CYP71A12 (At2g30750) is a membrane-anchored heme-thiolate cytochrome P450 monooxygenase of the CYP71 clan that metabolizes the tryptophan-derived intermediate indole-3-acetaldoxime (IAOx). It is the tandem paralog of CYP71A13 (about 89% identity) and, like CYP71A13, oxidizes IAOx via an alpha-hydroxy-indole-3-acetonitrile (indole cyanohydrin) intermediate, but it partitions products differently, releasing mainly indole-3-carbaldehyde and cyanide rather than the cysteine-indole-3-acetonitrile adduct that is channelled to camalexin. In vivo CYP71A12 is the principal enzyme for pathogen- and stress-inducible indole-3-carboxylic acid derivatives and the first enzyme of the cyanogenic 4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN) pathway, acting upstream of the flavin oxidase FOX1 and CYP82C2, whereas its contribution to camalexin is minor in leaves but substantial in flagellin-elicited roots; it associates with CYP71B15 (PAD3) and the other camalexin P450s in an ER-associated metabolon. The gene is strongly induced by pathogens, microbe-associated molecular patterns and leaf microbiota (a general non-self response gene); loss of CYP71A12 increases susceptibility to Pseudomonas syringae and, together with CYP71A13, alters leaf microbiota colonization. In fungus-infected leaves it is induced at infection sites mainly in epidermal cells, in contrast to the vasculature-restricted induction of CYP71A13.
Definition: Catalysis of the NADPH-P450 reductase- and O2-dependent alpha-hydroxylation of (E)-indol-3-ylacetaldoxime to 2-hydroxy-2-(1H-indol-3-yl)acetonitrile (indole-3-cyanohydrin).
Justification: 18O2 labelling shows that CYP71A12 and CYP71A13 incorporate O2-derived oxygen via an alpha-hydroxy-IAN intermediate, so GO:0047720 (IAOx = IAN + H2O) describes the net conversion but not the chemistry. CYP71A12 releases mostly the cyanohydrin breakdown products indole-3-carbaldehyde and cyanide, and UniProt describes its function as conversion of IAOx to indole cyanohydrin.
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004497 monooxygenase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based assignment of generic P450 monooxygenase activity. CYP71A12 is an intact heme-thiolate P450 and 18O2 labelling shows it incorporates molecular oxygen into its IAOx-derived products. Reason: Correct and directly supported by biochemistry (O2-derived oxygen incorporated via an alpha-hydroxy-IAN intermediate). The specific substrate-level activity is captured separately by the NEW indoleacetaldoxime dehydratase annotation. Supporting Evidence: PMID:24151049 With 97% 18O2, both CYP71A13 and CYP71A12 catalyze incorporation of the isotopic label into IAL (74% and 81% 18O incorporation, respectively, Figure S10), providing clear evidence for an Ξ±-hydroxy-IAN intermediate. |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Generic P450 InterPro mapping for the heme iron (axial Cys439 ligand). Reason: The heme iron is coordinated by the conserved cysteine (UniProt BINDING 439, Fe), so the statement is true, but the iron is part of the heme cofactor already captured by heme binding, and the term adds no functional information. Kept as non-core rather than accepted (same treatment as the CYP71A13 review; the independent review had marked it over-annotated, as for CYP71B15). |
| GO:0005515 protein binding | IPI PMID:31511315 The Formation of a Camalexin Biosynthetic Metabolon. | REMOVE | Summary: Interaction with CYP71B15/PAD3 (Q9LW27) detected in the camalexin biosynthetic metabolon study (co-IP/FRET-FLIM; IntAct). Reason: Generic protein binding conveys no functional information. The interaction with PAD3 is likely genuine and reflects assembly of an ER-associated camalexin biosynthetic enzyme complex, but there is no specific MF term that captures P450-P450 association in a metabolon; the complex aspect is represented by the catalytic complex annotation. Removal does not imply the interaction is false. Supporting Evidence: PMID:31511315 we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. |
