High Levels Of H2O2 Research Articles

CATALASE2 functions for seedling postgerminative growth by scavenging H2 O2 and stimulating ACX2/3 activity in Arabidopsis.

Increased fatty acid β-oxidation is essential for early postgerminative growth in seedlings, but high levels of H2 O2 produced by β-oxidation can induce oxidative stress. Whether and how catalase (CAT) functions in fine-tuning H2 O2 homeostasis during seedling growth remain unclear. Here, we report that CAT2 functions in early seedling growth. Compared to the wild type, the cat2-1 mutant, with elevated H2 O2 levels, exhibited reduced root elongation on sucrose (Suc)-free medium, mimicking soils without exogenous sugar supply. Treatment with the H2 O2 scavenger potassium iodide rescued the mutant phenotype of cat2-1. In contrast to the wild type, the cat2-1 mutant was insensitive to the CAT inhibitor 3-amino-1,2,4-triazole in terms of root elongation when grown on Suc-free medium, suggesting that CAT2 modulates early seedling growth by altering H2 O2 accumulation. Furthermore, like cat2-1, the acyl-CoA oxidase (ACX) double mutant acx2-1 acx3-6 showed repressed root elongation, suggesting that CAT2 functions in early seedling growth by regulating ACX activity, as this activity was inhibited in cat2-1. Indeed, decreased ACX activity and short root of cat2-1 seedlings grown on Suc-free medium were rescued by overexpressing ACX3. Together, these findings suggest that CAT2 functions in early seedling growth by scavenging H2 O2 and stimulating ACX2/3 activity.

Dietary resveratrol confers apoptotic resistance to oxidative stress in myoblasts

High levels of reactive oxygen species (ROS) contribute to muscle cell death in aging and disuse. We have previously found that resveratrol can reduce oxidative stress in response to aging and hindlimb unloading in rodents in vivo, but it was not known if resveratrol would protect muscle stem cells during repair or regeneration when oxidative stress is high. To test the protective role of resveratrol on muscle stem cells directly, we treated the C2C12 mouse myoblast cell line with moderate (100 μM) or very high (1 mM) levels of H2O2 in the presence or absence of resveratrol. The p21 promoter activity declined in myoblasts in response to high ROS, and this was accompanied a greater nuclear to cytoplasmic translocation of p21 in a dose-dependent matter in myoblasts as compared to myotubes. Apoptosis, as indicated by TdT-mediated dUTP nick-end labeling, was greater in C2C12 myoblasts as compared to myotubes (P<.05) after treatment with H2O2. Caspase-9, -8 and -3 activities were elevated significantly (P<.05) in myoblasts treated with H2O2. Myoblasts were more susceptible to ROS-induced oxidative stress than myotubes. We treated C2C12 myoblasts with 50 μM of resveratrol for periods up to 48 h to determine if myoblasts could be rescued from high-ROS-induced apoptosis by resveratrol. Resveratrol reduced the apoptotic index and significantly reduced the ROS-induced caspase-9, -8 and -3 activity in myoblasts. Furthermore, Bcl-2 and the Bax/Bcl-2 ratio were partially rescued in myoblasts by resveratrol treatment. Similarly, muscle stem cells isolated from mouse skeletal muscles showed reduced Sirt1 protein abundance with H2O2 treatment, but this could be reversed by resveratrol. Reduced apoptotic susceptibility in myoblasts as compared to myotubes to ROS is regulated, at least in part, by enhanced p21 promoter activity and nuclear p21 location in myotubes. Resveratrol confers further protection against ROS by improving Sirt1 levels and increasing antioxidant production, which reduces mitochondrial associated apoptotic signaling, and cell death in myoblasts.

