Ascorbate-glutathione cycle and H2O2 detoxification in elongating leaf bases of ryegrass: effect of inhibition of glutathione reductase activity on foliar regrowth

The role of the ascorbate-glutathione cycle and AOS detoxification was investigated during leaf growth of defoliated and undefoliated plants of ryegrass ( Lolium perenne L. cv. Bravo). Antioxidants and related enzymatic activities were located in elongating leaf bases (ELBs) of undefoliated plants, following a decreasing gradient from basal (meristem) to distal segments, inverse to H₂O₂ levels. In the meristematic zone, the intense activity of the ascorbate-glutathione cycle and the supply of reducing power by the oxidative pentose phosphate pathway allowed the maintenance of both antioxidant reduction and H₂O₂ detoxification. BCNU (1–3 bis(2-chloroethyl)- N -nitrosourea), a glutathione reductase inhibitor, induced an increase in the meristematic zone in both H₂O₂ and antioxidant levels and a decrease in reduced/oxidized ratios of glutathione and ascorbate. These changes were associated with a reduced foliar regrowth activity. In the absence of BCNU, defoliation did not modify the ratios of reduced/oxidized antioxidants, although it triggered a temporary increase in H₂O₂ level. The results are discussed on the basis of a possible control of leaf growth by glutathione and ascorbate.     

Phosphate Induces Rapid H2O2 Generation in Soybean Suspension Cells

Abstract: Involvement of reactive oxygen species has been implicated in plant defence against pathogens. We report here a novel pathway of H₂O₂ generation induced by the addition of phosphate in soybean ( Glycine max L.) cell suspension cultures. This H₂O₂ generation was initiated shortly after the addition of phosphate, and lasted only approximately one hour, as opposed to several hours observed during an attack by an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg). In addition, when cell cultures were treated with both phosphate and the avirulent pathogen, two distinct oxidative burst events were observed. In contrast to DPI-sensitive Psg -induced H₂O₂ generation, phosphate-induced H₂O₂ generation was insensitive to this NADPH oxidase inhibitor. This suggests that an NADPH oxidase-independent pathway may be involved in the phosphate-induced H₂O₂ accumulation, which could be involved in sensing of phosphate availability in the environment.     

Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide

Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H₂O₂, can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non-mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H₂O₂. This study demonstrated that 661W cells, a retina-derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well-established regulators. The roles of Nox2 and Nox4 in producing pro-survival H₂O₂ were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time-lapse imaging of dichlorofluorescein fluorescence (generated upon H₂O₂ production) in retinal explants was established and it showed that explants also produce a burst of H₂O₂. The increase in H₂O₂ production was partly blocked by an inhibitor of Nox proteins. Overall, this study demonstrates a pro-survival role of Nox2 and Nox4 in retina-derived cells, elucidates some of the regulatory mechanisms and reveals that a similar phenomenon exists in retinal tissue as a whole.     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.     

Effects of catalase on the accumulation of H2O2 in rice cells inoculated with rice blast fungus, Magnaporthe oryzae

Roles of H₂O₂ in the infection process of Magnaporthe oryzae on rice were investigated. In a leaf sheath assay for up to 48 h post-inoculation, the absence or presence of catalase in the conidia suspension was correlated with the level of accumulated H₂O₂ in infected leaf cells, as observed by staining with 3',3-diaminobenzidine tetrahydrochloride. In the incompatible interaction, the appearance of autofluorescence or frequency of cell death characterized by granulation (symptoms characteristic of hypersensitive responses) was not significantly affected by the presence of catalase in the conidia suspension. In the leaf blade assay, inoculation of compatible conidia in the presence of catalase produced more severe symptoms than that of conidia in the absence of catalase at 6 days post-inoculation. These results suggest that, in this host–parasite interaction, the primary role of host-produced H₂O₂ is in limiting hyphal growth after penetration through toxic action. Furthermore, in incompatible interactions, H₂O₂ is implied not to be a major mediator of hypersensitive cell death.     

