Early Reactive Oxygen Species Research Articles

The critical role of CmCIPK1-CmRbohD1/D2 complexes in generating H2O2 signals for enhancing salt tolerance in pumpkins

Pumpkin is of great economic importance not only as food resources but also as the most widely used rootstock to graft cucurbit crops. Pumpkin rootstock improves salt tolerance of cucumber scion through respiratory burst oxidase homolog protein D (CmRbohD)-mediated reactive oxygen species (ROS) burst, which further enhances Na+ export and K+ uptake. RbohD activation requires calcium signaling. However, the underlying mechanism remains largely unknown. In this study, we discovered two RbohD members from pumpkin involved in the ROS burst at the early stage of salt stress. CmRbohD1 and CmRbohD2 were functionally redundant and double mutation significantly impaired salt tolerance in pumpkin. Overexpression of CmRbohD1 and CmRbohD2 mitigated salinity-induced damage and maintained a relatively low Na+ content and high K+ content. We screened the potential calcineurin B-like interacting protein kinases (CmCIPKs) which could bind with CmRbohD1 and CmRbohD2. Our results revealed that two RbohDs formed complexes specifically with CmCIPK1, thereby bursting ROS production. Overexpression of CmCIPK1 promoted the early ROS burst under salt stress condition and maintained a relatively balanced Na+/K+ homeostasis. Altogether, we proposed a CmCIPK1-CmRbohD1/D2 complex for pumpkin salt stress signaling transduction, which regulates the ROS burst and Na+/K+ homeostasis. Our findings offer unprecedented insights into the general mechanism of pumpkin salt tolerance.

Unraveling the complex dynamics of signaling molecules in cellular signal transduction.

Signaling molecules in cellular responses to foreign stimuli are described as static up- or down-concentration changes during signal transduction. This is because analytical methods for transducing molecules are much slower than the signaling events. In this study, we develop a dynamic cell model and reveal the temporal regulation of signal transduction events in response to reactive oxygen species (ROS). The model contained a set of 10 batches of redox-modified cells that mimic the temporal ROS accumulation events. Validating this dynamic cell model, we discover that cells survive early ROS attacks by activating the Nrf2/polysulfide/p62/CDK1 pathway. Nearly all signaling molecules exhibit time-dependent V-shape or inverse V-shape activation/feedback regulation dynamics in response to ROS accumulation. The results show that the dynamic cell model approach is invaluable for revealing complex signal intensity- and time-dependent cell signaling events.

Abstract P2121: Relative Importance Of Reactive Oxygen Species From Reverse Electron Transfer At Mitochondrial Complex I In Reperfusion Injury

The metabolite succinate accumulates during tissue ischemia. In early reperfusion, succinate is rapidly metabolized, driving a burst of mitochondrial reactive oxygen species (ROS) generation that triggers cell death. It is widely believed that the source of succinate-driven ROS in early reperfusion is reverse electron transfer (RET) at respiratory complex I (Cx-I). However, evidence for Cx-I RET ROS is largely based on experimental evidence from isolated mitochondria with succinate as the sole substrate. We hypothesized that numerous other conditions present during early reperfusion may impact Cx-I RET ROS. Thus, experimental perturbations were applied to mitochondria to model early reperfusion, with measurement of ROS via the fluorescent probe PHPA. These conditions included: (i) A high concentration of NADH; (ii) A high concentration of lactate; (iii) A mildly acidic pH; (iv) A non-zero concentration of ADP buffered by a creatine / creatine phosphate / creatine kinase system; (v) Presence of purine nucleotide breakdown products; (vi) Presence of calcium. These studies revealed that under early reperfusion-like conditions mitochondrial ROS generation still occurred via Cx-I RET, but at a magnitude only half that previously reported. Furthermore, residual ROS that was insensitive to a specific Cx-I RET inhibitor (S1QEL) was slightly elevated under early-reperfusion like conditions, and this residual ROS was mostly inhibited by S3QEL, a blocker of ROS from respiratory complex III (Cx-III). Together, these data suggest that the relative contribution of Cx-I RET ROS to reperfusion injury may be overestimated, and that of Cx-III ROS underestimated.

Chemiluminescence-Based Assay to Monitor Early Oxidative Bursts in Soybean (Glycine max) Lateral Roots.

