Peroxidase Isoforms Research Articles

Petunia × hybrida during transition to flowering as affected by light intensity and quality treatments

Petunia × hybrida was grown under high (H), medium (M) and low (L) light intensity [photoperiod; 16 h d−1, photosynthetic photon flux density (PPFD); 360, 120 and 40 μmol m−2 s−1, respectively] as well as under end-of-day (EOD) red (R) and far-red (FR) light quality treatments [photoperiod; 14.5 h d−1, PPFD; 30 μmol m−2 s−1 EOD; 15 min, Control (C) light; without EOD light treatment]. Shoot growth, leaf anatomical and photosynthetic responses as well as the responses of peroxidase (POD) isoforms and their specific activities following transition to flowering (1–6 weeks) were evaluated. Flower bud formation of Petunia × hybrida was achieved at the end of the 4th week for H light treatment and on the end of the 6th week for FR light treatment. No flower bud formation was noticed in the C and R light treatments. H and M light treatments induced lower chlorophyll (Chla, Chlb, Chla+b) concentrations in comparison to L light. On the other hand R and FR light chlorophyll content were similar to C light. Photosynthetic parameters [CO2 assimilation rate (A), transpiration rate (E) and stomatal conductance (gs) values] were higher in the H light treated plants in comparison to M and L light treated plants. A, E and gs values of R and FR light were similar to C light plants. Leaf anatomy revealed that total leaf thickness, thickness of the contained tissues (epidermis, palisade and spongy parenchyma) and relative volume percentages of the leaf histological components were differently affected within the light intensity and the light quality treatments. POD specific activities increased from the 1st to the 6th week during transition to flowering. Native-PAGE analysis revealed the appearance of four anionic POD (A1–A4) isoforms in all light treatments. On the basis of the leaf anatomical, photosynthetic and plant morphological responses, the production of high quality Petunia × hybrida plants with optimal flowering times could be achieved through the control of both light intensity and light quality. The appearance of A1 and A2 anionic POD isoforms could be also used for successful scheduling under light treatments.

Induction of PR proteins and resistance by the biocontrol agent Clonostachys rosea in wheat plants infected with Fusarium culmorum

Clonostachys rosea (CR) is a common worldwide saprophyte with destructive effect against several plant pathogenic fungi showing antagonistic features against a wide variety of pathogens. We recently isolated a strain of C. rosea, named CR47, from wheat crown infected with Fusarium culmorum (FC); this strain proved to be effective against Fusarium seed borne diseases of cereals under field condition. In this paper the function of C. rosea applied as seed treatment on wheat seedling growth was investigated. In addition, we investigated the expression pattern of peroxidases and chitinases as well as PR4 proteins following both CR treatments of seeds and FC infection and also in the three-component system pathogen–antagonist–wheat. Several chitinase isoforms were induced by CR-treatment both in coleoptiles and roots, whereas some peroxidase isoforms were induced only in the presence of both antagonist and pathogen. In the latter case, it seems that CR-treatment by itself promotes plant growth and reduces the peroxidase expression, while enhances some chitinase isoforms probably involved in cell wall disruption. Moreover, both the antagonist and the pathogen studied induced PR4 protein expression, which probably exerts its role on the invading microorganisms by a translation-inhibitory process that could be ascribed to their ribonuclease activity.

Role of anionic isoforms of peroxidase during phytopathogenic infection of plants from the family Malvaceae

An increase of the activity and the appearance of new isoforms (high-and low-molecular-weight) in the isospectrum of peroxidase were noted after the action of the phytopathogen Verticillium dahliae on plants from the family Malvaceae. The temperature was shown to have an effect on the resistance to infection of the studied plants. Isoelectric focusing showed that anionic isoforms of peroxidase had isopoints 4.7 and 3.5.

Purification of lignin peroxidase isoforms from Streptomyces viridosporus T7A by hydrophobic based chromatographies

Five lignin peroxidases ALiP-P3 isoenzymes from Streptomyces viridosporus T7A were separated by hydrophobic interaction chromatography. The most abundant isoenzyme was purified by reversed phase chromatography and it was analyzed by mass spectrometry.

Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors

The molecular and biochemical mechanism(s) of polyamine (PA) action remain largely unknown. Transgenic tobacco plants overexpressing polyamine oxidase (PAO) from Zea mays exhibited dramatically increased expression levels of Mpao and high 1,3-diaminopropane (Dap) content. All fractions of spermidine and spermine decreased significantly in the transgenic lines. Although Dap was concomitantly generated with H(2)O(2) by PAO, the latter was below the detection limits. To show the mode(s) of H(2)O(2) scavenging, the antioxidant machinery of the transgenics was examined. Specific isoforms of peroxidase, superoxide dismutase and catalase were induced in the transgenics but not in the wild-type (WT), along with increase in activities of additional enzymes contributing to redox homeostasis. One would expect that because the antioxidant machinery was activated, the transgenics would be able to cope with increased H(2)O(2) generated by abiotic stimuli. However, despite the enhanced antioxidant machinery, further increase in the intracellular reactive oxygen species (ROS) by exogenous H(2)O(2), or addition of methylviologen or menadione to transgenic leaf discs, resulted in oxidative stress as evidenced by the lower quantum yield of PSII, the higher ion leakage, lipid peroxidation and induction of programmed cell death (PCD). These detrimental effects of oxidative burst were as a result of the inability of transgenic cells to further respond as did the WT in which induction of antioxidant enzymes was evident soon following the treatments. Thus, although the higher levels of H(2)O(2) generated by overexpression of Mpao in the transgenics, with altered PA homeostasis, were successfully controlled by the concomitant activation of the antioxidant machinery, further increase in ROS was detrimental to cellular functions and induced the PCD syndrome.

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa ( Medicago sativa L.)

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a pI value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing patterns revealed the induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alfalfa tissues.

Differential expression of sunflower peroxidase isoforms and transcripts during necrotrophic interaction with Alternaria helianthi

Sunflower leaf spot caused by the necrotrophic pathogen Alternaria helianthi is one of the most important biotic constraints in sunflower production. The biochemical basis of resistance to infection by A. helianthi in sunflower is poorly elucidated. Differential activation of early defense responses like increased peroxidase activity predisposes the host plant resistance. In the present study, the active role of peroxidase in resistant and susceptible interactions of sunflower with the leaf spot pathogen was demonstrated using native PAGE analysis and Northern blotting. Differential time-dependent induction of peroxidase isoforms was evident in resistant and susceptible genotypes after pathogen inoculation. In the resistant interaction, three peroxidase (PO) isoforms (PO1, PO2, and PO3) were found to be expressed at 2 and 12 h after inoculation, but in susceptible interaction, PO2 was present at 24 and 48 h and PO3 was found only at 24 h after inoculation. However, in the susceptible genotype, expression intensity was lower than in the resistant interaction at all the time intervals. Molecular expression profiling by Northern blotting using heterologous cDNA probes showed rapid up-regulation of peroxidase at 2 and 12 h in resistant interaction. These results demonstrate that peroxidase isoforms are involved in defense against A. helianthi, and genetic regulation in the form of transcript accumulation is important for expression of disease resistance.

Peroxidase Activity, Isozyme Patterns and Electrolyte Leakage in Roots of Cocoyam Infected with Pythium myriotylum

AbstractKnowledge of host defence response in cocoyam against Pythium myriotylum is essential in understanding disease resistance and the development of effective control measures for the cocoyam root rot disease. Four cocoyam accessions were assessed for root rot disease reaction by inoculating the roots of intact plants in the greenhouse and detached roots in the laboratory with P. myriotylum isolate ‘Mamu’ from Cameroon. Electrolyte leakage in detached infected roots and peroxidase activities and isozyme expressions in crude protein extracts collected from healthy roots of inoculated and non‐inoculated plants at 0, 2, 4, 6 and 8 days post inoculation (dpi) were used independently to assess resistance/tolerance. Based on the results, cocoyam accessions RO1054 and RO3015 were rated tolerant/moderately resistant while accessions RO2063 and ‘Local White’ (LW) were rated susceptible to P. myriotylum isolate ‘Mamu’. The time‐course spectrophotometric analysis of peroxidase activity showed an increase in activity in the roots of the tolerant RO1054, 8 days after inoculation. One peroxidase isoform (Rf 0.66) was induced only in roots of the susceptible inoculated accessions ‘Local White’ and R02063 at 4 and 6 dpi. One acidic peroxidase isozyme (pI 4.5) and one basic isozyme (pI 7.4) were detected only in roots of inoculated plants. Our results suggest that electrolyte leakage in infected roots can be used to assess tolerance/susceptibility in cocoyam and that tolerance to the root rot disease in cocoyam may be associated in part with an increased generation of peroxidase activity in the roots.

