Ribonuclease activities in bean roots

Vicia faba meristematic and elongating root cells (zones 0–4 and 10–20 mm) contained one nuclease (A₁) and four ribonucleases (A₂, A₃, C₁, C₂). When the overall activity of each enzyme was expressed per cell, the elongating cells contained 4-, 4-, 4-, 10- and 17-fold more activity than meristematic cells for A₁, C₁, C₂, A₂ and A₃, respectively.     

Suppressive effect of H2O2 on increase in activities of acid invertases in rye roots

Although various effects of H₂O₂ on plant cellular functions have been reported, the effects of H₂O₂ on glycolytic enzymes remain unknown. It was shown that H₂O₂ has a suppressive effect on increase in activities of soluble and insoluble acid invertases among a number of glycolytic enzymes during a growth stage of rye roots.     

Exogenous H2O2 increased catalase and peroxidase activities and proline content in Nitraria tangutorum callus

Antioxidative responses and proline accumulation induced by exogenous H₂O₂ were investigated in the callus from halophyte Nitraria tangutorum Bobr. H₂O₂-treated callus exhibited higher H₂O₂ content than untreated callus. The activities of catalase (CAT) and peroxidase (POD) significantly increased in the callus treated with H₂O₂, while ascorbate peroxidase (APX) activity decreased. In addition, significantly enhanced proline content was observed in the callus treated by H₂O₂, which could be alleviated by H₂O₂ scavenger dimethylthiourea and calcium (Ca) chelator ethylene glycol bis-(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (EGTA). Moreover, γ-glutamyl kinase (GK) activity increased in H₂O₂-treated callus, but proline dehydrogenase (PDH) activity decreased significantly, and the reduction was partly abolished by EGTA or Ca channel blocker verapamil. Assays using a scanning electron microscope showed significantly enhanced Ca content in H₂O₂-treated callus.     

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.     

Pyruvate accumulation during phosphate deficiency stress of bean roots

Culture of bean plants (Phaseolus vulgaris L. cv., Złota Saxa) for 16 d on phosphate-deficient nutrient medium resulted in an over twofold increase of pyruvate concentration in the root tissues. In a variety of plant tissues, the marked decline in cellular concentrations of adenylates and inorganic phosphate (Pi) influences the activity of pyruvate producing enzymes, which are dependent on the availability of ADP. In bean roots after 16 d of phosphate starvation pyruvate producing enzymes: phosphoenolpyruvate phosphatase (EC 3.1.3.2) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) had higher activities compared to those of control plants. The observed decrease of alanine and ethanol concentration and also alcohol dehydrogenase (EC 1.1.1.1) activity in phosphate-deficient roots may be the effect of the restrictions in pyruvate utilizing pathways. The increased activity of mitochondrial NAD-malic enzyme (EC 1.1.1.40) as well as the lower consumption of pyruvate during respiration of phosphate-deficient roots indicate that pyruvate concentration in mitochondria may be elevated. It is proposed that pyruvate accumulation in phosphate-deficient roots and alternative oxidase participation in respiration are important aspects of plant metabolic adaptations to Pi limitation, and may play a role in reducing oxidative stress induced by phosphate deficiency.     

Effect of variable glutathione peroxidase activity on H2O2-related cytotoxicity in cultured aortic endothelial cells

Primary cultures of porcine aortic endothelial cells were used to assess the effects of O2 intermediates produced by 10-40 mU/ml xanthine oxidase (XO; +2 mM hypoxanthine) or 25-100 mU/ml glucose oxidase (GO; +5 mM glucose). A 60-min incubation in the presence of the enzyme systems resulted in a dose-dependent toxic effect with evidence of cytolysis (increased LDH release) and cell loss (decrease in DNA and protein content), when these indexes were measured 24 hr after completion of the enzyme reaction. Decreased [3H]thymidine incorporation into DNA was the most sensitive index of cell dysfunction for both enzyme systems. The effects of various scavengers and enzymes indicated that H2O2 was the main O2 intermediate involved in the cytotoxicity resulting from the XO-hypoxanthine reaction. Increased glutathione peroxidase activity associated with the addition of 2 X 10(-7) M selenomethionine to culture medium had a partial protective effect which could be related to an increased rate of H2O2 degradation. Evidence for increased DNA synthesis after injury was found in cells previously exposed to XO-hypoxanthine, the degree of increase in [3H]thymidine incorporation being dependent on the intensity of the initial cytotoxicity. Cultured endothelial cells provide a useful tool to evaluate the role of O2 intermediates in endothelial cell injury, to test the effects of protective agents, and to study the repair process.     

