Accumulation Of Reactive Oxygen Intermediates Research Articles

Nitric oxide signaling in deconvolution

Changes in cellular redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signaling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide (NO) and a parallel accumulation of reactive oxygen intermediates (ROIs). The emerging data suggests these small, redox-active molecules orchestrate a plethora of immune responses. In this context, we are principally exploring S-nitrosylation, the addition of an NO moiety to a rare, highly reactive protein cysteine thiol to form an S-nitrosothiol (SNO). This key redox-based, post-translational modification is becoming established as a central mechanism to regulate plant immune responses, including: control of ROI synthesis, accumulation of the immune activator, salicylic acid, pathogen-triggered, hypersensitive cell death and also defense strategies at the plant cell wall. I will present some of our recent work associated with this research area.

MEDU-05. THE ROLE OF GABA METABOLISM IN MEDULLOBLASTOMA

Abstract BACKGROUND: Brain tumors are the most common cause of childhood oncological death, and medulloblastoma (originating in the cerebellum) is the most common malignant pediatric brain tumor. In the microenvironment of the brain, especially the cerebellum, variables related to GABA, the major inhibitory neurotransmitter in the nervous system, are particularly prominent. Abnormal GABAergic Receptor activation has been described in in aggressive MYC-driven Group 3 medulloblastoma. However these studies did not look at the metabolic contribution of GABA for the development of medulloblastomas. In addition to its role in neurotransmission through GABA receptor, GABA can act as a trophic factor during nervous system development to influence cellular events including proliferation, migration, differentiation, synapse maturation, and cell death. Under conditions that inhibit the tricarboxylic acid cycle (TCA), impair respiration, and enhance the accumulation of reactive oxygen intermediates, GABA can be used as an NADH energy source for growth through the GABA-shunt pathway regulators (ABAT, SSADH, GAT-1, GAT-3). Therefore, we hypothesize that blocking GABA-metabolic-shunt will lead to growth suppression and invasiveness of medulloblastoma in the cerebellar GABA-rich microenvironment. RESULTS: Our results show RNA microarray from patient medulloblastoma tissue have high expression of GABA-shunt regulators with ~3-fold increase in the expression of ABAT in MYC amplified versus non-amplified MYC tumors. When medulloblastomas were supplemented with GABA, there was a significant fold change in expression of GABA-shunt mediators and induction of large and stable tumor spheres with Epithelial-Mesenchymal Transition gene expression signature. We next investigated whether a novel perrilyl alcohol-based small molecule NEO216 targeted the GABA-shunt metabolic pathway. NEO216 administration significantly reduced GABA-mediated NADH levels, reversed EMT-profiling, leading to loss of sphere formation. CONCLUSION: Thus, the expression of GABA-metabolic shunt in medulloblastoma could be a malignant microenvironmental adaptation for growth and metastasis that could potentially be exploited through targeted therapy for patients benefit.

S-nitrosylation of NADPH oxidase regulates cell death in plant immunity

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.

Protective Role of HO-1 for Alcohol-Dependent Liver Damage

Background/Aims: Alcoholic liver disease is continuously increasing in developed countries being a leading cause of death worldwide. Chronic ethanol consumption induces oxidative stress by accumulation of reactive oxygen intermediates (ROI) while reducing the cellular antioxidant defense. Induction of heme oxygenase-1 (HO-1) may protect primary human hepatocytes (hHeps) from such damage. Thus, the aim of this study was to investigate the potential of polyphenols to protect hHeps from ethanol-dependent oxidative damage. Methods: hHeps were isolated by collagenase perfusion. ROI and cellular glutathione (GSH) were measured by fluorescent-based assays. Cellular damage was determined by lactate dehydrogenase (LDH) leakage and staining for apoptosis and necrosis. Nuclear translocation of Nrf2 and HO-1 expression were analyzed by Western blot. Results: Ethanol and TGF-β rapidly increase ROI and reduce GSH in hHeps, causing apoptosis with a release of approximately 40% total LDH after 72 h. Similar to incubation with hemin preincubation and co-incubation of cells with nifedipine, verapamil and quercetin significantly reduce oxidative stress and resulting cellular damage, in a dose-dependent manner, by initiating nuclear translocation of Nrf2 which in turn induces HO-1 under the control of p38 and ERK. Blocking of HO-1 activity with ZNPP9 reverses the protective effect of all three substances. Conclusion: Our results suggest that increasing HO-1 activity in hHeps protects them from oxidative stress-dependent damage. As polyphenols have great potential to induce HO-1 expression, they may play an important role for future therapeutic strategies to protect liver from oxidative stress-dependent damage observed during chronic alcohol consumption.

