Studies on the autoxidation of dopamine: interaction with ascorbate

An oxygen electrode was used to monitor the reaction between dopamine (DA, 1-20 mM) and oxygen at pH 7.4 and 37 degrees C, in both the presence and absence of ascorbate (10 mM). The selected concentrations approximate levels within DA neurons. Diethylenetriaminepentaacetic acid (DTPA, 0.1 mM) was used to suppress catalysis by trace metals in the reagents. Separate experiments with catalase showed that oxygen consumption could be equated with the formation of hydrogen peroxide. Depending upon the experimental conditions, ascorbate acted either as an antioxidant, suppressing oxygen consumption (H2O2 production) to 6-8% of the expected rate, or as a prooxidant, amplifying oxygen consumption by 640%. The antioxidant action is consistent with the scavenging of superoxide radicals by ascorbate. The prooxidant action is probably the result of redox cycling of a pre-melanin oxidation product derived from DA. Analyses conducted by high-performance liquid chromatography with electrochemical detection revealed formation of a product with a very low oxidation potential; the product was not 6-hydroxydopamine. These observations may be relevant to concepts of toxicity mediated by DA within neuronal systems.     

Effects of scavengers for active oxygen species on cell death by cryptogein

The hypersensitive reaction is a type of programmed cell death in plants. Cryptogein is a proteinaceous elicitor secreted from Phythophthora cryptogea. In one current model, active oxygen species (AOS) trigger programmed cell death in plants. In this study, we examined a variety of AOS scavengers to elucidate the function of AOS in the death program. Most of these AOS scavengers, including tiron, a scavenger for superoxide radical, catalase for hydrogen peroxide, and hydroquinone, sodium ascorbate and propyl gallate for free radicals, almost completely removed extracellular AOS. However, none of the reagents completely blocked the cell death process. Other reagents, such as histidine and dimethylfuran, scavengers for singlet oxygen, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase, showed significant toxicity in BY-2 cells. These results indicate that AOS produced in the extracellular space do not play a role in hypersensitive cell death.     

Generation of superoxide anion radicals and hydrogen peroxide in the auto-oxidation of caffeic acid

Caffeic acid (5-200 mkM) reduces cytochrome c during autoxidation in potassium phosphate buffer, pH 7-8. The reduction is inhibited by superoxide dismutase, which suggests generation of superoxide anion radicals. The generation rate is 0.028-0.115 mkmoles O2- per min. Superoxide appears to be a side product of the reaction, since the autoxidation of caffeic acid itself (followed by A420) is not inhibited by superoxide dismutase. The autoxidation is accompanied by oxygen of consumption. An addition of catalase results in liberation of some part of consumed oxygen, this being indicative of accumulation of hydrogen peroxide. Caffeic acid is known to be responsible for the resistance of plants to parasites because of its toxicity. This function presumably depends on superoxide or other reactive oxygen species.     

Active oxygen-associated control of rice blast disease by riboflavin and roseoflavin

Exogenous riboflavin and its dimethylated amino(nor)-derivative roseoflavin were studied in their ability to protect susceptible rice plants from blast disease and to induce fungitoxicity mediated by active oxygen. Both compounds, either added to the inoculum (10 microg/ml) or to soil (40 mg/kg, two days prior to inoculation), induced disease resistance, i.e., diminished the frequency of compatible-type lesions on infected leaves, mainly at the expense of the appearance of hypersensitive spots. Leaf diffusates of untreated plants possessed a weak fungitoxicity that increased slightly after leaf infection or illumination of diffusate. The flavins added to inoculum, to soil, or to a collected diffusate augmented significantly the light-activated part of the diffusate toxicity. In some instances, the light-independent part was stimulated as well. The effect was not due to direct fungitoxicity of flavins as they alone did not interfere with spores regardless of illumination. Antioxidant reagents (superoxide dismutase, catalase, scavengers of hydroxyl radical, and the iron ion chelator desferrioxamine) protected spores from intoxication in almost all cases. This implies the involvement of active oxygen in the toxic and, probably, disease-controlling effects of the flavins. Roseoflavin was a better inducer of disease resistance than riboflavin but was similar in stimulation of diffusate toxicity. However, roseoflavin did not produce superoxide and exhibited only weak fungitoxicity if substituted for riboflavin in the well-known O2--generating model photosystem containing methionine. Therefore, the superoxide generation due to photo-oxidation of methionine or similar substrates is not the cause of the increase of leaf diffusate fungitoxicity and of disease resistance of plants supplied with roseoflavin. It is suggested that the rise in active oxygen production favors a forthcoming hypersensitive reaction, and both phenomena contribute to resistance induced by flavo-compounds. The light-driven activation of oxygen may be of interest as a mode of action of novel fungicides.     