| GO:0005788 endoplasmic reticulum lumen | IDA PMID:33831160 The Formation of a Camalexin Biosynthetic Metabolon. | MODIFY | Summary: TAIR IDA (2022-02-03) for ER lumen. The cited PMID:33831160 is a deleted duplicate of PMID:31511315 (Mucha et al. 2019), whose evidence is confocal colocalization of CYP71A12 fusions with the ER lumenal marker RFP-HDEL. That shows ER localization but cannot distinguish the lumen from the membrane. Reason: The ER localization is experimentally sound and is kept. The lumen term is not: CYP71A12 is a signal-anchored class II P450 with a single N-terminal transmembrane helix, and the same paper states that such P450s face the cytosol. Mucha et al. 2019 (PMID:31511315; full text read on PMC, PMC6881122, not cacheable) states "Typically, eukaryotic P450 enzymes are anchored to the membrane of the endoplasmic reticulum (ER) with their catalytic centers facing the cytosolic side" and reports that CYP71A12, CYP71A13 and CYP71B15 "were localized to the ER and showed colocalization with the ER lumenal marker RFP-HDEL" (Supplemental Figure 4). Colocalization with a lumenal marker at confocal resolution identifies the organelle, not the side of the membrane, so endoplasmic reticulum membrane is the correct term. The Mucha full text could not be cached (PMC returns abstract only), so its sentences are quoted here rather than in supported_by. Proposed replacements: endoplasmic reticulum membrane Supporting Evidence: file:ARATH/CYP71A12/CYP71A12-uniprot.txt SUBCELLULAR LOCATION: Membrane {ECO:0000255}; Single-pass membrane PMID:21712415 three of the four known enzymes in the pathway (including the first and the last one) are cytochromes P450, which are associated with the endoplasmic reticulum, having their catalytic domain facing the cytosol |
| GO:0006952 defense response | NAS PMID:31511315 The Formation of a Camalexin Biosynthetic Metabolon. | MODIFY | Summary: Author statement that camalexin pathway enzymes act in defense. CYP71A12 products (camalexin, ICA derivatives, 4-OH-ICN) are inducible antimicrobial defense metabolites. Reason: Correct but very general. Mutant evidence specifically shows increased susceptibility of cyp71A12 to Pseudomonas syringae, and loss of the 4-OH-ICN pathway compromises antibacterial defense, so defense response to bacterium is the better-supported term. Either way this is a downstream (non-core) consequence of the biosynthetic activity; the independent review kept the general term as non-core instead. Proposed replacements: defense response to bacterium Supporting Evidence: PMID:26352477 Using surface inoculation to mimic the natural infection process, we found that compared to WT, the adult leaves of cyp71A12 and cyp82C2 are more susceptible to the virulent bacterial hemibiotroph Pst (Pseudomonas syringae pv. |
| GO:0009617 response to bacterium | IEP PMID:17573535 Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes th... | KEEP AS NON CORE | Summary: Expression-based annotation from the CYP71A13 characterization paper (cached abstract only), presumably reflecting pathogen induction of CYP71A12 alongside CYP71A13. Reason: CYP71A12 is well documented as strongly induced by bacteria and MAMPs (a general non-self response gene), so the expression annotation is correct, but transcriptional induction is not a core function. The full text of PMID:17573535 is not cached; deferring to the curator on the specific data. Supporting Evidence: PMID:39627368 Notable among them was CYP71A12, which encodes a cytochrome P450 monooxygenase that contributes to the synthesis of defence-associated tryptophan derivatives and is upregulated in response to pathogen encounter26 and beneficial microorganisms in induced systemic resistance5,25. |
| GO:0009617 response to bacterium | IMP PMID:23073694 Metabolic and transcriptomic changes induced in Arabidopsis ... | KEEP AS NON CORE | Summary: cyp71A12 mutant was among the mutants that failed to show Pseudomonas fluorescens SS101-induced resistance to Pst. Reason: Supported phenotype, but the response-to-bacterium framing is broad; the more informative process is the defense response to bacterium (see MODIFY of defense response and the IEP row). Kept as non-core. Supporting Evidence: PMID:23073694 Subsequent bioassays with seven Arabidopsis mutants ( myb51 , cyp79B2cyp79B3 , cyp81F2 , pen2 , cyp71A12 , cyp71A13 , and myb28myb29 ) disrupted in the biosynthesis pathways for these plant secondary metabolites showed that camalexin and glucosinolates are indeed required for the induction of Pst resistance by Pf |
