Effect of Heavy Metals on Wheat Seedlings: Activation of Antioxidant Enzymes

Accumulation of heavy metals in wheat grain exposed to multicomponent pollutants (industrial wastewater) was studied. The absolute content of metals (Zn, Cd, Cu, Cr, Ni, Co, Pb, and Mn) was found to be determined by the extent of purification of wastewater. An increase in the degree of grain contamination with heavy metals was accompanied by activation of antioxidant enzymes (superoxide dismutase, EC 1.15.1.1; catalase, EC 1.11.1.6; and peroxidase, EC 1.11.1.7) in leaves and activation of superoxide dismutase and peroxidase in roots. The ratio of activity of membrane enzymes to activity of cytosol enzymes was demonstrated to be high. It was concluded that the membranotropic effect of multicomponent contaminants was due to accumulation of heavy metals capable of inducing the antioxidant protection in the next generation of wheat seedlings.     

Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus

Nitric oxide (NO) is a bioactive molecule, which in plants was found to function as prooxidant as well as antioxidant. In the present study, we found that NO donor sodium nitroprusside (SNP) stimulates seed germination and root growth of lupin (Lupinus luteus L. cv. Ventus). Seed germination is promoted at concentrations between 0.1 and 800 μM SNP in a dose-dependent manner. The stimulation was most pronounced after 18 and 24 h and ceased after 48 h of imbibition. The promoting effect of NO on seed germination persisted even in the presence of heavy metals (Pb, Cd) and sodium chloride. Pretreatment of lupin seedlings for 24 h with 10 μM SNP resulted in efficient reduction of the detrimental effect of the abiotic stressors on root growth and morphology. The inhibitory effect of heavy metals on root growth was accompanied by increased activity of superoxide dismutase (SOD, EC 1.15.1.1.), which in roots preincubated with SNP was significantly higher. Some changes in the activity of other antioxidant enzymes, peroxidase (POX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) were also detected. Using the superoxide anion (O₂^•–)-specific indicator dihydroethidium (DHE), we found intense DHE-derived fluorescence in heavy metal-stressed roots, whereas in those pretreated with SNP the fluorescence was very low, comparable to the level in unstressed roots. On the basis of the above data, we conclude that the protective effect of NO in stressed lupin roots may be at least partly due to the stimulation of SOD activity and/or direct scavenging of the superoxide anion.     

Role of Antioxidant Systems in Wheat Genotypes Tolerance to Water Stress

The role of plant antioxidant systems in stress tolerance was studied in leaves of three contrasting wheat genotypes. Drought imposed at two different stages after anthesis resulted in an increase in H2O2 accumulation and lipid peroxidation and decrease in ascorbic acid content. Antioxidant enzymes like superoxide dismutase, ascorbate peroxidase and catalase significantly increased under water stress. Drought tolerant genotype C 306 which had highest ascorbate peroxidase and catalase activity and ascorbic acid content also showed lowest H2O2 accumulation and lipid peroxidation (malondialdehyde content) under water stress in comparison to susceptible genotype HD 2329 which showed lowest antioxidant enzyme activity and ascorbic acid content and highest H2O2 content and lipid peroxidation. HD 2285 which is tolerant to high temperature during grain filling period showed intermediate behaviour. Superoxide dismutase activity, however, did not show significant differences among the genotypes under irrigated as well as water stress condition. It seems that H2O2 scavenging systems as represented by ascorbate peroxidase and catalase are more important in imparting tolerance against drought induced oxidative stress than superoxide dismutase alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Occurrence of powdery mildew disease in wheat fertilized with increasing silicon doses: a chemometric analysis of antioxidant response