Endothelial nitric oxide synthase and cyclooxygenase are activated by hydrogen peroxide in renal hypertensive rat aorta

In this work, we hypothesized that cyclooxygenase (COX) activity can be regulated by nitric oxide (NO) and hydrogen peroxide (H2O2). In the renal hypertension (2K-1C), phenylephrine (PE)-induced contraction was lower than in normotensive (2K) rat aortas. This impaired contraction is due to NO/H2O2- induced vasodilation. We evaluated the effects of H2O2 on the activity of COX and endothelial NO-Synthase (eNOS) in 2K-1C rat aortas stimulated with PE. Responses for PE or H2O2 were evaluated in 2K-1C and 2K rat aortas, without or with inhibitors for COX (Indomethacin) or eNOS (L-NAME). COX isoforms expression was evaluated by Western blotting. eNOS inhibition was tested on thromboxane A2 (TXA2) and prostacyclin (PGI2) production. PE-induced contraction was lower in 2K-1C than in 2K. Indomethacin reduced PE-induced contraction in 2K, but it had no effect in 2K-1C. L-NAME reversed indomethacin-induced effect in 2K and it normalized PE-induced contraction in 2K-1C to the normotensive levels. COX-1 and COX-2 expression, TXA2 and PGI2 production were higher in 2K-1C than in 2K. eNOS inhibition did no modify TXA2/PGI2 production. In low concentrations, H2O2 induced relaxation only in 2K that was abolished by L-NAME while the contractions induced by high concentrations were abolished by indomethacin in both 2K and 2K-1C. The activity/expression of COX, and TXA2/PGI2 production were increased in 2K-1C, which were not modified by eNOS. High levels of H2O2 increased the endothelial COX activity, which induced contraction. Therefore, an high increase in H2O2 production may increase COX-induced vasoconstriction rather than eNOS-induced relaxation, which might contribute to aggravate hypertension.

Influence of chitosan coating combined with spermidine on anthracnose disease and qualities of ‘Nam Dok Mai’ mango after harvest

Chitosan (CTS) combined with spermidine (SPD) was applied as fruit coating for ‘Nam Dok Mai’ mango (Mangifera indica L.) compared with CTS and SPD treatments after harvest and stored at 25±2°C for 9days. Influence of all treatments on anthracnose disease and qualities of mango fruit was investigated after inoculation fruit with Colletotrichum gloeosporioides. Inoculated fruit coated with 1% CTS combined with 0.1ppm SPD exhibited the smallest area of lesion development (0–1cm); while non-coating inoculated fruit presented the most severe fungal decay (4–5cm). Furthermore, inoculated fruit coated with 1% CTS combined with 0.1ppm SPD showed higher plant defense mechanisms than control and other treated fruits. These phenomena were represented by the production of high levels of H2O2 and phenolic compounds during storage and the induction of defense enzyme activities including chitinase, β-1,3-glucanase and peroxidase whereas fruit treated with only CMS or SPD expressed lower effect on induction of plant defense mechanisms. Therefore, synergistic effect of chitosan and spermidine combination can increase the ability to inhibit anthracnose disease development on ‘Nam Dok Mai’ mango fruit. Delayed mango fruit softening by 1% CTS combined with 0.1ppm SPD was in correlation with reduced soluble pectin content during ripening stage. The results obtained suggested that 1% CTS combined with 0.1ppm SPD had potential to improve firmness and delay deterioration processes of ‘Nam Dok Mai’ mango fruit after harvest.

Distinct Levels of Reactive Oxygen Species Coordinate Metabolic Activity with Beta-cell Mass Plasticity

The pancreatic beta-cells control glucose homeostasis by secreting insulin in response to nutrient intake. The number of beta-cells is under tight metabolic control, as this number increases with higher nutrient intake. However, the signaling pathways matching nutrition with beta-cell mass plasticity remain poorly defined. By applying pharmacological and genetic manipulations, we show that reactive oxygen species (ROS) regulate dose-dependently beta-cell proliferation in vivo and in vitro. In particular, reducing ROS levels in beta-cells blocks their proliferation in response to nutrients. Using a non-invasive genetic sensor of intracellular hydrogen peroxide (H2O2), we reveal that glucose can directly increase the levels of H2O2. Furthermore, a moderate increase in H2O2 levels can stimulate beta-cell proliferation. Interestingly, while high H2O2 levels are inhibitory to beta-cell proliferation, they expand beta-cell mass in vivo by inducing rapid beta-cell neogenesis. Our study thus reveals a ROS-level-dependent mechanism linking nutrients with beta-cell mass plasticity. Hence, given the requirement of ROS for beta-cell mass expansion, antioxidant therapies should be applied with caution in diabetes.

