BGLU26, also called PENETRATION 2 (PEN2), is an atypical myrosinase of glycoside hydrolase family 1 in Arabidopsis thaliana. It hydrolyses the thioglucosidic bond of tryptophan-derived indole glucosinolates, notably indol-3-ylmethyl glucosinolate and its pathogen-induced, CYP81F2-dependent derivative 4-methoxyindol-3-ylmethyl glucosinolate, releasing glucose and an unstable aglycone that is further processed (with GSTU13 and PCS1) into indole metabolites exported by the ABC transporter PEN3. The enzyme also hydrolyses artificial O-glucosides in vitro, but this activity is separable from its biological role. PEN2 is a C-terminally tail-anchored protein dually targeted to the outer membranes of peroxisomes and mitochondria, with its catalytic domain facing the cytosol; during attempted fungal penetration, PEN2-bearing mitochondria become immobilised beneath the entry site, and the mitochondrial pool alone is sufficient for function. PEN2-dependent indole glucosinolate metabolism mediates pre-invasion, broad-spectrum resistance to nonadapted filamentous pathogens such as powdery mildews and Colletotrichum, and is also required for flg22-triggered callose deposition and contributes to defense against bacteria.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro GH1 (IPR001360) mapping to the generic O-glycosyl hydrolase term. PEN2 is a GH1 enzyme and does hydrolyse the O-glucoside 4-methylumbelliferyl-beta-D-glucoside in vitro, so the term is technically correct, but it is a parent of the experimentally supported beta-glucosidase activity and does not capture the physiologically relevant S-glycosyl (thioglucoside) hydrolysis. Reason: Correct but uninformative family-level inference. The biologically relevant activity is thioglucosidase (myrosinase) activity on indole glucosinolates; the O-glucosidase activity is separable from immune function (E183D variant retains 4MUG hydrolysis, loses I3G hydrolysis and fails to complement pen2). Supporting Evidence: file:ARATH/BGLU26/BGLU26-deep-research-falcon.md Replacing catalytic Glu183 with Asp eliminated detectable I3G hydrolysis while retaining 4MUG hydrolysis file:ARATH/BGLU26/BGLU26-deep-research-falcon.md generic Ξ²-glucosidase activity alone does not explain PEN2 |
| GO:0005739 mitochondrion | ISM GO_REF:0000122 | MODIFY | Summary: AtSubP sequence-based prediction of mitochondrial localization. Experimentally confirmed: PEN2 is a tail-anchored protein dually targeted to peroxisomes and the mitochondrial outer membrane, and the mitochondrial pool alone is sufficient for entry control. Reason: The prediction is borne out by Fuchs et al. 2016, which places PEN2 specifically on the outer membrane of mitochondria with its catalytic domain facing the cytosol. The more precise term mitochondrial outer membrane should be used. Proposed replacements: mitochondrial outer membrane Supporting Evidence: PMID:26721862 PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria PMID:26721862 Exclusive targeting of PEN2 to the outer membrane of mitochondria complements the pen2 mutant phenotype file:ARATH/BGLU26/BGLU26-deep-research-falcon.md C-terminally tail-anchored protein dually targeted to peroxisomal and mitochondrial outer membranes, with its large catalytic domain facing the cytosol |
| GO:0005777 peroxisome | IDA PMID:16293760 Pre- and postinvasion defenses both contribute to nonhost re... | ACCEPT | Summary: Lipka et al. 2005 showed PEN2-GFP localizing to peroxisomes. Later work confirmed peroxisomal association (as a membrane tail-anchored protein) in addition to mitochondria. Reason: Direct experimental localization, confirmed by Fuchs et al. 2016. Peroxisome is a valid location, although the mitochondrial pool appears to be the functionally sufficient one for pathogen entry control and the protein is anchored in the peroxisomal membrane facing the cytosol rather than in the matrix. Supporting Evidence: PMID:16293760 The PEN2 glycosyl hydrolase localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism PMID:26721862 PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria |
| GO:0005777 peroxisome | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location (Peroxisome) mapped to GO; derived from the same Lipka et al. 2005 experimental observation. Reason: Consistent with the IDA annotation and with Fuchs et al. 2016 dual peroxisome/mitochondria targeting. Supporting Evidence: PMID:16293760 The PEN2 glycosyl hydrolase localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism PMID:26721862 PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria |
| GO:0005975 carbohydrate metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro GH1 mapping to carbohydrate metabolic process. PEN2 hydrolyses indole glucosinolates (thioglucosides), releasing glucose and an unstable aglycone; its biological role is glucosinolate catabolism for defense, not carbohydrate metabolism. Reason: Generic family-level process term. The relevant process for PEN2 is indole glucosinolate catabolic process (GO:0042344), which is supported experimentally; carbohydrate metabolism is not a meaningful description of its role. Supporting Evidence: PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense PMID:19095898 supporting the hypothesis that PEN2 acts as a myrosinase catalyzing the hydrolysis of 4-methoxy-I3G |
| GO:0008422 beta-glucosidase activity | EXP PMID:19095900 A glucosinolate metabolism pathway in living plant cells med... | KEEP AS NON CORE | Summary: Bednarek et al. 2009 measured hydrolysis of 4-methylumbelliferyl-beta-D-glucoside by recombinant PEN2 (UniProt: Km 150 uM, Vmax 0.76 umol/min/mg, roughly tenfold lower Vmax than for I3G). Reason: The in vitro O-glucosidase activity is genuine and was assayed by the curators, so it should be retained, but it is not the physiological activity: the E183D variant keeps 4MUG hydrolysis, loses glucosinolate hydrolysis and does not restore entry resistance. The core activity is thioglucosidase (myrosinase) activity. Supporting Evidence: file:ARATH/BGLU26/BGLU26-deep-research-falcon.md Replacing catalytic Glu183 with Asp eliminated detectable I3G hydrolysis while retaining 4MUG hydrolysis file:ARATH/BGLU26/BGLU26-deep-research-falcon.md generic Ξ²-glucosidase activity alone does not explain PEN2 PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense |
| GO:0008422 beta-glucosidase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference of beta-glucosidase activity across the GH1 family node PTN000039458, which includes many characterized plant and animal beta-glucosidases. Reason: PEN2 sits inside the GH1 clade and retains O-glucosidase activity in vitro, so the node placement is sound. Within this clade, however, PEN2 has acquired atypical myrosinase activity that is the biologically relevant function; beta-glucosidase activity is retained but non-core. Supporting Evidence: file:ARATH/BGLU26/BGLU26-deep-research-falcon.md Replacing catalytic Glu183 with Asp eliminated detectable I3G hydrolysis while retaining 4MUG hydrolysis PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense |
| GO:0008422 beta-glucosidase activity | IEA GO_REF:0000003 | KEEP AS NON CORE | Summary: EC 3.2.1.21 to GO mapping. UniProt assigns EC 3.2.1.21 (beta-glucosidase) to PEN2 based on the 4MUG assay, rather than EC 3.2.1.147 (thioglucosidase / myrosinase). Reason: Consistent with the in vitro O-glucosidase activity, but the EC assignment underlying this mapping under-describes the enzyme; myrosinase activity on indole glucosinolates is the physiological reaction. Supporting Evidence: file:ARATH/BGLU26/BGLU26-deep-research-falcon.md Replacing catalytic Glu183 with Asp eliminated detectable I3G hydrolysis while retaining 4MUG hydrolysis PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense |
| GO:0009617 response to bacterium | IMP PMID:23073694 Metabolic and transcriptomic changes induced in Arabidopsis ... | KEEP AS NON CORE | Summary: van de Mortel et al. 2012 found that pen2, among other indole glucosinolate and camalexin pathway mutants, failed to show Pseudomonas fluorescens SS101-induced resistance against P. syringae. Reason: Mutant phenotype showing PEN2-dependent glucosinolate metabolism is required for a rhizobacterium-induced response; this is a downstream/necessity role, not a core function of the enzyme. Supporting Evidence: PMID:23073694 camalexin and glucosinolates are indeed required for the induction of Pst resistance by Pf .SS101 |
| GO:0009682 induced systemic resistance | IMP PMID:23073694 Metabolic and transcriptomic changes induced in Arabidopsis ... | KEEP AS NON CORE | Summary: Same experiment as above: pen2 mutants were impaired in Pf.SS101-induced systemic resistance to P. syringae. Reason: Experimental, curator-made annotation from a mutant bioassay; PEN2 contributes indole glucosinolate hydrolysis products required for the induced resistance. Pleiotropic downstream role, non-core. Supporting Evidence: PMID:23073694 camalexin and glucosinolates are indeed required for the induction of Pst resistance by Pf .SS101 |