| GO:0009682 induced systemic resistance | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT propagation from a node whose experimental grounding is the Pf.SS101 IMP data on CYP71A12 itself and CYP71A13. Reason: The node's only experimental grounding is the Pf.SS101 IMP data on CYP71A12 and CYP71A13, and those rows are themselves marked over-annotated. GO:0009682 is defined as a response to non-pathogenic bacteria conferring systemic resistance "that does not depend upon salicylic acid signaling", whereas the only experimental source (van de Mortel et al. 2012) states that the Pf.SS101-induced resistance to Pst is salicylic acid-dependent and not jasmonic acid/ethylene-dependent. The term therefore misdescribes the observed response. The mutant phenotype reflects a requirement for indolic defense metabolites in the effector phase of resistance, which is captured by the defense response to bacterium annotation; the node placement merits PAINT re-examination (see suggested questions). CYP71A12 appearing among the WITH/FROM sources is expected (its own IMP) and is not a criticism. Propagation Review Root cause: SOURCE BAD Sources checked: AGI_LocusCode:AT2G30750 Β· CYP71A12 SOURCE BAD IMP from van de Mortel et al. 2012; the observed resistance is salicylic acid-dependent, outside the GO:0009682 definition. AGI_LocusCode:AT2G30770 Β· CYP71A13 SOURCE BAD Same paper and same definitional mismatch as CYP71A12. PANTHER:PTN005384924 Β· PTN005384924 SOURCE BAD The node's only experimental grounding is the two IMP rows above. Supporting Evidence: PMID:23073694 the Pf .SS101 -induced resistance response to Pst is dependent on salicylic acid signaling and not on jasmonic acid and ethylene signaling. |
| GO:0009682 induced systemic resistance | IMP PMID:23073694 Metabolic and transcriptomic changes induced in Arabidopsis ... | MARK AS OVER ANNOTATED | Summary: cyp71A12 mutants lost Pf.SS101-induced resistance against Pst. Reason: The mutant phenotype is real, but the term does not fit it. GO:0009682 is defined as a response to non-pathogenic bacteria conferring systemic resistance "that does not depend upon salicylic acid signaling", whereas the only experimental source (van de Mortel et al. 2012) states that the Pf.SS101-induced resistance to Pst is salicylic acid-dependent and not jasmonic acid/ethylene-dependent. The term therefore misdescribes the observed response. The same treatment is applied to the identical rows on CYP71A13 and MYB51. The requirement of CYP71A12 for resistance to Pst is captured by defense response to bacterium. Supporting Evidence: PMID:23073694 Subsequent bioassays with seven Arabidopsis mutants ( myb51 , cyp79B2cyp79B3 , cyp81F2 , pen2 , cyp71A12 , cyp71A13 , and myb28myb29 ) disrupted in the biosynthesis pathways for these plant secondary metabolites showed that camalexin and glucosinolates are indeed required for the induction of Pst resistance by Pf |
| GO:0010120 camalexin biosynthetic process | NAS PMID:31511315 The Formation of a Camalexin Biosynthetic Metabolon. | ACCEPT | Summary: CYP71A12 catalyzes the IAOx-to-indole-cyanohydrin/IAN step of camalexin biosynthesis, partially redundant with CYP71A13 in leaves and a major contributor in roots. Reason: Directly supported by multiple primary studies - in vitro reconstitution (CYP71A12 can substitute for CYP71A13), the cyp71a12 cyp71a13 double mutant (only traces of camalexin), and the strong root camalexin defect of the cyp71A12 single mutant. CYP71A12 catalyses a step of the pathway, so involved_in is appropriate, though its quantitative role in leaf camalexin is minor relative to CYP71A13. Supporting Evidence: PMID:25953104 Based on the comparison of cyp71a13 and cyp71a12 cyp71a13 phenotypes, the contribution of CYP71A12 to camalexin formation in leaves is significant but minor in relation to CYP71A13. PMID:20348432 Indeed, a cyp71A12 insertion mutant was dramatically impaired for camalexin accumulation in the roots ( Figure 3 ). PMID:24151049 Substituting CYP71A12 in place of CYP71A13 also resulted in camalexin production, albeit at lower levels (Figure S11). |