The reactive oxygen species (ROS) burst assay is a valuable tool for studying pattern-triggered immunity (PTI) in plants. During PTI, the interaction between pathogen recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) leads to the rapid production of ROS in the apoplastic space. The resultant ROS can be measured using a chemiluminescent approach that involves the usage of horseradish peroxidase and luminol. Although several methods and protocols are available to detect early ROS bursts in leaf tissues, no dedicated method is available for root tissues. Here, we have established a reliable method to measure the PAMP-triggered ROS burst response in soybean lateral roots. In plants, lateral roots are the potential entry and colonization sites for pathogens in the rhizosphere. We have used important PAMPs such as chitohexaose, flagellin 22 peptide fragment, and laminarin to validate our method. In addition, we provide a detailed methodology for the isolation and application of fungal cell wall components to monitor the oxidative burst in soybean lateral roots. Furthermore, we provide methodology for performing ROS burst assays in soybean leaf discs with laminarin and fungal cell walls. This approach could also be applied to leaf and root tissues of other plant species to study the PTI response upon elicitor treatment. © 2023 Wiley Periodicals LLC. Basic Protocol: Reactive oxygen species (ROS) burst assay in soybean lateral root tissues Alternate Protocol: ROS burst assay in soybean leaf discs Support Protocol: Isolating fungal cell wall fractions.

Non-specific LIPID TRANSFER PROTEIN 1 enhances immunity against tobacco mosaic virus in Nicotiana benthamiana.

Plant non-specific lipid transfer proteins (nsLTPs) are small, cysteine-rich proteins that play significant roles in biotic and abiotic stress responses; however, the molecular mechanism of their functions against viral infections remains unclear. In this study, we employed virus-induced gene-silencing and transgenic overexpression to functionally analyse a type-I nsLTP in Nicotiana benthamiana, NbLTP1, in the immunity response against tobacco mosaic virus (TMV). NbLTP1 was inducible by TMV infection, and its silencing increased TMV-induced oxidative damage and the production of reactive oxygen species (ROS), compromised local and systemic resistance to TMV, and inactivated the biosynthesis of salicylic acid (SA) and its downstream signaling pathway. The effects of NbLTP1-silencing were partially restored by application of exogenous SA. Overexpressing NbLTP1 activated genes related to ROS scavenging to increase cell membrane stability and maintain redox homeostasis, confirming that an early ROS burst followed by ROS suppression at the later phases of pathogenesis is essential for resistance to TMV infection. The cell-wall localization of NbLTP1 was beneficial to viral resistance. Overall, our results showed that NbLTP1 positively regulates plant immunity against viral infection through up-regulating SA biosynthesis and its downstream signaling component, NONEXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), which in turn activates pathogenesis-related genes, and by suppressing ROS accumulation at the later phases of viral pathogenesis.

Omics analyses indicate sdhC/D act as hubs of early response of E. coli to antibiotics.

In recent years, the phenomenon of microbial resistance has become increasingly serious. The generation of reactive oxygen species (ROS) during the bactericidal process of antibiotics has attracted great interest, but little research has been done on the generation of ROS in the early stage of antibiotic action. We confirmed the rapid production of ROS by flow cytometry and transmission electron microscopy (TEM). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis indicated that the oxidative phosphorylation pathway is the key pathway of ROS production. Protein-protein interaction (PPI) network results indicate that sdhC/D are key genes in the oxidative phosphorylation pathway. The overexpression of sdhC/D resulted in a lower survival rate than the control strain after antibiotic treatments, which might be due to excess ROS induced by sdhC/D overexpression. The production of superoxide anion in the overexpress strain was significantly higher than that in the control strain, which further verified the importance of sdhC/D in the ROS release of bacteria. Current results showed that bacteria produce large amounts of ROS in the early stage of gentamicin and ampicillin action, and the regulation patterns of genes in the key pathway were consistent. sdhC/D are key genes in the early ROS release process of bacteria. Our study provides a basis for the search of ROS-related enhancers of antimicrobial action.

The Effect of Astaxanthin on Motility, Viability, Reactive Oxygen Species, Apoptosis, and Lipid Peroxidation of Human Spermatozoa During the Freezing-Thawing Process.

