Plasma Membrane NADPH Oxidase Research Articles

ROS in biotic interactions

Production of reactive oxygen species (ROS) is a hallmark of successful recognition of infection and activation of plant defenses. ROS play multifaceted signaling functions mediating the establishment of multiple responses and can act as local toxins. Controversy surrounds the origin of these ROS. Several enzymatic mechanisms, among them a plasma membrane NADPH oxidase and cell wall peroxidases, can be responsible for the ROS detected in the apoplast. However, high levels of ROS from metabolic origins and/or from downregulation of ROS-scavenging systems can also accumulate in different compartments of the plant cell. This compartmentalization could contribute to the specific functions attributed to ROS. Additionally, ROS interact with other signals and phytohormones, which could explain the variety of different scenarios where ROS signaling plays an important part. Interestingly, pathogens have developed ways to alter ROS accumulation or signaling to modify plant defenses. Although ROS have been mainly associated with pathogen attack, ROS are also detected in other biotic interactions including beneficial symbiotic interactions with bacteria or mycorrhiza, suggesting that ROS production is a common feature of different biotic interactions. Here, we present a comprehensive review describing the newer views in ROS signaling and function during biotic stress.

Reactive oxygen species derived from impaired quality control of Photosystem II are irrelevant to plasma-membrane NADPH oxidases

Protein quality control plays an important role in the photosynthetic apparatus because its components receive excess light energy and are susceptible to photooxidative damage. In chloroplasts, photodamage is targeted to the D1 protein of Photosystem II (PSII). The coordinated PSII repair cycle (PSII disassembly, D1 degradation and synthesis, and PSII reassembly) is necessary to mitigate photoinhibition. A thylakoid protease FtsH, which is formed predominantly as a heteromeric complex with two isoforms of FtsH2 and FtsH5 in Arabidopsis, is the major protease involved in PSII repair. A mutant lacking FtsH2 (termed var2) shows compromised D1 degradation. Furthermore, var2 accumulates high levels of chloroplastic reactive oxygen species (cpROS), reflecting photooxidative stress without functional PSII repair. To examine if the cpROS produced in var2 are connected to a ROS signaling pathway mediated by plasma membrane NADPH oxidase (encoded by AtRbohD or AtRbohF), we generated mutants in which either Rboh gene was inactivated under var2 background. Lack of NADPH oxidases had little or no impact on cpROS accumulation. It seems unlikely that cpROS in var2 activate plasma membrane NADPH oxidases to enhance ROS production and the signaling pathway. Mutants that are defective in PSII repair might be valuable for investigating cpROS and their physiological roles.

H2O2 localization in the green alga Micrasterias after salt and osmotic stress by TEM-coupled electron energy loss spectroscopy

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are constantly generated as by-products of normal metabolic cellular pathways and can be overproduced in response to stress. In this study, we investigated ROS production and localization of H2O2 after salt (200 mM KCl) and osmotic (iso-osmotic sorbitol concentration) stress in the unicellular green alga Micrasterias. By means of the dye H2DCFDA and confocal laser scanning microscopy, most ROS production could be detected in KCl-treated cells when compared to sorbitol-exposed cells and controls. For ultrastructural detection of H2O2, CeCl3, which reacts with H2O2 and produces cerium perhydroxide deposits, has been used. Cerium was identified by transmission electron microscopy (TEM)-coupled electron energy loss spectroscopy (EELS) in organelles of KCl- and sorbitol-treated cells and in controls. Statistical measurements of the presence of the cerium M4,5 edge were performed in mitochondria, chloroplasts, cell walls, and cytoplasmic sites of five individual cells after each treatment. The most pronounced increase in H2O2 production was found in chloroplasts of KCl- and sorbitol-treated cells. This shows that the chloroplast reveals the strongest response in H2O2 production after stress induction in Micrasterias. Significant elevation of H2O2 production also occurred in mitochondria and cytoplasm, whereas H2O2 levels remained unchanged or even slightly decreased in cell walls of treated cells. Additionally, TEM micrographs and EELS analyses provided indirect evidence for an increased H2O2 production at the plasma membrane of KCl-treated cells, indicating an involvement of the plasma membrane NADPH oxidase in H2O2 generation.

Chitosan-induced programmed cell death in plants.

Chitosan, CN(-), or H(2)O(2) caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H(2)O(2), or chitosan + H(2)O(2) on EC. H(2)O(2) formation in EC and GC mitochondria that was determined from 2',7'-dichlorofluorescein fluorescence was inhibited by CN(-) and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN(-)-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN(-)-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H(2)O(2) on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H(2)O(2) as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.

