A dissection of the effects of ethylene,H2O2 and high irradiance on antioxidants and several genes associated with stress and senescence in tobacco leaves

Ethylene and hydrogen peroxide are involved in the modulation of stress responses in plants, but their interrelation is not well understood. This work was designed to find differences between the actions of ethylene and H₂O₂ on antioxidants and senescence markers. Leaves of Nicotiana tabacum were sprayed with H₂O₂ or with ethephon (precursor of ethylene). To find the possible modulation of responses to acute abiotic stress, ethephon- and H₂O₂-sprayed leaves were further subjected to high irradiance (HL). The application of H₂O₂ strongly stimulated ethylene synthesis (ACC). Ethylene and H₂O₂, as single factors, stimulated the trolox equivalent antioxidant capacity (TEAC) and the activity of catalase (CAT), in contrast to HL alone (stimulation of nonspecific peroxidases and the total glutathione pool). However, after combined treatments (ethylene + HL and H₂O₂ + HL), the stimulatory action of H₂O₂ was related to TEAC and CAT activity, while the application of ethylene stimulated the total glutathione pool. Hydrogen peroxide enhanced the expression of the three CAT genes (Cat1, Cat2 and Cat3), in contrast to ethylene (Cat2 and Cat3) and HL (Cat1). In regard to the markers of senescence and pathogenesis the most pronounced difference between the actions of ethylene and H₂O₂, as single factors, was related to NPR1, whereas when leaf spraying was combined with HL, differences were found at WRKY53 and PR1a. HL reversed the stimulatory effects of H₂O₂/ethylene-driven enhancements of the expression of several genes (Cat1, Cat2, NPR1, WRKY53). These results show that multiple stressors, as usually encountered by plants in nature, may largely change those expression patterns of genes determined in a single factor analysis. Moreover, the actions of HL (often considered the internal H₂O₂ trigger) and of exogenous H₂O₂ on gene expression are clearly different.     

Partial purification and characterization of a copper-induced anionic peroxidase of sunflower roots

Treatment of 14-day-old sunflower seedlings with a toxic amount of copper (50 μM of CuSO₄) during 5 days caused significant increase in peroxidase activity in roots. Qualitative analysis of soluble proteins using native anionic PAGE followed by detection of peroxidase activity with guaïacol as electron donor in the presence of H₂O₂ revealed five stimulated peroxidases, named A1, A2, A3, A4, and A5. These peroxidases had differential behavior during the period of treatment. A1, A2, A3 and A4 were stimulated in the first period of stress, but rapidly suppressed at 72 h. A5 showed a progressive stimulation which was even increased at 120 h. A1 was partially purified, identified using liquid chromatography coupled to mass spectrometry (LC-MS/MS), and characterized. Effects of pH and temperature on its activity were determined with guaïacol as electron donor. Optima were obtained at pH 8 and at 40 °C. Analysis of substrate specificity showed that A1 was active on coniferyl alcohol but not on IAA. Enzymatic activity was inhibited by a high concentration of H₂O₂.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Modulation of antioxidant enzyme activities and metabolites ratios by nitric oxide in short-term salt stressed soybean root nodules

Several abiotic factors cause molecular damage to plants either directly or through the accumulation of reactive oxygen species such as hydrogen peroxide (H₂O₂). We investigated if application of nitric oxide (NO) donor 2,2′-(hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) could reduce the toxic effect resulting from short-term salt stress. Salt treatment (150 mM NaCl) alone and in combination with 10 μM DETA/NO or 10 μM DETA were given to matured soybean root nodules for 24 h. Salt stress resulted in high H₂O₂ level and lipid peroxidation while application of DETA/NO effectively reduced H₂O₂ level and prevented lipid peroxidation in the soybean root nodules. NO treatment increased the activities of ascorbate peroxidase and dehydroascorbate reductase under salt stress. Whereas short-term salt stress reduced AsA/DHAsA and GSH/GSSG ratios, application of the NO donor resulted in an increase of the reduced form of the antioxidant metabolites thus increasing the AsA/DHAsA and GSH/GSSG ratios. Our data suggests a protective role of NO against salt stress.     

