Implication of the oep16-1 mutation in a flu-independent, singlet oxygen-regulated cell death pathway in Arabidopsis thaliana

Singlet oxygen is a prominent form of reactive oxygen species in higher plants. It is easily formed from molecular oxygen by triplet–triplet interchange with excited porphyrin species. Evidence has been obtained from studies on the flu mutant of Arabidopsis thaliana of a genetically determined cell death pathway that involves differential changes at the transcriptome level. Here we report on a different cell death pathway that can be deduced from the analysis of oep16 mutants of A. thaliana. Pure lines of four independent OEP16-deficient mutants with different cell death properties were isolated. Two of the mutants overproduced free protochlorophyllide (Pchlide) in the dark because of defects in import of NADPH:Pchlide oxidoreductase A (pPORA) and died after illumination. The other two mutants avoided excess Pchlide accumulation. Using pulse labeling and polysome profiling studies we show that translation is a major site of cell death regulation in flu and oep16 plants. flu plants respond to photooxidative stress triggered by singlet oxygen by reprogramming their translation toward synthesis of key enzymes involved in jasmonic acid synthesis and stress proteins. In contrast, those oep16 mutants that were prone to photooxidative damage were unable to respond in this way. Together, our results show that translation is differentially affected in the flu and oep16 mutants in response to singlet oxygen.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His 6 -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1 D299A non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death.

Translocation and Functional Analysis of Pseudomonas savastanoi pv. savastanoi NCPPB 3335 Type III Secretion System Effectors Reveals Two Novel Effector Families of the Pseudomonas syringae Complex

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants.

IκB Is a Substrate for a Selective Pathway of Lysosomal Proteolysis

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor κ B (IκB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IκB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IκB is directly transported into isolated lysosomes in a process that requires binding of IκB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IκB uptake by lysosomes. Ubiquitination and phosphorylation of IκB are not required for its targeting to lysosomes. The lysosomal degradation of IκB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IκB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IκB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IκB is increased. There are both short- and long-lived pools of IκB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IκB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IκB degradation in lysosomes. This selective pathway of lysosomal degradation of IκB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor κ B. In addition, the response of nuclear factor κ B to several stimuli increases when this lysosomal pathway of proteolysis is activated.

BAX-induced cell death may not require interleukin 1β-converting enzyme-like proteases

Expression of BAX, without another death stimulus, proved sufficient to induce a common pathway of apoptosis. This included the activation of interleukin 1β-converting enzyme (ICE)-like proteases with cleavage of the endogenous substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation inhibitor for the rho family), as well as the fluorogenic peptide acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) successfully blocked this protease activity and prevented FAS-induced death but not BAX-induced death. Blocking ICE-like protease activity prevented the cleavage of nuclear and cytosolic substrates and the DNA degradation that followed BAX induction. However, the fall in mitochondrial membrane potential, production of reactive oxygen species, cytoplasmic vacuolation, and plasma membrane permeability that are downstream of BAX still occurred. Thus, BAX-induced alterations in mitochondrial function and subsequent cell death do not apparently require the known ICE-like proteases.

Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human–hamster hybrid (AL) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5′,6′-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in AL cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

The Involvement of Hydrogen Peroxide in the Differentiation of Secondary Walls in Cotton Fibers1

H2O2 is a widespread molecule in many biological systems. It is created enzymatically in living cells during various oxidation reactions and by leakage of electrons from the electron transport chains. Depending on the concentration H2O2 can induce cell protective responses, programmed cell death, or necrosis. Here we provide evidence that H2O2 may function as a developmental signal in the differentiation of secondary walls in cotton (Gossypium hirsutum) fibers. Three lines of evidence support this conclusion: (a) the period of H2O2 generation coincided with the onset of secondary wall deposition, (b) inhibition of H2O2 production or scavenging the available H2O2 from the system prevented the wall differentiation process, and (c) exogenous addition of H2O2 prematurely promoted secondary wall formation in young fibers. Furthermore, we provide support for the concept that H2O2 generation could be mediated by the expression of the small GTPase Rac, the accumulation of which was shown previously to be strongly induced during the onset of secondary wall differentiation. In support of Rac's role in the activation of NADPH oxidase and the generation of reactive oxygen species, we transformed soybean (Glycine max) and Arabidopsis cells with mutated Rac genes. Transformation with a dominantly activated cotton Rac13 gene resulted in constitutively higher levels of H2O2, whereas transformation with the antisense and especially with dominant-negative Rac constructs decreased the levels of H2O2.