Blumeria graminis (Bgt) is a pathogenic fungus that affects severally wheat plants provoking high losses in wheat production. Biochemical parameters like enzyme activity of catalase, superoxide dismutase, or peroxidases can be used to detect changes of metabolism in response to pathogen infection. We evaluated different biochemical and biometrics parameters to assess the effect of silicon, a widely recognized beneficial nutrient, in wheat infected naturally with Bgt. Integral study and interpretation of results obtained by multivariate analysis is the challenge of present work. Wheat plants growing in hydroponic solution were fertilized with increasing concentration of silicon. Responses of wheat plants to silicon treatments were assessed through the analysis of lipid peroxidation and antioxidant enzymes activity (catalase, ascorbate peroxidase and superoxide dismutase). Furthermore, biometric measures such as Pathogen Index, dry weight of shoot and roots, tiller height, spike length, spike mass, grain number and grain mass production were assessed. Pathogen Index decreased while mass and number of grain, dry weight of shoots were significantly increased, proportionally to silicon concentration increasing in culture media; biochemical parameters were also influenced by the concentration of silicon. Multivariate analysis indicated correspondence between increase of silicon treatments and decrease of antioxidant activities of APX, CAT and SOD. Relationships between the applied doses of silicon and decreasing Bgt expansion in foliar surfaces were also observed. Chemometrics proved to be an optimal tool for integrating data about metabolic status and demonstrate, in present case, that increasing concentration of silicon and the reduction of antioxidant enzyme activity are closely related to decreasing of powdery mildew disease.     

Effect of transition metals on stress, lipid peroxidation and antioxidant enzyme activities in tobacco cell cultures

to-baccoBright Yellow 2 (BY-2) suspension culture to understand the mechanisms of metal resistance in plant cells.We have analysed superoxide dismutase, catalase, and ascorbate peroxidase enzyme activities and superoxidedismutase-isoforms by isoelectric focusing gels in tobacco cells grown at two different toxic concentrations ofeach of the transition metals: copper, iron, manganese and zinc. Exposure of tobacco cells to these metals causedchanges in total superoxide dismutase activity in a different manner, depending on the metal assayed: after cop-perand manganese treatments, total superoxide dismutase activity was enhanced, while it was reduced after ironand zinc exposure. Superoxide dismutase-isoforms were affected by the metal used, and a Fe-SOD band with thesame isoelectric point as a Cu, Zn-SOD from non-treated cells, was induced after iron and zinc treatments. Cu,Zn-SODs were present in all metal-treatments whereas Mn-SOD was not detected in any case. Concerning otherantioxidant enzymes tested, such as catalase and ascorbate peroxidase, the latter showed a remarkable increase inactivity in response to copper treatments and catalase activity was enhanced after iron and with the lowest man-ganeseconcentration. Lipid peroxidation was increased after each metal treatment, as an indication of the oxi-dativedamage caused by metal concentration assayed in tobacco cells. These results suggest that an activation ofsome antioxidant enzymes in response to oxidative stress induced by transition metals is not enough to confertolerance to metal accumulation.     

Differential response of antioxidant enzymes in leaves of necrotic wheat hybrids and their parents

The leaves of necrotic hybrid of wheat ( Triticum aestivum L.) exhibited high superoxide content associated with increased lipid peroxidation and membrane damage in earlier studies (Khanna-Chopra et al. 1998, Biochem Biophys Res Commun 248: 712–715; Dalal and Khanna-Chopra 1999, Biochem Biophys Res Commun 262: 109–112). In the present study, we investigated the activities of the antioxidant enzymes in the leaves of necrotic wheat hybrids, Kalyansona×C306 (K×C) and WL711×C306 (WL×C) and their parents at different developmental stages. The K×C hybrid exhibited more severe necrosis than WL×C. In K×C, superoxide dismutase (SOD) activity showed no increase over the parents, while WL×C showed an early increase, but it was possibly insufficient to scavenge increased superoxide. Activities of guaiacol peroxidase, ascorbate peroxidase and glutathione reductase were enhanced, while catalase exhibited a decrease in activity, with the appearance of visible necrosis in both the hybrids. The isozyme profile of the antioxidant enzymes was similar in the hybrids and their parents. One existing isoform of guaiacol peroxidase showed an early appearance in the hybrid and increased in intensity with the progression of necrosis. The results reveal a differential response of antioxidant enzymes in necrotic wheat hybrids as compared to their parents. The response differed in magnitude at developmental stages of the leaves, which might be related to the intensity of necrosis expressed by the hybrids.     

Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury

The effect of ischemia-reperfusion on activity, protein and m-RNA levels of catalase, copper-zinc and manganese containing superoxide dismutases and glutathione peroxidase, the enzymes that are involved in free radical detoxification was studied in rat kidney. Ischemia alone did not alter either the activities or protein levels of superoxide dismutase and glutathione peroxidase. However, catalase activity was found to be inhibited to 82% of control. The inhibition of catalase was due to the inactivation of the enzyme as there was no significant change in enzyme protein level. Reperfusion following ischemia, however, led to a significant decrease in both the activities as well as the protein levels of all the antioxidant enzymes. The observed overall decrease in total superoxide dismutase activity was the net effect of a decrease in copper-zinc superoxide dismutase while manganese superoxide dismutase activity was found to be increased following reperfusion. This observed increased manganese superoxide dismutase activity was the result of its increased protein level. The mRNA levels for catalase, superoxide dismutases, and glutathione peroxidase were observed to be increased (100–145% of controls) following ischemia; reperfusion of ischemic kidneys, however, resulted in a significant decrease in the levels of mRNAs coding for all the enzymes except manganese superoxide dismutase which remained high. These results suggest that in tissue, the down regulation of the antioxidant enzyme system could be responsible for the pathophysiology of ischemia-reperfusion injury.     

Effect of sodium nitroprusside on heat resistance of wheat coleoptiles: Dependence on the formation and scavenging of reactive oxygen species

The effect of nitric oxide donor sodium nitroprusside (SNP) on resistance of coleoptiles of 4-day-old etiolated seedlings of wheat (Triticum aestivum L., cv. Elegiya) to damaging heating (10 min at 43°C) and possible dependence of this effect on changes in the activities of enzymes producing and scavenging reactive oxygen species (ROS) were studied. Treatment of coleoptiles with 500 μM SNP considerably boosted generation of superoxide anion radical therein. This effect was substantially suppressed by blocker of calcium channels (lanthanum chloride), calmodulin antagonist (chlorpromazine), and inhibitor of NADPH-oxidase (imidazole) but not by peroxidase inhibitor (salicylhydroxamic acid). NO donor activated antioxidant enzymes (superoxide dismutase, catalase, and soluble peroxidase) and elevated heat resistance of wheat coleoptiles. NO scavenger methylene blue, antioxidant agent ionol, calcium antagonists, and NADPH-oxidase inhibitor imidazole substantially reduced the elevation of heat resistance of wheat coleoptiles induced by NO donor. It was concluded that SNP-induced heat resistance of coleoptiles depended on calcium and ROS, whose production is probably boosted by activation of NADPH-oxidase.     

Changes in antioxidant enzyme activity in the fine roots of black poplar (Populus nigra L.) and cottonwood (Populus deltoides Bartr. ex Marsch) in a heavy-metal-polluted environment

The effect of heavy metal deposition onto soil from a copper smelter on lipid peroxidation and antioxidant enzyme activity in the fine roots of two poplars (Populus nigra L. and Populus deltoides Bartr. ex Marsch) was analyzed. The subjects were mature trees growing in real environments. In both analyzed species, heavy metals stimulated the overproduction of free radicals in fine roots (measured as malondialdehyde level), which was directly proportional to advancing senescence. In young fine roots, heavy metals caused a decrease in guaiacol peroxidase activity and presumably disturbed the lignification process. Catalase was highly sensitive to the presence of heavy metals in the soil. In contrast, ascorbate peroxidase and glutathione reductase activities were unaffected by heavy metals. In the case of superoxide dismutase, a clear increase in enzyme activity was observed only in P. nigra under drought conditions, whereas it was inhibited in polluted stands.     

Changes in activity of antioxidative enzymes in wheat (Triticum aestivum) seedlings under cold acclimation