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.     

Tannic acid inhibits in vitro iron-dependent free radical formation

The antioxidant activity of tannic acid (TA), a plant polyphenol claimed to possess antimutagenic and anticarcinogenic activities, was studied by monitoring (i) 2-deoxyribose degradation (a technique for OH detection), (ii) ascorbate oxidation, (iii) ascorbate radical formation (determined by EPR analysis) and (iv) oxygen uptake induced by the system, which comprised Fe(III) complexes (EDTA, nitrilotriacetic acid (NTA) or citrate as co-chelators), ascorbate and oxygen. TA removes Fe(III) from the co-chelators (in the case of EDTA, this removal is slower than with NTA or citrate), forming an iron-TA complex less capable of oxidizing ascorbate into ascorbate radical or mediating 2-deoxyribose degradation. The effectiveness of TA against 2-deoxyribose degradation, ascorbate oxidation and ascorbate radical formation was substantially higher in the presence of iron-NTA (or iron-citrate) than with iron-EDTA, which is consistent with the known formation constants of the iron complexes with the co-chelators. Oxygen uptake and 2-deoxyribose degradation induced by Fe(II) autoxidation were also inhibited by TA. These results indicate that TA inhibits OH formation induced by Fe(III)/ascorbate/O(2) mainly by arresting Fe(III)-induced ascorbate oxidation and Fe(II) autoxidation (which generates Fe(II) and H(2)O(2), respectively), thus limiting the production of Fenton reagents and OH formation. We also hypothesize that the Fe(II) complex with TA exhibits an OH trapping activity, which explains the effect of TA on the Fenton reaction.     

Salt Stress-induced Programmed Cell Death in Rice Root Tip Cells

Salt stressed rice root tips were used to investigate the changes of reactive oxygen species （ROS） and antioxidant enzymes at the early stages of programmed cell death （PCD）. The results indicated that 500 mmol/L NaCI treatment could lead to specific features of PCD in root tips, such as DNA ladder, nuclear condense and deformation, and transferase mediated dUTP nick end labeling positive reaction, which were initiated at 4 h of treatment and pro- gressed thereafter. Cytochrome c release from mitochondria into cytoplasm was also observed, which occurred at 2 h and was earlier than the above nuclear events. In the very early phase of PCD, an immediate burst in hydrogen peroxide and superoxide anion production rate was accompanied by two-phase changes of superoxide dismutases and ascorbate peroxidase. A short period of increase in the activity was followed by prolonged impairment. Thus, we conclude that salt can induce PCD in rice root tip cells, and propose that in the early phase of rice root tip cell PCD, salt stress-induced oxidative burst increased the antioxidant enzyme activity, which, in turn, scavenged the ROS and abrogated PCD. Also, when the stress is prolonged, the antioxidant system is damaged and accumulated ROS induces the PCD process, which leads to cytochrome c release and nuclear change.     

Esr Detection of Free Radical Intermediates During Autoxidation Of 5-Hydroxyprimaquine

Autoxidation of 5–hydroxyprimaquine, a putative metabolite of the antimalarial primaquine, was studied by oxygen consumption and ESR spectroscopy. 5–Hydroxyprirnaquine undenvent fast autoxidation under mild conditions (pH 7.4-8. 5, 25°C. and presence of I mM diethylenetriamine pentaacetic acid); each mol of the drug consumed 0.75 mol of oxygen and formed 0.5 mol of hydrogen peroxide. Direct-ESR experiments demonstrated that 5–hydroxyprimaquine autoxidation was accompanied by generation of a drug-derived free radical that is oxygen sensitive. Generation of hydroxyl radical was also established by spin-trapping experiments in the presence of 5,5–dimethyl-l-pyrroline N-oxide. The effect of antioxidant enzymes on hydroxyl radical adduct yield and analysis of autoxidation stoichiometry suggest that the main route for hydroxyl radical generation is the iron-catalyzed reaction between the drug-derived free radical and hydrogen peroxide.     

