Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi

Aims: The purpose of this study was to investigate the role of H₂O₂ and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H₂O₂‐specific scopoletin fluorometric assay and the CAT‐dependent H₂O₂ consumption assays, it was found that the production rate of intra/extracellular H₂O₂ and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H₂O₂ is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H₂O₂ is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H₂O₂ levels.     

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71–82.73% reduction of lesion length in a detached stem assay (T₃ and T₄ generations) and 76.67–82.0% reduction of lesion area in a detached leaf assay (T₄ generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T₃ and T₄ transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H₂O₂) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H₂O₂ at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H₂O₂ levels, and eliciting defense responses mediated by multiple signaling pathways.

     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

Characterization of a putative thioredoxin peroxidase prx1 of <Emphasis Type="Italic">Candida albicans</Emphasis>

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H₂O₂ scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H₂O₂, menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H₂O₂. These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H₂O₂, but its cellular function is specified to t-BOOH.     

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998     

Hydrogen sulfide alleviated chromium toxicity in wheat

Effects of H₂S on seed germination under chromium (Cr) stress were investigated in wheat (Triticum aestivum L.). Under Cr stress, the percentage of germination of wheat seeds decreased, but this decrease could be alleviated by pretreatment with NaHS, an H₂S donor, in a dose-dependent manner. Furthermore, NaHS significantly enhanced the activities of amylase, esterase, superoxide dismutase, catalase, ascorbate peroxidase, and guaiacol peroxidase in Cr-stressed germinating seeds, whereas reduced the Cr-induced increase in lipoxygenase activity and over-production of malondialdehyde (MDA) and H₂O₂, and sustained slightly higher content of endogenous H₂S.     

Increase in glutathione peroxidase activity in erythrocytes from trisomy 21 subjects.

Glutathione peroxidase activity has been measured in erythrocytes from normal subjects and from trisomy 21 patients. The latter cases show about 50 % increase of this enzyme similar to the increase observed for superoxide dismutase (erythrocuprein) suggesting either localisation of the gene for glutathione peroxidase on chromosome 21 (as is the case for erythrocuprein) or regulation of this enzyme by intracellular levels of O₂^$\text{?}$, H₂O₂ or superoxide dismutase.     

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO₂ and H₂O₂. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H₂O₂ levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum. Xiangbai Dong and Ruiqin Ji contributed equally to this paper.     

Enzymic Pathways Involved in Cell Response to H2O2

An influence of possible interaction of glutathione peroxidase and cyclooxygenase on the clonogenic survival of epithelial cells exposed in vitro to H₂O₂ was investigated. Indomethacin served as the inhibitor of cyclooxygenase, and the use of alkaline (7.5) or acidic (6.5) pH combined with controlled supply of glucose modified glutathione peroxidase activity. Indomethacin affected survival of cells exposed to H₂O₂ in a biphasic manner, enhancing cytotoxicity at lower hydrogen peroxide concentrations, and diminishing it at higher concentrations. The turning point moved gradually to higher concentrations of H₂O₂ corresponding to the augmented decomposition of hydrogen peroxide caused by increased activity of glutathione peroxidase. The data revealed that both enzymic pathways interact in the presence of H₂O₂, resulting in the overall cell survival different from that obtained after inhibition of either.     

Antibacterial effect of hydrogen peroxide-titanium dioxide suspensions in the decontamination of rough titanium surfaces

The chemical decontamination of infected dental implants is essential for the successful treatment of peri-implantitis. The aim of this study was to assess the antibacterial effect of a hydrogen peroxide-titanium dioxide (H₂O₂–TiO₂) suspension against Staphylococcus epidermidis biofilms. Titanium (Ti) coins were inoculated with a bioluminescent S. epidermidis strain for 8 h and subsequently exposed to H₂O₂ with and without TiO₂ nanoparticles or chlorhexidine (CHX). Bacterial regrowth, bacterial load and viability after decontamination were analyzed by continuous luminescence monitoring, live/dead staining and scanning electron microscopy. Bacterial regrowth was delayed on surfaces treated with H₂O₂–TiO₂ compared to H₂O₂. H₂O₂-based treatments resulted in a lower bacterial load compared to CHX. Few viable bacteria were found on surfaces treated with H₂O₂ and H₂O₂–TiO₂, which contrasted with a uniform layer of dead bacteria for surfaces treated with CHX. H₂O₂–TiO₂ suspensions could therefore be considered an alternative approach in the decontamination of dental implants.     

