Relationship between active oxygen species and cardenolide production in cell cultures of Digitalis thapsi: effect of calcium restriction

Elimination of calcium ions from the medium of undifferentiated cell cultures of Digitalis thapsi increased cardenolide production and induced extracellular H₂O₂ accumulation, as measured by the quenching of pyranine fluorescence. The addition of catalase reduced the response and the inclusion of superoxide dismutase enhanced the loss of fluorescence. This suggested that, besides H₂O₂, the superoxide anion was also formed before dismutating to H₂O₂. Additionally, exogenous H₂O₂ or superoxide dismutase stimulated cardenolide production whereas the addition of catalase markedly reduced it. These results point to a connection between H₂O₂ and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products; however, changes in the antioxidant system of D. thapsi cells were observed. Catalase activity was extremely low in control cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase activity was not modified in calcium-free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutathione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol-bis-β-aminoethyl ether and LaCl₃. All these results indicate that the depletion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. thapsi cultures.     

Drought acclimation confers oxidative stress tolerance by inducing co-ordinated antioxidant defense at cellular and subcellular level in leaves of wheat seedlings

Wheat ( Triticum aestivum L.) seedlings of a drought-resistant cv. C306 were subjected to severe water deficit directly or through stress cycles of increasing intensity with intermittent recovery periods (drought acclimation). The antioxidant defense in terms of redox metabolites and enzymes in leaf cells, chloroplasts, and mitochondria was examined in relation to ROS-induced membrane damage. Drought-acclimated seedlings modulated growth by maintaining favorable turgor potential and RWC and were able to limit H₂O₂ accumulation and membrane damage as compared with non-acclimated plants during severe water stress conditions. This was due to systematic upregulation of H₂O₂-metabolizing enzymes especially ascorbate peroxidase (APX, EC 1.11.1.11) and by maintaining ascorbate–glutathione redox pool in acclimated plants. By contrast, failure in the induction of APX and ascorbate–glutathione cycle enzymes makes the chloroplast susceptible to oxidative stress in non-acclimated plants. Non-acclimated plants protected the leaf mitochondria from oxidative stress by upregulating superoxide dismutase (SOD, EC 1.15.1.1), APX, and glutathione reductase (GR, EC 1.6.4.2) activities. Rewatering led to rapid enhancement in all the antioxidant defense components in non-acclimated plants, which suggested that the excess levels of H₂O₂ during severe water stress conditions might have inhibited or downregulated the antioxidant enzymes. Hence, drought acclimation conferred enhanced oxidative stress tolerance by well-co-ordinated induction of antioxidant defense both at the chloroplast and at the mitochondrial level.     

Metabolism of hydrogen peroxide by the scavenging system in Chlamydomonas reinhardtii

The metabolism of hydrogen peroxide by the scavenging system was studied in Chlamydomonas grown in a selenium-lacking and a selenium-containing medium. In cells of the former, 40% of external hydrogen peroxide (H₂O₂) was scavenged by ascorbate peroxidase (AsAP; EC 1.11.1.11) and the residual H₂O₂ by catalase (EC 1.11.1.6). The enzymes involved in the ascorbate-glutathione cycle including AsAP. were localized in the chloroplast. In cells of the latter, glutathione peroxidase (GSHP; EC 1.11.1.9) functioned primarily in the removal of external H₂O₂. GSHP was located solely in the cytosol. The Chlamydomonas AsAP was relatively stable in ascorbate-depleted medium as compared with chloroplast AsAP of higher plants. No inactivation of the enzyme was found upon its incubation with hydroxyurea, an inhibitor of the chloroplast enzyme of higher plants. The enzyme showed higher specificity with pyrogallol than with ascorbate. The amino acid sequences in the N-terminal region of Chlamvdomonas AsAP showed no significant similarity to any other AsAP from higher plants and Euglena . The enzyme had a molecular mass of 34 kDa. The K_m values of the enzyme for ascorbate and H₂O₂ were 5.2±0.3 and 25±3.4 μ M , respectively. Hydrogen peroxide was generated at a rate of 6.1±0.8 μmol mg^-1 chlorophyll h^-1 in intact chloroplasts isolated from Chlamydomonas cells grown in the presence of Na-selenite, and it diffused from the organelles into the medium.     