Rice isoflavone reductase‐like protein is associated with root development and ROS stress resistance

Isoflavone phytoalexins and isoflavone reductase gene in the rice has not been identified and characterized. We characterized OsIRL protein using Northern, Western, 2‐DE, immunohistochemical analysis, and transgenic plant in rice. The transcript and proteins were more abundant in stems and roots than in flower and leaves. Expression of OsIRL was more accumulated in the root than in the shoot and seed. To investigate the release of reactive oxygen intermediates (ROI) by seed during germination, we utilized the apoplastic oxidation of 2¡‐,7¡‐dichlorofluorescin to fluorescent 2¡‐7¡‐dichloroflurescein and DAB staining as an in vivo assay. Expression of OsIRL was increased in the root elongation and maturation position carrying accumulation of ROI. Expression of OsIRL was increased in the oxidants (H2O2, FeCl3, MV) and decreased in the antioxidants such as reduced glutathione in the root and rice suspension cell. OsIRL was accumulated in the root sclerenchyma, outside of cortex, endodermis, and metaphloem cell. A certain concentration of H2O2 was necessary for root elongation, suggesting that OsIRL protein may play a role as antioxidant. The OsIRL transcripts levels were highly increased in callus, leaves, stem, and roots by treatment with ABA. The transgenic OsIRL plants have methyl viologen resistance, ROS‐generator, indicating that OsIRL have involved in root growth and resistance to oxidative stress.

Mutants of GABA Transaminase (POP2) Suppress the Severe Phenotype of succinic semialdehyde dehydrogenase (ssadh) Mutants in Arabidopsis

BackgroundThe γ-aminubutyrate (GABA) shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD), the mitochondrial enzymes GABA transaminase (GABA-T; POP2) and succinic semialdehyde dehydrogenase (SSADH). We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs) and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported.Principal FindingsTo elucidate the role of succinic semialdehyde (SSA), γ-hydroxybutyrate (GHB) and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants.SignificanceWe conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.

Serum Apoptosis Markers in Acute Liver Failure: A Pilot Study

We sought to determine whether circulating apoptotic markers are altered in acute liver failure (ALF), differ with etiology, or predict clinical outcome in this condition. Serum levels of soluble Fas (sFas), tumor necrosis factor-alpha (TNF-alpha), hepatocyte growth factor (HGF), and interleukin-6 (IL-6) were measured in 67 acute liver failure patients, as well as controls. In a subset of the groups, we measured serum M-30 antigen, an exposed neoepitope from caspase cleavage. We also assessed M-30 immunoreactivity in liver tissue of ALF patients and controls. Median levels for TNF-alpha, HGF, IL-6, and M-30 antigen were at least 10-fold greater in ALF than in hepatitis C virus or normal controls (P < .0001). Median day 1 sFas, day 3 sFas, and day 1 HGF levels varied according to etiology of acute liver failure (P = .004, P = .011, and P = .019, respectively), with values for drug-induced liver injury and acetaminophen-related ALF higher than other etiologies. Median M-30 antigen levels were significantly higher in patients who were transplanted and/or died (2183 U/L) than spontaneous survivors (1004 U/L) (P = .026). M-30 immunoreactivity in liver tissue was significantly greater in ALF patients than HCV controls (P = .004). TNF-alpha, HGF, IL-6, and M-30 antigen were significantly elevated in ALF. High levels of sFas and HGF might help to confirm a diagnosis of drug-induced liver injury or acetaminophen-related ALF. Higher levels of M-30 antigen are associated with poor clinical outcomes in ALF.