Improved Histochemical Test for In Situ Detection of Hydrogen Peroxide in Cells Undergoing Oxidative Burst or Lignification

An improved version of a simple histochemical test for the in situ assaying the production of hydrogen peroxide in living plant tissue was demonstrated. The test solution containing 50 mM KI in a 4 % potato starch solution was directly applied to the fresh cut surface of the tissue to be tested. Incorporation of an enhancer potassium permanganate (1 % final concentration) into the test reagent resulted in a ten times greater hydrogen peroxide mediated oxidation of iodide ions to iodine, especially in the case when, e.g. suboptimal concentration of H₂O₂ is present or endogenous catalase decomposes the H₂O₂ in tissue as quickly as it is evolved. Subsequently, iodine is complexed by the starch to form a coloured product. H₂O₂ production by wound-induced oxidative burst or lignification can be easily discriminated due to the dual colour response. This revised version was published online in July 2006 with corrections to the Cover Date.     

CYTOCHEMICAL LOCALIZATION OF PEROXIDATIC ACTIVITY OF CATALASE IN RAT HEPATIC MICROBODIES (PEROXISOMES)

Prominent staining of rat hepatic microbodies was obtained by incubating sections of aldehyde-fixed rat liver in a modified Graham and Karnovsky's medium for ultrastructural demonstration of peroxidase activity. The electron-opaque reaction product was deposited uniformly over the matrix of the microbodies. The microbodies were identified by their size, shape, presence of tubular nucleoids, and other morphologic characteristics, and by their relative numerical counts. The staining reaction was inhibited by the catalase inhibitor, aminotriazole, and by KCN, azide, high concentrations of H₂O₂, and by boiling of sections. These inhibition studies suggest that the peroxidatic activity of microbody catalase is responsible for the staining reaction. In the absence of exogenous H₂O₂ appreciable staining of microbodies was noted only after prolonged incubation. Addition of sodium pyruvate, which inhibits endogenous generation of H₂O₂ by tissue oxidases, or of crystalline catalase, which decomposes such tissue-generated H₂O₂, completely abolished microbody staining in the absence of H₂O₂. Neither diaminobenzidine nor the product of its oxidation had any affinity to bind nonenzymatically to microbody catalase and thus stain these organelles. The staining of microbodies was optimal at alkaline pH of 8.5. The biological significance of this alkaline pH in relation to the similar pH optima of several microbody oxidases is discussed. In addition to staining of microbodies, a heat-resistant peroxidase activity is seen in some of the peribiliary dense bodies. The relation of this reaction to the peroxidase activity of lipofuscin pigment granules is discussed.     

Hydrogen Sulfide Maintains Good Nutrition and Delays Postharvest Senescence in Postharvest Tomato Fruits by Regulating Antioxidative Metabolism

As a signaling molecule, hydrogen sulfide (H₂S) plays an indispensable role in the modulation of ripening and senescence in fruits and vegetables. To explore the role of H₂S in regulating metabolism of postharvest tomato, ripening-related physiological parameters, activities of antioxidant enzymes and gene expression were analyzed in H₂S-fumigated tomato fruits. These results show that H₂S significantly delayed the color transition and softening of tomato fruit, and maintained higher level of flavonoids and lower level of anthocyanin during storage. Besides, H₂S could maintain higher level of nutritional-related metabolites, such as reducing sugar, ascorbic acid during postharvest storage. Moreover, H₂S decreased the rate of O₂⁻ production, inhibited the production of H₂O₂ and malondialdehyde (MDA), enhanced the activities of antioxidant enzymes including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) in tomato fruits, while reduced the activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and lipoxygenase (LOX). Besides, the expression of the antioxidant-encoding genes SlCAT2, SlPOD12 was generally upregulated with H₂S fumigation. Principal component analysis (PCA) suggests that H₂S induced significant discrepancy mainly to the differences in firmness, anthocyanin, flavonoid and the activity of guaiacol peroxidase (POD), and the correlation analysis further shows that H₂S affected pigment metabolism and nutritional quality. In conclusion, H₂S could maintain better appearance and nutritional quality, and prolong the storage period of postharvest tomato fruits through activating the antioxidative system.

     

In situ Inactivation of the Oxidase Activity of Xylem Peroxidases by H2O2 in the H2O2-Producing Xylem of Zinnia elegans

Zinnia elegans stems with 3,3′, 5, 5′-tetramethylbenzidine (TMB) in the presence and in the absence of catalase reveals the presence of xylem oxidase activities in the H₂O₂-producing lignifying xylem cells. This staining of lignifying xylem cells with TMB is the result of two independent mechanisms: one is the catalase-sensitive (H₂O₂-dependent) peroxidase-mediated oxidation of TMB, and the other the catalase-insensitive (H₂O₂-independent) oxidation of TMB, probably due to the oxidase activity of xylem peroxidases. The response of this TMB-oxidase activity of xylem peroxidases to different exogenous H₂O₂ concentrations was studied, and the results showed that H₂O₂ at high concentrations (100–1,000 mM) clearly acted as an inactivator of this xylem TMB-oxidase activity, although some inhibitory effect could still be appreciated at 10 mM H₂O₂. This xylem TMB-oxidase activity resided in a strongly basic cell wall-bound peroxidase (pl about 10.5). Given such a scenario, it may be concluded that this TMB-oxidase activity of peroxidase is located in tissues capable of sustaining H₂O₂ production, and that the in situ oxidase activity shown by this enzyme is inactivated by high H₂O₂ concentrations. Received 20 April 1999/ Accepted in revised form 16 August 1999     