CodY Regulates Thiol Peroxidase Expression as Part of the Pneumococcal Defense Mechanism against H2O2 Stress.

Streptococcus pneumoniae is a facultative anaerobic pathogen. Although it maintains fermentative metabolism, during aerobic growth pneumococci produce high levels of H2O2, which can have adverse effects on cell viability and DNA, and influence pneumococcal interaction with its host. The pneumococcus is unusual in its dealing with toxic reactive oxygen species (ROS) in that it neither has catalase nor the global regulators of peroxide stress resistance. Previously, we identified pneumococcal thiol peroxidase (TpxD) as the key enzyme for enzymatic removal of H2O2, and showed that TpxD synthesis is up-regulated upon exposure to H2O2. This study aimed to reveal the mechanism controlling TpxD expression under H2O2 stress. We hypothesize that H2O2 activates a transcription factor which in turn up-regulates tpxD expression. Microarray analysis revealed a pneumococcal global transcriptional response to H2O2. Mutation of tpxD abolished H2O2-mediated response to high H2O2 levels, signifying the need for an active TpxD under oxidative stress conditions. Bioinformatic tools, applied to search for a transcription factor modulating tpxD expression, pointed toward CodY as a potential candidate. Indeed, a putative 15-bp consensus CodY binding site was found in the proximal region of tpxD-coding sequence. Binding of CodY to this site was confirmed by EMSA, and genetic engineering techniques demonstrated that this site is essential for TpxD up-regulation under H2O2 stress. Furthermore, tpxD expression was reduced in a ΔcodY mutant. These data indicate that CodY is an activator of tpxD expression, triggering its up-regulation under H2O2 stress. In addition we show that H2O2 specifically oxidizes the 2 CodY cysteines. This oxidation may trigger a conformational change in CodY, resulting in enhanced binding to DNA. A schematic model illustrating the contribution of TpxD and CodY to pneumococcal global transcriptional response to H2O2 is proposed.

Hydrogen Peroxide-Responsive Nanoprobe Assists Circulating Tumor Cell Identification and Colorectal Cancer Diagnosis.

In the clinic, numeration of circulating tumor cells (CTCs) plays a critical role in cancer diagnosis and treatment, but conventional CTC identification and counting that rely on specific antibodies to characterize a cell's surface antigens are costive and with limitations. Importantly, false positive or negative results may occur due to the high heterogeneity and epithelial-mesenchymal transition (EMT) of CTCs. Herein we demonstrate a novel and effective CTC detecting nanoprobe that could rapidly respond to the high level of endogenous H2O2 of CTCs and report the signal through fluorescence emission. Briefly, a hydrophobic coumarin-benzene boronic acid pinacol ester (Cou-Bpin) was grafted onto hydrophilic glycol chitosan (GC) to form an amphiphilic molecule, which further assembled into micellar nanoparticles in aqueous solution. This new nanoprobe was highly sensitive to H2O2 with a detection limit of 0.1 μM and could rapidly enter the cells within 30 min. Upon exposure to intracellular H2O2, the nanoprobe exhibited remarkable one-photon and two-photon luminescent characteristics, which were suitable for imaging of endogenous H2O2 of various human colorectal cancer cells and assist the identification of CTCs. Compared to a conventional CTC counting assay, the nanoprobe-based CTC numeration could overcome the false-negative findings due to the low expression of cytokeratin 19 (CK19). In a clinic test, CTC counting results based on the new nanoprobe match better to the postoperative pathological results of four clinic patients who had colorectal cancer at different stages.