Reactive oxygen intermediates and oxalic acid in the pathogenesis of the necrotrophic fungus Sclerotinia sclerotiorum

An effective colonization of the host plant tissue by the necrotrophic fungus Sclerotinia sclerotiorum requires the secretion of the non-host specific toxin oxalic acid (OA), which is known to suppress the generation of reactive oxygen intermediates (ROI). A full-length cDNA coding for an oxalate decarboxylase (TOXDC), which converts OA into CO2 and formate, was isolated from the basidiomycete Trametes versicolor. It was overexpressed in tobacco plants to study the role of ROI and OA in the interaction between tobacco and S. sclerotiorum. The transgenic plants contained less OA and showed a delayed colonization of S. sclerotiorum; furthermore a strong ROI accumulation and nearly no catalase activity compared to the wild type (WT) plants could be detected. In addition, inoculation experiments with transgenic catalase-deficient plants (CAT1AS) and in vitro studies showed that S. sclerotiorum copes with strong ROI stress. Our results indicate that OA supports the infection process caused by S. sclerotiorum and the fungus itself is able to tolerate high ROI concentrations.

Regulatory Mechanisms of ROI Generation are Affected by Rice spl Mutations

Reactive oxygen intermediates (ROIs) play a pivotal role in the hypersensitive response (HR) in disease resistance. NADPH oxidase is a major source of ROI; however, the mechanisms of its regulation are unclear. Rice spl mutants spontaneously form lesions which resemble those occurring during the HR, suggesting that the mutations affect regulation of the HR. We found that spl2, spl7 and spl11 mutant cells accumulated increased amounts of H(2)O(2) in response to rice blast fungal elicitor. Increased accumulation of ROIs was suppressed by inhibition of NADPH oxidase in the spl cells, and was also observed in the ozone-exposed spl plants. These mutants have sufficient activities of ROI-scavenging enzymes compared with the wild type. In addition, spl7 mutant cells accumulated higher amounts of H(2)O(2) when treated with calyculin A (CA), an inhibitor of protein phosphatase. Furthermore, spl2 mutant plants exhibited accelerated accumulation of H(2)O(2) and increased rates of cell death in response to wounding. These results suggest that the spl2, spl7 and spl11 mutants are defective in the regulation of NADPH oxidase, and the spl7 mutation may give rise to enhancement of the signaling pathway which protein dephosphorylation controls, while the spl2 mutation affects both the pathogen-induced and wound-induced signaling pathways.

GABA shunt deficiencies and accumulation of reactive oxygen intermediates: insight from Arabidopsis mutants

In plants, succinic semialdehyde dehydrogenase (SSADH)-deficiency results in the accumulation of reactive oxygen intermediates (ROI), necrotic lesions, dwarfism, and hypersensitivity to environmental stresses [Bouché, N., Fait, A., Moller, S.G. and Fromm, H. (2003) Proc. Natl. Acad. Sci. USA. 100, 6843–6848]. We report that Arabidopsis ssadh knockout mutants contain five times the normal level of γ-hydroxybutyrate (GHB), which in SSADH-deficient mammals accounts for phenotypic abnormalities. Moreover, the level of GHB in Arabidopsis is light dependent. Treatment with γ-vinyl-γ-aminobutyrate, a specific γ-aminobutyrate (GABA)-transaminase inhibitor, prevents the accumulation of ROI and GHB in ssadh mutants, inhibits cell death, and improves growth. These results provide novel evidence for the relationship between the GABA shunt and ROI, which may, in part, explain the phenotype of SSADH-deficient plants and animals.

