDA reductase (MDHAR) ensures its reduction. We found that the extractable leaf NADPH-dependent MDHAR activity was strongly activated by TRX y2. Moreover, activity of recombinant plastid Arabidopsis MDHAR isoform (MDHAR6) was specifically increased by reduced TRX y, and not by other plastidial TRXs. Overall, these results reveal a new function for y-type TRXs and highlight their role as major antioxidants in plastids and their importance in plant stress tolerance.

Glutathione provides antioxidative defence and promotes microspore-derived embryo development in isolated microspore cultures of triticale (\xd7\u2009Triticosecale Wittm.)

Key messageDepending on the capability for stress adaptation, the role played by glutathione in microspore embryogenesis consists of both antioxidative activity and stimulation of embryo-like structure development.The efficiency of microspore embryogenesis (ME) is determined by the complex network of internal and environmental factors. Among them, the efficient defence against oxidative stress seems to be one of the most important. The present study confirms this hypothesis showing the positive effect of glutathione—the most abundant cellular antioxidant—on ME in isolated microspore cultures of triticale (× Triticosecale Wittm.). For the first time, low temperature (LT) pre-treatment of tillers was combined with the exogenous application of glutathione and associated with the total activity of low-molecular weight antioxidants, the endogenous content and redox status of glutathione, and the effectiveness of ME. The results indicate that efficient antioxidative defence is the first, although not the only, prerequisite for effective ME. In responsive genotypes, LT alone stimulated antioxidative defence and decreased cell redox status, which was associated with increased cell viability and high frequency (ca. 20%) of microspore reprogramming. Application of glutathione had no effect either on the microspore viability or on the initial number of embryogenic microspores. However, it increased the number of embryo-like structures, probably by stimulating the next phases of its development. In recalcitrant genotypes, the main role of glutathione seems to be its participation in cell protection from oxidative stress. However, even enhanced antioxidative activity, which sustained cell viability and increased the number of embryogenic microspores, was insufficient for efficient haploid/doubled haploid plant production. Evidently, there are still other defective elements in the complex network of factors that regulate the process of ME.

281 - Valproic acid inhibits superoxide dismutase activity in mouse P19 cells

Valproic Acid (VPA) is an antiepileptic drug that is also used now for many other indications, including bipolar and mood disorders. A serious drawback of VPA is that it can induce neural tube defects (NTD) to those exposed in utero. The mechanisms of VPA-induced NTD remain incomplete, but oxidative stress has been implicated. To better understand, the cause of oxidative stress, we measured the activity of superoxide dismutase (SOD) following VPA treatment. SOD is an antioxidant enzyme that when inhibited may also affect the intracellular ROS tone and redox status.P19 cells are embryo carcinoma cells that retain some differentative properties. In cells treated with VPA (5 mM), dichloroflouroscein (DCF) fluorescence as an indicator of ROS production was significantly increased and was similar to ROS generated in cells treated with an SOD inhibitor, LCS-1 (100 µM). In SOD activity assays, VPA caused a decrease (20%) in activity after 6 h. Loss of activity was not due to changes in SOD protein concentrations as immunoblot analysis showed SOD expression to be virtually unchanged with VPA treatment. Also at 6 h of exposure, glutathione redox states (Eh) were highly oxidized as compared to untreated controls. As such, data support that VPA inhibits SOD activity and thereby may serve as a mechanism by which exposure to increases ROS availability and alters intracellular redox states. Understanding these effects may shed some light on embryotoxicity of VPA and its role in NTD pathogenesis.

Drought tolerance in alfalfa (Medicago sativa L.) varieties is associated with enhanced antioxidative protection and declined lipid peroxidation

Drought stress is considered the most adverse factor restricting plant survival, growth, and productivity. The identification of the key adaptive mechanisms to drought stress is essential to enhance the drought resistance of plants. In this study, differential responses of three alfalfa varieties to drought, including Medicago sativa L. cv. Longzhong (drought-tolerant), Longdong (moderate drought-tolerant), and Gannong No. 3 (drought-sensitive), were comparatively studied at morphological, physio-biochemical, and transcriptional levels after a 12-day period of drought stress simulated by −1.2 MPa polyethylene glycol (PEG-6000). The results showed that prolonged drought stress dramatically decreased growth and photosynthetic capacity of three alfalfa varieties while it increased the accumulation of malondialdehyde (MDA), reactive oxygen species (ROS), osmolytes and antioxidants including reduced ascorbate and glutathione, ascorbate peroxidase (APX) activities, and gene expression of antioxidative enzymes (MsCu/Zn-SOD, MsFeSOD, MtPOD, MsGPX, MsAPX, MsMDAR, MtDHAR, and MsGR). Nine days of treatment and some key traits, including the maximum quantum yield of photosystem II (Fv/Fm), the levels of MDA, O2−, and H2O2, the redox states of ascorbate and glutathione, APX activity, and the transcript levels of MsFeSOD, MsGR, and MsMDAR, might contribute to differentiating the drought stress tolerance in alfalfa. Overall, drought-tolerant Longzhong showed the highest water retention, photosynthetic performance, and osmoregulation capacity, the lowest lipid peroxidation, and the highest antioxidant enzyme activities and gene expression, which were mainly involved in the ascorbate-glutathione cycle to maintain the balance between the generation and scavenging of intracellular ROS. These findings highlight that enhanced antioxidative protection and declined lipid peroxidation play an important role in alfalfa tolerance against drought.

Analyzing the Function of Catalase and the Ascorbate-Glutathione Pathway in H2O2 Processing: Insights from an Experimentally Constrained Kinetic Model.