Irreversible cross‐linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2

Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid membrane of chloroplasts play a central role in scavenging reactive oxygen species generated by photosystems. These enzymes are inactivated within minutes by H2O2 when the reducing substrate, ascorbate, is depleted. We found that, when the enzyme is inactivated by H2O2, a heme at the catalytic site of a stromal APX isoform is irreversibly cross-linked to a tryptophan residue facing the distal cavity. Mutation of this tryptophan to phenylalanine abolished the cross-linking and increased the half-time for inactivation from <10 to 62 s. In contrast with H2O2-tolerant peroxidases, rapid formation of the cross-link in APXs suggests that a radical in the reaction intermediate tends to be located in the distal tryptophan so that heme is easily cross-linked to it. This is the first report of a mutation that improves the tolerance of chloroplast APXs to H2O2.

Biological responses of wheat (Triticum aestivum) plants to the herbicide chlorotoluron in soils

Chlorotoluron is a phenylurea herbicide that is widely used for controlling grass weeds in the land of cereal, cotton and fruit production. However, extensive use of this herbicide may lead to its accumulation in ecosystems, thus inducing the toxicity to crops and vegetables. To assess chlorotoluron-induced toxicity in plants, we performed the experiment focusing on the metabolic adaptation of wheat plants (Triticum aestivum) to the chlorotoluron-induced oxidative stress. The wheat plants were cultured in the soils with chlorotoluron at concentrations of 0-25mg/kg. Chlorotoluron accumulation in plants was positively correlated with the external chlorotoluron concentrations, but negatively with the plant growth. Treatment with chlorotoluron induced the accumulation of O(2)(-) and H(2)O(2) in leaves and resulted in the peroxidation of plasma membrane lipids in the plant. We measured the endogenous proline level and found that it accumulated significantly in chlorotoluron-exposed roots and leaves. To understand the biochemical responses to the herbicide, activities of the antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were assayed. Analysis of SOD activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) revealed that there were three isoforms in the roots and leaves, but the isoforms in the tissues showed different patterns. Also, using the native PAGE, 6 isoforms of root POD and 10 in leaves were detected. The total activity of POD in roots was significantly enhanced. Activities of APX in roots and leaves showed a similar pattern. The CAT activities were generally suppressed under the chlorotoluron exposure.

Talc-based formulation of Pseudomonas fluorescens-induced defense genes against powdery mildew of grapevine

The efficacy of plant growth-promoting rhizobacteria (PGPR) was tested against the powdery mildew of grapevine caused by Uncinula necator (Schw.) Burn. Foliar application of talc-based Pseudomonas fluorescens strain Pf1 at 2% significantly reduced the incidence of powdery mildew under glasshouse conditions through the induction of defense gene products such as peroxidase (PO), polyphenol oxidase (PPO) and chitinase. The isoforms of peroxidases were observed in Native Poly acrylamide gel electrophorosis (PAGE). Plants challenged with Pf1 against U. necator expressed three isoforms of peroxidase (PO1 – PO3) while only one isoform of PO was expressed in control with less intensity. The expression pattern of PPO showed that there was no extra isoform except the level of induction in Pf1-treatment against the powdery mildew pathogen. Native gel electrophoresis detected four isoforms of chitinase in plants challenged with Pf1.

Change in phenylalanine ammonia lyase activity and isozyme patterns of polyphenol oxidase and peroxidase by salicylic acid leading to enhance resistance in cowpea against Rhizoctonia solani

In this paper, we describe a study concerning the determination of phenylalanine ammonia lyase (PAL) activities leading to decline in disease progress caused by Rhizoctonia solani after application of salicylic acid (SA). Two applications of 1.4 mM SA (pH 6.5) followed by inoculation with Rhizoctonia solani resulted in a quantitative change in polyphenol oxidase (PPO), peroxidase (PRX) isoforms and increase in PAL activities from 4.38 to 19.48 unit g−1 (FM) h−1 in Bundel-1, UPC-4200 and IFC-902 cowpea genotypes. Increase in PAL activities was further observed specifically in UPC-4200 when plants were exposed with Rhizoctonia solani spores. Total soluble proteins did not change after SA treatment, however they were significantly increased in SA sprayed-inoculated UPC-4200 genotype. Of the ten detected isoforms of polyphenol oxidase, isoforms 7 and 10, and isoform 4 of peroxidase showed increased activities by SA application. The disease symptoms measured as areas under progress curve (AUDPC) indicated less A value in SA sprayed Bundel-1 and UPC-4200 genotypes over their controls.

