ESP (epithiospecifier protein; also called ESR, EPITHIOSPECIFYING SENESCENCE REGULATOR; At1g54040) is a cytoplasmic Kelch-repeat protein that folds as a six-bladed beta-propeller, forms homodimers, and binds an iron cofactor through a conserved EXXXDXXXH-type triad in the loops at the base of the propeller. It belongs to the specifier-protein family of the glucosinolate-myrosinase defence system. ESP has no hydrolytic activity on intact glucosinolates; instead, when tissue damage brings glucosinolates into contact with myrosinase, ESP acts on the unstable thiohydroximate-O-sulfate aglucone released by myrosinase and redirects its breakdown away from the default isothiocyanate. With alkenyl glucosinolates that carry a terminal double bond (e.g. allylglucosinolate) it forms epithionitriles by intramolecular sulfur transfer to the terminal alkene, and with other aliphatic and indolic glucosinolates it forms simple nitriles. It is now regarded as an Fe(II)-dependent sulfolyase (EC 4.8.1.6 epithionitrile-forming; EC 4.8.1.5 nitrile-forming) rather than a myrosinase cofactor. ESP is expressed in the epidermis of aerial organs and in glucosinolate-rich S-cells, is induced by jasmonate, and is a major natural polymorphism among accessions: Landsberg erecta expresses functional ESP and produces mainly nitriles, whereas Columbia-0 has little or no basal leaf ESP expression under the commonly tested conditions and produces mainly isothiocyanates; ESP expression and localization are also condition-dependent. Its ecological role is ambiguous, as ESP-derived nitriles increased feeding by the generalist Trichoplusia ni compared with isothiocyanates. A second, WRKY53-linked role has been reported, in which ESP is recruited to the nucleus by WRKY53, inhibits WRKY53 DNA binding and delays leaf senescence, and in which overexpression increases resistance to bacterial and fungal pathogens.
Definition: Catalysis of the reaction: a (Z)-N-(sulfonatooxy)alkenimidothioate (a glucosinolate aglucone with a terminally unsaturated side chain) = an epithionitrile + sulfate (EC 4.8.1.6; RHEA:69228). The thiol sulfur is transferred intramolecularly to the terminal double bond of the side chain.
Justification: Epithiospecifier proteins are characterized Fe(II)-dependent catalysts with a curated EC number and Rhea reaction. GO currently stops at the carbon-sulfur lyase parent (GO:0016846), so the specific epithionitrile-forming activity that defines ESP cannot be expressed. A sibling term for thiocyanate-forming activity was proposed in the Thlaspi TFP review.
Parent term: carbon-sulfur lyase activity
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:17369373 The antagonist function of Arabidopsis WRKY53 and ESR/ESP in... | MODIFY | Summary: IPI with WRKY53 (Q9SUP6) from yeast two-hybrid, BiFC and EMSA work in Miao and Zentgraf 2007. The paper goes beyond binding: ESP (called ESR there) inhibits WRKY53 DNA binding in vitro and antagonises WRKY53 in leaf senescence. Reason: Generic protein binding is uninformative. The cited paper supports a more specific molecular function, which is inhibition of a transcription factor (WRKY53) by direct binding. EMSA showed that increasing amounts of ESR protein block WRKY53 DNA binding. GO:0140416 transcription regulator inhibitor activity ("inhibits the activity of a transcription regulator via direct binding") captures this. It is a non-core, moonlighting-type function. This is a separately assayed conditional function; low basal leaf ESP expression in Col-0 is not evidence that every inducible or nuclear ESP function is absent. Proposed replacements: transcription regulator inhibitor activity Supporting Evidence: PMID:17369373 We found that the senescence-specific WRKY53 transcription factor interacts with the JA-inducible protein EPITHIOSPECIFYING SENESCENCE REGULATOR (ESR/ESP). PMID:17369373 In vitro analyses by electrophoretic mobility shift assay (EMSA) revealed that in the presence of increasing amounts of ESR protein, the DNA binding activity of WRKY53 was inhibited |