Antioxidant response of a novel Streptomyces sp. M3004 isolated from legume rhizosphere to H2O2 and paraquat

This paper aims to investigate the effect of H₂O₂ and paraquat on the activities of superoxide dismutase (SOD) and catalase (CAT), and membrane lipid peroxidation (LPO) levels in newly isolated Streptomyces sp. M3004. SOD activities of Streptomyces sp. M3004, grown in 10 mM and 30 mM H₂O₂, were significantly lower than the control cultures. On the other hand, as an antioxidant enzyme, CAT activity in both H₂O₂ treatment conditions increased significantly compared with the control. These activity values in 10 mM and 30 mM H₂O₂ treatment on the 48th hour of incubation were 3.8- and 6.6-fold higher than the control, respectively. SOD activity decreased significantly with respect to paraquat concentration, which was added at the start of the incubation. CAT activities increased significantly in 1.0 mM and 3.0 mM paraquat treatments compared to control. As an indicative marker of membrane damage, LPO levels of the novel isolate Streptomyces sp. M3004 treated with H₂O₂, and paraquat stress conditions were significantly higher than the control. Nevertheless, compared with the 30 mM H₂O₂ in both treatment conditions, LPO levels in 10 mM H₂O₂ were significantly higher. The decreases in SOD activities in paraquat and H₂O₂ treatment conditions resulted in the increases in the LPO levels although it increases in CAT activities.     

Low stomatal density and reduced transpiration facilitate strawberry adaptation to salinity

Water and soil salinization are major constraints to agricultural productions because plant adaptation to hyperosmotic environments is generally associated to reduced growth and ultimately yield loss. Understanding the physiological/molecular mechanisms that link adaptation and growth is one of the greatest challenges in plant stress research since it would allow us to better define strategies to improve crop salt tolerance. In this study we attempted to establish a functional link between morphological and physiological traits in strawberry in order to identify margins to “uncouple” plant growth and stress adaptation. Two strawberry cultivars, Elsanta and Elsinore, were grown under 0, 10, 20 and 40 mM NaCl. Upon salinization Elsanta plants maintained a larger and more functional leaf area compared to Elsinore plants, which were irreversibly damaged at 40 mM NaCl. The tolerance of Elsanta was correlated with a constitutive reduced transpirational flux due to low stomatal density (173 vs. 234 stomata mm⁻² in Elsanta and Elsinore, respectively), which turned out to be critical to pre-adapt plants to the oncoming stress. The reduced transpiration rate of Elsanta (14.7 g H₂O plant⁻¹ h⁻¹) respect to Elsinore (17.7 g H₂O plant⁻¹ h⁻¹) most likely delayed the accumulation of toxic ions into the leaves, preserved tissues dehydration and consented to adjust more effectively to the hyperosmotic environment. Although we cannot rule out the contribution of other physiological and molecular mechanisms to the relatively higher tolerance of Elsanta, here we demonstrate that low stomatal density may be beneficial for cultivars prescribed to be used in marginal environments in terms of salinity and/or drought.     

Acute effects of cocaine,morphine and their combination on bioenergetic function and susceptibility to oxidative stress of rat liver mitochondria

AimsCocaine and heroin are frequently co-abused in a combination known as speedball. Despite the relevance of the liver in the metabolism and detoxification of these drugs, little is known about the impact of speedball on liver function.Main methodsIn this work, we evaluated the effects of cocaine, morphine and morphine + cocaine (Mor + Coc) combination (1:1) in isolated rat liver mitochondria, upon glutamate/malate or succinate energization, on bioenergetics and oxidative stress-related parameters by using Clark O₂, Ca^2 +, TPP⁺ and pH electrodes and by measuring thiobarbituric acid reactive substances (TBARS) and H₂O₂ production.Key findingsCocaine and Mor + Coc at the higher concentrations (1 mM) similarly increased O₂ consumption at state 2, state 4 and state oligomycin. In these conditions, maximum respiration was decreased only upon glutamate/malate energization, suggesting an involvement of complex I. Morphine (1 mM) only increased state 2 respiration. Cocaine and Mor + Coc induced a similar decrease in maximum mitochondrial membrane potential and in ADP-induced depolarization, whereas morphine had no effect. The drugs and their combination similarly decreased mitochondrial ATPase activity and had no effect on Ca^2 +-induced permeability transition. Morphine and Mor + Coc prevented lipid peroxidation, since in these conditions there was a decrease in O₂ consumption and in TBARS upon ADP/Fe^2 + stimulus, and a decrease in H₂O₂ formation, suggesting an antioxidant effect. Interestingly, heroin did not share morphine antioxidant properties.SignificanceOur results show that the sequential direct exposure of liver mitochondria to morphine and cocaine does not alter the effects observed in the presence of each drug alone.     

