RBOHD

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0004601 peroxidase activity	IEA GO_REF:0000002	REMOVE	Summary: RBOHD is a superoxide-generating NADPH oxidase, not a peroxidase. This IEA derives from the gp91phox/cytochrome-b245 (Cyt_b245) InterPro signature (IPR013623) and the associated UniProt "Peroxidase" keyword, which is misleading for RBOH proteins. The enzyme reduces O2 to superoxide using electrons from NADPH; it does not reduce hydrogen peroxide. Reason: The catalytic activity of RBOHD is electron transfer from NADPH to O2 to generate superoxide (and downstream H2O2 by dismutation), not peroxidase activity. The InterPro-derived peroxidase keyword is a known mis-mapping for the gp91phox family and over-annotates the gene. The informative catalytic term is GO:0016175 (added as NEW). Supporting Evidence: PMID:11756663 our demonstration that an NADPH oxidase subunit is required for ROI production confirms Doke's original suggestion that O is the first ROI produced
GO:0005509 calcium ion binding	IEA GO_REF:0000002	ACCEPT	Summary: RBOHD contains two canonical N-terminal EF-hand calcium-binding domains (UniProt residues 253-288 and 297-332, with Ca2+ coordinated by residues 266/268/270/272/277) and is a calcium-dependent NADPH oxidase. Calcium binding to these EF-hands is integral to its activation. Reason: Well supported by conserved EF-hand domains/Ca2+ binding sites in UniProt and by the calcium-dependence of RBOH oxidase activity. This is a core molecular function contributing to activity regulation. Supporting Evidence: PMID:24629339 Here we show that the receptor-like cytoplasmic kinase BIK1, a component of the FLS2 immune receptor complex, not only positively regulates flg22-triggered calcium influx but also directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation. file:ARATH/RBOHD/RBOHD-deep-research-falcon.md RBOHD is activated by direct Ca2+ binding to EF-hands and by phosphorylation
GO:0006952 defense response	IEA GO_REF:0000117	ACCEPT	Summary: RBOHD generates the apoplastic ROS burst central to plant immune responses; rbohD mutants fail to accumulate ROS during pathogen defense. This electronic annotation is consistent with strong experimental evidence (see also the IMP defense response annotation below). Reason: Defense response is a well-established core biological process for RBOHD, supported by mutant phenotypes during pathogen interactions. Supporting Evidence: PMID:11756663 AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response file:ARATH/RBOHD/RBOHD-deep-research-falcon.md major NADPH oxidase responsible for pathogen-triggered ROS
GO:0016020 membrane	IEA GO_REF:0000120	ACCEPT	Summary: RBOHD is a multi-pass (six transmembrane helix) integral membrane protein, specifically of the plasma membrane. This generic membrane term is correct but less informative than the plasma membrane annotation. Reason: True location but generic; the more specific plasma membrane annotation (GO:0005886) captures the functional compartment. Retained as accurate but non-core relative to plasma membrane. Supporting Evidence: PMID:24629339 directly phosphorylates the NADPH oxidase RbohD
GO:0016491 oxidoreductase activity	IEA GO_REF:0000002	ACCEPT	Summary: RBOHD is an oxidoreductase that transfers electrons from NADPH to O2. This is a very general parent of the specific superoxide-generating NADPH oxidase activity. Reason: Correct but high-level; acceptable as a broad IEA parent of the informative term GO:0016175. Not the core descriptor on its own. Supporting Evidence: PMID:11756663 AtrbohD and AtrbohF, encoding probable components of a plant NADPH oxidase file:ARATH/RBOHD/RBOHD-deep-research-falcon.md catalyzes electron transfer from cytosolic NADPH to molecular oxygen (O2) to generate superoxide (O2•−) in the apoplast, which can subsequently form H2O2
GO:0050664 oxidoreductase activity, acting on NAD(P)H, oxygen as acceptor	IEA GO_REF:0000002	ACCEPT	Summary: This term correctly describes RBOHD as an NAD(P)H-dependent oxidoreductase using oxygen as electron acceptor, which is precisely the RBOH oxidase chemistry. It is the immediate parent of the superoxide-generating term GO:0016175. Reason: Accurate and reasonably specific; consistent with the NADPH-binding ferredoxin-reductase module and O2-reducing oxidase activity. The product-specific child term GO:0016175 is added as NEW. Supporting Evidence: PMID:11756663 an NADPH oxidase subunit is required for ROI production file:ARATH/RBOHD/RBOHD-deep-research-falcon.md a catalytic C-terminal core with FAD- and NADPH-binding domains