| GO:0016020 membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Membrane; single-pass, predicted N-terminal anchor). Reason: Correct but general; CYP71A12 is a microsomal (ER-anchored) P450 with an N-terminal transmembrane helix. The specific compartment, endoplasmic reticulum membrane (GO:0005789), is captured by the MODIFY of the ER lumen IDA from Mucha et al. 2019, which shows ER localization by confocal colocalization with an ER marker. |
| GO:0016020 membrane | NAS PMID:31511315 The Formation of a Camalexin Biosynthetic Metabolon. | ACCEPT | Summary: Author statement in the metabolon paper that camalexin P450s are membrane-associated. Reason: Consistent with the N-terminal membrane anchor and with the membrane-associated metabolon model. |
| GO:0016491 oxidoreductase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Deep-node PAINT propagation of oxidoreductase activity across CYP71-clan P450s. Reason: Correct (CYP71A12 is an active P450 monooxygenase), though general; more specific activities are captured by monooxygenase activity and the NEW indoleacetaldoxime dehydratase activity. |
| GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen | IEA GO_REF:0000002 | ACCEPT | Summary: Generic P450 InterPro mapping. Reason: Correct class for a NADPH/P450 reductase-dependent P450 monooxygenase that incorporates O2-derived oxygen into its substrate. |
| GO:0020037 heme binding | IEA GO_REF:0000002 | ACCEPT | Summary: Heme-thiolate cofactor binding inherent to the P450 fold. Reason: Correct; heme is the catalytic cofactor (UniProt heme binding at Cys439). |
| GO:0042742 defense response to bacterium | IEP PMID:39627368 Plant microbiota feedbacks through dose-responsive expressio... | KEEP AS NON CORE | Summary: CYP71A12 is a general non-self response gene induced by leaf microbiota and MAMPs; the cyp71a12 cyp71a13 double mutant supports much higher Pst and commensal colonization. Reason: Defense against bacteria is a real biological role of CYP71A12 through its antimicrobial indolic products (4-OH-ICN, camalexin), supported by single-mutant susceptibility to Pst (Rajniak et al.) and by the double-mutant colonization phenotype here, so the term is correct. The IEP basis is induction by bacteria, and the mutant data are necessity evidence downstream of the biosynthetic activity, which is the core function. Kept as non-core, consistent with the defense response to bacterium IMP rows in the CYP71A13 and CYP79B2 reviews. Supporting Evidence: PMID:39627368 Pst exhibited increased abundances ranging from 7-fold in chx16 and mlo12 to up to 120-fold in cyp71a12 cyp71a13 after two days of colonization, relative to wild-type plants (Fig. PMID:26352477 Using surface inoculation to mimic the natural infection process, we found that compared to WT, the adult leaves of cyp71A12 and cyp82C2 are more susceptible to the virulent bacterial hemibiotroph Pst (Pseudomonas syringae pv. |
| GO:1902494 catalytic complex | NAS PMID:31511315 The Formation of a Camalexin Biosynthetic Metabolon. | KEEP AS NON CORE | Summary: Membership in the proposed camalexin biosynthetic metabolon (CYP79B2, CYP71A13, CYP71B15, ATR1, GSTU4). Reason: The metabolon evidence is centred on CYP71A13 and CYP71B15 baits; CYP71A12 was found among copurifying camalexin P450s, but the cached record is abstract-only and the complex is a dynamic enzyme assembly rather than a stable defined complex. Retained as non-core. Supporting Evidence: PMID:31511315 indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. file:ARATH/CYP71A12/CYP71A12-deep-research-falcon.md Targeted co-immunoprecipitation, fluorescence-lifetime measurements, and infected-Arabidopsis microsomal proteomics support association with an |
| GO:0047720 indoleacetaldoxime dehydratase activity | IDA PMID:25953104 TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Gene... | NEW | Summary: Recombinant CYP71A12 (yeast microsomes) converts indole-3-acetaldoxime with a catalytic efficiency slightly higher than CYP71A13, the enzyme that carries this activity in GO (IDA, PMID:17573535); both paralogs release IAN, and CYP71A12 further converts the cyanohydrin/IAN to indole-3-carbaldehyde and cyanide. Reason: The core catalytic activity of CYP71A12 is absent from its GO annotations, which carry only generic P450 activities. Two primary full-text studies assayed CYP71A12 directly. Klein et al. 2013 report persistent production of the dehydration product IAN from