Response of antioxidant systems to NaCl stress in the halophyte Cakile maritima

The effects of salt stress on antioxidative activities were investigated in a coastal halophyte, Cakile maritima . Two Tunisian accessions, Jerba and Tabarka, were compared. Plants were subjected to 100, 200, or 400 m M NaCl for 20 days. Parameters of oxidative stress [malondialdehyde (MDA), electrolyte leakage (EL), and hydrogen peroxide (H₂O₂) concentration], activities of several enzymes [superoxide dismutase (SOD), catalase (CAT), peroxydase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR)], and antioxidant molecules (ascorbate, ASC, and glutathione, GSH) were determined. Growth of Jerba plants was improved at 100 m M NaCl as compared to that of control. Tabarka growth was inhibited by salt at all NaCl concentrations. The relative salt tolerance of Jerba was associated with high antioxidant enzyme activities and glutathione content, together with low MDA content, EL, and H₂O₂ concentration. Lower antioxidant activities and higher MDA content, EL, and H₂O₂ concentration were found in Tabarka. As a whole, these data suggest that the capacity to limit oxidative damage is important for salt tolerance of C. maritima .     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

Drought-induced spikelet sterility is associated with an inefficient antioxidant defence in rice panicles

Water stress-induced spikelet sterility limits rice production under upland conditions. The causes of spikelet sterility under drought stress are poorly understood. In this study the role of antioxidant defence management in drought-induced spikelet sterility was investigated in two rice ( Oryza sativa ) genotypes differing in drought resistance. Drought-resistant N22 genotype showed less water stress-induced spikelet sterility when compared to the susceptible N118 genotype under upland conditions. The N22 panicles maintained higher RWC and turgor potential and lower H₂O₂ levels across the developmental stages under water stress than that of N118 panicles. Drought-induced enhancement in superoxide dismutase (SOD, EC 1.15.1.1) activity coupled with higher ascorbate (AsA), glutathione (GSH) content and enhanced ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities resulted in lower H₂O₂ levels in N22 panicles. In contrast, insufficient enhancement in SOD, APX and GR activities resulted in relatively higher H₂O₂ levels under water stress in N118 panicles. The N22 panicles exhibited a higher number of SOD and APX isozymes in comparison with N118 panicles that might provide better reactive oxygen species scavenging. Hence it is concluded that well-equipped antioxidant defence plays an important role in minimizing water stress-induced spikelet sterility in upland rice.     

NaCl treatment markedly enhances H2O2-scavenging system in leaves of halophyte Suaeda salsa

The C₃ halophyte Suaeda salsa L. grown under the high concentration of NaCl (200 m M ) was used to investigate the role of the hydrogen peroxide (H₂O₂)-scavenging system [catalase, ascorbate peroxidase, glutathione reductase (GR), ascorbic acid, and glutathione (GSH)] in removal of reactive oxygen species. The activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and GR (EC 1.6.4.2) increased significantly after 7 days of NaCl treatment. The isoform patterns of CAT and GR were not affected, but the staining intensities were significantly increased by NaCl treatment. Activities of both the thylakoid-bound APX or GR and stromal APX (S-APX) or GR in the chloroplasts were markedly enhanced under high salinity. Fifty percent of APX in the chloroplasts is thylakoid-bound APX. S-APX and GR activity represented about 74–78 and 64–71% of the total soluble leaf APX and GR activity, respectively. Salt treatment increased the contents of ascorbic acid and GSH. By contrast, a decreased content of H₂O₂ was found in the leaves of NaCl-treated S . salsa . The level of membrane lipid peroxidation decreased slightly after NaCl treatment. The plants grew well with high rate of net photosynthesis under high salinity. These data suggest that upregulation of the H₂O₂-scavenging system in plant cells, especially in the chloroplasts, is at least one component of the tolerance adaptations of halophytes to high salinity.     

Carbaryl-induced effects on glutathione content, glutathione reductase and superoxide dismutase activity of the cyanobacterium Nostoc muscorum

The carbamate insecticide carbaryl, at concentrations of 10 mg/l and above, significantly stimulated glutathione reductase (GR) and superoxide dismutase (SOD) activity in the cyanobacterium Nostoc muscorum. A low content of total glutathione (GSH + GSSG), decreased photosynthetic activity, and an increased level of H₂O₂ was observed in pesticide treated cyanobacteria. As no glutathione peroxidase was observed in this species, stimulation of GR and SOD activity, higher production of H₂O₂, and low glutathione level was attributed to the utilization of GSH to remove H₂O₂ spontaneously and nonenzymatically under conditions of pesticide toxicity.     