NADPH Oxidase-Mediated Reactive Oxygen Species Production: Subcellular Localization and Reassessment of Its Role in Plant Defense

Chemiluminescence detection of reactive oxygen species (ROS) triggered in tobacco BY-2 cells by the fungal elicitor cryptogein was previously demonstrated to be abolished in cells transformed with an antisense construct of the plasma membrane NADPH oxidase, NtrbohD. Here, using electron microscopy, it has been confirmed that the first hydrogen peroxide production occurring a few minutes after challenge of tobacco cells with cryptogein is plasma membrane located and NtrbohD mediated. Furthermore, the presence of NtrbohD in detergent-resistant membrane fractions could be associated with the presence of NtrbohD-mediated hydrogen peroxide patches along the plasma membrane. Comparison of the subcellular localization of ROS in wild-type tobacco and in plants transformed with antisense constructs of NtrbohD revealed that this enzyme is also responsible for the hydrogen peroxide production occurring at the plasma membrane after infiltration of tobacco leaves with cryptogein. Finally, the reactivity of wild-type and transformed plants to the elicitor and their resistance against the pathogenic oomycete Phytophthora parasitica were examined. NtrbohD-mediated hydrogen peroxide production does not seem determinant for either hypersensitive response development or the establishment of acquired resistance but it is most likely involved in the signaling pathways associated with the protection of the plant cell.

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+channels

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Fungal elicitor Pep-25 increases cytosolic calcium ions, H2O2 production and activates the octadecanoid pathway in Arabidopsis thaliana

Transgenic Arabidopsis thaliana plants expressing the protein aequorin were used to investigate the transient change in cytosolic calcium ions caused by stimulation through the fungal elicitor Pep-25. Our results show that the elicitor Pep-25 derived from Phytophthora sojae can induce H(2)O(2) production and an increase in cytosolic calcium ions. The transcription of LOX, OPR3, PED, AOS, AOC genes and the protein accumulation of AOS were induced by Pep-25 treatment. Pep-25 also induced an accumulation of jasmonic acid (JA). Blocking the production of H(2)O(2) and the increase of cytosolic calcium ions both suppressed the transcription of LOX, OPR3, PED, AOS, AOC genes, the accumulation of AOS, and the accumulation of JA. These results indicated that the production of H(2)O(2) derived from the plasma-membrane NADPH oxidase and the subsequently increase of cytosolic calcium ions are both required for the activation of the octadecanoid pathway by Pep-25 treatment in A. thaliana.

Relationship between H2O2 and jasmonic acid in pea leaf wounding response

The relationship of H2O2 and jasmonic acid (JA) in wound-induced defense response was investigated in the leaves of pea (Pisum sativum L.) plants. The results showed that both wounding and JA treatment led to a significant increase in activities of plasma membrane NADPH oxidase and phenylalanine ammonialyase. However, such an increase was blocked by the pretreatment with plasma membrane NADPH oxidase inhibitors, O2−scavengers, or H2O2 scavenger, implying that H2O2 functions downstream of JA. Furthermore, wounding treatment activated two key enzymes of JA biosynthesis, lipoxygenase and allene oxide synthase, while JA biosynthetic inhibitors impaired the wounding-induced H2O2 burst. Thus, it is suggested that H2O2 burst depends on JA production in plant wounding response.

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels.

Experimental evidence in the literature suggests that O(2)(*-) produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O(2)(*-). Stress treatments were imposed using 150 mm NaCl or 300 mM sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O(2)(*-) was determined using nitro blue tetrazolium, and H(2)O(2) was determined using 2', 7'-dichlorofluorescin. In non-stressed plants, the distribution of accelerating growth and highest O(2)(*-) levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O(2)(*-) levels increased in sorbitol-treated roots and decreased in NaCl-treated roots. The lack of association between apoplastic O(2)(*-) levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O(2)(*-) may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.

Origin of cadmium‐induced reactive oxygen species production: mitochondrial electron transfer versus plasma membrane NADPH oxidase