GO:0098869 cellular oxidant detoxification	IEA GO_REF:0000108	REMOVE	Summary: This term is inferred logically from the spurious peroxidase MF (GO:0004601 to GO:0098869). RBOHD generates reactive oxygen species for signaling; it does not detoxify cellular oxidants. The annotation is essentially backwards with respect to the protein's biology. Reason: RBOHD is a ROS-producing enzyme, not an antioxidant/detoxifying enzyme. The inference depends on the incorrect peroxidase activity assignment, which is itself being removed. Supporting Evidence: PMID:19726575 Functional RBOHD causes marked extracellular hydrogen peroxide accumulation
GO:0005794 Golgi apparatus	HDA PMID:22430844 Isolation and proteomic characterization of the Arabidopsis ...	MARK AS OVER ANNOTATED	Summary: A Golgi assignment from high-throughput organellar proteomics. For a multi-pass plasma-membrane oxidase, a Golgi signal most plausibly reflects transit through the secretory pathway or co-fractionation rather than a functional Golgi pool. The functional location is the plasma membrane. Reason: High-throughput proteomic localization without functional support; inconsistent with the established plasma-membrane site of RBOHD action. Likely reflects biosynthetic trafficking or proteomic co-fractionation. Supporting Evidence: PMID:22430844 Isolation and proteomic characterization of the Arabidopsis Golgi
GO:0005886 plasma membrane	HDA PMID:22923678 Putative glycosyltransferases and other plant Golgi apparatu...	ACCEPT	Summary: The plasma membrane is the functional location of RBOHD. It is a multi-pass plasma-membrane NADPH oxidase that releases superoxide into the apoplast, where it acts together with plasma-membrane receptor complexes (FLS2/BIK1) in immune signaling. Reason: Strongly supported by proteomics and by the abundant literature placing RBOHD in plasma-membrane PRR complexes; this is the core cellular location for its function. Supporting Evidence: PMID:24629339 BIK1, a component of the FLS2 immune receptor complex PMID:22923678 plant Golgi apparatus proteins are revealed by LOPIT proteomics file:ARATH/RBOHD/RBOHD-deep-research-falcon.md RBOHD is a plasma membrane-localized NADPH oxidase that produces ROS into the apoplast
GO:0071456 cellular response to hypoxia	HEP PMID:31519798 Integrative Analysis from the Epigenome to Translatome Uncov...	KEEP AS NON CORE	Summary: RBOHD expression/regulation is captured in an integrative epigenome-to-translatome study of transient stress that includes hypoxia. Consistent with a role for RBOH-derived ROS in low-oxygen responses (cf. HRU1/ROP2/RbohD module under anoxia), this peripheral, expression-pattern based annotation is plausible but not a core function. Reason: Expression-pattern (HEP) evidence from a high-throughput stress study; supports involvement in hypoxia responses but as a peripheral process rather than a defining function. Concordant with independent anoxia evidence (PMID:27251529). Supporting Evidence: PMID:27251529 HRU1 interacts with proteins that induce ROS production, the GTPase ROP2 and the NADPH oxidase RbohD, pointing to the existence of a low-oxygen-specific mechanism for the modulation of ROS levels
GO:0009536 plastid	HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati...	REMOVE	Summary: A plastid assignment from a global membrane-protein correlation-profiling study. RBOHD is a plasma-membrane oxidase; a plastid location is not supported by any functional study and most likely reflects co-fractionation/contamination in the high-throughput dataset. Reason: No functional or targeted experimental support for a plastid pool of RBOHD; inconsistent with its plasma-membrane topology and apoplastic ROS output. Likely a proteomic artifact. Supporting Evidence: PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling
GO:0005634 nucleus	ISM GO_REF:0000122	REMOVE	Summary: A purely computational subcellular-localization prediction (AtSubP). A six-transmembrane plasma-membrane oxidase is not expected to localize to the nucleus, and no experimental evidence supports a nuclear RBOHD. Reason: Sequence-based prediction (ISM) contradicted by the membrane topology and experimentally established plasma-membrane localization. No supporting functional evidence. Supporting Evidence: PMID:22923678 plant Golgi apparatus proteins are revealed by LOPIT proteomics