Role of chloroplast trienoic fatty acids in plant disease defense responses

Trienoic fatty acids (TAs) are the major polyunsaturated fatty acid species in the membrane lipids in plant cells. TAs are crucial for the adaptation to abiotic stresses, especially low- or high-temperature stress. We show that TAs in chloroplast membrane lipids are involved in defense responses against avirulent bacterial pathogens. Avirulent pathogen invasion of plants induces a transient production of reactive oxygen intermediates (ROI), programmed cell death and subsequent disease resistance. The Arabidopsis fad7fad8 mutation, which prevents the synthesis of TAs in chloroplast lipids, caused the reduction in ROI accumulation in leaves inoculated with Pseudomonas syringae pv. tomato DC3000 (avrRpm1). Linolenic acid, the most abundant TA, activated the NADPH oxidase that is responsible for ROI generation. TAs were transferred from chloroplast lipids to extrachloroplast lipids coincident with ROI accumulation after inoculation with Pst DC3000 (avrRpm1). Furthermore, the fad7fad8 mutant exhibited reduced cell death and was compromised in its resistance to several avirulent P. syringae strains. These results suggest that TAs derived from chloroplast lipids play an important role in the regulation of plant defense responses.

Mitochondrial succinic-semialdehyde dehydrogenase of the gamma-aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants.

The gamma-aminobutyrate (GABA) shunt is a metabolic pathway that bypasses two steps of the tricarboxylic acid cycle, and it is present in both prokaryotes and eukaryotes. In plants the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase and the mitochondrial enzymes GABA transaminase and succinic-semialdehyde dehydrogenase (SSADH). The activity of the GABA shunt in plants is rapidly enhanced in response to various biotic and abiotic stresses. However the physiological role of this pathway remains obscure. To elucidate its role in plants, we analyzed Arabidopsis T-DNA knockout mutants of SSADH, the ultimate enzyme of the pathway. Four alleles of the ssadh mutation were isolated, and these exhibited a similar phenotype. When exposed to white light (100 micromol of photons per m2 per s), they appear dwarfed with necrotic lesions. Detailed spectrum analysis revealed that UV-B has the most adverse effect on the mutant phenotype, whereas photosynthetic active range light has a very little effect. The ssadh mutants are also sensitive to heat, as they develop necrosis when submitted to such stress. Moreover, both UV and heat cause a rapid increase in the levels of hydrogen peroxide in the ssadh mutants, which is associated with enhanced cell death. Surprisingly, our study also shows that trichomes are hypersensitive to stresses in ssadh mutants. Our work establishes a role for the GABA shunt in preventing the accumulation of reactive oxygen intermediates and cell death, which appears to be essential for plant defense against environmental stress.

Protective effect of resveratrol on beta-amyloid-induced oxidative PC12 cell death.

Beta-amyloid peptide is considered to be responsible for the formation of senile plaques that accumulate in the brains of patients with Alzheimer's disease. There has been compelling evidence supporting the idea that beta-amyloid-induced cytotoxicity is mediated through the generation of reactive oxygen intermediates (ROIs). Considerable attention has been focused on identifying phytochemicals that are able to scavenge excess ROIs, thereby protecting against oxidative stress and cell death. Resveratrol (3,5,4'-trihydroxy-trans-stilbene), a phytoalexin found in the skin of grapes, has strong antioxidative properties that have been associated with the protective effects of red wine consumption against coronary heart disease ("the French paradox"). In this study, we have investigated the effects of resveratrol on beta-amyloid-induced oxidative cell death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with beta-amyloid exhibited increased accumulation of intracellular ROI and underwent apoptotic death as determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL staining). Beta-amyloid treatment also led to the decreased mitochondrial membrane potential, the cleavage of poly(ADP-ribose)polymerase, an increase in the Bax/Bcl-X(L) ratio, and activation of c-Jun N-terminal kinase. Resveratrol attenuated beta-amyloid-induced cytotoxicity, apoptotic features, and intracellular ROI accumulation. Beta-amyloid transiently induced activation of NF-kappaB in PC12 cells, which was suppressed by resveratrol pretreatment.