* Cadmium (Cd(2+)) is an environmental pollutant that causes increased reactive oxygen species (ROS) production. To determine the site of ROS production, the effect of Cd(2+) on ROS production was studied in isolated soybean (Glycine max) plasma membranes, potato (Solanum tuberosum) tuber mitochondria and roots of intact seedlings of soybean or cucumber (Cucumis sativus). * The effects of Cd(2+) on the kinetics of superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and hydroxyl radical ((*OH) generation were followed using absorption, fluorescence and spin-trapping electron paramagnetic resonance spectroscopy. * In isolated plasma membranes, Cd(2+) inhibited O2*- production. This inhibition was reversed by calcium (Ca(2+)) and magnesium (Mg(2+)). In isolated mitochondria, Cd(2+) increased and H(2)O(2) production. In intact roots, Cd(2+) stimulated H(2)O(2) production whereas it inhibited O2*- and (*)OH production in a Ca(2+)-reversible manner. * Cd(2+) can be used to distinguish between ROS originating from mitochondria and from the plasma membrane. This is achieved by measuring different ROS individually. The immediate (<or= 1 h) consequence of exposure to Cd(2+) in vivo is stimulation of ROS production in the mitochondrial electron transfer chain and inhibition of NADPH oxidase activity in the plasma membrane.

Hydrogen peroxide‐induced translocation of glycolipid‐anchored (c)AMP‐hydrolases to lipid droplets mediates inhibition of lipolysis in rat adipocytes

The insulin-independent inhibition of lipolysis by palmitate, the anti-diabetic sulphonylurea glimepiride and H2O2 in rat adipocytes involves stimulation of the glycosylphosphatidylinositol (GPI)-specific phospholipase-C (GPI-PLC) and subsequent translocation of the GPI-anchored membrane ectoproteins (GPI-proteins), Gce1 and cluster of differentiation antigen (CD73), from specialized plasma membrane microdomains (DIGs) to cytosolic lipid droplets (LDs). This results in cAMP degradation at the LD surface and failure to activate hormone-sensitive lipase. Reactive oxygen species (ROS) may trigger this sequence of events in response to palmitate and glimepiride. The effects of various inhibitors of ROS production on the release of H2O2, GPI anchor cleavage and translocation of the photoaffinity-labelled or metabolically labelled Gce1 and CD73 from DIGs to LD and inhibition of lipolysis by different fatty acids and sulphonylureas were studied with primary rat adipocytes. Glimepiride and palmitate induced the production of H2O2 via the plasma membrane NADPH oxidase and mitochondrial complexes I and III, respectively. Inhibition of ROS production was accompanied by the loss of (i) GPI-PLC activation, (ii) Gce1 and CD73 translocation and (iii) lipolysis inhibition in response to palmitate and glimepiride. Non-metabolizable fatty acids and the sulphonylurea drug tolbutamide were inactive. NADPH oxidase and GPI-PLC activities colocalized at DIGs were stimulated by glimepiride but not tolbutamide. The data suggest that ROS mediate GPI-PLC activation at DIGs and subsequent GPI-protein translocation from DIGs to LD in adipocytes which leads to inhibition of lipolysis by palmitate and glimepiride. This insulin-independent anti-lipolytic mechanism may be engaged by future anti-diabetic drugs.

Involvement of glucose-6-phosphate dehydrogenase in reduced glutathione maintenance and hydrogen peroxide signal under salt stress

Cellular redox homeostasis is essential for plant growth, development as well as for the resistance to biotic and abiotic stresses, which is governed by the complex network of prooxidant and antioxidant systems. Recently, new evidence has been published that NADPH, produced by glucose-6-phosephate dehydrogenase enzyme (G6PDH), not only acted as the reducing potential for the output of reduced glutathione (GSH), but was involved in the activity of plasma membrane (PM) NADPH oxidase under salt stress, which resulted in hydrogen peroxide (H2O2) accumulation. H2O2 acts as a signal in regulating G6PDH activity and expression, and the activities of the enzymes in the glutathione cycle as well, through which the ability of GSH regeneration was increased under salt stress. Thus, G6PDH plays a critical role in maintaining cellular GSH levels under long-term salt stress. In this addendum, a hypothetical model for the roles of G6PDH in modulating the intracellular redox homeostasis under salt stress is presented.

Signaling the Signal, Cyclic AMP-dependent Protein Kinase Inhibition by Insulin-formed H2O2 and Reactivation by Thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

Topological organization of NADPH‐oxidase in haematopoietic stem cell membrane: preliminary study by fluorescence near‐field optical microscopy

The aim of this study was to characterize the local distribution and organization of the plasma membrane NADPH-oxidase (NOX) in human haematopoietic stem cell (HSC) by means of the fluorescence scanning near-field optical microscopy approach. The presence of NOX in haematopoietic stem cells is thought to have a functional role as O(2) sensor and/or as low-level reactive oxygen species (ROS) producer to be used as redox messenger for controlling cell growth and differentiation. Given the harmful potential of ROS, a fine-tuning of NOX activity is needed. The high resolution imaging of haematopoietic stem cell membrane obtained in this study combined with the immunodetection of NOX indicates for this the occurrence of a cluster-organized structure. These membrane 'rafts'-like micro-compartments may constitute localized protein aggregates whereby the assembly/activation of the NOX components are functionally integrated with upstream factors constituting signal-transduction platforms.