Loss of Vacuolar Proton-translocating ATPase Activity in Yeast Results in Chronic Oxidative Stress

Yeast mutants lacking vacuolar proton-translocating ATPase (V-ATPase) subunits (vma mutants) were sensitive to several different oxidants in a recent genomic screen (Thorpe, G. W., Fong, C. S., Alic, N., Higgins, V. J., and Dawes, I. W. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6564-6569). We confirmed that mutants lacking a V(1) subunit (vma2Delta), V(o) subunit, or either of the two V(o) a subunit isoforms are acutely sensitive to H(2)O(2) and more sensitive to menadione and diamide than wild-type cells. The vma2Delta mutant contains elevated levels of reactive oxygen species and high levels of oxidative protein damage even in the absence of an applied oxidant, suggesting an endogenous source of oxidative stress. vma2Delta mutants lacking mitochondrial DNA showed neither improved growth nor decreased sensitivity to peroxide, excluding respiration as the major source of the endogenous reactive oxygen species in the mutant. Double mutants lacking both VMA2 and components of the major cytosolic defense systems exhibited synthetic sensitivity to H(2)O(2). Microarray analysis comparing wild-type and vma2Delta mutant cells grown at pH 5, permissive conditions for the vma2Delta mutant, indicated high level up-regulation of several iron uptake and metabolism genes that are part of the Aft1/Aft2 regulon. TSA2, which encodes an isoform of the cytosolic thioredoxin peroxidase, was strongly induced, but other oxidative stress defense systems were not induced. The results indicate that V-ATPase activity helps to protect cells from endogenous oxidative stress.

A role of glycosyl moieties in the stabilization of bitter gourd ( Momordica charantia) peroxidase

The possible role of carbohydrate moieties in the stabilization of proteins has been investigated by using bitter gourd peroxidase as a model system. A comparative study of glycosylated and non-glycosylated isoenzymes of bitter gourd peroxidase was performed at various temperatures, pH, water-miscible organic solvents, detergents and chaotropic agent like urea. The pH-optima and temperature-optima of both glycosylated and non-glycosylated isoforms of bitter gourd peroxidase remained unchanged. The probes employed were changes in the enzyme activity and fluorescence. The glycosylated form of peroxidase retained greater fraction of enzyme activity against the exposure caused by various physical and chemical denaturants. The unfolding of both forms of enzyme in the presence of high urea concentrations, studied by fluorescence, indicated greater perturbations in the conformation of non-glycosylated preparation. The different properties examined thus indicated that glycosylation plays an important role in the stabilization of native conformation of proteins against the inactivation caused by various types of denaturants.

Variability of antioxidant enzyme activity and isoenzyme profile in needles of Serbian spruce ( Picea omorika (Panč.) Purkinye)

Variations were studied of the activity and isoenzyme patterns of soluble peroxidase, catalase, catechol oxidase and superoxide dismutase, in needles of the Balkan endemic conifer Serbian spruce, Picea omorika (Panč.) Purkinye. The samples were collected from the natural habitat of the species, Mt. Tara. Seasonal changes were found to affect enzymatic activities and isoenzyme profiles. Total protein content was significantly lower in the summer than in other seasons. Several isoforms of peroxidase, catechol oxidase and superoxide dismutase (SOD), as well as two catalase isoenzymes were detected. The number of peroxidase isoenzymes was greatest during the vegetative season. Catalase and catechol oxidase peaked in summer and spring, respectively. Total SOD and Mn-SOD activities were significantly higher in the winter samples than the summer ones.

Dimorphic DNA variation in the anionic peroxidase gene AtPrx53 of Arabidopsis thaliana

The DNA polymorphism in the AtPrx53 gene which encodes anionic peroxidase was analyzed in 20 Arabidopsis thaliana accessions. There are two divergent sequence types (Col and Dj-like haplotypes) in the AtPrx53 gene that differ by 2 indel and 16 non-singleton nucleotide polymorphisms including 5 nucleotide polymorphic sites responsible for 4 deduced amino acid replacements. Two of the amino acid substitutions (Phe/Ser180and Asp/Asn270) could be responsible for the difference in electrophoretic mobility of AtPrx53 allozymes. One of them (Phe/Ser180) lies within the hypervariable region, indicating that this amino acid polymorphism is subjected to balancing selection. The revealed difference between deduced allozymes is related to the dimorphism in mobility of three major anionic peroxidase isoforms which according to previously established data encoded by AtPrx53 gene. The haplotype Col which included 12 accessions from three different continents is characterized by faster mobility of three isoforms in comparison with the Dj haplotype represented by eight accessions. There is a significant association between the haplotype and several developmental traits: leaf number, flowering time, main stem height etc. Lines of the Dj haplotype have shorter duration of vegetative stages and flower earlier than most of Col haplotype accessions. The reasons of this association are discussed.