| GO:0005634 nucleus | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from PANTHER node PTN002355837. The only experimental donor is ESP (AT1G54040) itself, so ESP's own IDA nucleus annotation grounds this row. That is expected, not circular. Reason: For ESP itself the nuclear pool is experimentally reported. ESP is recruited to the nucleus by WRKY53 and is exclusively cytoplasmic in wrky53 knockout cells. The nucleus is therefore a conditional, secondary location. The main site of specifier activity is the cytoplasm. Kept as non-core. Whether this node placement should extend to the NSP paralogs is addressed in those reviews. Supporting Evidence: PMID:17369373 ESR inhibits WRKY53 DNA binding in vitro, and their interaction is localized to the nucleus in vivo; however, ESR is exclusively in the cytoplasm in W53-KO cells, indicating that ESR is brought to the nucleus by the interaction. |
| GO:0005634 nucleus | IDA PMID:17369373 The antagonist function of Arabidopsis WRKY53 and ESR/ESP in... | KEEP AS NON CORE | Summary: BiFC and GFP-fusion localization show ESP-WRKY53 complexes in the nucleus. ESP enters the nucleus only in the presence of WRKY53. Reason: Direct experimental localization from a full-text paper, so the annotation is retained. The nuclear pool is WRKY53-dependent and relates to the proposed senescence-regulatory role, not to the core specifier activity. Non-core. Supporting Evidence: PMID:17369373 as cytoplasmic epithiospecifier and as negative regulator of WRKY53 in the nucleus |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Mapped from the UniProt subcellular-location annotation (Nucleus, from PMID:17369373; the note says ESP is brought to the nucleus by WRKY53). Reason: Consistent with the experimental data, but a conditional secondary location. Supporting Evidence: PMID:17369373 indicating that ESR is brought to the nucleus by the interaction |
| GO:0005634 nucleus | ISM GO_REF:0000122 | KEEP AS NON CORE | Summary: AtSubP sequence-based prediction of nuclear localization. Reason: The prediction happens to agree with the experimentally observed WRKY53-dependent nuclear pool. ESP has no nuclear localization signal annotated. The nucleus is a secondary location, so non-core. Supporting Evidence: PMID:17369373 their interaction is localized to the nucleus in vivo |
| GO:0005737 cytoplasm | EXP PMID:17369373 The antagonist function of Arabidopsis WRKY53 and ESR/ESP in... | ACCEPT | Summary: GFP-ESP is cytoplasmic, and exclusively so in wrky53 knockout protoplasts. The cytoplasm is where ESP meets myrosinase-released aglucones after tissue damage. Reason: Experimental localization consistent with the specifier function. This is the principal location of ESP activity. Supporting Evidence: PMID:17369373 as cytoplasmic epithiospecifier and as negative regulator of WRKY53 in the nucleus |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Mapped from the UniProt subcellular-location annotation (Cytoplasm, from PMID:17369373). Reason: Consistent with the experimental data. Supporting Evidence: PMID:17369373 ESR is exclusively in the cytoplasm in W53-KO cells |
| GO:0005737 cytoplasm | ISM GO_REF:0000122 | ACCEPT | Summary: AtSubP prediction of cytoplasmic localization. Reason: Agrees with experimental localization. ESP has no signal peptide or transit peptide. Supporting Evidence: PMID:17369373 ESR is exclusively in the cytoplasm in W53-KO cells |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | MARK AS OVER ANNOTATED | Summary: The AtSubP chloroplast prediction lacks corroborating localization evidence. Reason: This ISM row is an AtSubP prediction (GO_REF:0000122), not an inaccessible experimental publication. The established localization evidence in PMID:17369373 supports cytoplasmic epithiospecifier activity and a WRKY53-dependent nuclear pool. With no corroborated plastid targeting or plastid-localization experiment, the positive chloroplast assignment exceeds the evidence. MARK_AS_OVER_ANNOTATED does not assert that plastid localization is impossible in every condition. Supporting Evidence: PMID:17369373 as cytoplasmic epithiospecifier and as negative regulator of WRKY53 in the nucleus |
| GO:0009753 response to jasmonic acid | IMP PMID:17369373 The antagonist function of Arabidopsis WRKY53 and ESR/ESP in... | KEEP AS NON CORE | Summary: ESP expression and its WRKY53-associated function respond to jasmonate. Reason: PMID17369373 directly supports jasmonate-responsive ESP biology. PMID11752388 does not supply that experiment, but a miscited row does not make the same biological claim false when independently demonstrated. Retain the term consistently across sources and record the source-specific limitation here. Supporting Evidence: PMID:17369373 The result of the RNA gel blot analyses revealed that expression of ESR is reduced by SA and activated by JA PMID:17369373 These results demonstrate that ESR is a target of the JAR1- mediated and COI1- mediated JA signaling pathways and is downstream of JAR1 and COI1 . |