Cryopreservation of spermatozoa is a general procedure to preserve viable sperm for an indefinite period. Despite the efficiency of sperm cryopreservation, excessive reactive oxygen species (ROS) production during cryopreservation can induce structural and functional changes in spermatozoa. Also, cryopreservation has been shown to decrease the spermatozoa's antioxidant activity inducing them to be more sensitive to damage caused by ROS. Experimental evidence suggests that astaxanthin (AXT) has essential activities such as antioxidant, antibacterial, and antithrombotic properties. Therefore, this study aimed to evaluate the effect of AXT on the sperm quality of healthy men during freezing-thawing. In the first phase, 10 semen samples with different concentrations of AXT (0.0, 0.5, 1, and 2 μM) were cryopreserved to achieve an optimal dose of AXT. Then, motility, viability, and phosphatidylserine (PS) externalization were evaluated. In the second phase, 25 samples were collected and divided into 3 groups: fresh group, control group (untreated frozen-thawed samples), and AXT group (treated frozen-thawed with AXT). Then, samples were cryopreserved in freezing media supplemented with or without the optimal concentration of AXT (1 μM). After thawing, the levels of sperm parameters, including motility (using a computer-assisted sperm analyzer), viability (eosin-nigrosin), early apoptotic change (annexin V/propidium iodide), ROS (flow cytometry), and lipid peroxidation (LPO) (using enzyme-linked immunosorbent assay), were evaluated. Our results showed that the addition of 1 μM AXT to sperm freezing media improved all parameters of sperm motility and viability (p ≤ 0.05). Furthermore, it could reduce the levels of ROS parameters (intracellular hydrogen peroxide and superoxide) compared with the control group (p ≤ 0.05). Also, AXT significantly decreased the level of PS externalization (p ≤ 0.05) and LPO (p ≤ 0.05) after the freezing-thawing process. In conclusion, our findings demonstrated that human semen treatment with 1 μM AXT before the freezing-thawing process has protective effects against oxidative stress and could diminish the destructive effects of this process on sperm quality.

Intracellular reactive oxygen species trafficking participates in seed dormancy alleviation in Arabidopsis seeds.

Reactive oxygen species (ROS) release seed dormancy through an unknown mechanism. We used different seed dormancy-breaking treatments to decipher the dynamics and localization of ROS production during seed germination. We studied the involvement of ROS in the breaking of Arabidopsis seed dormancy by cold stratification, gibberellic acid (GA3 ) and light. We characterized the effects of these treatments on abscisic acid and gibberellins biosynthesis and signalling pathways. ROS, mitochondrial redox status and peroxisomes were visualized and/or quantified during seed imbibition. Finally, we performed a cytogenetic characterization of the nuclei from the embryonic axes during seed germination. We show that mitochondria participate in the early ROS production during seed imbibition and that a possible involvement of peroxisomes in later stages should still be analysed. At the time of radicle protrusion, ROS accumulated within the nucleus, which correlated with nuclear expansion and chromatin decompaction. Taken together, our results provide evidence of the role of ROS trafficking between organelles and of the nuclear redox status in the regulation of seed germination by dormancy.

Induction of Reactive Oxygen Species Is Necessary for Efficient Onset of Cyprinid Herpesvirus 2 Replication: Implications for Novel Antiviral Strategy With Antioxidants

Cyprinid herpesvirus 2 (CyHV-2) has caused great economic loss to the crucian carp breeding industry. Upon viral stimulation, eukaryotic cells generally activate the expression of anti-oxidative genes to maintain the intracellular oxidative balance and resist viral infection. Here, intracellular reactive oxygen species (ROS) levels in CyHV-2-infected cells were monitored to show that CyHV-2 induced the increase of intracellular ROS during early infection, and intracellular excessive accumulation of ROS was ameliorated during late infection, which was accompanied by activated expression of genes related to Nrf2 signaling pathway. In order to explore the interaction between CyHV-2 infection and ROS production, RyuF-2 cells were treated with either antioxidant epigallocatechin-3-gallate (EGCG) or berberine hydrochloride (BBH) and then infected with CyHV-2. Both BBH and EGCG could effectively inhibit the amplification of CyHV-2 while inhibiting the accumulation of intracellular ROS. Consistent with this, the oxidant stress-related genes were up-regulated by CyHV-2 infection and down-regulated in cells treated with either BBH or EGCG, through which the production of intracellular ROS was modulated. These results collectively demonstrated that early ROS accumulation favored the replication of CyHV-2, while antioxidants (BBH and EGCG) could inhibit the amplification of CyHV-2 by inhibiting ROS induction.

Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts β-Cell Function and Survival

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in β-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2•− production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts β-cells’ function and survival during insulitis, NOX2 might be a potential target for designing therapies against early β-cell dysfunction in the context of T1D onset.

Abstract LB103: Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells

Abstract Background: Nuclear events play a well-established role in the cytotoxicity of doxorubicin (DOX). Other reported mechanisms include redox cycling for reactive oxygen species (ROS) production, which is getting recognized for a larger role than previously recognized. In addition, ROS production may be implicated in lysosome membrane permeabilization (LMP) and subsequent cell death. We have previously reported DOX-induced LMP in breast cancer cells. The goal of this study is to establish the role of LMP in the cytotoxic process and whether ROS are the initiator of this LMP. Methods: Studies were conducted using DOX concentrations ranging from 0.1 μM to 10 μM for 2, 6, and 24 h exposure periods in MCF-7 and MDA-MB-231 breast cancer cells. Intracellular localization of DOX was examined by fluorescence microscopy using lysosome and nuclear markers. ROS production was determined by the DCFDA assay using a fluorescence plate reader. LMP was demonstrated using fluorescence microscopy as the cytosolic release of a 10 kDa AlexaFluor-488 Dextran conjugate that was preloaded into lysosomes. Mitochondrial membrane potential (MMP) was determined by JC-1 assay. Viability was determined by MTT. Cathepsin B activity will be determined by Z-Arg-Arg-AMC cathepsin B substrate. Experiments will be conducted in the presence of E-64 cathepsin B inhibitor and Tiron superoxide scavenger to establish the importance of LMP and ROS towards DOX cytotoxicity. Results: Nuclear uptake was seen in both cell lines as early as 2 h at a concentration of 10 μM. Furthermore, lysosome accumulation of DOX, although small, was visible by 2 h in MDA cells, increasing in degree with time and concentration. ROS levels were found to be highest at 2 h in MCF-7 cells, increasing by up to 100% of the untreated control, and occurring concurrently with LMP at higher concentrations. Preliminary results suggest much lower ROS levels in MDA-MB-231 cells. In MCF-7 cells, ROS production decreased at 6 h, but increased by 24 h with concurrent increases in LMP. Although less than the MCF-7 cells, MDA cells demonstrated notable LMP for the first time at 5 and 10 μM conditions, occurring concurrently with MMP. By 24 h, viability was substantially reduced for both cell lines, with MCF-7s demonstrating a greater reduction in viability. Significant MMP was not observable until 24 h in MCF-7 cells. Conclusions: The early and strong ROS occurring with LMP suggest a previously unrecognized DOX cytotoxic combination in MCF-7 cells. LMP does not appear to result from lysosomal accumulation of DOX. A reduced role of ROS and LMP may account for the lesser cytotoxicity in MDA-MB-231 cells. Both ROS and LMP appear to be necessary for cytotoxicity in MCF-7 cells. Citation Format: Rachel E. Nicoletto, Clyde M. Ofner. Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB103.

Nitrogen Dioxide Inhalation Exposures Induce Cardiac Mitochondrial Reactive Oxygen Species Production, Impair Mitochondrial Function and Promote Coronary Endothelial Dysfunction.

Traffic air pollution is a major health problem and is recognized as an important risk factor for cardiovascular (CV) diseases. In a previous experimental study, we showed that diesel exhaust (DE) exposures induced cardiac mitochondrial and CV dysfunctions associated with the gaseous phase. Here, we hypothesized that NO2 exposures to levels close to those found in DE induce a mitochondrial reactive oxygen species (ROS) production, which contribute to an endothelial dysfunction, an early indicator for numerous CV diseases. For this, we studied the effects of NO2 on ROS production and its impacts on the mitochondrial, coronary endothelial and cardiac functions, after acute (one single exposure) and repeated (three h/day, five days/week for three weeks) exposures in Wistar rats. Acute NO2 exposure induced an early but reversible mitochondrial ROS production. This event was isolated since neither mitochondrial function nor endothelial function were impaired, whereas cardiac function assessment showed a reversible left ventricular dysfunction. Conversely, after three weeks of exposure this alteration was accompanied by a cardiac mitochondrial dysfunction highlighted by an alteration of adenosine triphosphate (ATP) synthesis and oxidative phosphorylation and an increase in mitochondrial ROS production. Moreover, repeated NO2 exposures promoted endothelial dysfunction of the coronary arteries, as shown by reduced acetylcholine-induced vasodilatation, which was due, at least partially, to a superoxide-dependent decrease of nitric oxide (NO) bioavailability. This study shows that NO2 exposures impair cardiac mitochondrial function, which, in conjunction with coronary endothelial dysfunction, contributes to cardiac dysfunction. Together, these results clearly identify NO2 as a probable risk factor in ischemic heart diseases.