Reactive oxygen intermediates in plant-microbe interactions: who is who in powdery mildew resistance?

Reactive oxygen intermediates (ROIs) such as hydrogen peroxide (H(2)O(2)) and the superoxide anion radical (O*(2)(-)) accumulate in many plants during attack by microbial pathogens. Despite a huge number of studies, the complete picture of the role of ROIs in the host-pathogen interaction is not yet fully understood. This situation is reflected by the controversially discussed question as to whether ROIs are key factors in the establishment and maintenance of either host cell inaccessibility or accessibility for fungal pathogens. On the one hand, ROIs have been implicated in signal transduction as well as in the execution of defence reactions such as cell wall strengthening and a rapid host cell death (hypersensitive reaction). On the other hand, ROIs accumulate in compatible interactions, and there are reports suggesting a function of ROIs in restricting the spread of leaf lesions and thus in suppressing cell death. Moreover, in situ analyses have demonstrated that different ROIs may trigger opposite effects in plants depending on their spatiotemporal distribution and subcellular concentrations. This demonstrates the need to determine the particular role of individual ROIs in distinct stages of pathogen development. The well-studied interaction of cereals with fungi from the genus Blumeria is an excellent model system in which signal transduction and defence reactions can be further elucidated in planta. This review article gives a synopsis of the role of ROI accumulation, with particular emphasis on the pathosystem Hordeum vulgare L.- Blumeria graminis.

Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response

Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response

Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response.

Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The origin of these ROI has been controversial. Arabidopsis respiratory burst oxidase homologues (rboh genes) have been proposed to play a role in ROI generation. We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes. Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000(avrRpm1) and the oomycete parasite Peronospora parasitica. We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000(avrRpm1) inoculation. However, enhanced cell death is observed after infection of mutant lines with P. parasitica. Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.

Pulmonary induction of proinflammatory mediators following the rat exposure to benzo(a)pyrene-coated onto Fe 2O 3 particles

Epidemiological evidence firmly implicated an interactive effect between Fe 2O 3 and benzo(a)pyrene (B(a)P) in causing lung cancer. However, despite intensive investigation, the mechanism involved is not precisely established. Since the accumulation of reactive oxygen intermediates (ROI)-mediated damage and/or immune-induced injury might be a possible cause of lung cancer, we studied the oxidative and the inflammatory effects of Fe 2O 3 (3 mg), B(a)P (3 mg) or B(a)P (3 mg)-coated onto Fe 2O 3 (3 mg) particles on this relevant organ target in Sprague–Dawley rats. We investigated lipid peroxidation (malondialdehyde; MDA) and secretion of some inflammatory mediators (tumor necrosis factor-alpha, TNF-α; interleukin-1 beta, IL-1β; nitric oxide, NO) in lungs. In addition, mRNA expressions of TNF-α, IL-1β and inductible nitric oxide synthase (iNOS) were evaluated. Our results show that exposure to Fe 2O 3 and B(a)P, alone or in association, induced 2-fold increases in MDA production suggesting thereby oxidative stress conditions ( P<0.01). Exposure to Fe 2O 3, B(a)P or B(a)P-coated onto Fe 2O 3 particles significantly increased both mRNA expression and/or synthesis of inflammatory mediators. The main findings of this work were that the association of Fe 2O 3 and B(a)P induces more pronounced induction of inflammatory mediators (IL-1β secretion, P<0.01; IL-1β mRNA expression, P<0.01; iNOS mRNA expression, P<0.05) than B(a)P by itself. Hence, our results may explain why concurrent exposure to Fe 2O 3 and B(a)P is more deleterious in lungs than exposure to B(a)P alone.

Barley Mla and Rar mutants compromised in the hypersensitive cell death response against Blumeria graminis f.sp. hordei are modified in their ability to accumulate reactive oxygen intermediates at sites of fungal invasion.