Image-Based Measurement of H2O2 Reaction-Diffusion in Wounded Zebrafish Larvae

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100–300 μm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H2O2) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells. But before it can oxidize cellular proteins, H2O2 must get past the antioxidant barriers that protect the cellular proteome. To test whether, or on which length scales this occurs during physiological wound signaling, we developed a computational method based on reaction-diffusion principles that infers H2O2 degradation rates from intravital H2O2-biosensor imaging data. Our results indicate that at high tissue H2O2 levels the peroxiredoxin-thioredoxin antioxidant chain becomes overwhelmed, and H2O2 degradation stalls or ceases. Although the wound H2O2 gradient reaches deep into the tissue, it likely overcomes antioxidant barriers only within ∼30 μm of the wound margin. Thus, Duox-mediated long-range signaling may require other spatial relay mechanisms besides extracellular H2O2 diffusion.

Overexpression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.).

Overexpressing miR529a can enhance oxidative stress resistance by targeting OsSPL2 and OsSPL14 genes that can regulate the expression of their downstream SOD and POD related genes. MicroRNAs are involved in the regulation of plant developmental and physiological processes, and their expression can be altered when plants suffered environment stresses, including salt, oxidative, drought and Cadmium. The expression of microRNA529 (miR529) can be induced under oxidative stress. However, its biological function under abiotic stress responses is still unclear. In this study, miR529a was overexpressed to investigate the function of miR529a under oxidative stress in rice. Our results demonstrated that the expression of miR529a can be induced by exogenous H2O2, and overexpressing miR529a can increase plant tolerance to high level of H2O2, resulting in increased seed germination rate, root tip cell viability, reduced leaf rolling rate and chlorophyll retention. The expression of oxidative stress responsive genes and the activities of superoxide dismutase (SOD) and peroxidase (POD) were increased in miR529a overexpression plant, which could help to reduce redundant reactive oxygen species (ROS). Furthermore, only OsSPL2 and OsSPL14 were targeted by miR529a in rice seedlings, repressing their expression in miR529aOE plants could lead to strengthen plant tolerance to oxidation stress. Our study provided the evidence that overexpression of miR529a could strengthen oxidation resistance, and its target genes OsSPL2 and OsSPL14 were responsible for oxidative tolerance, implied the manipulation of miR529a and its target genes regulation on H2O2 related response genes could improve oxidative stress tolerance in rice.

Inducible Sequential Oxidation Process in Water-Soluble Copper Nanoclusters for Direct Colorimetric Assay of Hydrogen Peroxide in a Wide Dynamic and Sampling Range.

Direct and fast detection methods for H2O2 have great demand in materials science, biology, and medicine. Colorimetric assay of H2O2 has been regarded as one versatile approach that can avoid tedious operation and complicated setup. In this report, we provided a cost-effective and time-saving H2O2 colorimetric assay strategy based on a mercaptosuccinic acid (MSA)-stabilized Cu nanocluster (NC) probe without using any chromogenic reagent. Direct and fast colorimetric detection of H2O2 was realized based on the color change of MSA-capped Cu NCs in aqueous medium. It was found that the Cu NCs presented eligible resistance to natural oxidation either in concentrated solution or in the powder state. However, the dissolved oxygen in a highly diluted solution of the Cu NCs could trigger the aggregation of the Cu NCs and their further fusion into small Cu nanoparticles (NPs). When this diluted solution served as a probe solution for detecting H2O2, a sequential oxidation process occurred in the newly formed Cu NPs, including the cleavage of MSAs on the surface and conversion of Cu into Cu2O, leading to the probe with capacity for H2O2 assay in a wide dynamic and sampling range. The sensitive solution color change was attributed to the growth of the Cu NPs (fading of plasmonic absorption) upon the addition of low levels of H2O2 and the transition of the valence states of Cu (color reactions) upon the addition of high levels of H2O2. A concentration range of H2O2 from 1 μM to 1 M could be detected by a small dose of the probe. Moreover, the Cu NCs powder subsequent to storage for 10 months could maintain a similar sensitivity for H2O2 assay, which provides possibilities for a wide range of practical applications in water samples.