Ascorbate-glutathione cycle and H2O2 detoxification in elongating leaf bases of ryegrass: effect of inhibition of glutathione reductase activity on foliar regrowth

The role of the ascorbate-glutathione cycle and AOS detoxification was investigated during leaf growth of defoliated and undefoliated plants of ryegrass ( Lolium perenne L. cv. Bravo). Antioxidants and related enzymatic activities were located in elongating leaf bases (ELBs) of undefoliated plants, following a decreasing gradient from basal (meristem) to distal segments, inverse to H₂O₂ levels. In the meristematic zone, the intense activity of the ascorbate-glutathione cycle and the supply of reducing power by the oxidative pentose phosphate pathway allowed the maintenance of both antioxidant reduction and H₂O₂ detoxification. BCNU (1–3 bis(2-chloroethyl)- N -nitrosourea), a glutathione reductase inhibitor, induced an increase in the meristematic zone in both H₂O₂ and antioxidant levels and a decrease in reduced/oxidized ratios of glutathione and ascorbate. These changes were associated with a reduced foliar regrowth activity. In the absence of BCNU, defoliation did not modify the ratios of reduced/oxidized antioxidants, although it triggered a temporary increase in H₂O₂ level. The results are discussed on the basis of a possible control of leaf growth by glutathione and ascorbate.     

Plant glutathione peroxidases

Oxidative stress in plants causes the induction of several enzymes, including superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The first two are responsible for converting superoxide to H₂O₂ and its subsequent reduction to H₂O, and the third is involved in recycling of ascorbate. Glutathione peroxidases (GPXs, EC 1.11.1.9) are a family of key enzymes involved in scavenging oxyradicals in animals. Only recently, indications for the existence of this enzyme in plants were reported. Genes with significant sequence homology to one member of the animal GPX family, namely phospholipid hydroperoxide glutathione peroxidase (PHGPX), were isolated from several plants. Cit-SAP, the protein product encoded by the citrus csa gene, which is induced by salt-stress, is so far the only plant PHGPX that has been isolated and characterized. This protein differs from the animal PHGPX in its rate of enzymatic activity and in containing a Cys instead of selenocysteine (Sec) as its presumed catalytic residue. The physiological role of Cit-SAP and its homologs in other plants is not yet known.     

Cytoprotective effect of polypeptide from Chlamys farreri on neuroblastoma (SH-SY5Y) cells following H2O2 exposure involves scavenging ROS and inhibition JNK phosphorylation

Oxidative stress has long been linked to cell death in many neurodegenerative conditions. Treatment with antioxidants is a promising approach for slowing disease progression. In this study, we used the neuroblastoma SH-SY5Y cells as an in vitro model to first assess the effect of polypeptide from Chlamys farreri (PCF), a natural marine antioxidant, on H₂O₂-induced neuronal cell death. Pre-treatment of SH-SY5Y cells with PCF inhibited H₂O₂-induced cell death in a concentration-dependent manner. In parallel, intracellular reactive oxygen species generation and lipid peroxidation were inhibited by PCF. Under severe H₂O₂ insult, PCF promoted endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, and glutathione. PCF also protected DNA from oxidative damage and enhanced the removal of 8-oxo-7,8-dihydro-2'-deoxyguanosine from DNA. Further, we found that PCF potentially prevented H₂O₂–induced cell apoptosis. When investigated mitogen-activated protein kinase signaling pathway, we found that pre-treatment of cells with PCF significantly blocked H₂O₂–induced phosphorylation of c- Jun N-terminal kinase of the mitogen-activated protein kinase family. However, PCF had little inhibitory effect on the H₂O₂–induced activation of extracellular signal-regulated kinase. Taken together, these data demonstrate that PCF prevents oxidative stress-induced reactive oxygen species production and c- Jun N-terminal kinase activation and may be useful in the treatment of neurodegenerative diseases.     

Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings

Vigna cutjang Endl. cv. Pusa Barsati seedlings, subjected to increasing degrees of water stress (−0.5, −1.0, −1,5 MPa), produced an approximately proportional increase in glycolate oxidase activity, hydrogen peroxide (H₂O₂) and proline content but a decrease in catalase activity, ascorbic acid and protein content. Leaf water potential (leaf ψ) and relative water content (RWC) were also lowered with increasing stress. Pretreatment with l -cysteine and reduced glutathione (10-3 M) decreased glycolate oxidase activity, H₂O₂ content, ascorbic acid oxidase activity, proline content and also slightly improved the water status of leaves stressed (−1.0 MPa) for 2 days. Pretreatment of non-stressed seedlings with these antioxidants had little or no effect. These studies indicate that treatment with antioxidants makes the plant tolerant against water stress by modulating the endogenous levels of H₂O₂ and ascorbic acid in stressed tissue.     