| GO:0009941 chloroplast envelope | HDA PMID:12938931 Proteomic study of the Arabidopsis thaliana chloroplastic en... | MARK AS OVER ANNOTATED | Summary: Detection of PEN2 in a mass-spectrometry survey of "mixed" chloroplast envelope membranes. Reason: Targeted live-cell imaging, fractionation and retargeting studies place PEN2 on peroxisomal and mitochondrial outer membranes, not plastids. Detection in a high-throughput envelope preparation most likely reflects co-purifying organellar membranes; this location should not be used to describe PEN2. Supporting Evidence: PMID:12938931 "mixed" envelopes were subsequently isolated using sucrose step gradients PMID:26721862 PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria |
| GO:0016020 membrane | IDA PMID:16293760 Pre- and postinvasion defenses both contribute to nonhost re... | ACCEPT | Summary: Lipka et al. 2005 localization data; PEN2 is a C-terminally tail-anchored membrane protein. Reason: Correct, if general. More specific membrane locations are mitochondrial outer membrane and peroxisomal membrane (Fuchs et al. 2016). Supporting Evidence: PMID:16293760 The PEN2 glycosyl hydrolase localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism PMID:26721862 PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria |
| GO:0019137 thioglucosidase activity | IDA PMID:19095900 A glucosinolate metabolism pathway in living plant cells med... | ACCEPT | Summary: Bednarek et al. 2009 showed recombinant PEN2 hydrolyses the indole glucosinolates I3G and 4-methoxy-I3G (atypical myrosinase, a beta-thioglucoside glucohydrolase). Reason: This is the core molecular function of PEN2, supported by direct enzyme assays, the catalytic-residue mutant, and in vivo accumulation of 4-methoxy-I3G in pen2 mutants. Supporting Evidence: PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense PMID:19095898 supporting the hypothesis that PEN2 acts as a myrosinase catalyzing the hydrolysis of 4-methoxy-I3G PMID:19095898 the IGS profile of the pen2-1 mutant shows increased accumulation of 4-methoxy-I3G upon Flg22 treatment |
| GO:0019760 glucosinolate metabolic process | IMP PMID:19095900 A glucosinolate metabolism pathway in living plant cells med... | MODIFY | Summary: pen2 mutants accumulate the indole glucosinolate 4-methoxy-I3G and fail to produce downstream hydrolysis products after pathogen challenge. Reason: Correct but general. The evidence specifically shows catabolism (hydrolysis) of indole glucosinolates; indole glucosinolate catabolic process (GO:0042344) is the precise term. Proposed replacements: indole glucosinolate catabolic process Supporting Evidence: PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense PMID:19095898 the IGS profile of the pen2-1 mutant shows increased accumulation of 4-methoxy-I3G upon Flg22 treatment |
| GO:0019762 glucosinolate catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of glucosinolate catabolic process at node PTN002570733, with PEN2 itself and TGG1 among the experimental donors. Reason: PEN2 catalyses glucosinolate hydrolysis directly; the IBA is supported by PEN2 own experimental data and is consistent with the more specific indole glucosinolate catabolic process. Supporting Evidence: PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense |
| GO:0042344 indole glucosinolate catabolic process | IMP PMID:19095898 Glucosinolate metabolites required for an Arabidopsis innate... | ACCEPT | Summary: Clay et al. 2009 showed pen2 mutants accumulate 4-methoxy-I3G after flg22 treatment, consistent with PEN2 catalysing its hydrolysis. Reason: Core process: PEN2 performs the hydrolytic step of indole glucosinolate breakdown, as shown biochemically (Bednarek et al. 2009) and genetically. Supporting Evidence: PMID:19095898 supporting the hypothesis that PEN2 acts as a myrosinase catalyzing the hydrolysis of 4-methoxy-I3G PMID:19095898 the IGS profile of the pen2-1 mutant shows increased accumulation of 4-methoxy-I3G upon Flg22 treatment PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense |
| GO:0042742 defense response to bacterium | IMP PMID:19095898 Glucosinolate metabolites required for an Arabidopsis innate... | KEEP AS NON CORE | Summary: pen2-1 seedlings were slightly but significantly more susceptible to P. syringae pv. tomato DC3000 in a flg22-dependent growth suppression assay. Reason: Modest phenotype; PEN2 indole glucosinolate hydrolysis contributes to MAMP-triggered defense against bacteria, but its principal role is pre-invasion antifungal defense. Supporting Evidence: PMID:19095898 the IGS hydrolytic mutant pen2-1 are slightly but significantly more susceptible to wild-type PtoDC3000 |