| GO:0009753 response to jasmonic acid | TAS PMID:11752388 The Arabidopsis epithiospecifier protein promotes the hydrol... | KEEP AS NON CORE | Summary: ESP expression and its WRKY53-associated function respond to jasmonate. Reason: PMID17369373 directly supports jasmonate-responsive ESP biology. PMID11752388 does not supply that experiment, but a miscited row does not make the same biological claim false when independently demonstrated. Retain the term consistently across sources and record the source-specific limitation here. Supporting Evidence: PMID:17369373 The result of the RNA gel blot analyses revealed that expression of ESR is reduced by SA and activated by JA |
| GO:0010150 leaf senescence | IMP PMID:17369373 The antagonist function of Arabidopsis WRKY53 and ESR/ESP in... | KEEP AS NON CORE | Summary: ESR knockout (SALK_055029) accelerates, and overexpression retards, leaf senescence. The knockout phenotype was complemented by ESR under its own promoter, and epistasis places the effect through WRKY53. Reason: Experimental IMP with complementation in a full-text paper, so it is retained. It is not a core function. The mechanism (WRKY53 inhibition) is unrelated to specifier chemistry. The study was done in Col-0, which other groups describe as a natural ESP knockout with no detectable leaf ESP activity (PMID:27990154). That tension is flagged in suggested_questions. Supporting Evidence: PMID:17369373 Leaf senescence is accelerated in ESR knockout plants (ESR-KO) but retarded in ESR overexpressors (ESR-OE), with the reverse true for WRKY53. PMID:17369373 In these plants, the phenotype of the wild-type plants was completely restored PMID:27990154 Due to an insertion upstream of the ESP coding sequence, Col-0 is a natural knockout of ESP and ESP activity is not detectable in leaf extracts |
| GO:0019762 glucosinolate catabolic process | IDA PMID:11752388 The Arabidopsis epithiospecifier protein promotes the hydrol... | ACCEPT | Summary: Recombinant ESP converts glucosinolates to epithionitriles and simple nitriles in the presence of myrosinase. It directly processes the glucosinolate aglucone during glucosinolate breakdown. Reason: Core process. ESP performs a step of glucosinolate breakdown, the post-myrosinase conversion of the aglucone to (epithio)nitrile. Supporting Evidence: PMID:11752388 The heterologously expressed Arabidopsis ESP was able to convert glucosinolates both to epithionitriles and to simple nitriles in the presence of myrosinase |
| GO:0019762 glucosinolate catabolic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA-based electronic annotation to glucosinolate catabolic process. Reason: Correct and consistent with experimental data for ESP. Supporting Evidence: PMID:11752388 The heterologously expressed Arabidopsis ESP was able to convert glucosinolates both to epithionitriles and to simple nitriles in the presence of myrosinase |
| GO:0019762 glucosinolate catabolic process | IMP PMID:23999604 Molecular models and mutational analyses of plant specifier ... | ACCEPT | Summary: Mutational analysis of specifier proteins, including an ESP, altered glucosinolate breakdown product profiles. Abstract-only in cache. Reason: Consistent with the well-established role of ESP in glucosinolate breakdown. The abstract confirms that an epithiospecifier protein was modelled and mutated, and the curator saw the full text. Supporting Evidence: PMID:23999604 We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site |