Elicitation of H2O2 production in cucumber hypocotyl segments by oligo-1,4-α-D-galacturonides and an oligo-β-glucan preparation from cell walls of Phythophthora megasperma f. sp. glycinea

The production of H₂O₂ by cucumber hypocotyl segments ( Cucumis sativus L. cv. Wisconsin SMR 58) in response to α-1,4-linked oligomers of galacturonic acid and oligo-β-glucans from the cell walls of Phytophthora megasperma f. sp. glycinea was studied. Oligogalacturonides with degrees of polymerization of 9 to 13 elicited H₂O₂ production, the most effective being the deca-, undeca- and dodecamers. A similar relationship between size and effect was previously obtained when oligogalacturonides were tested for their ability to elicit lignification in cucumber hypocotyls. The oligogalacturonide-induced increase in H₂O₂ concentration was detected after 4 h, reaching a maximum after 10 h of incubation. The glucan elicitor induced lignification at a 100-fold lower concentration than the oligogalacturonides, but yielded only 10% of the maximum H₂O₂ accumulation seen with oligogalacturonides. The glucan elicitor-induced H₂O₂ production was detectable after 2 h, and reached a maximum after 4 to 6 h. Catalase abolished the elicitation of both phenol red oxidation and lignification in cucumber hypocotyls. At least part of the oligogalacturonide-induced H₂O₂ production appeared to be dependent upon de novo protein synthesis.     

Changes in catalase activity and hydrogen peroxide concentration in plants in response to low temperature

Taxicity of oxygen species such as free radicals and H₂O₂ has been invoked to explain a number of degradative processes in plants, most involving photo-oxidation. Since catalase is a major protectant against accumulation and toxicity of H₂O₂, we examined alterations in catalase activity in several plant species ( Pisum sativum L. cv. Greenfeast, Vigna radiata (L.) R. Wilcz, Cucumis sativus L. cv. Heinz Pickling, and Passiflora spp.) during chilling, and compared this change to change in H₂O₂ content. Catalase activity was reduced in a range of chilling sensitive and tolerant species by exposure to low temperature. This reduction in catalase activity correlated better with the onset of visible symptoms than with the treatment itself. Visible injury in turn was dependent on light and temperature differences. Hydrogen peroxide concentrations invariably decreased with low temperatures.
Reduction in catalase activity therefore does not necessarily imply accumulation of H₂O₂ to damaging levels. The absence of a clear inverse relationship between catalase activity and H₂O₂ concentration suggests the continued activity of other reactions that remove H₂O₂ and these may be important in the tolerance of plants to oxidative attack. Loss of catalase activity may result from the inability of damaged peroxisomal membranes to transport catalase precursors into the peroxisome.     

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.     

A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta)

A hurst of hydrogen peroxide has been found in the red macroalga Eucheuma platycladwn Schmitz as a response to mechanical stress. After exposure of pieces of thalli (2 cm) broken from the plant and stirred with a magnetic bar an oxidative burst was registered, as measured by luminol dependent chemiluminescence (LDC). The burst was totally inhibited by cataluse (EC 1.11.1.6). showing the generation of H_2:O_2; Ten g of seaweed in 300 ml sea water caused a maximal medium concentration of LDC corresponding to 7 u .M H₂O_2; The burst decayed after about 30 min. The decay is probably caused by increased catalase aciivity of the sea water. due to leakage of catalasc from the seaweed. Addition of NaN₃ caused a dramatic increase in LDC. probably due to inhibition of catalase. Similar bursts of active oxygen, involving active oxygen species such as O₂, H₂O₂ and OH. have been reported as pan of the hypcrsensitive reaction in some higher plants, e.g. tobacco. potato and soybean. Exposure of plants or cell suspension cultures to some pathogenic bacteria, fungi, inorganic elicitors or physical damage causes an oxidalive burst that is often followed by necrosis. The production ot active oxygen is thought to he a first defence against invading pathogens. We assume that the oxidative burst from E. platycladum is of a defensive nature, providing a protection against grazers and pathogenic organisms. To our knowledge this is the first repoil of an oxidalive burst from seaweeds.     