Superoxide Dismutase in Arabidopsis: An Eclectic Enzyme Family with Disparate Regulation and Protein Localization1

A number of environmental stresses can lead to enhanced production of superoxide within plant tissues, and plants are believed to rely on the enzyme superoxide dismutase (SOD) to detoxify this reactive oxygen species. We have identified seven cDNAs and genes for SOD in Arabidopsis. These consist of three CuZnSODs (CSD1, CSD2, and CSD3), three FeSODs (FSD1, FSD2, and FSD3), and one MnSOD (MSD1). The chromosomal location of these seven SOD genes has been established. To study this enzyme family, antibodies were generated against five proteins: CSD1, CSD2, CSD3, FSD1, and MSD1. Using these antisera and nondenaturing-polyacrylamide gel electrophoresis enzyme assays, we identified protein and activity for two CuZnSODs and for FeSOD and MnSOD in Arabidopsis rosette tissue. Additionally, subcellular fractionation studies revealed the presence of CSD2 and FeSOD protein within Arabidopsis chloroplasts. The seven SOD mRNAs and the four proteins identified were differentially regulated in response to various light regimes, ozone fumigation, and ultraviolet-B irradiation. To our knowledge, this is the first report of a large-scale analysis of the regulation of multiple SOD proteins in a plant species.

Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca2+ channels. Inhibition of the oxidative response impaired Cl− efflux, K+ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits.

Reactive oxygen species play a central role in vascular inflammation and atherogenesis, with enhanced superoxide (O2.-) production contributing significantly to impairment of nitric oxide (.NO)-dependent relaxation of vessels from cholesterol-fed rabbits. We investigated potential sources of O2.- production, which contribute to this loss of endothelium-dependent vascular responses. The vasorelaxation elicited by acetylcholine (ACh) in phenylephrine-contracted, aortic ring segments was impaired by cholesterol feeding. Pretreatment of aortic vessels with either heparin, which competes with xanthine oxidase (XO) for binding to sulfated glycosaminoglycans, or the XO inhibitor allopurinol resulted in a partial restoration (36-40% at 1 muM ACh) of ACh-dependent relaxation. Furthermore, O2.(-)-dependent lucigenin chemiluminescence, measured in intact ring segments from hypercholesterolemic rabbits, was decreased by addition of heparin, allopurinol or a chimeric, heparin-binding superoxide dismutase. XO activity was elevated more than two-fold in plasma of hypercholesterolemic rabbits. Incubation of vascular rings from rabbits on a normal diet with purified XO (10 milliunits/ml) also impaired .NO-dependent relaxation but only in the presence of purine substrate. As with vessels from hypercholesterolemic rabbits, this effect was prevented by heparin and allopurinol treatment. We hypothesize that increases in plasma cholesterol induce the release of XO into the circulation, where it binds to endothelial cell glycosaminoglycans. Only in hypercholesterolemic vessels is sufficient substrate available to sustain the production of O2.- and impair NO-dependent vasorelaxation. Chronically, the continued production of peroxynitrite, (ONOO-) which the simultaneous generation of NO and O2.- implies, may irreversibly impair vessel function.

Ozone-induced responses in Arabidopsis thaliana: the role of salicylic acid in the accumulation of defense-related transcripts and induced resistance.

Exposure of Arabidopsis thaliana to ozone results in the expression of a number of defense-related genes that are also induced during a hypersensitive response. A potential common link between the activation of defense gene expression during a hypersensitive response and by ozone treatment is the production of active oxygen species and the accumulation of hydrogen peroxide. Here we report that salicylic acid accumulation, which can be induced by hydrogen peroxide and is required for the expression of both a hypersensitive response and systemic acquired resistance, is also required for the induction of some, but not all, ozone-induced mRNAs examined. In addition, we show that ozone exposure triggers induced resistance of A. thaliana to infection with virulent phytopathogenic Pseudomonas syringae strains. Infection of transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of salicylic acid, or npr1 mutant plants, which are defective in the expression of systemic acquired resistance at a step downstream of salicylic acid, demonstrated that the signaling pathway activated during ozone-induced resistance overlaps with the systemic acquired resistance activation pathway and is salicylic acid dependent. Interestingly, plants expressing salicylate hydroxylase exhibited increased sensitivity to ozone exposure. These results demonstrate that ozone activates at least two distinct signaling pathways, including a salicylic acid dependent pathway previously shown to be associated with the activation of pathogen defense reactions, and that this latter pathway also induces a protective response to ozone.

beta-Amyloid toxicity in organotypic hippocampal cultures: protection by EUK-8, a synthetic catalytic free radical scavenger.