Changes in antioxidant and lignifying enzyme activities in sunflower roots (Helianthus annuus L.) stressed with copper excess

Treatment with 50 microM CuSO4 for five days caused significant decrease in dry-matter production and protein level of ten-day-old sunflower seedling roots. An increase of lipoperoxidation product rate was also observed. The involvement of some enzyme activities in the sunflower root defence against Cu-induced oxidative stress was studied. Copper treatment induced several changes in antioxidant enzymes. SOD (superoxide dismutase, EC 1.15.1.1) activity was reduced but CAT (catalase, EC 1.11.1.6) and GPX (guaiacol peroxidase, EC 1.11.1.7) activities were significantly enhanced. The lignifying peroxidase activities, assayed using coniferyl alcohol and syringaldazine, were also stimulated. Analysis by native gel electrophoresis of syringaldazine peroxidase activity showed the stimulation of an isoform (A2) and the induction of another one (A1) under cupric stress conditions. On the other hand, the activity of PAL (phenylalanine ammonia lyase, EC 4.3.1.5), which plays an important role in plant defence, was also activated. The possible mechanisms by which Cu-induced growth delay and changes in enzymatic activities involved in plant defence processes are discussed.     

Phenol removal using Brassica juncea hairy roots: role of inherent peroxidase and H(2)O(2)

Removal of phenol, a major pollutant in aqueous effluents was studied using plant hairy root cultures. Among four different species of hairy roots tested, Brassica juncea showed the highest potential for phenol removal. The effect of phenol concentration and reuse in a batch system was studied using B. juncea hairy root cultures. Unlike most of the studies reported earlier, phenol removal by the hairy roots was seen to take place without the need for addition of external hydrogen peroxide (H(2)O(2)). To understand the mechanism of phenol removal, levels of peroxidase and phenol oxidase were monitored in the hairy roots. Peroxidase activity in the roots was enhanced when exposed to phenol, while phenol oxidase remained constant. Since peroxidase has a pre-requisite for H(2)O(2), the levels of H(2)O(2) were monitored for its in situ synthesis. H(2)O(2) levels were seen to increase in the presence of phenol. Thus, a mechanism wherein hairy roots also produce H(2)O(2) besides peroxidase, as a protection strategy of plant against xenobiotic stress is plausible.     

Modulation of reactive oxygen species activities and H2O2 accumulation during compatible and incompatible tomato-root-knot nematode interactions

Here, the interaction of Melodoigyne incognita virulent and avirulent pathotypes with susceptible and Mi-resistant tomato (Solanum lycopersicon) has been studied. Significant differences in nematode penetration occurred 2 days postinoculation (dpi) and became stable from 3 dpi onwards. The hypersensitive cell response (HR) in resistant plants prevented the installation of the avirulent pathotype. The virulent pathotype overcame the Mi (nematode) resistance and induced feeding sites in root cells without triggering HR. Reactive oxygen species (ROS), visualized by subcellular reduction of nitroblue tetrazolium, accumulated in nematode penetrated cells. Quantitative analyses with dichlorofluorescein indicated that the oxidative burst occurred very early with both pathotypes, with an enhanced rate in hyper-responsive cells. Hydrogen peroxide (H(2)O(2)), detected by cerium chloride reaction, accumulated in the cell walls and especially in cells neighbouring HR. The apoplastic location of cerium perhydroxide indicated that either the plasma membrane or the cell wall was the primary site of the superoxide/H(2)O(2) generator. The data provide evidence, for the first time, for ROS-generated signals and their spatiotemporal expression in the host and nonhost interaction of tomato with nematodes.     

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H₂O₂ and decreased the ratio of scopoletin (consumed):H₂O₂ (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O₂ of 2:1. On step-wise addition of a small concentration of H₂O₂ a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H₂O₂ ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H₂O₂ in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H₂O₂ during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.     

Scavenging of H2O2 and production of oxygen by horseradish peroxidase

Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the "oxidative burst" associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2-scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed.     

Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities

H₂O₂ can freely crosses membranes and in the presence of Fe²⁺ (or Cu⁺) it is prone to participate in Fenton reaction. This study evaluated the concentration and time-dependent effects of H₂O₂-induced oxidative stress on MnSOD, Se:GPx and catalase and on aconitase. Acute and chronic H₂O₂ treatments were able to induce oxidative stress in HeLa cells as they significantly decreased aconitase activity and also caused a very significant decrease on antioxidant enzyme activities. The inhibition of enzyme activities was time- and concentration-dependent. Chronic treatment with 5 µM H₂O₂/h after 24 h was able to decrease all enzyme activities almost at the same level as the acute treatment. Acute and chronic treatments on antioxidant enzyme activities were prevented by cell treatment with ascorbic acid or N-acetylcysteine. These results indicate that antioxidant enzymes can also be affected by the same ROS they produce or neutralize if the time of exposure is long enough.     

Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase

Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H(2)O(2) exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris-HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35 min at 80 °C) and T(m) (from 73 to 77.5 °C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H(2)O(2) stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H(2)O(2) tolerance in concentrated buffer.     