Influence of vitamin C and vitamin E on redox signaling: Implications for exercise adaptations

The exogenous antioxidants vitamin C (ascorbate) and vitamin E (α-tocopherol) often blunt favorable cell signaling responses to exercise, suggesting that redox signaling contributes to exercise adaptations. Current theories posit that this antioxidant paradigm interferes with redox signaling by attenuating exercise-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. The well-documented in vitro antioxidant actions of ascorbate and α-tocopherol and characterization of the type and source of the ROS/RNS produced during exercise theoretically enable identification of redox-dependent mechanisms responsible for the blunting of favorable cell signaling responses to exercise. This review aimed to apply this reasoning to determine how the aforementioned antioxidants might attenuate exercise-induced ROS/RNS production. The principal outcomes of this analysis are (1) neither antioxidant is likely to attenuate nitric oxide signaling either directly (reaction with nitric oxide) or indirectly (reaction with derivatives, e.g., peroxynitrite); (2) neither antioxidant reacts appreciably with hydrogen peroxide, a key effector of redox signaling; (3) ascorbate but not α-tocopherol has the capacity to attenuate exercise-induced superoxide generation; and (4) alternate mechanisms, namely pro-oxidant side reactions and/or reduction of bioactive oxidized macromolecule adducts, are unlikely to interfere with exercise-induced redox signaling. Out of all the possibilities considered, ascorbate-mediated suppression of superoxide generation with attendant implications for hydrogen peroxide signaling is arguably the most cogent explanation for blunting of favorable cell signaling responses to exercise. However, this mechanism is dependent on ascorbate accumulating at sites rich in NADPH oxidases, principal contributors to contraction-mediated superoxide generation, and outcompeting nitric oxide and superoxide dismutase isoforms. The major conclusions of this review are: (1) direct evidence for interference of ascorbate and α-tocopherol with exercise-induced ROS/RNS production is lacking; (2) theoretical analysis reveals that both antioxidants are unlikely to have a major impact on exercise-induced redox signaling; and (3) it is worth considering alternate redox-independent mechanisms.     

Exogenous salicylic acid alleviates effects of long term drought stress and delays leaf rolling by inducing antioxidant system

Salicylic acid (SA) is one of the important signal molecules modulating plant responses to environmental stress. In this study, the effects of exogenous SA on leaf rolling, one of drought avoidance mechanisms, and antioxidant system were investigated in Ctenanthe setosa during long term drought stress. The plants were subjected to 38-day drought period and they were treated with or without SA (10⁻⁶ M) on the 25th, 27th and 29th days of the period. Leaf samples were harvested on the 30th, 34th and 38th days. Some antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase), reactive oxygen species (hydrogen peroxide and superoxide) and lipid peroxidation were determined during the drought period. Treatment with SA prevented water loss and delayed leaf rolling in comparison with control leaves. Exogenous SA induced all antioxidant enzyme activities more than control leaves during the drought. Ascorbate and glutathione, α-tocopherol, carotenoid and endogenous SA level were induced by the SA treatment. Levels of reactive oxygen species were higher in SA treated plants than control ones on the 34th day. Their levels on the 38th day, however, fastly decreased in SA treated plants. SA treatment prevented lipid peroxidation while the peroxidation increased in control plants. The results showed that exogenous SA can alleviate the damaging effect of long term drought stress by decreasing water loss and inducing the antioxidant system in the plant having leaf rolling, alternative protection mechanism to drought.     

Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation

Hematoxylin, a natural dye commonly used as a histological stain, generates superoxide upon oxidation to its quinonoid product, hematein. The parameters affecting this reaction were assessed in developing a new and versatile assay for superoxide dismutase. The autoxidation of hematoxylin to hematein was accompanied by an increase in absorbance between 400 and 670 nm. The autoxidation rate was proportional to hematoxylin concentration and increased with pH above 6.55. Trace metals accelerated the autoxidation and this effect was eliminated by EDTA. Superoxide dismutase inhibited the autoxidation 90-95% below pH 7.8, but above pH 8.1 the rate was augmented by superoxide dismutase. The rate inhibition at low pH was proportional to the superoxide dismutase concentration up to 70% inhibition. The rate acceleration at high pH was proportional to superoxide dismutase concentration up to approximately 200% acceleration. The autoxidation rate was not significantly affected by ethanol, cyanide, azide, hydrogen peroxide, or catalase. However, the reaction was inhibited by the reducing agents NADH, reduced glutathione, ascorbate, and dithiothreitol, and by undialyzed extracts of Escherichia coli B. When cell extracts were dialyzed prior to assay, the degree of inhibition observed was proportional to the concentration of superoxide dismutase in the extract. These observations form the basis for negative and positive assays of superoxide dismutase which are inexpensive and simple to perform. The negative assay has the added advantage of being applicable at physiological pH.     

