RBOHD (Q9FIJ0) curation notes

Gene: RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD / AtrbohD), Arabidopsis thaliana, locus AT5G47910.

Identity and architecture (UniProt Q9FIJ0)

• Plasma-membrane NADPH oxidase of the RBOH (TC 5.B.1.3) family; gp91phox/NOX homolog. 921 aa, six transmembrane helices, N-terminal cytoplasmic region (~1-376) carrying two canonical EF-hand Ca2+-binding domains (residues 253-288 and 297-332) plus EF-hand-like regions, and a C-terminal cytoplasmic FAD-binding (FR-type) + NADPH-binding ferredoxin-reductase module.
• EC=1.6.3.- (NAD(P)H:oxygen oxidoreductase) and EC=1.11.1.- listed; BRENDA cross-reference 1.6.3.1.
• Ca2+ binding sites at residues 266, 268, 270, 272, 277 (PROSITE EF-hand). [UniProt FT BINDING Ca(2+)]
• Phosphoserines: Ser-8, Ser-9, Ser-26, Ser-39, Ser-339, Ser-343, Ser-347. Ser-39/Ser-343/Ser-347 phosphorylated by BIK1 upon flg22; Ser-347 (+Ser-8/9/339) by SIK1 — phosphorylation activates ROS production. [UniProt PTM]
• UniProt FUNCTION: "Calcium-dependent NADPH oxidase that generates superoxide. Involved in the generation of reactive oxygen species (ROS) during incompatible interactions with pathogens, in response to pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) signaling and in UV-B and abscisic acid ROS-dependent signaling."
• Subcellular location: Membrane; multi-pass membrane protein. Tissue: more abundant in roots; expressed in mesophyll and guard cells.

Core molecular function

RBOHD is the catalytic subunit of a plasma-membrane NADPH oxidase that transfers electrons from cytosolic NADPH across the membrane to molecular O2, producing apoplastic superoxide (O2.-), which dismutates to H2O2. The proximal product is superoxide:
- PMID:11756663
- The correct catalytic MF GO term is GO:0016175 "superoxide-generating NAD(P)H oxidase activity" (verified via OLS; def "NAD(P)H + O2 = NAD(P)+ + O2-"). The seeded annotations use GO:0016174 "NAD(P)H oxidase H2O2-forming activity" (def NAD(P)H + O2 = NAD(P)+ + H2O2) — H2O2 is formed only after dismutation of the primary superoxide product, so GO:0016175 is more accurate for RBOH proteins.

Activation / regulation

• Calcium binds the EF-hands; activity is calcium-dependent (UniProt). GO:0005509 calcium ion binding is well supported by EF-hand domains.
• BIK1, a receptor-like cytoplasmic kinase in the FLS2 complex, directly phosphorylates RBOHD at Ser-39/Ser-343/Ser-347 in a Ca2+-independent manner to enhance ROS. PMID:24629339
• SIK1 (MAP4K) binds RBOHD upon flagellin perception and activates it by phosphorylation (Ser-347). [UniProt; PubMed:30212650]
• PBL13 (a Ser/Thr RLCK) associates with RBOHD before pathogen perception and negatively regulates ROS; the association is disrupted by flg22. PMID:26432875
• LecRK-IX.2 induces RBOHD phosphorylation, likely via CPKs, in pattern-triggered immunity. PMID:28696275
• HRU1 (universal stress protein) interacts with RBOHD and ROP2 to modulate ROS under anoxia. PMID:27251529
• MPK8 (Ca2+/CaM-MKK3 pathway) negatively regulates ROS via control of RbohD expression in wound signaling. PMID:21419340

Biological processes

• PAMP/PTI immunity and the apoplastic oxidative (respiratory) burst; defense response. [PMID:11756663 "AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response"; "AtrbohD gene is required for most of the ROI observed after inoculation with avirulent Pst"]
• Control/limitation of cell death: RBOHD-derived ROS suppress spread of hypersensitive cell death in surrounding cells (antagonize SA-dependent pro-death signals). PMID:16170317 Context-dependent (dual roles) in Alternaria pathosystem. PMID:19726575
• Defense response to fungus (Alternaria brassicicola). PMID:19726575
• ROS metabolic process / ROS gene network. [PMID:15608336; PMID:15705948 (ozone, G-protein)]
• ABA/stomatal: RBOHD-derived ROS required for ABA-induced stomatal closure and drought responses. [PMID:26704641 "HY1-ABI4 signaling ... involved in stomatal closure was dependent on the RbohD-derived ROS production"; UniProt disruption phenotype "impaired in abscisic acid-induced stomatal closing"]
• Osmosensing / carbohydrate homeostasis under cellulose-biosynthesis inhibition: rbohDF impaired in osmosensitive metabolic changes. PMID:22422940
• Response to wounding (via MPK8 control of RbohD). PMID:21419340
• Response to heat / thermotolerance: atrbohD shows weaker thermotolerance defects. PMID:15923322
• Cellular response to hypoxia: RBOHD transcript regulation under transient hypoxia stress (HEP, translatome/epigenome study). PMID:31519798

Localization

• Plasma membrane is the functional location (HDA proteomics PMID:22923678; abundant literature). Multi-pass PM protein.
• Golgi (HDA, PMID:22430844 / LOPIT proteomics PMID:22923678) and plastid (HDA, PMID:28887381 protein-correlation profiling) signals are high-throughput organellar-proteomics assignments. For a multi-pass PM oxidase these most likely reflect endomembrane/secretory trafficking or co-fractionation rather than a distinct functional compartment; mark as over-annotated / non-core.
• Nucleus (ISM, GO_REF:0000122, AtSubP prediction) is a computational subcellular-localization prediction with no experimental support for a nuclear RBOHD; a transmembrane PM oxidase is not expected in the nucleus. Remove.

