scholarly journals The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256217
Author(s):  
Christina Krönauer ◽  
Thomas Lahaye
Keyword(s):  
Nadph Oxidase ◽  
Oxidation State ◽  
Transcriptional Activation ◽  
Oxidase Activity ◽  
Plant Immunity ◽  
Striking Similarity ◽  
In Planta ◽  
Nadph Oxidase Activity ◽  
Wide Range

The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding effector protein AvrBs3 from the bacterial pathogen Xanthomonas. Expression of Bs3 in yeast inhibited proliferation, demonstrating that Bs3 function is not restricted to the plant kingdom. The Bs3 sequence shows striking similarity to flavin monooxygenases (FMOs), an FAD- and NADPH-containing enzyme class that is known for the oxygenation of a wide range of substrates and their potential to produce H2O2. Since H2O2 is a hallmark metabolite in plant immunity, we analyzed the role of H2O2 during Bs3 HR. We purified recombinant Bs3 protein from E. coli and confirmed the FMO function of Bs3 with FAD binding and NADPH oxidase activity in vitro. Translational fusion of Bs3 to the redox reporter roGFP2 indicated that the Bs3-dependent HR induces an increase of the intracellular oxidation state in planta. To test if the NADPH oxidation and putative H2O2 production of Bs3 is sufficient to induce HR, we adapted previous studies which have uncovered mutations in the NADPH binding site of FMOs that result in higher NADPH oxidase activity. In vitro studies demonstrated that recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3S211A also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H2O2 produced by Bs3 on its own is not sufficient to trigger HR.

scholarly journals The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2in vitro

2021 ◽  
Author(s):  
Christina Krönauer ◽  
Thomas Lahaye
Keyword(s):  
Cell Death ◽  
Nadph Oxidase ◽  
Transcriptional Activation ◽  
Oxidase Activity ◽  
Bacterial Pathogen ◽  
Histochemical Staining ◽  
In Planta ◽  
Transcription Activator ◽  
Nadph Oxidase Activity

ABSTRACTThe pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding transcription activator-like effector AvrBs3 from the bacterial pathogen Xanthomonas. Bs3 is homologous to flavin monooxygenases (FMOs), an enzyme class that has NADPH oxidase activity and can produce H2O2, a hallmark metabolite in plant immune reactions. Histochemical staining of infected pepper leaves and a translational fusion of Bs3 to the redox reporter roGFP2 both indicated that the Bs3-dependent HR induces a local increase in H2O2 levels in planta. Moreover, our in vitro studies with recombinant Bs3 protein confirmed its NADPH oxidase activity. To test if the NADPH oxidation of Bs3 induces HR, we adapted previous studies which have uncovered mutations in fungal FMOs that result in higher NADPH oxidase activity. We replicated one of these mutations and demonstrated that the generated recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3 in vitro. Translational fusions to roGFP2 showed that Bs3S211A also had increased NADPH oxidase activity in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta. Ultimately, this reveals that Bs3 produces H2O2in planta, but that the H2O2 produced by Bs3 on its own is not sufficient to trigger HR. We also demonstrated that expression of Bs3 not only triggered HR in plants, but also inhibited proliferation of yeast, which lends this model system to be utilized for the genetic dissection of Bs3 function in future studies.One sentence summaryThe executor-type resistance protein Bs3 from pepper (Capsicum annuum) acts as an NADPH oxidase but reactive oxygen species produced by Bs3 are not sufficient to trigger plant cell death

scholarly journals Attenuation of renal excretory responses to ANG II during inhibition of superoxide dismutase in anesthetized rats

2010 ◽  
Vol 298 (2) ◽  
pp. F401-F407 ◽  
Author(s):  
Md. Abdul Hye Khan ◽  
Mohammed Toriqul Islam ◽  
Alexander Castillo ◽  
Dewan Syed Abdul Majid
Keyword(s):  
Superoxide Dismutase ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Ang Ii ◽  
Sod Activity ◽  
Doppler Flowmetry ◽  
Nadph Oxidase Activity ◽  
Blood Flows ◽  
Renal Responses