Activated oxygen species such as superoxide radicals, singlet oxygen, hydrogen peroxide and hydroxyl radicals can be produced in plants exposed to low, non-freezing, non-injurious temperatures. To prevent or alleviate oxidative injury, plants have evolved several mechanisms which include scavenging by natural antioxidants and enzymatic antioxidant systems such as superoxide dismutases, catalase and peroxidases. Although overproduction of hydrogen peroxide and increased tolerance to oxidative stress can be induced in wheat by low-temperature treatments, data concerning changes in the enzymatic antioxidant systems are almost absent. With the aim to provide this information, antioxidant enzyme (superoxide dismutases, catalase and peroxidases) activities were analysed in leaves and roots of Triticum aestivum cvs Brasilia (frost resistant in field) and Eridano (less frost resistant in field) seedlings grown at day/night temperatures of 24/22°C (control treatment) and 12/5°C (low-temperature treatment). Our data showed that superoxide dismutase activities were unaffected by low-temperature treatment both in leaves and roots. Catalase activity in leaves and roots was decreased in 12/5°C-grown seedlings, but Brasilia maintained higher catalase activity than Eridano. Differences were also observed in guaiacol peroxidase activities between control and acclimated seedlings: Higher guaiacol peroxidase activities were found in the leaves of 12/5°C-grown seedlings while in roots these activities were lower. Moreover, Brasilia guaiacol peroxidase activities were higher than Eridano. Superoxide dismutase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by low-temperature treatment. Changes in the activities of antioxidant enzymes induced by cold acclimation support the hypothesis that a frost-resistant wheat cultivar, in comparison with a less frost-resistant one, maintains a better defence against activated oxygen species during low-temperature treatment.     

Conjugation resistance to Fusarium graminearum Schwabe with multiple molecular forms of some enzymes in winter wheat

Electrophoretic spectra of multiple molecular forms of peroxidase (EC 1.11.1.7), superoxide dismutase (EC 1.15.1.1), phenol oxidase (EC 1.10.3.1), and cytochrome oxidase (EC 1.9.3.1) in seedlings of two aegilops species and eleven genotypes of bread winter wheat differing in the level of their resistance to Fusarium infection are presented. Several izoforms of peroxidase, phenol oxidase, and superoxide dismutase correlate with the level of resistance to Fusarium. Infection of plants with the pathogen enhances expressiveness of some multiple forms of enzymes. Such response to infection in less pronounced in the sensitive genotypes as compared with that in the resistant ones.     

Induction of heat resistance of wheat coleoptiles by salicylic and succinic acids: Connection of the effect with the generation and neutralization of reactive oxygen species

The influence of salicylic (SaA) and succinic (SuA) acids on the generation of reactive oxygen species (ROS) and the heat resistance of wheat (Triticum aestivum L.) coleoptiles has been studied. The treatment of coleoptiles with 10 μM SaA or SuA results in the accumulation of hydrogen peroxide and enhanced formation of a superoxide anion radical. This effect was partially suppressed by both α-naphthol (the NADPH oxidase inhibitor) and salicylhydroxamic acid (peroxidase inhibitor). SaA and SuA cause an increase in the activity of antioxidant enzymes, such as superoxide dismutase, catalase, and soluble peroxidase, and improve the heat resistance of coleoptiles. Antioxidant ionol and inhibitors of the NADPH oxidase and peroxidase significantly reduce the positive influence of SaA and SuA on the heat resistance of wheat coleoptiles. ROS are considered to be intermediates for heat resistance induction in coleoptiles, treated with SaA and SuA; enhanced ROS generation can be caused by an increased activity of the NADPH oxidase and peroxidase.     

Molybdenum application enhances antioxidant enzyme activity and COR15a protein expression under cold stress in wheat

Under cold stress, reactive oxygen species (ROS) are considered the main source of damage to plant cells. Mechanisms of ROS scavenging in wheat are very important during stress and the antioxidant enzymes superoxide dismutase, Catalase and Glutathione peroxidase are key to facilitating ROS scavenging. Molybdenum (Mo) is involved in many plant physiological and biochemical processes including antioxidant enzymes. This study reports research to investigate the effect of Mo application in enhancing antioxidant enzymes in two wheat cultivars. The results confirmed that antioxidant defense is important in wheat that is exposed to abiotic stress and that changes in activities of antioxidant enzymes occurred during exposure of plants to low non-freezing temperatures and by adding Mo. Mo application had a positive effect on gene expression of both Cbf14 and COR15a protein expression, indicating upregulation of the stress response regulon. In addition, Mo enhanced antioxidant enzymes activity and improved frost tolerance.     

Activities of scavenging enzymes of oxygen radicals in early maturation stages of Paragonimus westermani.

