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.     

Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels

Numerous studies argue that salicylic acid (SA) is an important component of the plant signal transduction pathway(s) leading to disease resistance. The discovery that the SA-binding protein is a catalase, whose activity is blocked by SA, led to the proposal that one of SA's modes of action is to inhibit this H₂O₂-degrading enzyme and thus elevate H₂O₂ levels. To test this model, an attempt was made to mimic the action of SA by reducing the synthesis of catalase using antisense RNA technology. Analyses of transgenic tobacco plants that expressed the tobacco catalase 1 ( cat1 ) or catalase 2 ( cat2 ) gene in an antisense orientation indicate that there is no correlation between modest to high levels of reduction in catalase activity and activation of plant defenses such as pathogenesis-related (PR)-1 protein synthesis. However, three independent antisense catalase transgenic plants (ASCAT1 Nos 16, 17, and 28), which exhibited the most severe reduction in catalase activity (∼90% or more), developed chlorosis or necrosis on some of their lower leaves. These same leaves accumulated very high levels of PR-1 proteins and showed enhanced resistance to tobacco mosaic virus. Necrosis and elevated SA, which appear to result from severe depression of catalase levels, may be responsible for the induction of these defense responses.     

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.     

Protein dephosphorylation mediates salicylic acid-induced expression of PR-1 genes in tobacco

Several lines of evidence suggest that salicylic acid (SA) is an endogenous signal for the activation of several plant defense responses, including the expression of genes encoding pathogenesis-related (PR) proteins such as the acidic PR-1 proteins. During recent years, studies have suggested that interaction of SA with catalase and ascorbate peroxidase leads to two signals in tobacco - elevated H₂O₂ levels and lipid peroxides. However, to date, relatively little is known about the molecular and biochemical mechanisms that mediate transduction beyond these signals or through other SA-effector proteins. Using protein kinase and phosphatase inhibitors, this study demonstrates that PR-1 gene induction can be mediated by dephosphorylation of serine/threonine residue(s) of two or more unidentified phosphoproteins. The protein phosphatase inhibitors, okadaic acid and calyculin A blocked SA-mediated induction of PR-1 genes, implying the involvement of a phosphoprotein downstream of SA. The protein kinase inhibitors K-252a and staurosporine induced PR-1 gene expression. PR-1 gene induction by K-252a was suppressed by okadaic acid. Surprisingly, this induction was also suppressed in NahG transgenic tobacco plants which convert SA to catechol. Moreover, K-252a stimulated production of SA and its glucoside, suggesting that another phosphoprotein acts upstream of SA. Taken together, these results suggest that there are two (or more) phosphoproteins which function in the same signal transduction pathway leading to PR-1 gene induction. The SA-inducible acidic PR-2 genes were similarly affected by the inhibitors, while the genes for actin and phenylalanine ammonia lyase were not.     

Salicylic acid modulates ozone-induced hypersensitive cell death in tobacco plants

Ozone-tolerant Bel B and ozone-sensitive Bel W3 tobacco cultivars were subjected to acute ozone fumigation (200 p.p.b. for 3 h) and the subcellular localization of H₂O₂ was then studied. H₂O₂ accumulated on the cell walls and plasma membrane of both cultivars but the accumulation pattern differed greatly. H₂O₂ production was high in both cultivars immediately after fumigation, but, in the tolerant Bel B cultivar, after 7 h was only detected in some spongy cells adjacent to epidermal cells. Instead, in the sensitive Bel W3 cultivar, accumulation was still abundant in the cell walls of palisade, spongy and epidermal cells at this time. Significant changes in apoplastic ascorbate pool were noted in both cultivars in the first hours after fumigation. As the reduced ascorbate content remained unchanged, the marked increase in total ascorbate must have originated from the striking increase in dehydroascorbate, particularly in the ozone-sensitive Bel W3. Exposure of plants to ozone resulted in a marked transient increase in both free and conjugated salicylic acid (SA) as well as an increase in the activity of benzoic acid 2-hydroxylase which catalyses SA biosynthesis. SA induction differed greatly in the two cultivars, in that: (1) SA accumulation was far greater in the ozone-sensitive Bel W3 cv. and (2) the maximum SA peak was delayed in Bel W3 and observed only 7 h after fumigation ended. These results suggest that a high SA content, as documented in the ozone-sensitive Bel W3 cultivar, could trigger the production of ROS with subsequent SA-mediated cell-death.     

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.     

Role of Extracellular Polysaccharide (EPS) Slime of Plant Pathogenic Bacteria in Protecting Cells to Reactive Oxygen Species

Pseudomonas syringae pv. phaseolicola , a phytopathogenic bacterium, seemed very sensitive in planta to the adverse action of reactive oxygen species (ROS) produced by two chemical systems. The disease symptoms in host plants were also suppressed by ROS. Several other plant pathogenic bacteria ( P. syringae pv. pisi, Erwinia amylovora, Xanthomonas campestris pv. pelargonii ) as well as P. fluorescens were also sensitive in vitro to the inhibiting or killinig action of ROS. It was shown that O₂ and H₂O₂ were produced in our two chemical systems and were involved in the killing action. OH'however was not involved in the adverse action on bacteria of the ROS. Superoxide dismutase and catalase were able to reverse the killing action of ROS. When the EPS slime around bacteria was removed by washing and centrifuging the cells, bacteria were more sensitive to ROS. However, when the cells of EPS- mutants were washed and centrifuged, their sensitivity to the killing action of ROS did not change because the lack of slime around the mutant cells.
The EPS^- Tn5 mutants of P. syringae pv. phaseolicola and the natural EPS^- mutant of E. amylovora were more sensitive to ROS than the wild type strains. These results support the idea that the EPS slime protects bacteria from ROS (O^-₂ and H₂O₂).     