IAOx by these enzymes and, by 18O2 labelling, an O2-derived alpha-hydroxy-IAN (cyanohydrin) intermediate. MΓΌller et al. 2015 measured kinetics for both enzymes and conclude that both efficiently dehydrate IAOx, and that CYP71A12 catalyses dehydration of IAOx to IAN followed by conversion to indole-3-carbaldehyde and cyanide. The overall IAOx-to-IAN conversion is therefore demonstrated for CYP71A12, and the comparator CYP71A13 carries GO:0047720 by IDA and IEA. Caveat (from the independent review): the 18O2 data show that the reaction is a monooxygenation via a cyanohydrin rather than a simple hydro-lyase reaction, so GO:0047720 describes the net conversion, not the mechanism; a more exact monooxygenase term is proposed in proposed_new_terms and the question is raised in suggested_questions. Monooxygenase activity is accepted alongside. Supporting Evidence: PMID:25953104 For the reaction with IAOx , catalytic efficiency was approximately 0.066 Β΅ m β1 s β1 for CYP71A12 and approximately 0.029 Β΅ m β1 s β1 for CYP71A13. Besides the limitations regarding comparisons between catalytic efficiencies of different enzymes ( Eisenthal et al., 2007 ), it is clear that both enzymes are efficiently dehydrating IAOx . PMID:25953104 CYP71A12 catalyzed two consecutive reactions, the dehydration of IAOx to IAN , which is then further converted to ICHO and cyanide PMID:24151049 The persistent production of the dehydration product IAN alongside oxidized products from IAOx led us to examine whether IAN is an intermediate en route to camalexin or an off-pathway product. PMID:24151049 Although CYP71A12 turned over IAOx at a rate comparable to that of CYP71A13, the final products IAL and Cys-IAN accumulated in different ratios with these two enzymes |
| GO:0106148 4-hydroxyindole-3- carbonyl nitrile biosynthesis | IMP PMID:26352477 A new cyanogenic metabolite in Arabidopsis required for indu... | NEW | Summary: CYP71A12 catalyses the first oxidation (IAOx to indole-3-cyanohydrin) of the cyanogenic 4-OH-ICN pathway, upstream of FOX1 and CYP82C2; cyp71A12 mutants have about 10% of wild-type ICN derivatives and the pathway is reconstituted with CYP71A12, FOX1 and CYP82C2. Reason: Participation test - CYP71A12 performs a step of this pathway (the cyanohydrin-forming oxidation), shown by in vitro reconstitution as well as by the mutant metabolite profile, so it does part of the work rather than being merely required. GO:0106148 was created from this same paper (definition xref PMID:26352477) but currently has no annotations at all, including the downstream enzymes CYP82C2 (which carries only the GO:0106149 ICN 4-hydroxylase MF) and FOX1, so its absence on CYP71A12 reflects an unused new term rather than a curatorial convention. It is not an ancestor or descendant of camalexin biosynthetic process. This replaces the independent review's NEW GO:0042435 indole-containing compound biosynthetic process, which was dropped because it is an ancestor of the already-accepted GO:0010120, and its proposed new term for ICN biosynthesis, which already exists as GO:0106148. Note that the GO definition calls 4-OH-ICN a cyanogenic glucoside, which it is not (see suggested_questions). Supporting Evidence: PMID:26352477 all ICN derivatives with the exception of A6 are at ~10% of WT levels in the cyp71A12 mutant, but unaffected in the cyp71A13 and cyp71A18 mutants PMID:26352477 A combination of yeast microsomal CYP71A12 and CYP82C2 and N. benthamiana-expressed FOX1 was sufficient to catalyze the conversion of IAOx to ICN |
| GO:0050832 defense response to fungus | IMP PMID:31411742 The role of CYP71A12 monooxygenase in pathogen-triggered try... | NEW | Summary: cyp71A12 mutant combinations (with pen2) show defects in post-invasion resistance to the filamentous fungi Plectosphaerella cucumerina and Colletotrichum tropicale, and CYP71A12 is the major enzyme for pathogen-induced ICA accumulation. Reason: CYP71A12 does part of the work of this defense response - it catalyses the IAOx oxidation step that produces the inducible antifungal indolics (ICA derivatives and, partly, camalexin) - so it passes the participation test in the sense GO applies to phytoalexin biosynthetic enzymes. Comparator check - the paralog CYP71A13 and the downstream camalexin P450 PAD3/CYP71B15 