Toxic and Protective Effects of l-DOPA on Mesencephalic Cell Cultures

Abstract: The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 μ M ) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [³H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions. Ascorbate (200 μ M ), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [³H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, l-DOPA prevented cell death by L-BSO.     

Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase.

Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of autoxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, indicating the participation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide dismutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.     

新型真菌源激活蛋白诱导水稻抗病性及其生理机制

为明确新型真菌源激活蛋白对水稻抗病性的诱导作用及其生理机制,研究了激活蛋白对水稻稻瘟病和白叶枯病的诱导抗病性,监测了激活蛋白处理后水稻过氧化物酶（POD）、多酚氧化酶（PPO）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）活性及过氧化氢（H₂O₂）含量变化。结果表明,1～6 μg·mL^-1激活蛋白对稻瘟病和白叶枯病的诱抗效果分别为45.2%～71.4%和47.6%～66.3%,以6 μg·mL^-1激活蛋白的诱抗效果最好。与对照相比,2 μg·mL^-1激活蛋白处理水稻后3～15 d内不同程度诱导了防御酶POD、PPO和SOD活性,抑制CAT活性,提高H₂O₂含量。新型真菌源激活蛋白能够诱导水稻产生对稻瘟病和白叶枯病的抗病性,其诱导抗性机制与水稻体内的活性氧代谢密切相关。     

The spatial patterns of oxidative stress indicators co-locate with early signs of natural senescence in maize leaves

Reactive oxygen species play a crucial role for various physiological and developmental processes in plants. Here, we report a spatial pattern of oxidative stress and antioxidant defence within maize leaf. Localization of hydrogen peroxide in different region of leaf clearly exhibits well-defined increasing pattern of accumulation from the base to the leaf tip. Lipid peroxidation, an index of oxidative damage, also showed a similar pattern-like hydrogen peroxide that is lowest at the base and highest at the leaf tip. NADPH oxidase, an enzyme responsible for superoxide anion generation, showed highest activity in the leaf tip and least in the leaf base regions. Superoxide dismutase (SOD) activity was increased from the base to the leaf tip. Peroxidases, DAB-peroxidase (DAB-POD) and guaiacol-peroxidase (G-POD), catalase (CAT) and glutathione reductase (GR) also showed increases in their activities from the base to the leaf tip. Ascorbate peroxidase (APX), however, showed a reverse trend—highest at the base and least in the leaf tip. The decrease in APX and increases in the activities of other antioxidant enzymes SOD, CAT, DAB-POD, G-POD and GR along with H₂O₂ and lipid peroxidation, ascorbate/dehydroascorbate and non-protein thiol levels from the base to the leaf tip clearly exhibit a spatial pattern prior to the onset of visible signs of senescence in the maize leaf.     

Heme degradation during autoxidation of oxyhemoglobin

Two fluorescent heme degradation compounds are detected during autoxidation of oxyhemoglobin. These fluorescent compounds are similar to fluorescent compounds formed when hydrogen peroxide reacts with hemoglobin [E. Nagababu and J. M. Rifkind, Biochem. Biophys. Res. Commun. 247, 592-596 (1998)]. Low levels of heme degradation in the presence of superoxide and catalase are attributed to a reaction involving the superoxide produced during autoxidation. The inhibition of most of the degradation by catalase suggests that the hydrogen peroxide generated during autoxidation of oxyhemoglobin produces heme degradation by the same mechanism as the direct addition of hydrogen peroxide to hemoglobin. The formation of the fluorescent degradation products was inhibited by the peroxidase substrate, ABTS, which reduces ferrylhemoglobin to methemoglobin, indicating that ferrylhemoglobin is produced during the autoxidation of hemoglobin. It is the transient formation of this highly reactive Fe(IV) hemoglobin, which is responsible for most of the heme degradation during autoxidation.     

