RBOHD Antibody
Description
What is RBOHD Antibody?
RBOHD antibody is a polyclonal antibody raised against specific epitopes of the Arabidopsis thaliana RBOHD protein (UniProt: Q9FIJ0, TAIR: AT5G47910) . It is widely used to investigate RBOHD's role in plant immunity, stress responses, and ROS signaling. The antibody is validated for applications including Western blot (WB), immunofluorescence (IF), and immunoprecipitation (IP) .
Key Features of RBOHD Antibody:
Development and Validation
The antibody targets conserved regions of RBOHD, enabling cross-reactivity with homologs in multiple plant species . For example:
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Immunofluorescence in potato (Solanum tuberosum) revealed RBOHD localization in chloroplasts, Golgi apparatus, and mitochondria during compatible and incompatible interactions with Potato virus Y (PVY) .
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Western blot confirmed RBOHD accumulation in Arabidopsis mutants (pbl13, pire) with enhanced ROS production .
Research Applications
RBOHD antibody has been instrumental in elucidating:
A. ROS Regulation in Immunity
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RBOHD-generated ROS are critical for stomatal closure, callose deposition, and resistance to pathogens like Pseudomonas syringae and Colletotrichum higginsianum .
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Phosphorylation at residues S862 and T912 modulates RBOHD activity and stability. Mimicking phosphorylation (e.g., T912D) reduces protein abundance, linking post-translational modifications to ROS dynamics .
B. Pathogen Interactions
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In Arabidopsis, RBOHD mutants (rbohD) exhibit altered susceptibility to Turnip mosaic virus (TuMV) and bacterial pathogens .
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Immunofluorescence in potato showed RBOHD accumulation near necrotic cells during PVY infection, highlighting its role in hypersensitive responses .
Key Research Findings
Recent studies utilizing RBOHD antibody have revealed:
Technical Considerations
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Differential recovery between plant accessions was linked to the activity of three genes: RBOHD, SENESCENCE-ASSOCIATED GENE113, and ORESARA1. These genes function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence, and the hormones abscisic acid and ethylene. PMID: 29891679
- The induction of stress marker genes and reactive oxygen species (ROS)-scavenging enzyme genes under various stress conditions is dependent on the ERF74-RbohD-ROS signal pathway. PMID: 28164334
- The role of NADPH oxidases under cadmium (Cd) toxicity was investigated using Arabidopsis thaliana mutants AtrbohC, AtrbohD and AtrbohF. PMID: 26765289
- RBOHD plays a significant role in the systemic response to salt stress. PMID: 27261066
- Differential spatio-temporal expression of the Rboh genes contributes to fine-tuning RBOH/NADPH oxidase-dependent reactive oxygen species production and signaling in Arabidopsis immunity. PMID: 26798024
- The involvement of hydrogen peroxide (H2O2) generated by nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) in the antioxidant defense system was assessed in salt-challenged Arabidopsis thaliana seedlings. PMID: 25462961
- Systemic acquired acclimation in plants is stress specific, and the reactive oxygen species wave is associated with the production of systemic variation potentials, specifically linking the electric signals with RBOHD function in A. thaliana. PMID: 24038652
- Infection of Arabidopsis thaliana by the parasitic nematode Heterodera schachtii activated the NADPH oxidases RbohD and RbohF to produce ROS, which was necessary to restrict infected plant cell death and promote nurse cell formation. PMID: 24714570
- Research suggests that both AtrbohD and AtrbohF are essential for abscissic acid-promoted reactive oxygen species production in roots. PMID: 23963673
- Mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals. [RBOHD] PMID: 21964330
- Rhobd plays a role in the process of stomatal closure in response to salicylic acid. PMID: 23500715
- There is no additive effect of disease symptoms or bacterial growth in the AtrbohD x Atgox1 and AtrbohD x Atgox3 double mutants when compared with individual mutants. PMID: 22751316
- Data suggest that specific sphingobases can induce cell death and require respiratory burst oxidase-D (RBOHD) for early reactive oxygen species induction in plants. PMID: 21856300
- Mild salt stress triggers a rapid increase in RbohD expression in Arabidopsis. PMID: 21205037
- Reactive oxygen species generated by Atrbohs are closely related to the jasmonic acid signaling pathway. PMID: 21421415
- RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels. PMID: 19726575
- Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD. PMID: 18218618
- The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. PMID: 19690149
- Data show that a rapid systemic signal traveled at a rate of 8.4 centimeters per minute and was dependent on the respiratory burst oxidase homolog D (RbohD) gene. PMID: 19690331
Q&A
FAQs for RBOHD Antibody in Academic Research
Advanced Research Questions
How to resolve contradictory data on RBOHD’s role in viral vs. fungal pathogen responses?
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Methodological Answer:
What advanced techniques are needed to study RBOHD phosphorylation in ROS signaling?
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Methodological Answer:
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Use Phos-tag™ gels to separate phosphorylated RBOHD isoforms induced by extracellular ATP or pathogen elicitors .
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Combine co-immunoprecipitation with kinase assays (e.g., DORN1 or CPK5) to identify phosphorylation sites (e.g., Ser-39, Ser-343) .
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Validate findings using Arabidopsis lines expressing phospho-null RBOHD variants (e.g., Ser→Ala mutations) .
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How to integrate RBOHD antibody data with transcriptomic datasets for network analysis?
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Methodological Answer:
Data Tables
Table 1: RBOHD Functional Roles in Pathogen Responses
Table 2: RBOHD Phosphorylation Sites and Regulatory Kinases
| Phosphorylation Site | Kinase | Functional Outcome | Assay Used |
|---|---|---|---|
| Ser-39 | DORN1 | ATP-induced ROS production | Phos-tag™ gel |
| Ser-343 | CPK5 | Ca²⁺-dependent pathogen response | Kinase assay |
Methodological Notes for Contradictory Data
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Localization Discrepancies: Combine subcellular fractionation with confocal microscopy (e.g., GFP-tagged RBOHD) to clarify plasma membrane vs. endosomal localization .
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ROS Quantification: Use luminescent probes (e.g., L-012) for real-time ROS measurement in rbohD/F double mutants to dissect RBOHD/F redundancy .