Aspirin Induces Mitochondrial Ca2+ Remodeling in Tumor Cells via ROS‒Depolarization‒Voltage-Gated Ca2+ Entry

Aspirin (acetylsalicylic acid) and its metabolite salicylate, have an anti-melanoma effect by evoking mitochondrial dysfunction through poorly understood mechanisms. Depolarization of the plasma membrane potential leads to voltage-gated Ca2+ entry (VGCE) and caspase-3 activation. In the present study, we investigated the role of depolarization and VGCE in aspirin’s anti-melanoma effect. Aspirin and to a lesser extent, salicylate (≥2.5 mM) induced a rapid (within seconds) depolarization, while they caused comparable levels of depolarization with a lag of 2~4 h. Reactive oxygen species (ROS) generation also occurred in the two-time points, and antioxidants abolished the early ROS generation and depolarization. At the same concentrations, the two drugs induced apoptotic and necrotic cell death in a caspase-independent manner, and antioxidants and Ca2+ channel blockers prevented cell death. Besides ROS generation, reduced mitochondrial Ca2+ (Ca2+m) and mitochondrial membrane potential preceded cell death. Moreover, the cells expressed the Cav1.2 isoform of l-type Ca2+ channel, and knockdown of Cav1.2 abolished the decrease in Ca2+m. Our findings suggest that aspirin and salicylate induce Ca2+m remodeling, mitochondrial dysfunction, and cell death via ROS-dependent depolarization and VGCE activation.

Oncostatin M, A Profibrogenic Mediator Overexpressed in Non-Alcoholic Fatty Liver Disease, Stimulates Migration of Hepatic Myofibroblasts.

Background: Hepatic myofibroblasts (MFs) can originate from hepatic stellate cells, portal fibroblasts, or bone marrow-derived mesenchymal stem cells and can migrate towards the site of injury by aligning with nascent and established fibrotic septa in response to several mediators. Oncostatin M (OSM) is known to orchestrate hypoxia-modulated hepatic processes involving the hypoxia-inducible factor 1 (HIF-1). Methods. In vivo and in vitro experiments were performed to analyze the expression of OSM and OSM-receptor (OSMR) in three murine models of non-alcoholic-fatty liver disease (NAFLD) and -steatohepatitis (NASH) and in human NASH patients as well as the action of OSM on phenotypic responses of human MFs. Results: Hepatic OSM and OSMR levels were overexpressed in three murine NASH models and in NASH patients. OSM stimulates migration in human MFs by involving early intracellular ROS generation and activation of Ras/Erk, JNK1/2, PI3K/Akt as well as STAT1/STAT3 pathways and HIF-1α. OSM-dependent migration relies on a biphasic mechanism requiring early intracellular generation of reactive oxygen species (ROS) and late HIF1-dependent expression and release of VEGF. Conclusion: OSM is overexpressed in experimental and human progressive NAFLD and can act as a profibrogenic factor by directly stimulating migration of hepatic MFs.

Alloxan Disintegrates the Plant Cytoskeleton and Suppresses mlo-Mediated Powdery Mildew Resistance.