| GO:0030234 enzyme regulator activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA reflecting the historical view of specifier proteins as myrosinase cofactors that regulate the outcome of myrosinase catalysis. Reason: The PAINT node placement across specifier proteins is sound, but the MF term is outdated. Specifier proteins have no activity on intact glucosinolates. They act on the aglucone after myrosinase, bind an Fe(II) cofactor in their own active site, and are now regarded as Fe(II)-dependent lyases (catalysts), not regulators of myrosinase. UniProt curates ESP with EC 4.8.1.6 and EC 4.8.1.5. The appropriate available GO term is carbon-sulfur lyase activity (GO:0016846), consistent with the Thlaspi TFP review. No more specific GO term exists yet; see proposed_new_terms. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: ROLE CONFLATION Sources checked: PANTHER:PTN001509219 SUPPORTS TRANSFER Specifier-protein ancestral node. Placing the specifier activity at this node is sound, but the node carries the historical "myrosinase cofactor/regulator" MF term instead of the Fe(II)-dependent lyase activity. The fix is a term swap for all descendants, not a node change. AGI_LocusCode:AT1G54040 SUPPORTS TRANSFER ESP's own IDA (PMID:11752388) is the experimental seed. The data (myrosinase-dependent conversion of glucosinolates to nitriles) are sound, but the regulator interpretation is superseded by the lyase model. Proposed replacements: carbon-sulfur lyase activity Supporting Evidence: PMID:30900313 Based on these insights, we propose that specifier proteins are catalysts that might be classified as Fe2+ -dependent lyases. PMID:30395611 Furthermore, we demonstrate presence of iron in epithiospecifier protein and nitrile-specifier protein 3 from Arabidopsis thaliana (AtESP and AtNSP3). |
| GO:0030234 enzyme regulator activity | IDA PMID:11752388 The Arabidopsis epithiospecifier protein promotes the hydrol... | MODIFY | Summary: Based on the 2001 biochemical characterization, in which ESP required myrosinase for activity. The authors discussed whether ESP acts as an independent enzyme on the aglucone or as a proteinaceous cofactor of myrosinase. Reason: The experiment is sound, but the interpretation is superseded. Later structural, iron-quantification and mutagenesis work showed that ESP has its own iron-containing active site that converts the myrosinase-released aglucone. Mutating the AtESP iron-binding residues strongly reduces activity. The activity is therefore a lyase activity on the aglucone (EC 4.8.1.6 / 4.8.1.5), best captured by carbon-sulfur lyase activity, and not regulation of myrosinase. Proposed replacements: carbon-sulfur lyase activity Supporting Evidence: PMID:11752388 Because myrosinase is required for activity, ESP may simply use the aglycone produced by myrosinase as a direct substrate for nitrile formation PMID:30395611 Substitution of the corresponding iron binding residues of AtESP, E260 and D264, by Gln and Asn, respectively, resulted in strongly diminished activity PMID:30900313 Based on these insights, we propose that specifier proteins are catalysts that might be classified as Fe2+ -dependent lyases. |
| GO:0042742 defense response to bacterium | IMP PMID:17369373 The antagonist function of Arabidopsis WRKY53 and ESR/ESP in... | KEEP AS NON CORE | Summary: ESR-overexpressing plants restricted Pseudomonas syringae DC3000 growth more than ESR-KO plants, and ESR is induced early after infection. Reason: Experimental IMP in a full-text paper, retained. The mechanism is unresolved: it may come from altered glucosinolate breakdown products or from the WRKY53 link. The effect was measured in Col-0, a natural leaf ESP knockout (PMID:27990154). The defence role of ESP-derived nitriles is itself ambiguous: in the founding study, ESP-dependent nitrile formation increased feeding by T. ni. Defence against bacteria is therefore a plausible downstream consequence, not a core function. Supporting Evidence: PMID:17369373 ESR-OE showed higher resistance than ESR-KO to bacterial and fungal pathogens. PMID:11752388 The role of ESP in plant defense is uncertain, because the generalist herbivore Trichoplusia ni (the cabbage looper) was found to feed more readily on nitrile-producing than on isothiocyanate-producing Arabidopsis. |
| GO:0042802 identical protein binding | IDA PMID:27498030 Crystal structure of the Epithiospecifier Protein, ESP from ... | KEEP AS NON CORE | Summary: Reflects the dimeric state seen in the 2.3 A AtESP crystal structure, which shows a dimerization mode different from TaTFP. Reason: A genuine structural property, not the catalytic core function. The homodimerization term below is the more specific annotation. Handled as in the TFP review. Supporting Evidence: PMID:27498030 AtESP shows a dimerization pattern different from TaTFP |