Measurement of hydrogen peroxide and organic hydroperoxide concentrations during autumn in Beijing, China

Gaseous peroxides play important roles in atmospheric chemistry. To understand the pathways of the formation and removal of peroxides, atmospheric peroxide concentrations and their controlling factors were measured from 7:00 to 20:00 in September, October, and November 2013 at a heavily trafficked residential site in Beijing, China, with average concentrations of hydrogen peroxide (H2O2) and methyl hydroperoxide (MHP) at 0.55ppb and 0.063ppb, respectively. H2O2 concentrations were higher in the afternoon and lower in the morning and evening, while MHP concentrations did not exhibit a regular diurnal pattern. Both H2O2 and MHP concentrations increased at dusk in most cases. Both peroxides displayed monthly variations with higher concentrations in September. These results suggested that photochemical activity was the main controlling factor on variations of H2O2 concentrations during the measurement period. Increasing concentrations of volatile organic compounds emitted by motor vehicles were important contributors to H2O2 and MHP enrichment. High levels of H2O2 and MHP concentrations which occurred during the measurement period probably resulted from the transport of a polluted air mass with high water vapor content passing over the Bohai Bay, China.

Sanguinarine-induced oxidative stress and apoptosis-like programmed cell death(AL-PCD) in root meristem cells of Allium cepa

A vast number of studies on plant cell systems clearly indicate that various biotic and abiotic stresses give rise to the uncontrolled increase in the level of reactive oxygen species (ROS). Excess concentrations of ROS result in damage to proteins, lipids, carbohydrates, and DNA, which may lead, in consequence, to the apoptotic cell death. The current study investigates the effects of sanguinarine (SAN), a natural alkaloid derived from the roots of Sanguinaria canadensis, on root apical meristem cells of Allium cepa. It is shown that SAN treatment generated large amounts of hydrogen peroxide (H2O2) and superoxide anion (O2·−). Oxidative stress induced in SAN-treated cells was correlated with DNA fragmentation, formation of micronuclei (MN), altered and ‘degenerated’ chromatin structures characteristic of apoptosis-like programmed cell death (AL-PCD). The experiments with SAN + MG132 (a proteasome inhibitor engaged in Topo II-mediated formation of cleavable complexes) and SAN + ascorbic acid (AA; H2O2 scavenger) seem to suggest, however, that the high level of H2O2 is not the only factor responsible for changes observed at the chromatin level and for the consequent cell death. Our findings imply that Topo II-DNA covalent complexes and 26S proteasomes are also involved in SAN-induced DNA damage.

191 - Redox-Active Mn Porphyrins, MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+ But Not Redox-Inert MnTBAP3-Suppress Tumor Growth in an Environment Where H2O2 Is Produced