GO term decisions summary

• GO:0016175 superoxide-generating NAD(P)H oxidase activity = correct core MF (proposed replacement for GO:0016174).
• GO:0005509 calcium ion binding = ACCEPT (EF-hands, Ca2+-dependent).
• GO:0004601 peroxidase activity (IEA, InterPro/KW) = REMOVE/over-annotation: RBOHD is a superoxide-generating oxidase, not a peroxidase; the "Peroxidase" keyword derives from the gp91phox/cytochrome-b245 InterPro signature and is misleading.
• GO:0016491 oxidoreductase, GO:0050664 oxidoreductase acting on NAD(P)H O2 acceptor = generic parents of GO:0016175; keep as accept (true but less informative).
• GO:0098869 cellular oxidant detoxification (inferred from peroxidase activity) = REMOVE: RBOHD generates ROS for signaling, it does not detoxify oxidants; inference rests on the spurious peroxidase MF.
• GO:0005515 protein binding (BIK1/FLS2, PBL13, HRU1, LecRK) = uninformative; mark over-annotated, captured better by molecular_function regulator interactions.
• GO:0002679 respiratory burst involved in defense response = good candidate NEW BP term for the immune oxidative burst (verified via OLS).

Deep research synthesis (Falcon / Edison Scientific report)

Source: [file:ARATH/RBOHD/RBOHD-deep-research-falcon.md]. The Falcon deep research report corroborates and extends the existing review without contradicting any decisions.

• Catalytic core confirmed: RBOHD "catalyzes electron transfer from cytosolic NADPH to molecular oxygen (O2) to generate superoxide (O2.-) in the apoplast, which can subsequently form H2O2" — reinforces GO:0016175 (superoxide-generating NAD(P)H oxidase) as the proximal-product MF over the H2O2-forming GO:0016174. Domain logic: N-terminal two EF-hand Ca2+-binding motifs, "a catalytic C-terminal core with FAD- and NADPH-binding domains", six TM helices with two hemes (His axial ligands).
• Activation: "RBOHD is activated by direct Ca2+ binding to EF-hands and by phosphorylation" — supports GO:0005509 calcium ion binding as core. Residue-level phosphosites compiled: BIK1 -> S39/S343/S347; RIPK -> S343/S347; CPK16 -> S133/S148/S163/S347; SIK1/MAP4K -> S347; DORN1 -> S22/T24. PTI axis: "In PTI, PRRs such as FLS2/EFR activate downstream cytoplasmic kinases (e.g., BIK1) that phosphorylate RBOHD to drive a rapid ROS burst."
• Immunity: RBOHD is the "major NADPH oxidase responsible for pathogen-triggered ROS" in Arabidopsis — supports GO:0006952 defense response and GO:0002679 respiratory burst involved in defense response as core.
• Localization: "RBOHD is a plasma membrane-localized NADPH oxidase that produces ROS into the apoplast" — supports GO:0005886 plasma membrane as the functional compartment (consistent with marking Golgi/plastid/nucleus assignments as non-core/over-annotated/removed).
• Guard-cell ABA signaling: RBOHD (with RBOHF) is "a pivotal ROS source for guard cell ABA signaling, supporting ABA-induced stomatal closure"; persulfidation at Cys825/Cys890 enhances ROS (Shen et al. 2020) — consistent with the ROS/stomatal-closure annotations.
• New (2023-2024) negative regulators not in current GOA, captured as a suggested question rather than annotations: PB1CP (New Phytol 2024; competes with BIK1, promotes endocytosis/turnover, lowers RBOHD abundance); QSK1 (Plant Cell 2024; PRR-RBOHD complex-associated, downregulates FLS2/EFR, exploited by HopF2Pto); PBL13/PIRE ubiquitination + vacuolar degradation and C-terminal nitrosylation (Torres 2024 commentary); CBE1/MOB7 translational repression of RBOHD accumulation (JBC 2023). These provide mechanistic regulatory context; no new GO IDs were invented from them.

No annotation decisions were weakened. Falcon file: supporting_text entries were added to: GO:0005509 (Ca binding), GO:0006952 (defense, IEA), GO:0016491 (oxidoreductase), GO:0050664 (NAD(P)H O2-acceptor oxidoreductase), GO:0005886 (plasma membrane), GO:0005515 (BIK1/FLS2 interaction), GO:0002679 NEW, GO:0016175 NEW, and to the three core_functions.