Improved tolerance toward low temperature in banana (Musa AAA Group Cavendish Williams)

To further understand the physiological mechanisms of cold-tolerance in banana plants, the responses of four introducing cultivars (cv.) W811 (via long-term cold adaptation), PB, BJ10 and BJ11 to low-temperature stress (LT) were investigated. LT caused increased malondialdehyde (MDA) content, elevated contents of hydrogen peroxide (H₂O₂) and superoxide radical (O₂⁻), and decreased photochemical efficiency (F_v/F_m) and net photosynthetic rate (P_n) in the leaves of four banana cultivars, but cv. W811 showed better LT tolerance than the other three cultivars. After 72 h of LT, four key antioxidative enzymes in the four cultivars showed different responses. Compared to controls, superoxide dismutase (SOD) activities in the four cultivars showed a significant decrease and W811 had the smallest amount of decrease. Catalase (CAT) activities showed a significant decrease. Peroxidase (POD) activities kept relatively higher activities and showed no significant changes (P > 0.05) in W811, BJ10 and BJ11 whereas that in PB showed a significant increase (P < 0.001). Ascorbate peroxidase (APX) activities in W811 and PB showed no significant changes (P > 0.05). Our results showed that higher cold-tolerance in cv. W811 may correlate with the long-term cold adaptation of the antioxidative enzymes such as SOD, POD and APX that alleviate oxidative stress caused by LT.     

The mechanism(s) involved in the photoprotection of PSII at elevated CO2 in nodulated alfalfa plants

In a previous study, we found that enhanced CO₂ subjected to nodulated alfalfa plants grown at different temperatures (ambient and ambient + 4 °C) and water availability regimes could protect PSII from photodamage. The main objective of this study was to determine the mechanism(s) involved in the photoprotection of PSII at elevated CO₂ levels in this plant. Elevated CO₂ reduced carboxylation capacity-induced photosynthetic acclimation and reduced enzymatic and/or nonenzymatic antioxidant activities, suggesting that changes in electron flow did not cause any photooxidative damage (which was also confirmed by H₂O₂ and lipid peroxidation analyses). Enhanced nonphotochemical quenching and xanthophyll cycle pigments revealed that plants grown at 700 μmol mol⁻¹ CO₂ compensated for the reduction in energy sink with a larger capacity for nonphotochemical dissipation of excitation energy as heat, i.e., modulating the status of the VAZ components. Elevated CO₂ induced the de-epoxidation of violaxanthin to zeaxanthin, facilitating thermal dissipation and protecting the photosynthetic apparatus against the deleterious effect of excess excitation energy.     

The mechanism(s) involved in the photoprotection of PSII at elevated CO2 in nodulated alfalfa plants

In a previous study, we found that enhanced CO₂ subjected to nodulated alfalfa plants grown at different temperatures (ambient and ambient + 4 °C) and water availability regimes could protect PSII from photodamage. The main objective of this study was to determine the mechanism(s) involved in the photoprotection of PSII at elevated CO₂ levels in this plant. Elevated CO₂ reduced carboxylation capacity-induced photosynthetic acclimation and reduced enzymatic and/or nonenzymatic antioxidant activities, suggesting that changes in electron flow did not cause any photooxidative damage (which was also confirmed by H₂O₂ and lipid peroxidation analyses). Enhanced nonphotochemical quenching and xanthophyll cycle pigments revealed that plants grown at 700 μmol mol^?1 CO₂ compensated for the reduction in energy sink with a larger capacity for nonphotochemical dissipation of excitation energy as heat, i.e., modulating the status of the VAZ components. Elevated CO₂ induced the de-epoxidation of violaxanthin to zeaxanthin, facilitating thermal dissipation and protecting the photosynthetic apparatus against the deleterious effect of excess excitation energy.     

Phytochemical screening of flavonoids with their antioxidant activities from rapeseed (Brassica napus L.)