Cationic Mn(III) N-substituted pyridylporphyrins (MnPs), such as MnTnBuOE-2-PyP5+ and MnTE-2-PyP5+, initially optimized as powerful mimics of superoxide dismutase, also impact cellular redox-based signaling pathways, most so redox sensitive thiol bearing proteins such as NF-κB, whereby affecting cellular apoptotic and proliferative pathways. Such actions lead to S-glutathionylation of proteins and their subsequent inactivation. In addition to MnP catalyst, both H2O2 and glutathione are required for such process, which includes initial oxidation of Mn porphyrin by H2O2, followed by oxidation of protein thiols by high-valent MnP in a GPx-like fashion. While tumors already have higher H2O2 levels, they are sensitive to any further increase in H2O2 which strategy is used to induce tumor killing. Mn porphyrins do not differentiate tumor from normal tissue and thereby interact at identical rate constants with small reactive species and proteins in both types of tissues. Yet, the yield of reactions depends upon the MnPs levels and levels of reactive species. In the case of NF-kB, the higher are the levels of reactants (MnPs and reactive species) the larger would be the yield of its oxidative damage. Consequently, the larger would be the inhibition of NF-κB activation and in turn the suppression of anti-apoptotic pathways (for involvement of NF-κB pathways in MnP/Asc anticancer effects see Tovmasyan et al abstract on Redox Proteomics). Using LC-MS/MS we showed the preferential accumulation of 3 different MnPs in tumor over normal tissue in a 4T1 flank mouse tumor model. In such model, the already high tumor H2O2 levels were increased with either radiation (RT) or ascorbate or both. The higher tumor accumulation of MnTE-2-PyP5+ relative to MnTnBuOE-2-PyP5+ resulted in larger tumor suppression. Regardless of much higher tumor vs normal tissue accumulation, the redox-inert MnTBAP3- sensitized tumor neither to ascorbate nor to radiation. As anticipated the tumor GSSG levels were increased only when redox-active MnTE-2-PyP5+, but not MnTBAP3-, was combined with RT/Ascorbate treatment. Grant support NIH 1R03-NS082704-01, 5-P30-CA14236-29, CNPq and CAPES from Brazil.

Ergothioneine Biosynthesis and Functionality in the Opportunistic Fungal Pathogen, Aspergillus fumigatus.

Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H2O2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H2O2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H2O2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.

Leaf miner-induced morphological, physiological and molecular changes in mangrove plantAvicennia marina(Forsk.) Vierh

Avicennia marina (Forsk.) Vierh is a widespread mangrove species along the southeast coasts of China. Recently, the outbreak of herbivorous insect, Phyllocnistis citrella Stainton, a leaf miner, have impacted on the growth of A. marina. Little is reported about the responses of A. marina to leaf miner infection at the biochemical, physiological and molecular levels. Here, we reported the responses of A. marina to leaf miner infection from the aspects of leaf structure, photosynthesis, and antioxidant system and miner responsive genes expression. A. marina leaves attacked by the leaf miner exhibited significant decreases in chlorophyll, carbon and nitrogen contents, as well as a decreased photosynthetic rate. Scanning and transmission electron microscopic observations revealed that the leaf miner only invaded the upper epidermis and destroyed the epidermal cell, which lead to the exposure of salt glands. In addition, the chloroplasts of mined leaves (ML) were swollen and the thylakoids degraded. The maximal net photosynthetic rate, stomatal conductance (Gs), carboxylation efficiency (CE), dark respiration (Rd), light respiration (Rp) and quantum yields (AQE) significantly decreased in the ML, whereas the light saturation point (Lsp), light compensation point (Lcp), water loss and CO2 compensation point (Г) increased in the ML. Moreover, chlorophyll fluorescence features also had been changed by leaf miner attacks. Interestingly, higher generation rate of O2ˉ· and lower antioxidant enzyme expression in the mined portion (MP) were found; on the contrary, higher H2O2 level and higher antioxidant enzyme expression in the non-mined portion (NMP) were revealed, implying that the NMP may be able to sense that the leaf miner attacks had happened in the MP of the A. marina leaf via H2O2 signaling. Besides, the protein expression of glutathione S-transferase (GST) and the glutathione (GSH) content were increased in the ML. In addition, insect resistance-related gene expression such as chitinase 3, RAR1, topless and PIF3 had significantly increased in the ML. Taken together, our data suggest that leaf miners could significantly affect leaf structure, photosynthesis, the antioxidant system and miner responsive gene expression in A. marina leaves.

Contrasting Regulation of NO and ROS in Potato Defense-Associated Metabolism in Response to Pathogens of Different Lifestyles.