GO:0005515 protein binding	IPI PMID:28696275 A Lectin Receptor-Like Kinase Mediates Pattern-Triggered Sal...	MARK AS OVER ANNOTATED	Summary: Refers to functional interaction within pattern-triggered immunity signaling; the lectin receptor-like kinase LecRK-IX.2 induces RBOHD phosphorylation (likely via calcium-dependent protein kinases) to trigger ROS. "Protein binding" is uninformative about RBOHD's actual molecular function. Reason: Generic protein binding conveys no specific functional information. The biologically meaningful content (regulation of RBOHD ROS output by upstream immune kinases) is captured in the core_functions and process annotations rather than by GO:0005515. Supporting Evidence: PMID:28696275 LecRK-IX.2 is capable of inducing RbohD phosphorylation, likely by recruiting calcium-dependent protein kinases to trigger ROS production in Arabidopsis
GO:0005515 protein binding	IPI PMID:24629339 The FLS2-associated kinase BIK1 directly phosphorylates the ...	MARK AS OVER ANNOTATED	Summary: This IPI records the interaction of RBOHD with BIK1 and FLS2. BIK1 directly phosphorylates RBOHD to enhance ROS during immunity. While biologically important, the bare "protein binding" term is uninformative. Reason: The interaction with BIK1/FLS2 is a key activation mechanism, but GO:0005515 does not capture it usefully. The functional relationship (kinase-mediated activation of RBOHD) is documented in notes and core_functions; a specific kinase-binding term would be preferable but is not added here to avoid over-fitting. Supporting Evidence: PMID:24629339 the receptor-like cytoplasmic kinase BIK1, a component of the FLS2 immune receptor complex ... directly phosphorylates the NADPH oxidase RbohD at specific sites file:ARATH/RBOHD/RBOHD-deep-research-falcon.md In PTI, PRRs such as FLS2/EFR activate downstream cytoplasmic kinases (e.g., BIK1) that phosphorylate RBOHD to drive a rapid ROS burst
GO:0005515 protein binding	IPI PMID:26432875 PBL13 Is a Serine/Threonine Protein Kinase That Negatively R...	MARK AS OVER ANNOTATED	Summary: Records the interaction of RBOHD with the negative-regulatory kinase PBL13 (split-luciferase complementation), which is disrupted by flagellin treatment. Informative biologically but not captured by the generic term. Reason: Generic protein binding is uninformative. The PBL13 interaction is a negative-regulatory mechanism for RBOHD ROS production, documented in notes; GO:0005515 itself should not be treated as a core molecular function. Supporting Evidence: PMID:26432875 PBL13 is able to associate with the nicotinamide adenine dinucleotide phosphate, reduced oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) by split-luciferase complementation assay, and this association is disrupted by flagellin treatment
GO:0005515 protein binding	IPI PMID:27251529 Universal stress protein HRU1 mediates ROS homeostasis under...	MARK AS OVER ANNOTATED	Summary: Records interaction of RBOHD with the universal stress protein HRU1 (and ROP2) in modulating ROS production under anoxia. Biologically meaningful but uninformative as a bare protein-binding term. Reason: Generic protein binding lacks specificity. The HRU1/ROP2 interaction links oxygen sensing to RBOHD ROS output under low oxygen; documented in notes rather than via GO:0005515. Supporting Evidence: PMID:27251529 HRU1 interacts with proteins that induce ROS production, the GTPase ROP2 and the NADPH oxidase RbohD
GO:0072593 reactive oxygen species metabolic process	IGI PMID:26704641 Arabidopsis HY1-Modulated Stomatal Movement: An Integrative ...	ACCEPT	Summary: RBOHD-derived ROS are required for ABA/HY1-ABI4-dependent stomatal closure; genetic interaction places RBOHD upstream of the ROS levels that mediate stomatal movement. Consistent with RBOHD as a primary ROS generator. Reason: Well supported; RBOHD is a major contributor to cellular ROS metabolism, here in the context of ABA-dependent stomatal regulation. Supporting Evidence: PMID:26704641 the promotion of ABA-triggered up-regulation of RbohD abundance and reactive oxygen species (ROS) levels in the hy1 mutant was almost fully blocked by the mutation of ABI4
GO:0007231 osmosensory signaling pathway	IMP PMID:22422940 Osmosensitive changes of carbohydrate metabolism in response...	KEEP AS NON CORE	Summary: In cellulose-biosynthesis-inhibition experiments, rbohDF mutants fail to show the osmosensitive metabolic changes, implicating RBOHD/F-derived ROS in osmo/cell-wall-integrity signaling. Reason: Supported by mutant phenotype, but this is a specialized, peripheral signaling context relative to RBOHD's central immune/ROS-burst role. Retained as a genuine but non-core process. Supporting Evidence: PMID:22422940 osmotic support does not suppress CBI-induced metabolic changes in seedlings impaired in ... reactive oxygen species production (respiratory burst oxidase homolog DF [rbohDF])