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Characterisation and changes in the antioxidant system of chloroplasts and chromoplasts isolated from green and mature pepper fruits

Purification and characterisation of pepper ( Capsicum annuum L) chloroplasts and chromoplasts isolated from commercial green, red and yellow mature fruits were undertaken. Induction of the synthesis of several antioxidants in organelles isolated from mature fruits was found. The ultrastructure of organelles and the presence and activity of SOD isozymes and enzymes involved in the ASC-GSH cycle, together with the non-enzymatic antioxidant content and some oxidative parameters, were analysed. It was found that lipids, rather than proteins, seem to be a target for oxidation in the chromoplasts. The ascorbate and glutathione contents were elicited during differentiation of chloroplasts into chromoplasts in both red and yellow fruits. The activity of SOD and of components of the ASC-GSH cycle was up-regulated, suggesting that these enzymes may play a role in the protection of plastids and could act as modulators of signal molecules such as O₂^˙− and H₂O₂ during fruit maturation. The presence of an Mn-SOD in chromoplasts isolated from yellow pepper fruits was also investigated in terms of structural and antioxidant differences between the two cultivars.     

Inhibition of Dopamine Release by Prostaglandin EP3 Receptor via Pertussis Toxin-Sensitive and -Insensitive Pathways in PC12 Cells

Abstract: Hydrogen peroxide (H₂O₂) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H₂O₂ on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H⁺-ATPase (V-ATPase) in the vesicle membrane. We report here that the V-ATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H₂O₂. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H₂O₂-induced inhibition of the V-ATPase, and ATP or GTP partially protect the ATPase from inhibition by H₂O₂. These and other results suggest that the mechanism of inhibition of the V-ATPase by H₂O₂ involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V-ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down-regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.     

The effect of short-term H2S fumigation on water-soluble sulphydryl and glutathione levels in spinach

Abstract. Short-term fumigation of Spinacia oleracea with 380 μg m⁻³ H₂S (250 ppb) resulted in a rapid accumulation of water-soluble SH-compounds in the shoots. After 1 h exposure a substantial increase in the SH-content was already detectable and maximal accumulation, three- to four-fold that in control plants, was observed after 24 h of exposure. Irradiation during H₂S exposure only slightly affected the rate and level of SH-accumulation. H₂S fumigation did not affect the water-soluble SH-content of the roots. Glutathione was the sole water-soluble SH-compound accumulating upon exposure to H₂S. It was calculated that during the first hour of exposure to 380 μg m⁻³ H₂S 39% of the possible absorbed H₂S was converted into glutathione. The SH-content of the water-soluble proteins of the shoots was not affected by H₂S exposure. When fumigation was stopped, a rapid decrease in glutathione content was observed and after 48 h the content was comparable to that of the control plants. Contrary to H₂S, SO₂ fumigation did not result in a rapid accumulation of glutathione in spinach shoots. The possible role of glutathione accumulation during H₂S fumigation is discussed.     

Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit

The role of APX (ascorbate peroxidase) in protection against oxidative stress was examined using transgenic tobacco plants. The full length cDNA, coding Arabidopsis thaliana L. APX fused downstream to the chloroplast transit sequence from A. thaliana glutathione reductase, was cloned into appropriate binary vector and mobilized into Agrobacterium tumefaciens C58C2. Leaf discs were infected with the Agrobacterium and cultured on medium supplied with kanamycin. The incorporation of the gene in tobacco genome was confirmed by Southern dot blot hybridization. Transgenic lines were generated, and the line Chl-APX5 shown to have 3.8-fold the level of APX activity in the wild-type plants. The isolated chloroplasts from this line showed higher APX activity. During early investigation, this line showed enhanced tolerance to the active oxygen-generating paraquat and sodium sulphite. The first generation of this line, also, showed enhanced tolerance to salt, PEG and water stresses, as determined by net photosynthesis. The present data indicate that overproducing the cytosolic APX in tobacco chloroplasts reduces the toxicity of H₂O₂.     

Effects of cucumber mosaic virus infection on electron transport and antioxidant system in chloroplasts and mitochondria of cucumber and tomato leaves

We examined the responses of the photosynthetic and respiratory electron transport and antioxidant systems in cell organelles of cucumber ( Cucumis sativus L.) and tomato ( Lycopersicon esculentum Mill.) leaves to infection of cucumber mosaic virus (CMV) by comparing the gas exchange, Chl fluorescence, respiratory electron transport, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate–glutathione (AsA–GSH) cycle enzymes and the production of H₂O₂ in chloroplasts, mitochondria and soluble fraction in virus-infected and non-infected leaves. Long-term CMV infection resulted in decreased photosynthesis and respiration rates. Photosynthetic electron flux to carbon reduction, respiratory electron transport via both complex I and complex II and also the Cyt respiration rate all significantly decreased, while photosynthetic alternative electron flux and alternative respiration significantly increased. These changes in electron transport were accompanied by a general increase in the activities of SOD/AsA–GSH cycle enzymes followed by an increased H₂O₂ accumulation in chloroplasts and mitochondria. These results demonstrated that disturbance of photosynthetic and respiratory electron transport by CMV also affected the antioxidative systems, thereby leading to oxidative stress in various organelles.