Research note : Unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria

The effect of MRS broth on the stability of hydrogen peroxide (H₂O₂) has been studied. Known concentrations (1–100 μg ml⁻¹) of H₂O₂ were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H₂O₂ was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H₂O₂ in MRS broth (pH 6·2) could not be detected.     

Effect of oxygen and peroxide on Bacteroides fragilis cell and phage survival after treatment with DNA damaging agents

Abstract Bacteroides fragilis Bf-2 cells were more sensitive to far-UV radiation, N -methyl- N '-nitrosoguanidine, ethylmethane sulphonate, acriflavine and mitomycin C under aerobic conditions than under anaerobic conditions. The opposite effect was observed with H₂O₂-treated cells and exposure to O₂ enhanced the survival of H₂O₂-treated cells. Pretreatment of cells with sublethal concentrations of H₂O₂ also increased the survival of H₂O₂-treated cells. Reactivation of UV- and X-irradiated and methylmethane sulphonate and H₂O₂-treated phage b-1 was induced by O₂ and H₂O₂ in B. fragilis .     

Role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione S-transferase gene expression in maize (Zea mays L.)

The role of hydrogen peroxide (H₂O₂) and various antioxidants in the regulation of expression of the three Cat and Gst1 genes of maize ( Zea mays L.) has been investigated. Low concentrations of H₂O₂ appeared to inhibit Cat1 , Cat3 , and Gst1 gene expression, while higher doses strongly induced these genes. Time course experiments indicated that high concentrations of H₂O₂ induced Cat1 , Cat2 , and Gst1 gene expression to higher levels, and in less time, than lower H₂O₂ concentrations. Induction of Cat3 was superimposed on the circadian regulation of the gene. These results demonstrate a direct signaling action of H₂O₂ in the regulation of antioxidant gene responses in maize.The effects of the antioxidant compounds N-acetylcysteine, pyrrolidine dithiocarbamate, hydroquinone, and the electrophile antioxidant responsive element (ARE)-inducer β -naphthoflavone were quite different and specific for each gene/compound/concentration combination examined. The response of each gene to each antioxidant compound tested was unique, suggesting that the ability of these compounds to affect expression of the maize Cat and Gst1 genes may not be the result of a common (antioxidant) mode of action. A putative regulatory ARE motif involved in the regulation of antioxidant and oxidative stress gene responses in mammalian systems is present in the promoter of all three maize catalase genes and we tested its ability to interact with nuclear extracts prepared from 10 days post-imbibition senescing scutella. Protein-DNA interactions in the ARE motif and the U2 snRNA homologous regions of the Cat1 promoter were observed, suggesting that ARE may play a role in the high induction of Cat1 in a tissue which, due to senescence, is under oxidative stress.     

Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation

Suspension-cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells irradiated with UV-C (254 nm. 558 J m⁻²) showed a transient production of H₂O₂ as measured by chemiluminescence of luminol in the presence of peroxidase (EC 1.1 1.1.7). The peak concentration of H₂O₂, which occurred at about 60–90 min after irradiation, was 8–9 μ M . The time course for the appearance of H₂O₂ matched that for UV–induced K⁺ efflux. Treatments that inhibited the UV-induced efflux of K⁺, including heat and overnight incubation with cycloheximide and diethylmaleate, also inhibited the appearance of H₂O₂. The converse was not always true, since catalase (EC 1.11.1.6. and salicylhydroxamic acid, which inhibited luminescence, did not stop K⁺ efflux. We conclude that H₂O₂ synthesis depends on K⁺ efflux. Because H₂.O₂ in the extracellular space is required for lignin synthesis in many plant tissues, we suggest that the UV–stimulated production of H₂O₂ is an integral part of a defensive lignin synthesis.     

Induction of oxidative stress and GPX-like protein activation in tomato plants after mechanical stimulation

In Lycopersicon esculentum Mill. (cv. VFN8), mechanical stimulation induced a rapid and transient increase of with hydrogen peroxide (H₂O₂), a part of an oxidative burst. The reaction was followed by an antioxidative response, with the involvement of phospholipid hydroperoxide glutathione peroxidase (PHGPX)-like protein (EC 1.11.1.9). Induction of expression of two putative PHGPX genes was observed in rubbed internodes. To characterize the importance of this antioxidant gene, enzymatic activities of glutathione peroxidase (GPX) and PHGPX were measured, respectively, H₂O₂ and hydroperoxide lipid as oxidant. Only PHGPX activities were induced by the mechanical treatment, suggesting a major role of PHGPX in the mechanisms of antioxidant defence in plant.     