| GO:0050832 defense response to fungus | IMP PMID:16293760 Pre- and postinvasion defenses both contribute to nonhost re... | ACCEPT | Summary: Lipka et al. 2005 identified PEN2 as required for pre-invasion nonhost resistance to the powdery mildews Blumeria graminis f. sp. hordei and Erysiphe pisi; later shown for Colletotrichum gloeosporioides and other filamentous pathogens. Reason: PEN2 participates directly: it catalyses the hydrolysis of 4-methoxy-I3G that generates the antifungal products at attempted entry sites, and mitochondria carrying PEN2 accumulate beneath the fungal penetration site. This is participation by catalysis, not mere necessity, so the defense term is justified alongside the mechanistic indole glucosinolate catabolic process. Supporting Evidence: PMID:16293760 Arabidopsis PEN2 restricts pathogen entry of two ascomycete powdery mildew fungi PMID:19095900 activated by the atypical PEN2 myrosinase (a type of beta-thioglucoside glucohydrolase) for antifungal defense PMID:20605856 thereby demonstrating the involvement of PEN2 in nonhost resistance to this pathogen |
| GO:0052544 defense response by callose deposition in cell wall | IMP PMID:19095898 Glucosinolate metabolites required for an Arabidopsis innate... | KEEP AS NON CORE | Summary: Clay et al. 2009 found pen2 mutants lost flg22-induced callose deposition. Reason: PEN2 acts upstream: a hydrolysis product of 4-methoxy-I3G is proposed to act as a signal or co-activator for callose synthesis by PMR4/GSL5. PEN2 itself does not transport or deposit callose. Callose deposition and fungal entry resistance are distinct readouts. Supporting Evidence: PMID:19095898 Both pen2-1 and pen2-2 mutants exhibited a loss of the callose response to Flg22 |
| GO:0071472 cellular response to salt stress | IBA GO_REF:0000033 | REMOVE | Summary: PAINT inference from AT1G66270 and AT1G66280 (BGLU21 and BGLU22, ER-body beta-glucosidases) at node PTN002570733. Reason: The salt-stress donors are ER-body resident root/seedling glucosidases whose function is tied to ER bodies; PEN2 is a cytosol-facing, peroxisome/mitochondria tail-anchored myrosinase with no evidence of a role in salt stress. The process appears lineage-specific to the ER-body subclade and is over-propagated to PEN2. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN002570733 Β· PTN002570733 SUPPORTS SOURCE BUT NOT TARGET Salt-stress evidence at this node comes from the ER-body glucosidases AT1G66270/AT1G66280; PEN2 is a peroxisome/mitochondria tail-anchored myrosinase with no salt-stress evidence. Supporting Evidence: file:ARATH/BGLU26/BGLU26-deep-research-falcon.md C-terminally tail-anchored protein dually targeted to peroxisomal and mitochondrial outer membranes, with its large catalytic domain facing the cytosol |
| GO:0098542 defense response to other organism | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of defense response to other organism, with PEN2 itself as the experimental donor. Reason: General but correct; PEN2 has direct experimental support for defense against fungi and bacteria, and its own data seed this IBA. Supporting Evidence: PMID:16293760 Arabidopsis PEN2 restricts pathogen entry of two ascomycete powdery mildew fungi |
| GO:0005778 peroxisomal membrane | IDA PMID:26721862 Immobilized Subpopulations of Leaf Epidermal Mitochondria Me... | NEW | Summary: Fuchs et al. 2016 showed PEN2 is tail-anchored with dual targeting to peroxisomal and mitochondrial membranes. Reason: More precise than the existing peroxisome annotations: PEN2 is anchored in the peroxisomal membrane via its C-terminal tail with the catalytic domain facing the cytosol. Supporting Evidence: PMID:26721862 PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria file:ARATH/BGLU26/BGLU26-deep-research-falcon.md C-terminally tail-anchored protein dually targeted to peroxisomal and mitochondrial outer membranes, with its large catalytic domain facing the cytosol |
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Download this section (compressed HTML)Q: Should UniProt/EC assignments for PEN2 be revised from EC 3.2.1.21 (beta-glucosidase) to EC 3.2.1.147 (thioglucosidase), given that the O-glucosidase activity is separable from the immune function?
Q: What is the decisive bioactive product of PEN2-initiated 4-methoxy-I3G hydrolysis at fungal entry sites, and is it directly antifungal or a signal?
Q: Does the peroxisomal PEN2 pool have any function distinct from the mitochondrial pool that is sufficient for entry control?
Experiment: Compare kinetic parameters of purified PEN2 on 4-methoxy-I3G, 4-hydroxy-I3G and I3G at cytosolic pH, alongside the E183D variant, to define the native substrate preference.
Experiment: Peroxisome-only retargeted PEN2 (e.g. PEX-specific tail anchor) in pen2 to test whether the peroxisomal pool alone can complement entry resistance.
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