Plant stress involves redox signaling linked to reactive oxygen species such as hydrogen peroxide (H2O2), which can be generated at high rates in photosynthetic cells. The systems that process H2O2 include catalase (CAT) and the ascorbate-glutathione pathway, but interactions between them remain unclear. Modeling can aid interpretation and pinpoint areas for investigation. Recent Advances: Based on emerging data and concepts, we introduce a new experimentally constrained kinetic model to analyze interactions between H2O2, CAT, ascorbate, glutathione, and NADPH. The sensitivity points required for accurate simulation of experimental observations are analyzed, and the implications for H2O2-linked redox signaling are discussed. We discuss several implications of the modeled results, in particular the following. (i) CAT and ascorbate peroxidase can share the load in H2O2 processing even in optimal conditions. (ii) Intracellular H2O2 concentrations more than the low μM range may rarely occur. (iii) Ascorbate redox turnover is largely independent of glutathione until ascorbate peroxidation exceeds a certain value. (iv) NADPH availability may determine glutathione redox status through its influence on monodehydroascorbate reduction. (v) The sensitivity of glutathione status to oxidative stress emphasizes its potential suitability as a sensor of increased H2O2. Important future questions include the roles of other antioxidative systems in interacting with CAT and the ascorbate-glutathione pathway as well as the nature and significance of processes that achieve redox exchange between different subcellular compartments. Progress in these areas is likely to be favored by integrating kinetic modeling analyses into experimentally based programs, allowing each approach to inform the other.

Changes in tocochromanols and glutathione reveal differences in the mechanisms of seed ageing under seedbank conditions and controlled deterioration in barley

“Orthodox” seeds are desiccation tolerant, enabling dry storage for agricultural and conservational purposes. Protocols of “controlled deterioration” (CD) using elevated temperatures and seed water contents (WCs) are frequently used to study mechanisms of seed ageing. However, the relevance of using CD to understand how seeds age at lower water contents and temperatures has been disputed. Here, comparisons in antioxidant changes are drawn between barley seed stored for up to 15 years in a seedbank with seeds aged using CD (45 °C, 75% RH; WC = 13% FW) under normoxia (21%) or elevated (75%) O2. Regardless of ageing method, glutathione (GSH) levels decreased concomitantly with an oxidative shift in the glutathione redox state (EGSSG/2GSH) as viability was lost. An earlier oxidative shift in EGSSG/2GSH and more rapid loss of seed vigour was found in seeds aged by CD under 75% O2, compared to 21% O2, but longevity was not shortened by elevated O2. Seedbank-aged seeds contained more glutathione disulphide (GSSG) levels, leaked more electrolytes upon imbibition and contained less tocochromanols than non-stored seeds. In contrast, in response to CD, levels of GSSG fell, electrolyte leakage did not increase and tocochromanol levels increased (21% O2), or remained stable (75% O2). In a further set of 24 barley genotypes stored for up to 15 years at 20 °C (WC = 11% FW) and 0 °C (WC = 8% FW), tocochromanol levels positively (r = 0.42) correlated with total germination, which ranged between 1-78% (20 °C) and 65-97% (0 °C). Of all tocochromanol species, α-tocopherol correlated most with total germination (r = 0.49) and γ-tocotrienol least (r = 0.08). It is concluded that CD is not a perfect model for understanding mechanisms of ageing that occur under seedbank conditions.

Nitrate- and nitric oxide-induced plant growth in pea seedlings is linked to antioxidative metabolism and the ABA/GA balance

This study looks at the effects of potassium nitrate (KNO3) and sodium nitroprusside (SNP), a nitric oxide (NO)-donor, on the development, antioxidant defences and on the abscisic acid (ABA) and gibberellin (GA) levels in pea seedlings. Results show that 10 mM KNO3 and 50 μM SNP stimulate seedling fresh weight (FW), although this effect is not reverted by the action of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO-scavenger.The KNO3 treatment increased peroxidase (POX) and ascorbate oxidase (AOX) activities. SNP, on the other hand, reduced monodehydroascorbate reductase (MDHAR) activity and produced a significant increase in superoxide dismutase (SOD), POX and AOX activities. The “KNO3 plus cPTIO” treatment increased ascorbate peroxidase (APX), MDHAR, glutathione reductase (GR) and SOD activities, but POX activity decreased in relation to the KNO3 treatment. The “SNP plus cPTIO” treatment increased APX and MDHAR activities, whereas a huge decrease in POX activity occurred. Both the KNO3 and the SNP treatments increased reduced ascorbate (ASC) concentrations, which reached control values in the presence of cPTIO. All treatments increased the dehydroascorbate (DHA) level in pea seedlings, leading to a decrease in the redox state of ascorbate. In the “KNO3 plus cPTIO” treatment, an increase in the redox state of ascorbate was observed. Glutathione contents, however, were higher in the presence of SNP than in the presence of KNO3. In addition, KNO3 produced an accumulation of oxidised glutathione (GSSG), especially in the presence of cPTIO, leading to a decrease in the redox state of glutathione. The effect of SNP on reduced glutathione (GSH) levels was reverted by cPTIO, suggesting that NO has a direct effect on GSH biosynthesis or turnover.Both the KNO3 and SNP treatments produced an increase in GA4 and a decrease in ABA concentrations, and this effect was reverted in the presence of the NO-scavenger. Globally, the results suggest a relationship between antioxidant metabolism and the ABA/GA balance during early seedling growth in pea. The results also suggest a role for KNO3 and NO in the modulation of GA4 and ABA levels and antioxidant metabolism in pea seedlings. Furthermore, this effect correlated with an increase in the biomass of the pea seedlings.