Ten flavone compounds, including three new flavonoid glycosides, were isolated from defatted rapeseed, and their protective antioxidant effect on H₂O₂-induced oxidative damage in human umbilical vein endothelial cells (ECV-304) was investigated. Three new flavonoid glycosides were identified as kaempferol-3-O-[(6-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (8), kaempferol-3,7-di-O-β-d-glucopyranoside-4'-O-(6-O-sinapoyl)-β-d-glucopyranoside (9), and kaempferol-3-O-[(3-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (10). The protective effects of all of the isolated compounds on H₂O₂-induced oxidative damage were assessed, and the activities of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) were measured. All of compounds had a protective effect on H₂O₂-induced oxidative damage in ECV-304 cells and the presence of a substituted sinapoyl group and its position in the structures were used to elucidate the activity differences.     

Copper and zinc in Elodea canadensis from rivers with various pollution levels

The anthropogenic impact of xenobiotics contributes to environmental risk for the aquatic environment and thus, must be controlled. Elodea canadensis, a cosmopolitan aquatic macrophyte with an important role in the ecology of many littoral zones, may provide an integrated record of pollution. Therefore, it was interesting to investigate the accumulation of Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn in this species and in water and bottom sediments collected from rivers with various levels of contamination. Of these rivers one control and one polluted was selected for the collection of E. canadensis for an experiment to compare the ability of this species to accumulate Cu and Zn. These elements were supplemented at concentrations (mg L⁻¹) of 0.01, 0.02, 0.03, 0.05, 0.08 and 0.14 as CuSO₄·5H₂O, and 0.4, 0.6, 0.9, 1.4, 2.03 and 3.04 as ZnSO₄·7H₂O and in a mixture containing (mg L⁻¹) 0.01Cu + 0.4Zn, 0.02Cu + 0.6Zn, 0.03Cu + 0.9Zn, 0.05Cu + 1.4Zn, 0.08Cu + 2.03Zn and 0.14Cu + 3.04Zn. After the experiment, E. canadensis from the polluted river contained significantly higher Cu and Zn concentrations when applied separately and also significantly higher Cu and Zn concentrations when applied as a mixture compared to the control river. These higher concentrations in E. canadensis from the polluted river were found in all combinations in the experiment. Thus, E. canadensis habituated in polluted sites to the exposure, and long-term influence of elevated metal levels appeared to be better adapted, and it also exhibited a higher increase in biomass than plants from the control river in all the experimental Cu and Zn solutions. Younger leaves of E. canadensis were more resistant to the effects of Cu and Zn than older leaves. Both Cu and Zn negatively affected the cell structure of older leaves, although the influence of Cu on plasma membrane integrity and chloroplast distribution was stronger than that of Zn. The influence of the Cu + Zn mixture on E. canadensis resulted in less pronounced cell disintegration than the influence of Cu added separately.The explanation of differences in the E. canadensis biomass increase and metal concentrations under the binary Cu and Zn impact needs further examination.     

An amperometric H2O2 biosensor based on cytochrome c immobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H₂O₂ biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM. The sensor had a detection limit of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM^?1 cm^?2. The sensor gave accurate and satisfactory results, when employed for determination of H₂O₂ in different fruit juices.     

Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity

BackgroundHorseradish peroxidase (HRP) catalyzes H₂O₂ dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H₂O₂-induced inactivation, have been investigated.MethodsHRP reaction with H₂O₂ was studied by following H₂O₂ depletion, O₂ evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow.ResultsNitroxide protects HRP against H₂O₂-induced inactivation. The rate of H₂O₂ dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H₂O₂. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) > 4-OH-TPO > 3-carbamoyl proxyl > 4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III.ConclusionsNitroxide catalytically protects HRP against inactivation induced by H₂O₂ while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex.General SignificanceNitroxides catalytically protect heme proteins against inactivation induced by H₂O₂ revealing an additional role played by nitroxide antioxidants in vivo.     

Response surface analysis for enzymatic decolorization of Congo red by manganese peroxidase

The enzymatic decolorization process of manganese peroxidase (MnP) is a complex system, which is greatly affected by the concentrations of H₂O₂, Mn²⁺, dye and enzyme. This work aimed to study these factors and investigate the combined interactions between them by applying response surface methodology (RSM) for decolorization of Congo red with MnP from Schizophyllum sp. F17, meanwhile conventional one-factor-at-a-time analysis was carried out. Through the one-factor-at-a-time analysis the optimized H₂O₂, Mn²⁺, Congo red and MnP extract was 0.2 mM, 0.5 mM, 50 mg/l and 0.8 ml, respectively, and the maximum decolorization attained under such conditions was 24.2%. Response surface analysis was conducted through Box–Behnken design and a second-order polynomial model (R² = 0.8565) was generated to describe the combined effect and the interactions quantificationally. ANOVA analysis indicated that the interactions between H₂O₂ and MnP, between dye and MnP were significant; the optimum condition through RSM was found to be 0.35 mM H₂O₂, 0.5 mM Mn²⁺, 75 mg/l Congo red and 1.4 ml MnP extract, for maximum decolorization of 30.8%.     