To examine the functional interaction between superoxide dismutase (SOD) and NADPH oxidase activity, we assessed renal responses to acute intra-arterial infusion of ANG II (0.5 ng·kg−1·min−1) before and during administration of a SOD inhibitor, diethyldithiocarbamate (DETC, 0.5 mg·kg−1·min−1), in enalaprilat-pretreated (33 μg·kg−1·min−1) rats ( n = 11). Total (RBF) and regional (cortical, CBF; medullary; MBF) renal blood flows were determined by Transonic and laser-Doppler flowmetry, respectively. Renal cortical and medullary tissue NADPH oxidase activity in vitro was determined using the lucigenin-chemiluminescence method. DETC treatment alone resulted in decreases in RBF, CBF, MBF, glomerular filtration rate (GFR), urine flow (V), and sodium excretion (UNaV) as reported previously. Before DETC, ANG II infusion decreased RBF (−18 ± 3%), CBF (−16 ± 3%), MBF [−5 ± 6%; P = not significant (NS)], GFR (−31 ± 4%), V (−34 ± 2%), and UNaV (−53 ± 3%). During DETC infusion, ANG II also caused similar reductions in RBF (−20 ± 4%), CBF (−19 ± 3%), MBF (−2 ± 2; P = NS), and in GFR (−22 ± 7%), whereas renal excretory responses (V; −12 ± 2%; UNaV; −24 ± 4%) were significantly attenuated compared with those before DETC. In in vitro experiments, ANG II (100 μM) enhanced NADPH oxidase activity both in cortical [13,194 ± 1,651 vs. 20,914 ± 2,769 relative light units (RLU)/mg protein] and in medullary (21,296 ± 2,244 vs. 30,597 ± 4,250 RLU/mg protein) tissue. Application of DETC (1 mM) reduced the basal levels and prevented ANG II-induced increases in NADPH oxidase activity in both tissues. These results demonstrate that renal excretory responses to acute ANG II administration are attenuated during SOD inhibition, which seems related to a downregulation of NADPH oxidase in the deficient condition of SOD activity.

Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems

2007 ◽  
Vol 292 (1) ◽  
pp. C413-C422 ◽  
Author(s):  
Denise C. Fernandes ◽  
João Wosniak ◽  
Luciana A. Pescatore ◽  
Maria A. Bertoline ◽  
Marcel Liberman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Experimental Studies ◽  
Oxidation Products ◽  
Ang Ii ◽  
Specific Product ◽  
Nadph Oxidase Activity

Dihydroethidium (DHE) is a widely used sensitive superoxide (O2•−) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2•−, and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was ∼1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2•− production or NADPH oxidase activity in many vascular experimental studies.

scholarly journals Curcumin protects islet cells from glucolipotoxicity by inhibiting oxidative stress and NADPH oxidase activity both in vitro and in vivo

Islets ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 152-164 ◽  
Author(s):  
Jing Li ◽  
Ninghua Wu ◽  
Xiao Chen ◽  
Hongguang Chen ◽  
Xiaosong Yang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Islet Cells ◽  
Nadph Oxidase Activity

scholarly journals NADPH oxidase activity is independent of p47 in vitro

1997 ◽  
Vol 272 (11) ◽  
pp. 7566
Author(s):  
Jennifer L. Freeman ◽  
J. David Lambeth
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Nadph Oxidase Activity

Abstract 19182: Epicardial Adipose Tissue Volume Selectively Predicts Myocardial Redox State in Patients With Ischemic Heart Disease

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Alexios S Antonopoulos ◽  
Nikant Sabharwal ◽  
Cheerag Shirodaria ◽  
Andrew Kelion ◽  
Raman Uberoi ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Heart Disease ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Redox State ◽  
Epicardial Adipose Tissue ◽  
Specific Inhibitor ◽  
Volume Index ◽  
Nadph Oxidase Activity ◽  
Wide Range

Introduction: Myocardial redox state is a strong determinant of heart biology. Epicardial adipose tissue (EpAT) is in close contact with the human heart and it is likely that by secreting a wide range of adipokines it may affect the biology of the underlying myocardium. Hypothesis: We hypothesised that EpAT volume may predict myocardial redox state and consequently myocardial function in ischaemic heart disease (IHD). Methods: We recruited 38 patients undergoing coronary artery bypass grafting surgery. Patients underwent transthoracic echocardiography to assess cardiac function and cardiac computerised tomography to determine the volume of EpAT, pericardial (PerAT) and subcutaneous (ScAT) AT, by using a strictly pre-defined protocol. Samples of right atrium appendage were collected during surgery and myocardial NADPH oxidase activity was assessed by lucigenin-enhanced chemiluminescence (using its substrate -NAPDH 100uM +/- its specific inhibitor VAS2870 40uM). Results: Increased EpAT volume index (EpAT volume/body surface area), but not PerAT or ScAT, was strongly associated with increased myocardial NADPH oxidase activity (Figure). EpAT volume index was not related with left ventricle (LV) mass index or LV systolic/diastolic function (p=NS for all). EpAT volume index was also unrelated to risk factors or body mass index (p=NS for all). Conclusions: These findings introduce the concept of a local cross-talk between EpAT and the underlying myocardium in humans. EpAT volume can be used as a surrogate of myocardial redox state, and this may have direct implications in risk stratification of patients undergoing coronary bypass surgery.

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