Recessively inherited mutant alleles of Mlo genes (mlo) confer broad-spectrum penetration resistance to powdery mildew pathogens in angiosperm plants. Although a few components are known to be required for mlo resistance, the detailed molecular mechanism underlying this type of immunity remains elusive. In this study, we identified alloxan (5,5-dihydroxyl pyrimidine-2,4,6-trione) and some of its structural analogs as chemical suppressors of mlo-mediated resistance in monocotyledonous barley (Hordeum vulgare) and dicotyledonous Arabidopsis thaliana. Apart from mlo resistance, alloxan impairs nonhost resistance in Arabidopsis. Histological analysis revealed that the chemical reduces callose deposition and hydrogen peroxide accumulation at attempted fungal penetration sites. Fluorescence microscopy revealed that alloxan interferes with the motility of cellular organelles (peroxisomes, endosomes and the endoplasmic reticulum) and the pathogen-triggered redistribution of the PEN1/SYP121 t-SNARE protein. These cellular defects are likely the consequence of disassembly of actin filaments and microtubules upon alloxan treatment. Similar to the situation in animal cells, alloxan elicited the temporary accumulation of reactive oxygen species (ROS) in cotyledons and rosette leaves of Arabidopsis plants. Our results suggest that alloxan may destabilize cytoskeletal architecture via induction of an early transient ROS burst, further leading to the failure of molecular and cellular processes that are critical for plant immunity.

ANAC032 regulates root growth through the MYB30 gene regulatory network

Reactive oxygen species (ROS) play important roles as root growth regulators. We previously reported a comprehensive transcriptomic atlas, which we named ROS-map, that revealed ROS-responsible genes in Arabidopsis root tips. By using ROS-map, we have characterised an early ROS response key transcription factor, MYB30, as a regulator of root cell elongation under ROS signals. However, there are other ROS-responsible transcription factors which have the potential to regulate root growth. In the present study, we characterised the function of another early ROS-responsible transcription factor, ANAC032, that was selected from ROS-map. Overexpression of ANAC032 fused with the transcriptional activation domain, VP16, inhibited root growth, especially decreasing cell elongation. By transcriptome analysis, we revealed that ANAC032 regulated many stress-responsible genes in the roots. Intriguingly, ANAC032 upregulated MYB30 and its target genes. The upregulation of MYB30 target genes was completely abolished in the ANAC032-VP16x2 OX and ANAC032 estradiol-inducible line in myb30-2 mutants. Moreover, root growth inhibition was alleviated in ANAC032-OX in myb30-2 mutants. Overall, we characterised an upstream transcription factor, ANAC032, of the MYB30 transcriptional cascade which is a key regulator for root cell elongation under ROS signalling.

Papilla formation, defense gene expression and HR contribute to the powdery mildew resistance of the novel wheat line L699 carrying Pm40 gene

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive wheat diseases worldwide and causes severe yield losses in most wheat-growing areas. POWDERY MILDEW (Pm) genes are responsible for wheat resistance to Bgt. Recently, a wheat line L699 carrying a novel Pm40 gene which was originated from Elytrigia inermedium, was developed and showed high resistance to Bgt; however, the resistance mechanism of L699 is still unclear. In current study, a range of defense responses were examined in wheat line L699 inoculated with Bgt, using a highly susceptible cultivar CN26 without Pm40 gene as a control. We found that high levels of reactive oxygen species (ROS) were rapidly accumulated and abundant papilla were formed at the infection sites where Bgt conidiospores germinated with abnormal morphology at a high frequency on L699 compared to CN26. Local hypersensitive cell death and defense genes in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways were then rapidly induced in L699. As a consequence, L699 exhibited no typical disease symptoms, whereas CN26 were fully covered by white hyphae and conidia. These data provide evidence that for the resistance of the wheat line L699 with the dominant gene Pm40 against Bgt, early ROS accumulation and papilla formation acts as dominant penetration resistance, leading to morphological abnormality of Bgt, when defense gene expression in SA and JA signaling and HR response enhance the post-penetration resistance by arresting onset of Bgt.

Improved drought tolerance by α-naphthaleneacetic acid-induced ROS accumulation in two soybean cultivars