Relationship between active oxygen species and cardenolide production in cell cultures of Digitalis thapsi: effect of calcium restriction

Elimination of calcium ions from the medium of undifferentiated cell cultures of Digitalis thapsi increased cardenolide production and induced extracellular H₂O₂ accumulation, as measured by the quenching of pyranine fluorescence. The addition of catalase reduced the response and the inclusion of superoxide dismutase enhanced the loss of fluorescence. This suggested that, besides H₂O₂, the superoxide anion was also formed before dismutating to H₂O₂. Additionally, exogenous H₂O₂ or superoxide dismutase stimulated cardenolide production whereas the addition of catalase markedly reduced it. These results point to a connection between H₂O₂ and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products; however, changes in the antioxidant system of D. thapsi cells were observed. Catalase activity was extremely low in control cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase activity was not modified in calcium-free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutathione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol-bis-β-aminoethyl ether and LaCl₃. All these results indicate that the depletion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. thapsi cultures.     

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.     

Amyloid β-Mediated Oxidative and Metabolic Stress in Rat Cortical Neurons: No Direct Evidence for a Role for H2O2 Generation

Abstract: H₂O₂ and free radical-mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H₂O₂-scavenging enzyme catalase protects neurons in culture against Aβ-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H₂O₂. Aβ-mediated elevation in intracellular H₂O₂ production is suppressed by addition of a potent H₂O₂ scavenger without any significant neuroprotection. Three intracellular biochemical markers of H₂O₂-mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde-3-phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24-h exposure of neurons to Aβ. Our results challenge a pivotal role for H₂O₂ generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.     

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.     

Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis-related expression under high light

Transgenic tobacco deficient in either Cat1 (Cat1AS), Cat2 (Cat2AS), or both (CatGH) was generated through sense and antisense technology. Cat1AS, Cat2AS, and CatGH plants showed no visible phenotype when grown at low light (100 µmol m⁻² sec⁻¹. Under these conditions, deficiency in Cat1 and/or Cat2 did not lead to constitutive pathogenesis-related (PR-1) expression and did not potentiate PR-1 induction by exogenous salicylic acid. This demonstrates that catalase suppression per se is not a sufficient signal for PR-1 induction. In Cat1-deficient plants exposed to higher light intensities (250–1000 µmol m⁻² sec⁻¹), PR-1 expression was induced without pathogenic challenge and multiplication of Pseudomonas syringae pv. syringae was repressed. Yet, it is unlikely that Cat1 deficiency is mimicking the mode of action of salicylic acid in tobacco, because, concurrent with PR-1 induction, Cat1 deficiency at high light provoked severe leaf damage, characterized by white necrotic lesions. Taken together, these results do not support the model that catalase inactivation is the key route by which salicylic acid induces PR defense responses in healthy tissue. However, because catalase deficiency is potentially lethal to leaves, catalase inactivation by salicylic acid could be of importance in the establishment of hypersensitive responses.     

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.     

Hydrogen Peroxide Production by Rat Brain In Vivo

Abstract: H₂ O₂ production by rat brain in vivo was observed with a method based on the measurement of brain catalase. The administration to the rat of 3-amino-1, 2, 4-triazole, an H₂ O_2- dependent inhibitor of catalase, caused progressive inhibition of brain catalase activity in both the supernatant and pellet fractions of homogenates of the striatum and prefrontal cortex. The prevention of catalase inhibition by prior administration of ethanol confirmed that catalase inhibition in vivo was dependent upon H₂ O₂. A significant portion of the catalase (30-33%) appeared in the supernatant fraction from a slow-speed homogenization procedure and was not significantly contaminated by either erythrocytes or capillaries. In the whole homogenate, less than 6% of the catalase activity was attributed to erythrocytes. Modification of intracellular monoamine oxidase activity by either pargyline or reserpine did not change the rate of inhibition of catalase by aminotriazole. A probable interpretation of these data is that H₂ O₂ generated by mitochondrial monoamine oxidase does not reach the catalase compartment; the catalase is contained in particles described by other investigators as the microperoxisomes of brain. In studies in vitro , the production of H₂ O₂ by rat brain mitochondria with either dopamine or serotonin as substrate was confirmed.     

Inhibition of Dopamine Release by Prostaglandin EP3 Receptor via Pertussis Toxin-Sensitive and -Insensitive Pathways in PC12 Cells

Abstract: Hydrogen peroxide (H₂O₂) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H₂O₂ on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H⁺-ATPase (V-ATPase) in the vesicle membrane. We report here that the V-ATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H₂O₂. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H₂O₂-induced inhibition of the V-ATPase, and ATP or GTP partially protect the ATPase from inhibition by H₂O₂. These and other results suggest that the mechanism of inhibition of the V-ATPase by H₂O₂ involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V-ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down-regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.     