| GO:0042803 protein homodimerization activity | IDA PMID:27498030 Crystal structure of the Epithiospecifier Protein, ESP from ... | KEEP AS NON CORE | Summary: The AtESP crystal structure shows a homodimer. A later mechanistic study also describes AtESP as a homodimer. Reason: Supported structurally but is quaternary structure, not the core catalytic function. Kept as non-core. Supporting Evidence: PMID:30900313 Elucidation of crystal structures of TaTFP and ESP from Arabidopsis thaliana (AtESP) (which are homodimers) |
| GO:0080028 nitrile biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA to nitrile biosynthesis at the specifier-protein node. ESP and five NSP paralogs are experimental donors, ESP's own annotation among them. Reason: Correct and core. ESP forms simple nitriles and epithionitriles from glucosinolate aglucones, and nitrile formation is ancestral in the family. ESPs arose from NSPs. Supporting Evidence: PMID:11752388 Thus, the F15I1.12 gene product seemed to act as an ESP (converting alkenyl glucosinolates to epithionitriles) as well as converting nonalkenyl glucosinolates to simple nitriles. PMID:30900313 a monophyletic origin of epithiospecifier proteins (ESPs) from NSPs has been demonstrated |
| GO:0080028 nitrile biosynthetic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA-based electronic annotation to nitrile biosynthesis. Reason: Correct and consistent with experimental data. Supporting Evidence: PMID:11752388 The heterologously expressed Arabidopsis ESP was able to convert glucosinolates both to epithionitriles and to simple nitriles in the presence of myrosinase |
| GO:0080028 nitrile biosynthetic process | IMP PMID:23999604 Molecular models and mutational analyses of plant specifier ... | ACCEPT | Summary: Mutational analysis of specifier proteins, including an epithiospecifier protein, altered nitrile/epithionitrile product formation. Abstract-only in cache. Reason: Consistent with ESP's established nitrile- and epithionitrile-forming activity. Deferring to the curator, who had the full text. Supporting Evidence: PMID:23999604 In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. |
| GO:0005506 iron ion binding | IDA PMID:30395611 Iron is a centrally bound cofactor of specifier proteins inv... | NEW | Summary: Iron was detected in purified recombinant AtESP. Mutating the AtESP iron-binding triad residues E260 and D264 strongly reduces activity. UniProt also curates Fe(2+) as ESP's cofactor. Reason: A direct molecular property of ESP that is essential to its catalytic mechanism. The Fe(II) cofactor sits in the active site and coordinates the aglucone. It is not currently annotated in GOA. This is an MF binding term supported by direct iron quantification, so the process-participation concerns do not apply. Supporting Evidence: PMID:30395611 Furthermore, we demonstrate presence of iron in epithiospecifier protein and nitrile-specifier protein 3 from Arabidopsis thaliana (AtESP and AtNSP3). PMID:30395611 Substitution of the corresponding iron binding residues of AtESP, E260 and D264, by Gln and Asn, respectively, resulted in strongly diminished activity |
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Download this section (compressed HTML)Q: How can the WRKY53-dependent senescence and pathogen phenotypes reported for ESR/ESP knockouts in Col-0 be reconciled with Col-0 being a natural leaf ESP knockout with no detectable ESP activity? Are these phenotypes independent of specifier activity?
Q: Does ESP physically associate with myrosinase (TGG1/TGG2) in vivo to capture the short-lived aglucone, or does it act on freely diffusing aglucone released after tissue disruption?
Q: What is the net ecological effect of ESP-mediated epithionitrile/nitrile formation, given that it increases feeding by the generalist T. ni but may reduce attraction of glucosinolate-specialist herbivores?
Experiment: Compare Ler and Col-0 backgrounds carrying esp null alleles and catalytically dead ESP variants (iron-triad E260Q/D264N) for leaf senescence, WRKY53 target expression and P. syringae growth. This separates specifier-activity-dependent from WRKY53-interaction-dependent phenotypes.
Experiment: Run no-choice and choice bioassays with generalist (T. ni) and specialist (Pieris rapae, Plutella xylostella) herbivores on near-isogenic ESP-functional vs esp lines to test the specialist-crypsis hypothesis for ESP.
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