Effect of duration of exercise on excess postexercise O2 consumption

This study was undertaken to determine the effect of exercise duration on the time course and magnitude of excess postexercise O2 consumption (EPOC). Six healthy male subjects exercised on separate days for 80, 40, and 20 min at 70% of maximal O2 consumption on a cycle ergometer. A control experiment without exercise was performed. O2 uptake, respiratory exchange ratio (R), and rectal temperature were monitored while the subjects rested in bed 24 h postexercise. An increase in O2 uptake lasting 12 h was observed for all exercise durations, but no increase was seen after 24 h. The magnitude of 12-h EPOC was proportional to exercise duration and equaled 14.4 +/- 1.2, 6.8 +/- 1.7, and 5.1 +/- 1.2% after 80, 40, and 20 min of exercise, respectively. On the average, 12-h EPOC equaled 15.2 +/- 2.0% of total exercise O2 consumption (EOC). There was no difference in EPOC:EOC for different exercise durations. A linear decrease with exercise duration was observed in R between 2 and 24 h postexercise. No change was observed in recovery rectal temperature. It is concluded that EPOC increases linearly with exercise duration at a work intensity of 70% of maximal O2 consumption.     

Effect of a highly dispersed copper powder on superoxide dismutase and glutathione peroxidase activities in experimental myocardial infarct

Administration of highly dispersed copper powder in a dose 0.2 mg/kg three days before modelled coronary-occlusion myocardial infarction caused transitory increase in activity of antioxidant enzymes--superoxide dismutase and glutathione peroxidase in the necrotic zone of myocardium of rats, and also staunch increase in the activity of these enzymes in noninfarction region.     

Salinity-induced subcellular accumulation of H2O2 in leaves of rice

The localization of salt-induced H₂O₂ accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl₃ staining at ultrastructure level. When the 3-week-old rice plants were affected by 100 mM NaCl for 14 days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H₂O₂ accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl₃ were mainly observed especially around the swelling of thylakoids. H₂O₂ accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H₂O₂ accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H₂O₂ under salinity is responsible for the changes in chloroplast ultrastructure. H₂O₂ accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H₂O₂ and the production is light dependent under salinity.     

Tyrosinase and superoxide dismutase activities of peroxidase in the vacuoles of beet roots

In the absence of H₂O₂, isoforms of vacuolar phenol-dependent peroxidase (PO) in beet (Beta vulgaris) roots oxidized phenolic compounds like tyrosinases. Tyrosinase activity of PO manifested a clearly expressed pH-dependence with the optimum at pH of 8.0–9.0; peroxidase activity was the highest at pH 5.0–7.0. The inhibitory analysis confirmed a specificity of observed reactions. Along with tyrosinase activity, PO manifested SOD-like activity, which was also expressed in the absence of H₂O₂ and depended on some factors, for example, on the way of analyzed sample preparation. This activity appeared at a long-term dialysis and low temperature. SOD-like PO activity was observed in the presence of such substrates as 3,3′-diaminobenzidine and IAA. The results obtained allow a conclusion that vacuolar PO, as well as PO of other localization and origin is a polyfunctional enzyme, which, under definite conditions, can catalyze reactions of oxidase type.     

Polyoxometalates as peroxidase mimetics and their applications in H2O2 and glucose detection

Polyoxometalates (H(3)PW(12)O(40), H(4)SiW(12)O(40) and H(3)PMo(12)O(40)) have been proven to possess intrinsic peroxidase-like activity for the first time, which can catalyze oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) by H(2)O(2) to form a blue color in aqueous solution. Among them, H(3)PW(12)O(40) (PW(12)) exhibits higher catalytic activity to TMB than natural enzyme HRP and other two POMs. In addition, H(3)PW(12)O(40)/graphene exhibited higher activity than H(3)PW(12)O(40) in this catalytic oxidation reaction due to the effect of graphene in promoting the electron transfer between the substrate and catalyst. POMs/H(2)O(2)/TMB system provides a simple, accurate approach to colorimetric detection for H(2)O(2) or glucose. The colorimetric method based on POMs showed good response toward H(2)O(2) and glucose detection with a linear range from 1.34×10(-7) to 6.7×10(-5) mol/L and 1×10(-7) to 1×10(-4) mol/L, respectively. The results showed that it is a simple, cheap, more convenient, highly selective, sensitive, and easy handling colorimetric assay.     

Effect of morphactin on certain plant growth substances in bean roots

The effect of morphactin (methyl-2-chloro-9-hydroxyfluorene-9-carboxylate) on the content of several plant growth substances in bean roots was determined. Beans (Phaseolus vulgaris L. cv. Spartan) were soaked in aqueous solutions of morphactin, 1 x 10^-4, 1 x 10^-5, and 1 x 10^-6M and grown in moist vermiculite. As controls were used beans grown in water-moistened vermiculite either intact or having the root tips removed (decapped). The roots, morphactin-treated, controls, and the decapped ones were analyzed for indol-3-yl acetic acid (IAA), indol-3-yl acrylic acid (IAcA), indol-3-yl pyruvic acid (IPyA), indol-3-yl lactic acid (1LA), abscisic acid (ABA), gibberellins GA₁, GA₃, GA₄, and GA₉ using gas-liquid chromatographic methods. Morphactin, while affecting the geotropical responses, changed also the growth substance content of roots. IAA, ABA, GA₁, and GA₉ contents decreased, IPyA, IAeA, GA₃, and GA₄ contents were not affected and ILA content increased slightly with increasing dosages of morphactin. Growth substance pattern of decapped roots resembled that of the roots treated with the highest dose, 1 x 10^-4M, of morphactin.