The pathogenesis-related accumulation of superoxide radical anions (O2*-) and hydrogen peroxide (H2O2) was comparatively analyzed in a barley line (Hordeum vulgare L. cv Sultan-5) carrying the powdery mildew (Blumeria graminis f.sp. hordei, Speer, Bgh) resistance gene Mla12, and in susceptible mutants defective in Mla12 or in genes "required for Mla12-specified disease resistance" (Rar1 and Rar2). In-situ localization of reactive oxygen intermediates was performed both by microscopic detection of azide-insensitive nitroblue tetrazolium (NBT) reduction or diaminobenzidine (DAB) polymerization, and by an NBT-DAB double-staining procedure. The Mla12-mediated hypersensitive cell death occurred either in attacked epidermal cells or adjacent mesophyll cells of wild-type plants. Whole-cell H2O2 accumulation was detected in dying cells, while O2*- emerged in adjacent cells. Importantly, all susceptible mutants lacked these reactions. An oxalate oxidase, which is known to generate H2O2 and has been implicated in barley resistance against the powdery mildew fungus, was not differentially expressed between the wild type and all mutants. The results demonstrate that the Rar1 and Rar2 gene products, which are control elements of R-gene-mediated programmed cell death, also control accumulation of reactive oxygen intermediates but not the pathogenesis-related expression of oxalate oxidase.

Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me‐JA but is dependent on MAPKK activity

Recognition of avirulent microbial pathogens activates an oxidative burst leading to the accumulation of reactive oxygen intermediates (ROIs), which are thought to integrate a diverse set of defence mechanisms resulting in the establishment of plant disease resistance. A novel transgenic Arabidopsis line containing a gst1:luc transgene was developed and employed to report the temporal and spatial dynamics of ROI accumulation and cognate redox signalling in response to attempted infection by avirulent strains of Pseudomonas syringae pv. tomato (Pst). Strong engagement of the oxidative burst was dependent on the presence of functional Pst hrpS and hrpA gene products. Experiments employing pharmacological agents suggested that at least two distinct sources, including an NADPH oxidase and a peroxidase-type enzyme, contributed to the generation of redox cues. The analysis of gst1 and pal1 gene expression in nahG, coi1 and etr1 plants suggested that engagement of the oxidative burst and cognate redox signalling functioned independently of salicylic acid, methyl jasmonate and ethylene. In contrast, studies using a panel of protein kinase and phosphatase inhibitors and in-gel kinase assays in these mutant backgrounds suggested that a 48 kDa mitogen-activated protein kinase (MAPK) activity was required for the activation of gst1 and pal1 in response to redox cues. Thus the engagement of a bifurcating redox signalling pathway possessing a MAPK module may contribute both to the establishment of plant disease resistance, and to the development of cellular protectant mechanisms.

Ozone‐induced oxidative burst in the ozone biomonitor plant, tobacco Bel W3

Localized cell death is a common feature of ozone phytotoxicity and is generally thought to be initiated by the strong oxidant ozone itself as well as by ozone-derived reactive oxygen intermediates (ROIs). Here we report that ozone (150 nl l(-1), 5 h) elicits cellular ROI production in the ozone-sensitive tobacco cv. Bel W3, but not in the tolerant cv. Bel B. Both cultivars exhibited a transient first maximum of apoplastic ROI accumulation followed by a comparable induction of glutathione peroxidase transcript levels. During postcultivation in pollutant-free air, a second and sustained peak of apoplastic ROI accumulation was detected only in cv. Bel W3. Histochemical staining revealed a spot-like accumulation of H(2)O(2) and, to a lesser extent, of superoxide anion radicals in this cultivar. The H(2)O(2) spots ('burst initiation sites') occurred mainly in the vicinity of leaf veins and correlated in number and distribution with discrete sites of local cell death and with visible symptoms that evolved between 15 and 72 h. The results indicate that ozone effects are amplified in the sensitive tobacco cv. Bel W3 by an oxidative burst which participates in the generation of hypersensitive cell death-like lesions.