Preliminary study on flg22-induced defense responses in female gametophytes of Saccharina japonica (Phaeophyta)

Flg22, which is the most conserved 22-amino acid peptide in the N-terminal part of flagellin, functions as an effective elicitor in higher plants. Marine algae and higher plants share some conserved characteristics on defense response pathways. Flg22-induced defense responses were investigated in female gametophytes of Saccharina japonica. Condensation of chloroplasts and thickened cell walls, as well as relatively stable structures of mitochondria and nucleus indicated that there were hypersensitive programmed cell death occurred after induction by flg22. By using luminol-dependent luminescence detection method, rapid release of H₂O₂ was detected in the induced female gametophytes and reached a peak concentration of about 46 μM at 2 h. Reactive oxygen species production was also observed histologically using the fluorescent dye 2′,7′-dichlorofluorescein diacetate, showing a consistent result with quantitative analysis of H₂O₂. Furthermore, results of antioxidant enzyme activities indicated that there was a trend in the order of catalase > superoxide dismutase > glutathione peroxidase. Finally, high level phenol content of cell-free extracts was found after flg22 induction. According to our results, flg22 could be an effective elicitor which could induce defense responses in female gametophytes of S. japonica.     

Dynamics of enzymes generating hydrogen peroxide in solid-state fermentation ofPanus tigrinus on wheat straw

The relationship between the production of extracellular H₂O₂, hydrogen peroxide-producing enzymes and ligninolytic peroxidase was examined during solid-state cultivation ofPanus tigrinus on wheat straw. Glyoxal oxidase, Mn²⁺-dependent peroxidase and glucose oxidase, capable of H₂O₂ generation, were found in the extracellular enzyme preparation. The production of H₂O₂ has two maxima: the maximal production correlates well with the maximal activities of glyoxal oxidase and Mn²⁺-dependent peroxidase, while another, lower peak of H₂O₂ generation is related to the second peak of Mn²⁺-dependent peroxidase activity. The contribution of glucose oxidase to the production of hydrogen peroxide is probably only marginal. Comparison of the dynamics of these extracellular activities and the ligninolytic peroxidase showed good temporal correlation indicating an interrelation of the two processes.     

Effects of putrescine on oxidative stress induced by hydrogen peroxide in Salvinia natans L.

Salvinia natans L. response to hydrogen peroxide (H₂O₂) induced oxidative stress through physiological activities was evaluated. The plants were incubated with varying concentrations (0, 50, 100 µM) of H₂O₂ and 100 µM of H₂O₂ supplemented with 1 mM putrescine (Put) in hydroponic culture. This is observed with the decline in proline content and its biosynthetic enzymes viz. γ-glutamyl kinase and γ-glutamyl phosphate reductase activity. Protein carbamylated derivative by protein oxidation was another trait for oxidative damages by H₂O₂. The antioxidative enzymes like guaiacol peroxidase (GPX), glutathione reductase (GR), and catalase (CAT) recorded to express through in-gel staining with the H₂O₂ exposure. On nuclear level, plants were sensitive to H₂O₂ where the DNA disintegration was studied with comet assay and maximum comet tail observed at 100 µM H₂O₂ treatment. Application of Put reduced the generation of protein oxidation and comet tail length as well as moderated the enzyme activity as revealed through in-gel staining.     

Oxygen activation and defence against oxygen toxicity in a psychrophilic bacteroidaceae

When suddenly exposed to air the growth of the obligate anaerobic bacterium of the bacteroidaceae type, strain B6, continues for a few hours before coming to a complete stop. When air is shut off soon after growth has ceased, the organism is able to reestablish anaerobic conditions due to an ability to reduce O₂, and resumes normal growth after another few hours. The O₂ reducing ability of the organism is due to the presence in the cells of a particlebound NADH oxidase, a soluble NADPH oxidase and a soluble pyruvate oxidase. The two pyridine nucleotide oxidase reduce O₂ to H₂O₂, the pyruvate oxidase reduces O₂ to H₂O. Catalase and peroxidase were not detected in anaerobically grown cells. Kinetic studies with cell-free extracts showed that the pyruvate oxidase had a considerably greater affinity (smaller K _m) for O₂ and capacity (higher V _max) for O₂ reduction than the two other oxidases. It is postulated that the pyruvate oxidase acts as a scavenger for O₂, leading to the non-toxic reduction product H₂O, and thus functions as a defense mechanism against oxygen toxicity when the organism is exposed to aerobic condition.Abbreviations PY peptone-yeast extract - PYG PY-glucose - PN pyridine nucleotide - PNH reduced PN - CCCP carbonylcyanide m-chlorophenylhydrazone - DNP 2.4-dinitrophenol