Our research provides new insights into how the low and steady-state levels of nitric oxide (NO) and reactive oxygen species (ROS) in potato leaves are altered after the challenge with the hemibiotroph Phytophthora infestans or the necrotroph Botrytis cinerea, with the subsequent rapid and invader-dependent modification of defense responses with opposite effects. Mainly in the avirulent (avr) P. infestans–potato system, NO well balanced with the superoxide level was tuned with a battery of SA-dependent defense genes, leading to the establishment of the hypersensitive response (HR) successfully arresting the pathogen. Relatively high levels of S-nitrosoglutathione and S-nitrosothiols concentrated in the main vein of potato leaves indicated the mobile function of these compounds as a reservoir of NO bioactivity. In contrast, low-level production of NO and ROS during virulent (vr) P. infestans-potato interactions might be crucial in the delayed up-regulation of PR-1 and PR-3 genes and compromised resistance to the hemibiotrophic pathogen. In turn, B. cinerea triggered huge NO overproduction and governed inhibition of superoxide production by blunting NADPH oxidase. Nevertheless, a relatively high level of H2O2 was found owing to the germin-like activity in cooperation with NO-mediated HR-like cell death in potato genotypes favorable to the necrotrophic pathogen. Moreover, B. cinerea not only provoked cell death, but also modulated the host redox milieu by boosting protein nitration, which attenuated SA production but not SA-dependent defense gene expression. Finally, based on obtained data the organismal cost of having machinery for HR in plant resistance to biotrophs is also discussed, while emphasizing new efforts to identify other components of the NO/ROS cell death pathway and improve plant protection against pathogens of different lifestyles.

Requirement of aquaporin-3-mediated hydrogen peroxide for NF-κB cell signaling and psoriasis pathogenesis

Aquaporin 3 (AQP3), a water/glycerol channel protein, has been recently shown to transport hydrogen peroxide (H2O2). Here we show that AQP3-mediated H2O2 is involved in nuclear factor-κB (NF-κB) cell signaling in keratinocytes and in the pathogenesis of psoriasis. In in vitro studies with primary keratinocytes from wild-type and AQP3 knockout (AQP3−/−) mice, the extracellular H2O2 generated by membrane NADPH oxidase 2 (Nox2) activation in response to TNF-α was intracellularly transported via AQP3, which was indispensable for the activation of NF-κB. We found the association of AQP3 with Nox2 in membrane fraction that might facilitate the transient increase in intracellular H2O2 levels around membranes after TNF-α stimulation. Coincidentally, AQP3−/− mice was impaired in the development of psoriasis in vivo mouse model which was partially dependent on TNF-α-mediated cell signaling, in which continuous NF-κB activation and a higher H2O2 level in keratinocytes were found to be suppressed by AQP3 deficiency. These data reveal that AQP3-mediated H2O2 is required for NF-κB activation in keratinocytes during psoriasis development. Our findings provide novel insights into the pathogenesis of psoriasis and may provide a basis for new therapies.

Interaction between Cucumber mosaic virus 2b protein and plant catalase induces a specific necrosis in association with proteasome activity

Key messageCucumber mosaic virus(CMV) can induce a specific necrosis onArabidopsisthrough the interaction between the CMV 2b protein and host catalase, in which the ubiquitin–proteasome pathway may be involved.We previously reported that the CMV 2b protein, the viral RNA silencing suppressor, interacted with the H2O2 scavenger catalase (CAT3), leading to necrosis on CMV-inoculated Arabidopsis leaves. We here confirmed that CMV could more abundantly accumulate in the CAT3-knockout mutant (cat3), and that CAT3 makes host plants a little more tolerant to CMV. We also found that the necrosis severity is not simply explained by a high level of H2O2 given by the lack of CAT3, because the recombinant CMV, CMV-N, induced much milder necrosis in cat3 than in the wild type, suggesting some specific mechanism for the necrosis induction. To further characterize the 2b-inducing necrosis in relation to its binding to CAT3, we conducted the agroinfiltration experiments to overexpress CAT3 and 2b in N. benthamiana leaves. The accumulation levels of CAT3 were higher when co-expressed with the CMV-N 2b (N2b) than with CMV-Y 2b (Y2b). We infer that N2b made a more stable complex with CAT3 than Y2b did, and the longevity of the 2b–CAT3 complex seemed to be important to induce necrosis. By immunoprecipitation (IP) with an anti-ubiquitin antibody followed by the detection with anti-CAT3 antibodies, we detected a higher molecular-weight smear and several breakdown products of CAT3 among the IP-proteins. In addition, the proteasome inhibitor MG132 treatment could actually increase the accumulation levels of CAT3. This study suggests that the host proteasome pathway is, at least partially, responsible for the degradation of CAT3, which is manifested in CMV-infected tissues.