GO:0033500 carbohydrate homeostasis	IMP PMID:22422940 Osmosensitive changes of carbohydrate metabolism in response...	KEEP AS NON CORE	Summary: The same study links RBOHD/F-derived ROS to osmosensitive control of carbohydrate metabolism after cellulose biosynthesis inhibition. The effect on carbohydrate homeostasis is indirect and downstream of the osmo-signaling role. Reason: Indirect, context-specific phenotype; ROS from RBOHD feed into an osmosensitive regulatory circuit that affects carbohydrate metabolism, but this is peripheral to the protein's core ROS-generating immune function. Supporting Evidence: PMID:22422940 carbohydrate metabolism is responsive to changes in cellulose biosynthesis activity and turgor pressure
GO:0009611 response to wounding	IEP PMID:21419340 Calmodulin-dependent activation of MAP kinase for ROS homeos...	KEEP AS NON CORE	Summary: The Ca2+/CaM-MPK8-MKK3 wound-signaling pathway negatively regulates ROS accumulation through control of RbohD expression, linking RBOHD to wound-induced ROS homeostasis. Reason: RBOHD participates in wound-induced ROS homeostasis (as a regulated ROS source), but the evidence is expression-pattern based (IEP) and the process is peripheral to its central immune ROS-burst function. Supporting Evidence: PMID:21419340 The MPK8 pathway negatively regulates ROS accumulation through controlling expression of the Rboh D gene
GO:0072593 reactive oxygen species metabolic process	IMP PMID:11756663 Arabidopsis gp91phox homologues AtrbohD and AtrbohF are requ...	ACCEPT	Summary: rbohD insertion mutants eliminate the majority of pathogen-induced ROS, demonstrating that RBOHD is the principal generator of the defense oxidative burst. This is a core process annotation. Reason: Strong genetic evidence that RBOHD drives ROS metabolic process (the extracellular oxidative burst) during defense; a defining function. Supporting Evidence: PMID:11756663 The AtrbohD gene is required for most of the ROI observed after inoculation with avirulent Pst
GO:0072593 reactive oxygen species metabolic process	TAS PMID:15705948 Different signaling and cell death roles of heterotrimeric G...	ACCEPT	Summary: RBOHD is part of the Arabidopsis ROS gene network and contributes ROS in the oxidative stress response (including ozone/G-protein-mediated responses). Consistent with its established role in ROS production. Reason: TAS annotation consistent with the well-documented role of RBOHD as a generator of signaling ROS; duplicate aspect of the core ROS metabolic process annotation. Supporting Evidence: PMID:11756663 extracellular ROI production in Arabidopsis requires Atrboh function
GO:0043069 negative regulation of programmed cell death	IGI PMID:16170317 Pathogen-induced, NADPH oxidase-derived reactive oxygen inte...	ACCEPT	Summary: RBOHD-derived ROS suppress the spread of hypersensitive cell death into cells surrounding infection sites, antagonizing salicylic acid-dependent pro-death signals. Thus RBOHD limits cell-death spread even though its ROS can also trigger localized death. Reason: Supported by genetic evidence; an established (if context-dependent) role of RBOHD in restricting programmed cell death spread during the immune response. Supporting Evidence: PMID:16170317 the subsequent oxidative burst can suppress cell death in cells surrounding sites of NADPH oxidase activation PMID:19726575 functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells
GO:0016174 NAD(P)H oxidase H2O2-forming activity	IMP PMID:19726575 Dual roles of reactive oxygen species and NADPH oxidase RBOH...	MODIFY	Summary: RBOHD is a NADPH oxidase whose immediate enzymatic product is superoxide; the measured apoplastic H2O2 arises by (spontaneous or SOD-catalyzed) dismutation of that superoxide. The more accurate catalytic term is GO:0016175 "superoxide-generating NAD(P)H oxidase activity". The H2O2-forming term reflects the downstream detected species rather than the primary reaction. Reason: The proximal product is superoxide (PMID:11756663), so the product-specific MF should be the superoxide-generating activity. H2O2 is formed secondarily by dismutation, making GO:0016174 a less accurate descriptor of the catalyzed reaction. Proposed replacements: superoxide-generating NAD(P)H oxidase activity Supporting Evidence: PMID:11756663 O is the first ROI produced PMID:19726575 Functional RBOHD causes marked extracellular hydrogen peroxide accumulation
GO:0050832 defense response to fungus	IMP PMID:19726575 Dual roles of reactive oxygen species and NADPH oxidase RBOH...	ACCEPT	Summary: rbohD knockout alters ROS accumulation and cell-death patterns upon infection with the necrotrophic fungus Alternaria brassicicola, demonstrating involvement in antifungal defense (with dual, position- dependent effects on cell death). Reason: Supported by mutant phenotype in a defined fungal pathosystem; a genuine, if context-dependent, defense-against-fungus role for RBOHD. Supporting Evidence: PMID:19726575 a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola
GO:0009408 response to heat	IMP PMID:15923322 Heat stress phenotypes of Arabidopsis mutants implicate mult...	KEEP AS NON CORE	Summary: atrbohD mutants show (weaker) defects in acquired thermotolerance, implicating RBOHD-derived ROS/oxidative-burst signaling in the heat-stress response among multiple contributing pathways. Reason: Supported by mutant phenotype but the effect is relatively weak and this abiotic-stress role is peripheral to RBOHD's central function in the immune ROS burst. Supporting Evidence: PMID:15923322 Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D) ... showed weaker defects
GO:0016174 NAD(P)H oxidase H2O2-forming activity	TAS PMID:15608336 Cytosolic ascorbate peroxidase 1 is a central component of t...	MODIFY	Summary: As above, RBOHD is an NADPH oxidase; its primary product is superoxide, with H2O2 produced by dismutation. The superoxide-generating activity term (GO:0016175) is the more accurate molecular function. Reason: Same rationale as the IMP-supported GO:0016174 annotation; the proximal reaction generates superoxide. Replace with the superoxide-generating NAD(P)H oxidase activity term. Proposed replacements: superoxide-generating NAD(P)H oxidase activity Supporting Evidence: PMID:11756663 O is the first ROI produced
GO:0006952 defense response	IMP PMID:11756663 Arabidopsis gp91phox homologues AtrbohD and AtrbohF are requ...	ACCEPT	Summary: Loss of RBOHD eliminates most pathogen-induced ROS during incompatible (avirulent) interactions, establishing RBOHD as essential for the defense oxidative burst. This is a core biological process. Reason: Strong genetic (IMP) support; defense response via the ROS burst is the central biological role of RBOHD. Supporting Evidence: PMID:11756663 AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response
GO:0002679 respiratory burst involved in defense response	IMP PMID:11756663 Arabidopsis gp91phox homologues AtrbohD and AtrbohF are requ...	NEW	Summary: RBOHD is the principal enzyme producing the apoplastic oxidative ("respiratory") burst that accompanies plant immune responses; rbohD mutants lose most pathogen-induced extracellular ROS. This specific term captures the defining immune process better than the generic ROS metabolic process / defense response terms. Reason: A more precise BP term (verified in GO as GO:0002679) directly describing the RBOHD-generated immune oxidative burst, supported by mutant genetics and by the activation of RBOHD within the FLS2/BIK1 PRR complex. Supporting Evidence: PMID:11756663 extracellular ROI production in Arabidopsis requires Atrboh function PMID:24629339 directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation file:ARATH/RBOHD/RBOHD-deep-research-falcon.md major NADPH oxidase responsible for pathogen-triggered ROS
GO:0016175 superoxide-generating NAD(P)H oxidase activity	IDA PMID:11756663 Arabidopsis gp91phox homologues AtrbohD and AtrbohF are requ...	NEW	Summary: RBOHD is a gp91phox-homologous NADPH oxidase that transfers electrons from cytosolic NADPH across the plasma membrane to O2, producing superoxide as the proximal product (subsequently dismutated to H2O2). This is the informative, product-specific catalytic molecular function and the core activity of the protein. Reason: Captures the accurate catalytic activity of RBOHD (superoxide generation), replacing the less accurate H2O2-forming term and the spurious peroxidase term. Verified GO ID via OLS. Supporting Evidence: PMID:11756663 an NADPH oxidase subunit is required for ROI production confirms Doke's original suggestion that O is the first ROI produced file:ARATH/RBOHD/RBOHD-deep-research-falcon.md catalyzes electron transfer from cytosolic NADPH to molecular oxygen (O2) to generate superoxide (O2•−) in the apoplast, which can subsequently form H2O2

Gene Description

Existing Annotations Review

Core Functions

Binds calcium through its two N-terminal EF-hand motifs, conferring calcium dependence on oxidase activation; together with phosphorylation by immune kinases (e.g. BIK1), calcium binding switches RBOHD into its ROS-producing state during pattern-triggered immunity.

As the principal generator of pathogen-induced extracellular reactive oxygen species, RBOHD drives the immune oxidative burst and modulates the spatial spread of hypersensitive programmed cell death during plant defense.

References

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Notes

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