Oxygen free radicals have been proposed to mediate amyloid peptide (beta-AP)-induced neurotoxicity. To test this hypothesis, we evaluated the effects of EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, on neuronal injury produced by beta-AP in organotypic hippocampal slice cultures. Cultures of equivalent postnatal day 35 (defined as mature) and 14 (defined as immature) were exposed to various concentrations of beta-AP (1-42 or 1-40) in the absence or presence of 25 microM EUK-8 for up to 72 hours. Neuronal injury was assessed by lactate dehydrogenase release and semiquantitative analysis of propidium iodide uptake at various times after the initiation of beta-AP exposure. Free radical production was inferred from the relative increase in dichlorofluorescein fluorescence, and the degree of lipid peroxidation was determined by assaying thiobarbituric acid-reactive substances. Treatment of mature cultures with beta-AP (50-250 microg/ml) in serum-free conditions resulted in a reproducible pattern of damage, causing a time-dependent increase in neuronal injury accompanied with formation of reactive oxygen species. However, immature cultures were entirely resistant to beta-AP-induced neurotoxicity and also demonstrated no dichlorofluorescein fluorescence or increased lipid peroxidation after beta-AP treatment. Moreover, mature slices exposed to beta-AP in the presence of 25 microM EUK-8 were significantly protected from beta-AP-induced neurotoxicity. EUK-8 also completely blocked beta-AP-induced free radical accumulation and lipid peroxidation. These results not only support a role for oxygen free radicals in beta-AP toxicity but also highlight the therapeutic potential of synthetic radical scavengers in Alzheimer disease.

Thermosensitive phenotype of transgenic mice overproducing human glutathione peroxidases.

Exposure of humans and other mammals to hyperthermic conditions elicits many physiological responses to stress in various tissues leading to profound injuries, which eventually result in death. It has been suggested that hyperthermia may increase oxidative stress in tissues to form reactive oxygen species harmful to cellular functions. By using transgenic mice with human antioxidant genes, we demonstrate that the overproduction of glutathione peroxidase (GP, both extracellular and intracellular) leads to a thermosensitive phenotype, whereas the overproduction of Cu,Zn-superoxide dismutase has no effect on the thermosensitivity of transgenic mice. Induction of HSP70 in brain, lung, and muscle in GP transgenic mice at elevated temperature was significantly inhibited in comparison to normal animals. Measurement of peroxide production in regions normally displaying induction of HSP70 under hyperthermia revealed high levels of peroxides in normal mice and low levels in GP transgenic mice. There was also a significant difference between normal and intracellular GP transgenic mice in level of prostaglandin E2 in hypothalamus and cerebellum. These data suggest direct participation of peroxides in induction of cytoprotective proteins (HSP70) and cellular mechanisms regulating body temperature. GP transgenic mice provide a model for studying thermoregulation and processes involving actions of hydroxy and lipid peroxides in mammals.

Elicitins from Phytophthora and basic resistance in tobacco.

Elicitins are a family of small proteins secreted by species of Phytophthora. They are thought to be major determinants of the resistance response of tobacco against these oomycetes, since purified elicitins, alone and at low concentrations, can induce vigorous defense responses in tobacco (i.e., hypersensitive cell death and resistance against subsequent pathogen attack), and in vitro elicitin production by Phytophthora isolates is strongly negatively correlated with their pathogenicity on tobacco plants. A number of elicitins have been purified and their amino acid sequences have been determined and found to be conserved. A three-dimensional structure for elicitin is emerging from nuclear magnetic resonance studies. Two structural classes, alpha and beta, are distinguished by their biological effects when applied to decapitated stems or petioles; the beta class causes more necrosis on leaves and provides better subsequent protection against pathogen attack. However, both these classes of elicitins will similarly cause necrosis when each is, instead, directly infiltrated into tobacco leaf panels. Effects of elicitins on tobacco cells include rapid electrolyte leakage, changes in protein phosphorylation and amounts of active oxygen species, and later production of ethylene and capsidiol. The sites of initial interaction with tobacco cells are unknown, but the interaction appears to induce general defense-related responses.