2-Cys peroxiredoxin is required in successful blood-feeding, reproduction, and antioxidant response in the hard tick Haemaphysalis longicornis.

BackgroundTicks are obligate hematophagous arthropods that feed on vertebrate blood that contains iron. Ticks also concentrate host blood with iron; this concentration of the blood leads to high levels of iron in ticks. The host-derived iron reacts with oxygen in the tick body and this may generate high levels of reactive oxygen species, including hydrogen peroxide (H2O2). High levels of H2O2 cause oxidative stress in organisms and therefore, antioxidant responses are necessary to regulate H2O2. Here, we focused on peroxiredoxin (Prx), an H2O2-scavenging enzyme in the hard tick Haemaphysalis longicornis.MethodsThe mRNA and protein expression profiles of 2-Cys peroxiredoxin (HlPrx2) in H. longicornis were investigated in whole ticks and internal organs, and developmental stages, using real-time PCR and Western blot analysis during blood-feeding. The localization of HlPrx2 proteins in tick tissues was also observed by immunostaining. Moreover, knockdown experiments of HlPrx2 were performed using RNA interference to evaluate its function in ticks.ResultsReal-time PCR showed that HlPrx2 gene expression in whole ticks and internal organs was significantly upregulated by blood-feeding. However, protein expression, except in the midgut, was constant throughout blood-feeding. Knockdown of the HlPrx2 gene caused significant differences in the engorged body weight, egg weight and hatching rate for larvae as compared to the control group. Finally, detection of H2O2 after knockdown of HlPrxs in ticks showed that the concentration of H2O2 significantly increased before and after blood-feeding.ConclusionTherefore, HlPrx2 can be considered important for successful blood-feeding and reproduction through the regulation of H2O2 concentrations in ticks before and after blood-feeding. This study contributes to the search for a candidate target for tick control and further understanding of the tick’s oxidative stress coping mechanism during blood-feeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1748-2) contains supplementary material, which is available to authorized users.

CsPAO4 of Citrus sinensis functions in polyamine terminal catabolism and inhibits plant growth under salt stress.

Polyamine oxidase (PAO) is a key enzyme catalyzing polyamine catabolism leading to H2O2 production. We previously demonstrated that Citrus sinensis contains six putative PAO genes, but their functions are not well understood. In this work, we reported functional elucidation of CsPAO4 in polyamine catabolism and salt stress response. CsPAO4 was localized to the apoplast and used both spermidine (Spd) and spermine (Spm) as substrates for terminal catabolism. Transgenic plants overexpressing CsPAO4 displayed prominent increase in PAO activity, concurrent with marked decrease of Spm and Spd and elevation of H2O2. Seeds of transgenic lines displayed better germination when compared with wild type (WT) under salt stress. However, both vegetative growth and root elongation of the transgenic lines were prominently inhibited under salt stress, accompanied by higher level of H2O2 and more conspicuous programmed cell death (PCD). Exogenous supply of catalase (CAT), a H2O2 scavenger, partially recovered the vegetative growth and root elongation. In addition, spermine inhibited root growth of transgenic plants. Taken together, these data demonstrated that CsPAO4 accounts for production of H2O2 causing oxidative damages under salt stress and that down-regulation of a PAO gene involved in polyamine terminal catabolism may be an alternative approach for improving salt stress tolerance.