In early maturation stages of Paragonimus westermani (metacercariae, 4-, 8-, 12-week old worms), activities of antioxidant enzymes, such as superoxide dismutase, catalase, peroxidase and glutathione peroxidase, were examined. Specific activity of catalase was the highest in metacercariae and decreasing with age. That of superoxide dismutase was higher in metacercariae and 4-week worms. Specific activity of peroxidase was at its peak in 4-week worms while that of glutathione peroxidase was in 8-week worms. Specific activities of all these antioxidant enzymes were decreased to their lowest in 12-week old adults.     

Hepatoprotective and antioxidant property of Andrographis paniculata (Nees) in BHC induced liver damage in mice

Andrographis paniculata (AP) treatment prevents BHC induced increase in the activities of enzymes y-Glutamyl transpeptidase, glutathione-S-transferase and lipid peroxidation. The activities of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and the levels of glutathione were decreased following BHC effect. Administration of AP showed protective effects in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase as well the level of glutathione. The activity of lipid peroxidase was also decreased. The result indicate antioxidant and hepatoprotective action of A. paniculata.     

Superoxide,hydrogen peroxide,and the gelling of phloem sap from Cucurbita pepo

The possible involvement of superoxide and hydrogen peroxide in the oxidative gelling of phloem exudate from Cucurbita pepo. was investigated. Neither superoxide dismutase (EC 1.15.1.1) nor catalase (EC 1.11.1.6) inhibited the reaction. Although catalase could not be detected in exudate, both peroxidase (EC. 1.11.1.7) and superoxide dismutase were present in reasonable amounts. Polyacrylamide gel electrophoresis revealed one major and one minor isozyme of superoxide dismutase, both of which were adjudged to contain copper and zinc as their prosthetic metals, on the basis of cyanide inhibition and molecular weight.Abbreviations SOD superoxide dismutase     

Modification of Antioxidant Status of Host Cell in Response to Bougainvillea Antiviral Proteins

Bougainvillea xbuttiana antiviral proteins (AVPs) exhibited high antioxidant activity as measured by ferric reducing / antioxidant (FRAP) power assay. These AVPs were also found to modify activities of antioxidant enzymes like superoxide dismutase, peroxidase and catalase. The activities of superoxide dismutase and peroxidase increased, while the activity of catalase decreased in Tobacco mosaic virus (TMV) infected tobacco leaves. The trend was reversed when the leaves were treated with AVP alone. However, in TMV + AVP treated leaves, the activities of all the three enzymes were found to be midway between the activities obtained with other two treatments. It is therefore, suggested that Bougainvillea AVPs might be controlling viral diseases by scavenging reactive oxygen species as well as by altering host plant cell metabolism to maintain its antioxidant status.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

Localisation and metabolism of reactive oxygen species during Bremia lactucae pathogenesis in Lactuca sativa and wild Lactuca spp.

A plant's physiology is modified simultaneously with Oomycete pathogen penetration, starting with release and accumulation of reactive oxygen species (ROS). Localisation of superoxide, hydrogen peroxide, peroxidase and variation in their activity, and the isoenzyme profile of antioxidant enzymes peroxidase (1.11.1.7), catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) were studied in six genotypes of four Lactuca spp. (L. sativa, L. serriola, L. saligna and L. virosa) challenged with Bremia lactucae (race NL16). These factors were related to the differential expression of resistance during the course of 96h after inoculation (hai). Accumulation of hydrogen peroxide in infected cells together with enhanced activity of H(2)O(2)-scavenging enzymes in leaf extracts characterised resistant Lactuca spp. genotypes 6-12hai, and peaked at 48-96hai with expression of a hypersensitive reaction. Substantial changes of guaiacol peroxidase activity were detected only in the cytosolic enzyme; activities of the membrane-bound and the ion-bound enzymes were insignificant in the interactions of host genotypes and pathogen isolate examined. The most significant modifications of ROS metabolism were found in resistant L. virosa (NVRS 10.001 602), a genotype responding to pathogen ingress by a rapid and extensive hypersensitive reaction. Formation of the superoxide anion was not detected in either susceptible or resistant plants, and there was also no increase of superoxide dismutase activity or changes in its isozyme profile. The significance of precise balancing the intracellular level of hydrogen peroxide for variability of phenotypic expression of responses to B. lactucae infection in Lactuca spp. is discussed.