The activity of peroxidase in various cell fractions of wheat plants infected with Septoria nodorum berk.

The activities of peroxidase isoforms and hydrogen peroxide content in leaf cuttings of wheat (Triticum aestivum L., cv. Diamant) resistant to Septoria blotch were studied during aging and following the infection with Septoria nodorum Berk. The differential activation of peroxidase isoforms was regulated by hydrogen peroxide level in the tissue. At early stages of fungus development in plant tissues, the decrease in the activities of soluble, membrane and ion-bound fractions of peroxidase elevated the level of hydrogen peroxide in infected tissues and rapidly activated peroxidase isoforms in infected tissues as compared to the aging ones even before disease symptoms appeared. The anionic peroxidases, which were first to respond to the pathogen, seem to stand for wheat resistance to fungal infections and the protection of leaf tissues from oxidative stress.

Antioxidative Enzymes in the Response of Buckwheat (Fagopyrum esculentum Moench) to Ultraviolet B Radiation

The behavior of the enzymatic antioxidant defense system was studied in buckwheat leaves and seedlings subjected to short-term enhanced UV-B radiation. The effects of UV-B action were monitored immediately after irradiation as well as after recovery. The applied dose induced an increase in lipid peroxidation and total flavonoid content, a decrease in chlorophyll content, and a change in enzymatic digestibility of extracted DNA. The activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase, and soluble peroxidase, as well as the isoelectric focusing (IEF) pattern of peroxidase isoforms, was analyzed. In treated as well as recovered seedlings, soluble and ascorbate peroxidase activities were increased. The activity of SOD was not altered, whereas CAT activity was decreased. In contrast to seedlings, only CAT activity was increased in treated and recovered leaves.

Effects of selenium supplementation on expression of glutathione peroxidase isoforms in cultured human lung adenocarcinoma cell lines

Selenium is an essential nutrient, a component of several anti-oxidant enzymes, and a possible factor in cancer risk, including lung cancer. We determined the subtoxic range of selenium concentration (as sodium selenite) required to increase and maintain the expression of anti-oxidant selenoproteins gluthathione peroxidases GPX1 and GPX4 at a constant level in cultures of human lung adenocarcinoma cell lines (H460, H1703 and H1944) and in HPL1D, a non-transformed lung epithelial cell line. Selenium dose-dependently increased GPX1 protein expression 1.8-fold in HPL1D cells and approximately 40-fold in H460 and H1944 cancer cells, with maximum effects at 20-40 nM. GPX4 protein was also increased, but more so in HPL1D (five-fold) than in H460 or H1944 cells (two- to three-fold). GPX1 mRNA showed similar patterns but differences of lesser magnitude. GPX1 protein and activity level was not consistently detectable in H1703 cells, with or without Se supplementation; its mRNA was present but very low. GPX4 protein level was also low in H1703 cells, but was markedly increased by selenium supplementation (48-fold). These results confirm a role for selenium in risk of lung cancer and the independent regulation of GPX1 and GPX4. Characterization of individual tumors with regard to GPX1 and GPX4 levels and regulation might be useful for interpretation of clinical studies on effects of selenium in lung cancer risk.

Peroxidase activity and inducibility in the sea fan coral exposed to a fungal pathogen

The enzymatic defense mechanisms of Gorgonia ventalina to the fungal pathogen Aspergillus sydowii may play important roles in colony resistance to infection. In this study, we examined the role of the superfamily of peroxidase enzymes in the coral response to a naturally occurring pathogen. We examined the inducibility of peroxidases by experimentally exposing corals to A. sydowii and found that peroxidase activity was induced after an 8 day incubation period. In contrast, naturally infected corals collected from the reef had lower peroxidase activity when compared to healthy corals. Infected sea fans from the field also had less measurable protein in their tissues and increased purple sclerites near infection sites and it is likely that these infections are months old. Using native-PAGE activity gels, we detected 5 peroxidase isozymes in healthy corals, indicating that multiple isoforms of peroxidase with a plurality of possible functions are present in this coral. The role of the peroxidase enzymes in disease resistance was examined by testing anti-fungal activity of commercially available and partially purified sea fan peroxidases. In both cases there was significant, dose-dependent anti-fungal activity. While peroxidases are ubiquitous enzymes involved in many cellular pathways, we also hypothesize that G. ventalina utilizes these enzymes as an integral component in disease resistance pathways. As such, they may also contribute to the initiation of physiochemical defenses such as melanization and lipid soluble anti-fungal metabolites.