both carry defense response to fungus (IMP), and both are accepted as core in their reviews. Its paralog-specific role in ICA (not shared by CYP71A13) means the contribution is not redundant. The independent review declined this row as necessity-only evidence, and the CYP71A12-specific data are abstract-only (phenotypes largely in a pen2 background); the row is kept on the comparator convention but the disagreement is recorded in suggested_questions. Fungal-infection expression data (epidermal induction at Colletotrichum infection sites) are consistent but are not used as evidence. Supporting Evidence: PMID:31411742 We also show that both enzymes are key players in the resistance of Arabidopsis against selected filamentous pathogens after they invade. PMID:31411742 Our results indicate that CYP71A12, but not CYP71A13, is the major enzyme responsible for the accumulation of ICA in Arabidopsis in response to pathogen ingression. |
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Download this section (compressed HTML)Q: Should TAIR revise the ER lumen (GO:0005788) IDA rows from Mucha et al. 2019 (cited under the deleted duplicate PMID:33831160) on CYP71A12, CYP71A13 and CYP71B15 to endoplasmic reticulum membrane, given that the evidence is colocalization with a lumenal marker and the paper itself describes these P450s as membrane-anchored and cytosol-facing?
Q: Should CYP71A12 and CYP71A13 be described by indoleacetaldoxime dehydratase activity (GO:0047720, IAOx = IAN + H2O; net conversion shown and called dehydration by MΓΌller et al. 2015) or by a cyanohydrin-forming monooxygenase term, given that 18O2 labelling shows O2-derived oxygen in the products via an alpha-hydroxy-IAN intermediate (PMID:24151049)? The two independent reviews disagreed here; modules/camalexin_biosynthesis.yaml currently uses monooxygenase activity only.
Q: Should GO gain a specific term for indole-3-carboxylic acid biosynthesis, the in vivo process for which CYP71A12 is non-redundant, and should the definition of GO:0106148 4-hydroxyindole-3-carbonyl nitrile biosynthesis be corrected (it calls 4-OH-ICN a cyanogenic glucoside; it is an aglycone alpha-ketonitrile) and its label spacing fixed?
Q: How much of the pathogen-induced ICOOH/ICA pool depends on CYP71A12 producing it directly (via indole-3-carbaldehyde), and how much comes from non-enzymatic hydrolysis of ICN?
Q: Is defense response to fungus appropriate for CYP71A12 on the strength of mutant phenotypes that are largely shown in a pen2 background (PMID:31411742, abstract-only here), or is that necessity evidence that should be left to the metabolite biosynthesis terms? The two independent reviews disagreed; the combined review follows the CYP71A13/CYP71B15 convention.
Q: Is the PAINT node carrying induced systemic resistance for CYP71A12/CYP71A13 justified, given that the only descendant evidence concerns a salicylic acid-dependent rhizobacterial response?
Q: Does epidermis-biased induction of CYP71A12 at fungal infection sites (versus vascular CYP71A13) translate into cell-type-specific ICA versus camalexin output?
Experiment: Express CYP71A12-fluorescent protein fusions with ER membrane and lumen markers, and test catalytic-domain topology by protease protection of microsomes.
Hypothesis: CYP71A12 is anchored in the ER membrane with its catalytic domain facing the cytosol.
Type: subcellular localization
Experiment: Use cell-type-specific promoters to complement cyp71a12 cyp71a13 double mutants and quantify ICA derivatives, 4-OH-ICN and camalexin, plus fungal resistance, after infection.
Hypothesis: Epidermal CYP71A12 induction at Colletotrichum higginsianum infection sites drives local ICA/4-OH-ICN accumulation, whereas vascular CYP71A13 supports camalexin.
Type: cell-type-specific metabolite profiling
Experiment: Steady-state kinetics of purified, reconstituted CYP71A12 and CYP71A13 with IAOx under 16O2 and 18O2, trapping the cyanohydrin and quantifying IAN, to give product partitioning and settle the dehydratase versus monooxygenase assignment.
Hypothesis: CYP71A12 forms indole-3-cyanohydrin by monooxygenation, and IAN arises from its dehydration rather than by a direct hydro-lyase reaction.
Type: enzyme kinetics and product trapping
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