Tuning magnetic properties using targeted structural distortion: New additions to a family of Mn6 single-molecule magnets

We report the synthesis and magnetic properties of three hexametallic Mn clusters: [Mn₆O₂(Et-sao)₆(O₂C-Naphth)₂(EtOH)₄(H₂O)₂] (1) (HO₂C-Naphth = 1-naphthoic acid, Et-saoH₂ = 2-hydroxyphenylpropanone oxime), [Mn₆O₂(Et-sao)₆(O₂C-Anthra)₂(EtOH)₄(H₂O)₂] · 0.66EtOH · 0.33H₂O (HO₂C-Anthra = anthracene-9-carboxylic acid) (2 · 0.66EtOH · 0.33H₂O) and [Mn₆O₂(Et-sao)₆(O₂CPhCCH)₂(EtOH)₄(H₂O)₂] · 1.7EtOH · 0.3H₂O (HO₂CPhC  CH = 4-ethynylbenzoic acid) (3 · 1.7EtOH · 0.3H₂O). Clusters 1–3 exhibit ferromagnetic exchange between all six Mn^III centres resulting in S = 12 ground spin states. Ac magnetic susceptibility and single crystal micro-SQUID measurements on 1–3 confirm SMM behaviour with barriers to magnetisation reversal of 60.12 (1), 60.10 (2) and 66.79 (3) K.     

IBA-induced changes in antioxidant enzymes during adventitious rooting in mung bean seedlings: The role of H2O2

The changes in antioxidant enzyme activity during the induction of adventitious roots in mung bean seedlings treated with Indole-3-butyric acid (IBA), hydrogen peroxide (H₂O₂), ascorbic acid (ASA) and diphenylene iodonium (DPI) were investigated. As compared with the controls, treatments of seedlings with 10 μM IBA significantly decreased POD activity by 55% and 49.6% at 3 h and 12 h of incubation, respectively, and significantly increased by 49.8% at 36 h of incubation; treatments of seedlings with 10 mM H₂O₂ significantly decreased POD activity by 42%, 60%, 39% and 38% at 3 h, 12 h, 24 h and 48 h of incubation, respectively, the changes in POD activity were coincident with those in IBA-treated seedlings during the 0–12 h incubation period; treatments of seedlings with 2 mM ASA significantly decreased APX activities by 27% only at 3 h of incubation, the varying trend of POD activity was similar to incubation with water; 10 μM DPI treatments significantly decreased POD activity by 42%, 40%, 54% and 28% at 3 h, 6 h, 12 h and 48 h of treatment, respectively. CAT activities remained at relatively stable levels and no major changes occurred from 0 h to 48 h during the incubation phase of adventitious rooting. The results may imply that CAT, an H₂O₂-metabolizing enzyme, is inactivated by H₂O₂ during the formation of adventitious roots. As compared with the controls, IBA treatments significantly decreased APX activities by 48%, 53% and 66% at 3 h, 9 h and 12 h of treatment, respectively; H₂O₂ treatments significantly decreased APX activities by 59%, 51% and 57% at 3 h, 12 h and 36 h of incubation, respectively; ASA treatments significantly decreased APX activities by 37% only at 3 h of incubation; DPI treatments significantly decreased APX activities by 54%, 53% and 63% at 3 h, 6 h and 12 h of incubation, respectively, and significantly increased APX activity by 106% at 24 h. These results indicated that the influence of IBA, H₂O₂, ASA and DPI on the changes in APX activity were the same as on the changes in POD activity. Furthermore, similar trends in the changes of APX activity and POD activity were observed during the induction and initiation rooting phase. This finding implies that APX and POD serve the same functions, possibly related to the level of H₂O₂, during the formation of adventitious roots. The early decrease of POD and APX activities in the initiation phase of IBA- and H₂O₂-treated seedlings may be one mechanism underlying the IBA- and H₂O₂-mediated facilitation of adventitious rooting.