Silicon nutrition potentiates the antioxidant metabolism of rice plants under iron toxicity

Iron toxicity reduces growth of rice plants in acidic lowlands. Silicon nutrition may alleviate many stresses including heavy metal toxicity in plants. In the present study, the ameliorating effects of silicon nutrition on rice (Oryza sativa L.) plants under toxic Fe levels were investigated. Plants were cultivated in greenhouse in hydroponics under different Fe treatments including 10, 50, 100, and 250 mg L^?1 as Fe-EDTA and silicon nutrition including 0 and 1.5 mM sodium silicate. Iron toxicity imposed significant reduction in plant fresh weight, tiller, and leaf number. The activity of catalase, cell wall, and soluble peroxidases, and polyphenol oxidase in shoots decreased due to moderate Fe toxicity (50 and 100 mg L^?1), but increased at greater Fe concentration. Ascorbate peroxidase activity increased in both roots and shoots of Fe-stressed plants. Iron toxicity led to increased tissue hydrogen peroxide and lipid peroxidation. Silicon nutrition improved plant growth under all Fe treatments and alleviated Fe toxicity symptoms, probably due to lower Fe concentration of Si-treated plants. Silicon application could improve the activity of antioxidant enzymes such as catalase, ascorbate peroxidase, and soluble peroxidase under moderate Fe toxicity, which resulted in greater hydrogen peroxide detoxification and declined lipid peroxidation. Thus, silicon nutrition could ameliorate harmful effects of Fe toxicity possibly through reduction of plant Fe concentration and improvement of antioxidant enzyme activity.     

Imaging of photo-oxidative stress responses in leaves

High resolution digital imaging was used to identify sites of photo-oxidative stress responses in Arabidopsis leaves non-invasively, and to demonstrate the potential of using a suite of imaging techniques for the study of oxidative metabolism in planta. Tissue-specific photoinhibition of photosynthesis in individual chloroplasts in leaves was imaged by chlorophyll fluorescence microscopy. Singlet oxygen production was assessed by imaging the quenching of the fluorescence of dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole (DanePy) that results from its reaction with singlet oxygen. Superoxide and hydrogen peroxide accumulation were visualized by the reduction of nitroblue tetrazolium (NBT) to formazan deposits and by polymerization with 3,3'-diaminobenzidine (DAB), respectively. Stress-induced expression of a gene involved with antioxidant metabolism was imaged from the bioluminescence from leaves of an Arabidopsis APX2-LUC transformant, which co-expresses an ascorbate peroxidase (APX2) with firefly luciferase. Singlet oxygen and superoxide production were found to be primarily located in mesophyll tissues whereas hydrogen peroxide accumulation and APX2 gene expression were primarily localized in the vascular tissues.     

Alleviation of Oxidative Stress Induced by 24-Epibrassinolide in Soybean Plants Exposed to Different Manganese Supplies: UpRegulation of Antioxidant Enzymes and Maintenance of Photosynthetic Pigments

Adverse effects promoted by inadequate manganese (Mn) supply (deficiency or toxicity) causes inefficiency of the antioxidant system and degradation of chlorophylls. However, 24-epibrassinolide (EBR) is a natural steroid that exhibits beneficial effects on antioxidant metabolism, chlorophyll levels and stress indicators. Therefore, this research aims to evaluate whether EBR application via spray can alleviate oxidative stress in soybean plants exposed to different Mn concentrations and to determine possible contributions of the antioxidant enzymes and photosynthetic pigments. Experiment followed a completely randomized factorial design with two concentrations of 24-epibrassinolide (0 and 100 nM EBR, described as − EBR and + EBR, respectively) and three Mn doses (0.25, 25 and 2500 µM Mn, described as low, control and high supply of Mn, respectively). Plants treated with low and high concentrations of Mn + EBR exhibit significant increases in all enzymes evaluated (superoxide dismutase, catalase, ascorbate peroxidase and peroxidase). To superoxide dismutase (SOD), EBR spray promoted increments of 77%, 38% and 76% under low, control and high Mn supplementation, respectively, compared to same treatment in absence of EBR. Clearly intense activity is linked to SOD contributed by dismutation of superoxide into hydrogen peroxide, being subsequently decomposed by other enzymes (catalase, ascorbate peroxidase and peroxidase). Concomitantly, plants with Mn deficiency and toxicity sprayed with 100 nM EBR presented maintenance of chlorophylls and carotenoids due to reduction of superoxide and hydrogen peroxide and consequently reduced chloroplast membrane damages as indicated by malondialdehyde levels and electrolyte leakage.