Drought is a major abiotic factor limiting agricultural crop production. The objective of this study was to investigate whether α-naphthaleneacetic acid (NAA) confers drought tolerance to soybeans and if such tolerance is correlated with the early reactive oxygen species (ROS) accumulation in leaves. The plants of soybean (Glycine max [L.] Merr.) cv. Nannong 99-6 and cv. Kefeng 1 were foliar treated with 40 mg L−1 NAA at the beginning of bloom and then exposed to water stress for 10 d. We monitored changes in ROS levels, lipid peroxidation and antioxidant system as well as plant biomass during the drought treatment. The results showed that drought stress significantly depressed the growth and yield regardless of spraying NAA. However, drought-stressed plants treated with NAA showed much higher plant biomass and yield than those without NAA. The ROS levels increased in stressed Kefeng 1 but not in stressed Nannong 99-6 2–4 days after the treatment (DAT). During 6–10 DAT, stressed Kefeng 1 had greater increase in the levels of superoxide dismutase (SOD), guaiacol peroxidase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione peroxidase (GPX), γ-glutamylcysteine synthetase (γ-GCS), reduced ascorbate (AsA), and glutathione (GSH), smaller increase in ROS and malondialdehyde (MDA) as compared with stressed Nannong 99-6. Low ROS prevented ROS from directly reacting with membrane lipid during this stage and, consequently, reduced the cell damage. NAA application elevated ROS levels at 4 DAT, and then increased antioxidant capacity and blocked the increase in the MDA and ROS in stressed Nannong 99-6 and Kefeng 1. Overall, the results indicate that NAA application effectively alleviates the adverse effects of drought stress, which is partially attributable to increase in antioxidant ability and decrease in lipid peroxidation induced by the early ROS accumulation triggered by NAA.

Reactive oxygen species derived from NADPH oxidase 1 and mitochondria mediate angiotensin II-induced smooth muscle cell senescence

Cellular senescence has emerged as an important player in both physiology and pathology. Excessive reactive oxygen species (ROS) is known to mediate cellular senescence. NADPH oxidases are major sources for ROS production in the vascular wall; the roles of different NADPH oxidase isoforms in cellular senescence remain unclear, however. We investigated the roles of two NADPH oxidase isoforms in mitochondrial dysfunction during angiotensin II (Ang II)-induced cellular senescence of human aortic vascular smooth muscle cells (VSMCs). Ang II (10−7M) stimulated ROS generation, exhibiting early increases between 30 and 60min and sustained increases between 24h and 72h, and induced VSMCs senescence after 48h or 72h treatment as assessed with senescence-associated β-galactosidase activity and the expression of two cell cycle inhibitors, p21 and p16. ROS scavengers and membrane-permeable catalase (catalase-PEG) reduced Ang II-stimulated cellular senescence. Furthermore, small interfering RNA (siRNA) of NADPH oxidase catalytic subunit Nox1, but not that of another isoform Nox4, inhibited Ang II-induced cellular senescence. Nox1 siRNA inhibited both early and sustained ROS increases induced by Ang II. In addition, a mitochondrial-specific antioxidant, mitoQ10, effectively inhibited Ang II-induced ROS increases and cellular senescence. Ang II decreased ATP synthesis and induced mitochondrial membrane depolarization, which were attenuated by pre-treating cells with Nox1 siRNA, mitoQ10 or catalase-PEG. The effect of Ang II on the mitochondrial regulator peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream genes was examined. Ang II stimulated S570 phosphorylation of PGC-1α with concomitant decreases in catalase and uncoupling protein-2 (UCP-2) levels between 12h and 72h, which were inhibited by Nox1 siRNA. Knockdown of both catalase and UCP-2 mimicked Ang II-induced VSMC senescence. These results suggested that Ang II-stimulated Nox1 activation mediates mitochondrial dysfunction, probably by decreasing PGC-1α activity and increasing mitochondrial oxidative stress, and leads to cellular senescence of VSMCs.

Pomegranate peel extract polyphenols induced apoptosis in human hepatoma cells by mitochondrial pathway

This study was aimed to investigate the influence of pomegranate peel polyphenols (PPPs) on the proliferation and apoptosis of HepG2 cells (a kind of human hepatoma cells) and the related mechanism. The inverted fluorescence microscope and the flow cytometer (FCM) were used to test the changes of the cellular morphology, cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial transmembrane potential (Δψm). The kit was used to measure the activities of caspase-3/9, and Western Blot was used to detect the expressions of apoptosis-associated proteins including p53, Bcl-2/Bax, Cyt-c and PARP. The results showed that the cells cycle of HepG2 arrested at the S-phase by PPPs and the amount of the early apoptotic cells and ROS level were increased obviously, the level of Cyt-c and the activity of Caspase-3/9 markedly were also increased by PPPs, as well as the ratio of Bax/Bcl-2 and the protein expressions of P53. It was concluded that PPPs could inhibit the growth of HepG2 cells by blocking the cell cycle and inducing the mitochondrial apoptotic pathway in a dose-dependent manner.