Metabolism of hydrogen peroxide by the scavenging system in Chlamydomonas reinhardtii

The metabolism of hydrogen peroxide by the scavenging system was studied in Chlamydomonas grown in a selenium-lacking and a selenium-containing medium. In cells of the former, 40% of external hydrogen peroxide (H₂O₂) was scavenged by ascorbate peroxidase (AsAP; EC 1.11.1.11) and the residual H₂O₂ by catalase (EC 1.11.1.6). The enzymes involved in the ascorbate-glutathione cycle including AsAP. were localized in the chloroplast. In cells of the latter, glutathione peroxidase (GSHP; EC 1.11.1.9) functioned primarily in the removal of external H₂O₂. GSHP was located solely in the cytosol. The Chlamydomonas AsAP was relatively stable in ascorbate-depleted medium as compared with chloroplast AsAP of higher plants. No inactivation of the enzyme was found upon its incubation with hydroxyurea, an inhibitor of the chloroplast enzyme of higher plants. The enzyme showed higher specificity with pyrogallol than with ascorbate. The amino acid sequences in the N-terminal region of Chlamvdomonas AsAP showed no significant similarity to any other AsAP from higher plants and Euglena . The enzyme had a molecular mass of 34 kDa. The K_m values of the enzyme for ascorbate and H₂O₂ were 5.2±0.3 and 25±3.4 μ M , respectively. Hydrogen peroxide was generated at a rate of 6.1±0.8 μmol mg^-1 chlorophyll h^-1 in intact chloroplasts isolated from Chlamydomonas cells grown in the presence of Na-selenite, and it diffused from the organelles into the medium.     

Comparison of quantitative and qualitative methods of detecting hydrogen peroxide produced by human vaginal strains of lactobacilli

A quantitative method was developed for the measurement of micromolar quantities of H₂O₂ produced in Rogosa broth and peptonized milk broth by vaginal strains of lactobacilli isolated from women. The production of substantial amounts reproducibly was dependent on the growth of the organisms in acid media (pH ≤6.0) under anaerobic or micro-aerophilic conditions with continuous agitation. The addition to the media of the enzyme inhibitor, 3-amino-l,2,4-triazole, with or without catalase sometimes induced the production of H₂O₂ especially in non-agitated cultures. However, other agents such as concanavalin and o -dianisidine had no enhancing effect, and catalase or peroxidase alone completely inhibited H₂O₂ production.
The H₂O₂ produced in the acid media was stable for more than a month at 5°C but not in media at pH ≥ 7.0. Of five strains of lactobacilli tested by the quantitative method and by a chromogenic qualitative method (Rogosa-catalase or -peroxidase agar), three consistently produced H₂O₂ measurable by the former method, but none did so after growth of the organisms on Rogosa-catalase/peroxidase agar which suggested that the qualitative method was unreliable. The fact that H₂O₂ was produced in substantial quantities by some strains and not at all by others enabled H₂O₂-producers and non-producers to be distinguished easily.     

Hydrogen peroxide release from bacterial spores during germination

The release of free H₂O₂ from spores of Clostridium perfringens and Bacillus megaterium during germination has been demonstrated using the scopoletin fluorescence assay. Scopoletin oxidation was markedly inhibited when exogenous catalase was added, and was also influenced by the concentration of spores. H₂O₂ release into the germination medium was observed to parallel the O₂ consumption during germination, suggesting that the H₂O₂ may arise from certain O₂-dependent metabolism associated with initiation of spore germination.     

Detoxification of hydrogen peroxide maintains the water transport activity in figleaf gourd (Cucurbita ficifolia) root system exposed to low temperature

The figleaf gourd ( Cucurbita ficifolia Bouché) root system has the ability to take up water and nutrients at low soil temperatures, and in the present paper, we attempt to reveal some of the molecular mechanisms behind this low-temperature tolerance. Exposure of figleaf gourd root system to low temperature induced accumulation of H₂O₂ along the plasma membrane but not in the cytoplasm. H⁺-ATPase (EC 3.6.1.35) activity of isolated root plasma membranes and root hydraulic conductivity ( Lp_r ) were largely insensitive to externally applied H₂O₂. However, using bromocresol purple, it was shown that the acidification of the medium surrounding the root was strongly inhibited with low temperature- and H₂O₂-treated roots. Addition of catalase (EC 1.11.1.6) to the root medium during low-temperature exposure led to a recovery of H⁺-efflux along the root surface and increased Lp_r , demonstrating the importance of an H₂O₂ detoxification system in the root cells. Additional evidence for an increased Lp_r was obtained by the Fenton reaction wherein a warming of the solution increased the activity of the detoxification system. All available evidence suggests that the ability of figleaf gourd root system to maintain a low level of H₂O₂ in the cytoplasm and to detoxify reactive oxygen species is related to the maintenance of water transport activity at low temperatures.