Involvement of NADPH oxidase NtrbohD in the rapid production of H 2O 2 induced by ABA in cultured tobacco cell line BY-2

The mechanisms for the production of hydrogen peroxide (H 2O 2) induced by abscisic acid (ABA) were investigated in suspension culture cells of tobacco BY-2 cells. The results showed that the immediate generation of H 2O 2, which was mainly derived from superoxide dismutase-catalyzed dismutation of superoxide radical, was significantly induced by ABA. Furthermore, treatment of the cultured tobacco cells with ABA resulted in a time-dependent quick increase in plasma membrane (PM) NADPH oxidase activity, which coincided on time and magnitude with the elevation in ABA-induced accumulation of H 2O 2. Moreover, these enhanced effects were pronouncedly inhibited by two NADPH oxidase inhibitors, diphenylene iodonium and imidazole, suggesting that PM NADPH oxidase is involved in the rapid accumulation of H 2O 2 in cultured tobacco cells. In addition, analysis of the expression level of NtrbohD, a PM NADPH oxidase gene in tobacco, by RT-PCR and protein gel blot revealed that the gene at both mRNA and protein levels was upregulated by ABA, indicating that NtrbohD participates in the ABA-stimulated rapid production of H 2O 2 in tobacco culture cells. Taken together, these findings suggest that ABA induces the rapid accumulation of reactive oxygen species via NADPH oxidase in suspension culture cells of tobacco, and that NADPH oxidase and H 2O 2 appear to be important components in ABA signal transduction pathway in plants.

Inhibition of catalase activity as an early response of Arabidopsis thaliana cultured cells to the phytotoxin fusicoccin

In Arabidopsis thaliana cells, fusicoccin (FC) treatment induced an early and marked increase in the extracellular H(2)O(2) level. It also increased the huge hypo-osmotic stress-induced oxidative wave and, in addition, prevented the H(2)O(2) peak drop. These effects were apparently not linked to changes in either cytoplasmic pH or cytoplasmic free calcium concentration, since they occurred independently of the activity state of the plasma membrane (PM) H(+)-ATPase and neither influx nor efflux of (45)Ca(2+) was modified by FC. In the presence of diphenylene iodonium (DPI), inhibiting the PM NADPH oxidase presumably responsible for reactive oxygen species (ROS) production, no apoplastic H(2)O(2) development was detected either with or without FC. However, no increase in DPI-sensitive ferricyanide reduction, but rather a gradual decrease, occurred with FC. These results suggested that the H(2)O(2) increase observed with FC was not due to a overproduction of ROS but, more probably, to a reduced capability of FC-treated cells to degrade the H(2)O(2) formed. This view, at first supported by the finding that FC-treated cells failed to break down exogenously supplied H(2)O(2), was clearly confirmed by a series of measurements on exogenous catalase activity, tested in cell-free media of FC-treated samples. This assay, in fact, allowed ascertainment and partial characterization of an as yet unidentified factor increasingly accumulating in the incubation medium of FC-treated cells, behaving as a non-competitive catalase inhibitor and able to reduce markedly the cell's capability for H(2)O(2) scavenging.

Role of Nitric Oxide and Hydrogen Peroxide During the Salt Resistance Response

Ion homeostasis is essential for plant cell resistance to salt stress. Under salt stress, to avoid cellular damage and nutrient deficiency, plant cells need to maintain adequate K nutrition and a favorable K to Na ratio in the cytosol. Recent observations revealed that both nitric oxide (NO) and hydrogen peroxide (H2O2) act as signaling molecules to regulate K to Na ratio in calluses from Populus euphratica under salt stress. Evidence indicated that NO mediating H2O2 causes salt resistance via the action of plasma membrane H+-ATPase but that activity of plasma membrane NADPH oxidase is dependent on NO. Our study demonstrated the signaling transduction pathway. In this addendum, we proposed a testable hypothesis for NO function in regulation of H2O2 mediating salt resistance.

Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress

In the present study, we investigated the role of glucose-6-phosphate dehydrogenase (G6PDH) in regulating the levels of reduced form of glutathione (GSH) to the tolerance of calli from two reed ecotypes, Phragmites communis Trin. dune reed (DR) and swamp reed (SR), in a long-term salt stress. G6PDH activity was higher in SR callus than that of DR callus under 50-150 mM NaCl treatments. In contrast, at higher NaCl concentrations (300-600 mM), G6PDH activity was lower in SR callus. A similar profile was observed in GSH contents, glutathione reductase (GR) and glutathione peroxidase (GPX) activities in both salt-stressed calli. After G6PDH activity and expression were reduced in glycerol treatments, GSH contents and GR and GPX activity decreased strongly in both calli. Simultaneously, NaCl-induced hydrogen peroxide (H2O2) accumulation was also abolished. Exogenous application of H2O2 increased G6PDH, GR, and GPX activities and GSH contents in the control conditions and glycerol treatment. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted NaCl-induced H(2)O(2) accumulation, decreased these enzymes activities and GSH contents. Furthermore, exogenous application of H2O2 abolished the N-acetyl-L: -cysteine (NAC)-induced decrease in G6PDH activity, and DPI suppressed the effect of buthionine sulfoximine (BSO) on induction of G6PDH activity. Western-blot analyses showed that G6PDH expression was stimulated by NaCl and H2O2, and blocked by DPI in DR callus. Taken together, G6PDH activity involved in GSH maintenance and H2O2 accumulation under salt stress. And H2O2 regulated G6PDH, GR, and GPX activities to maintain GSH levels. In the process, G6PDH plays a central role.

Pharmacological inhibition of neuronal NADPH oxidase protects against 1-methyl-4-phenylpyridinium (MPP +)-induced oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells

Oxidative stress is widely recognized as a key mediator of degenerative processes in Parkinson's disease (PD). Recently, we demonstrated that the dopaminergic toxin MPP + initiates oxidative stress to cause caspase-3-dependent apoptotic cell death in mesencephalic dopaminergic neuronal (N27) cells. In this study, we determined the source of reactive oxygen species (ROS) produced during MPP +-induced apoptotic cell death. In addition to mitochondria, plasma membrane NADPH oxidase is considered a major producer of ROS inside the cell. Here, we show that N27 neuronal cells express key NADPH oxidase subunits gp91 phox and p67 phox. We used structurally diverse NADPH oxidase inhibitors, aminoethyl-benzenesulfonylfluoride (AEBSF, 100–1000 μM), apocynin (100–1000 μM), and diphenylene iodonium (DPI, 3–30 μM), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300 μM MPP +-induced ROS production for over 45 min in N27 cells, in a dose-dependent manner. Further treatment with DPI, apocynin, and SOD also blocked MPP +-induced ROS production. In Sytox cell death assays, co-treatment with AEBSF, apocynin, or DPI for 24 h significantly suppressed MPP +-induced cytotoxic cell death. Similarly, co-treatment with these inhibitors also significantly attenuated MPP +-induced increases in caspase-3 enzymatic activity. Furthermore, quantitative DNA fragmentation ELISA assays revealed that AEBSF, DPI, and apocynin rescue N27 cells from MPP +-induced apoptotic cell death. Together, these results indicate for the first time that intracellular ROS generated by NAPDH oxidase are present within the mesencephalic neuronal cells, and are a key determinant of MPP +-mediated dopaminergic degeneration in in vitro models of dopaminergic degeneration. This study supports a critical role of NADPH oxidase in the oxidative damage in PD; targeting this enzyme may lead to novel therapies for PD.

Involvement of NADPH oxidase in sulfur dioxide-induced oxidative stress in plant cells

Bisulfite, a major form of SO2 in aqueous phase of apoplast, may reduce photosynthesis rate and thereby crop yield through inducing reactive oxygen species (ROS). In this study, ROS production was directly detected in a living cell of leaf of spinach (Spinacia oleracea L.) using laser scanning confocal microscopes with the assistance of the fluorescence probe dichlorofluorescin diacetate (H2DCF-DA). Results showed that, under bisulfite stress, a large quantity of ROS indicated by DCF fluorescence was produced in epidermic tissue. The role of plasma membrane (PM) NADPH oxidase in bisulfite-induced ROS production was also investigated. Treatment with bisulfite resulted in a significant increase in the content of ROS and the activity of PM NADPH oxidase in spinach leaves. The effects caused by bisulfite were inhibited pronouncedly by pretreatment with two widely used NADPH oxidase inhibitors (diphenyleneiodonium and quinacrine). Moreover, the change patterns of the bisulfite-induced increase and inhibitor-caused decrease in the two parameters were quite similar. Additionally, only a small amount of ROS could be observed on in vitro chloroplasts under bisulfite stress. Based on all the results, we conclude that ROS is involved in bisulfite-induced stress, and the bisulfite-induced enhancements in levels of ROS originate mainly from PM NADPH oxidase.