Detection of hydrogen peroxide produced by meat lactic starter cultures

Twelve strains of meat lactic starter cultures (Pediococcus spp. and Lactobacillus plantarum) were found to produce hydrogen peroxide in vitro. The (cumulative) amounts of H₂O₂ produced were measured through the peroxidative action of catalase on H₂O₂ and oxidation of added formate to CO₂ by the H₂O₂-catalase complex formed. There was a problem in building a calibration curve for converting values of formate oxidation into amounts of H₂O₂, either by adding H₂O₂ directly to the assay mixture or having it produced via a glucose-glucose oxidase system.     

Infection biology and defence responses in sorghum against Colletotrichum sublineolum

Aims:  To investigate the infection biology of Colletotrichum sublineolum (isolate CP2126) and defence responses in leaves of resistant (SC146), intermediately resistant (SC326) and susceptible (BTx623) sorghum genotypes.
Methods and Results:  Infection biology and defence responses were studied quantitatively by light microscopy, H₂O₂ accumulation by DAB staining and HRGP accumulation by immunological methods. Inhibition of conidial germination and appressorium formation may represent prepenetration defence responses on the leaf surface. Inducible defence responses in the resistant genotypes included decreases in formation of appressoria as well as accumulation of H₂O₂, HRGPs and phytoalexins. Concomitant with these inducible responses, fungal growth was stopped during or just after penetration in genotypes SC146 and SC326. High levels of H₂O₂ accumulating at late infection stages (5 days after inoculation) in the susceptible genotype BTx623 correlated with necrosis and tissue degeneration.
Conclusions:  The early accumulation of H₂O₂ and HRGPs indicates roles in defence whereas the late accumulation in genotype BTx623 correlated with successful pathogenesis.
Significance and Impact of the Study:  The fact that there is a significant correlation between induced accumulation of H₂O₂, papilla formation and cell wall cross-linking, as evidenced by HRGP accumulation, and cessation of pathogen growth in resistant genotypes may help exploit host resistance in sorghum.     

Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi

The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm. elicited the production of H₂O₂ in cell suspension cultures of the resistant species Ulmus pumila L. This response was not observed in suspensions of the susceptible elm U. campestris Mill. H₂O₂ production started after a lag time of 30–40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h. Treatment of the suspensions with exogenously added H₂O₂ did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin. Spore germination and growth of O. novo-ulmi were significantly delayed with different amounts of H₂O₂ (0.1–1 m M ). These results suggest that H₂O₂ production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection. Whether H₂O₂ is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.     

Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction

Active oxygen species (AOS) are believed to have important roles in plants in general and in plant—pathogen interactions in particular. They are believed to be involved in signal transduction, cell wall reinforcement, hypersensitive response (HR) and phytoalexin production, and to have direct antimicrobial effects. Since current methods are inadequate for localizing AOS in intact plant tissue, most studies have been conducted using cell suspension culture/elicitors systems. 3,3-diaminobenzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H₂O₂ in the presence of peroxidase, and it was found that, by allowing the leaf to take up this substrate, in-vivo and in-situ detection of H₂O₂ can be made at subcellular levels. This method was successfully used to detect H₂O₂ in developing papillae and surrounding haloes (cell wall appositions) and whole cells of barley leaves interacting with the powdery mildew fungus. Thus, H₂O₂ can be detected in the epidermal cell wall subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium from 15 h. The earliest time point for observation of H₂O₂ in relation to epidermal cells undergoing HR is 15 h after inoculation, first appearing in the zones of attachment to the mesophyll cells underneath, and eventually in the entire epidermal cell. Furthermore, it was observed that proteins in papillae and HR cells are cross-linked, a process believed to be fuelled by H₂O₂. This cross-linking reinforces the apposition, presumably assisting the arrest of the pathogen.