CRTISO (carotenoid isomerase; prolycopene isomerase, EC 5.2.1.13; also CCR2) is a nuclear-encoded, plastid-targeted flavoprotein of the carotenoid/retinoid oxidoreductase family that catalyses the last step of the plant poly-cis route from phytoene to lycopene. In plants and cyanobacteria the desaturases PDS and ZDS introduce cis double bonds, so the product of ZDS is 7,7',9,9'-tetra-cis-lycopene (prolycopene) rather than all-trans-lycopene. CRTISO isomerises the adjacent 7,9 (and 7',9') cis pairs of prolycopene and of its precursor 7,9,9'-tri-cis-neurosporene to the trans configuration, yielding all-trans-lycopene, the substrate of the lycopene beta- and epsilon-cyclases and hence the precursor of all cyclic carotenes and xanthophylls (lutein, violaxanthin, neoxanthin, zeaxanthin) and of the apocarotenoid hormones derived from them. The enzyme is homologous to the bacterial CRTI-type desaturases and binds FAD in an N-terminal dinucleotide-binding fold, but it catalyses a non-redox reaction that requires the reduced flavin and a membrane environment; unlike CRTI it has no desaturase activity and it does not act on cis-zeta-carotenes, which are handled by Z-ISO. Because light-driven photoisomerisation can partly replace the enzyme in illuminated tissue, loss of CRTISO (the ccr2 mutant) reduces lutein in green leaves, whereas etiolated seedlings, roots and non-photosynthetic tissues accumulate prolycopene and other poly-cis carotenes in place of xanthophylls. Dark-grown ccr2 etioplasts also lack the prolamellar body and green slowly, a downstream consequence of a cis-carotene-derived apocarotenoid signal that represses prolamellar-body formation. The protein carries a 56-residue chloroplast transit peptide and is a peripheral membrane protein of plastid membranes; its transcription is controlled by the histone methyltransferase SDG8/CCR1.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0009507 chloroplast | ISM GO_REF:0000122 | ACCEPT | Summary: Sequence-based (AtSubP) prediction of chloroplast localization. Consistent with the experimentally mapped 56-residue chloroplast transit peptide (cleavage after Ser-56 with N-acetylation of Val-57 in the plant N-terminal acetylome), with UniProt's plastid/chloroplast membrane assignment, and with the enzyme's substrates and phenotype, which are entirely plastidial. The enzyme also functions in etioplasts and other non-green plastids, so 'chloroplast' describes the major but not the only plastid type in which it acts. Reason: Correct organelle for a carotenoid biosynthetic enzyme whose precursor carries a chloroplast transit peptide and whose loss changes the pigment composition of chloroplasts (lutein deficiency) and etioplasts (poly-cis carotene accumulation). Retained as a core location. Supporting Evidence: file:ARATH/CRTISO/CRTISO-uniprot.txt SUBCELLULAR LOCATION: Plastid, chloroplast membrane {ECO:0000250}; PMID:22223895 post-translational N-Ξ±-acetylation of the mature protein after removal of the transit peptide PMID:11884677 The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants |
| GO:0009662 etioplast organization | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT propagation of the Arabidopsis ccr2 phenotype (etioplasts lacking a prolamellar body) to the plant CRTISO clade at node PTN005296160. Q9M9Y8 in its own WITH/FROM is the expected marker that the IBD was seeded by this gene's IMP. The phenotype is real and mechanistically understood: it is caused by a cis-carotene-derived apocarotenoid signal that accumulates when CRTISO is missing and represses prolamellar-body formation, and it is reverted by blocking upstream cis-carotene synthesis (ziso) or carotenoid cleavage dioxygenase activity. Reason: CRTISO does none of the work of assembling an etioplast; it is a lycopene biosynthetic enzyme whose absence perturbs prolamellar-body formation indirectly, through the accumulation and cleavage of its poly-cis substrates. That is an upstream requirement (TAIR curated the underlying IMP with acts_upstream_of_or_within), not participation in etioplast organization, so the term is kept as a genuine but non-core, downstream process. The phylogenetic inference itself is reasonable because the same poly-cis pathway, and hence the same cis-carotene accumulation in the dark, operates in all plant CRTISO orthologs (tomato tangerine etiolated seedlings also accumulate cis-carotenes). Supporting Evidence: PMID:11884677 Etioplasts of dark-grown crtISO mutants accumulate acyclic poly-cis-carotenoids in place of cyclic all-trans-xanthophylls and also lack prolamellar bodies (PLBs), the lattice of tubular membranes that defines an etioplast. This demonstrates a requirement for carotenoid biosynthesis to form the PLB. PMID:32003746 chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis |
| GO:0009662 etioplast organization | IMP PMID:11884677 Identification of the carotenoid isomerase provides insight ... | KEEP AS NON CORE | Summary: Park et al. showed by electron microscopy that dark-grown ccr2 (crtISO) etioplasts accumulate acyclic poly-cis carotenoids instead of cyclic all-trans xanthophylls and lack prolamellar bodies, and that seedlings green more slowly on illumination. The cached record is abstract-only, but the claim is stated explicitly in the abstract and was reproduced and mechanistically dissected later (the PLB defect is due to a cis-carotene-derived apocarotenoid signal, and is rescued in ccr2 ziso double mutants and by carotenoid cleavage dioxygenase inhibitors). Reason: A robust, well-documented mutant phenotype, correctly annotated by TAIR as acts_upstream_of_or_within. CRTISO's own activity is prolycopene isomerisation; the prolamellar-body defect is a downstream consequence of altered carotenoid composition and signalling, not a step of etioplast assembly that CRTISO performs. Kept as non-core rather than accepted as a core function or removed. Supporting Evidence: PMID:11884677 The absence of PLBs in crtISO mutants demonstrates a function for this unique structure and carotenoids in facilitating chloroplast development during the first critical days of seedling germination and photomorphogenesis. PMID:32003746 ccr2 is similar to cop1/det1 mutants in that it lacks a PLB in etioplasts, yet it is unique among PLB-deficient mutants in having normal PChlide and POR protein levels |
| GO:0016116 carotenoid metabolic process | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from IPR045892 (prolycopene isomerase-like family). This broader family also contains the cyanobacterial CrtD C-3',4' desaturase of myxoxanthophyll biosynthesis, so InterPro mapped it to the generic parent 'carotenoid metabolic process'. For CRTISO the process is unambiguously biosynthetic: it converts prolycopene to all-trans-lycopene in the lycopene branch of carotenoid biosynthesis. Reason: Not wrong, but less specific than the experimental evidence supports. The more informative term is GO:0016117 carotenoid biosynthetic process (already carried by the gene from IPR014101, TAS and this review), with GO:1901177 lycopene biosynthetic process as the most specific pathway term (proposed as NEW below). Proposed replacements: carotenoid biosynthetic process Supporting Evidence: PMID:11884677 carotenoid desaturation in plants requires a third distinct enzyme activity, the carotenoid isomerase (CRTISO), which, unlike phytoene and zeta-carotene desaturases, apparently arose from a progenitor bacterial desaturase file:ARATH/CRTISO/CRTISO-uniprot.txt PATHWAY: Carotenoid biosynthesis; lycopene biosynthesis. |
| GO:0016117 carotenoid biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from IPR014101 (prolycopene isomerase family), an entry defined on the Arabidopsis CRTISO itself. CRTISO catalyses the terminal isomerisation of the poly-cis desaturation route to all-trans-lycopene, a committed step of carotenoid biosynthesis. Reason: Correct and appropriately specific for a domain-level inference; the experimental annotations (TAS, IMP) and the mutant pigment phenotypes agree. Supporting Evidence: PMID:21209101 CRTISO isomerizes all cis double bonds formed by the action of PDS and ZDS ( 14 ) yielding 7,9,9β²,7β²-tetra- cis -lycopene 3 (commonly referred to as prolycopene; 15 ) to finally form all- trans -lycopene |
| GO:0016117 carotenoid biosynthetic process | TAS PMID:11884677 Identification of the carotenoid isomerase provides insight ... | ACCEPT | Summary: Traceable author statement from the paper that identified Arabidopsis CRTISO by positional cloning of ccr2 and showed that the recombinant enzyme isomerises poly-cis carotenoids to all-trans carotenoids. ccr2 mutants have reduced lutein in light-grown tissue and accumulate poly-cis carotene precursors in the dark. Reason: The core biological process of this enzyme: it performs a step (prolycopene to all-trans-lycopene) of carotenoid biosynthesis. Supported by genetics and by in vitro enzymology in Arabidopsis and in the tomato ortholog. Supporting Evidence: PMID:11884677 The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants |
| GO:0016117 carotenoid biosynthetic process | TAS PMID:11884678 Cloning of tangerine from tomato reveals a carotenoid isomer... | ACCEPT | Summary: Traceable author statement from the companion paper that cloned the tomato tangerine locus as CRTISO and showed that tangerine fruit accumulate prolycopene instead of all-trans-lycopene. The paper concerns the tomato ortholog (Q8S4R4) but states the function for plant CRTISO generally, and the Arabidopsis enzyme was identified simultaneously with the same function. Reason: The process is correct for Arabidopsis CRTISO and independently established in Arabidopsis (PMID:11884677); the tomato paper is a legitimate supporting statement for the conserved plant function. Curator use of a companion ortholog paper as TAS is acceptable and is not a mis-attribution. Supporting Evidence: PMID:11884678 the tangerine gene, designated CRTISO, encodes an authentic carotenoid isomerase that is required during carotenoid desaturation PMID:11884678 The function of carotene isomerase in plants presumably is to enable carotenoid biosynthesis to occur in the dark and in nonphotosynthetic tissues. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from IPR002937, the amine oxidase domain, an FAD-binding fold shared by monoamine/polyamine oxidases, CRTI-type carotene desaturases and CRTISO. CRTISO retains the fold and binds FAD, but its reaction (EC 5.2.1.13, 7,7',9,9'-tetra-cis-lycopene = all-trans-lycopene) is a cis-trans isomerisation with no net change in oxidation state; the reduced flavin acts catalytically, and the enzyme has no desaturase activity. Reason: A fold-level paralog activity that this protein does not have. CRTISO is classified as an isomerase (EC 5.2.1.13) and its GO activity GO:0046608 sits under cis-trans isomerase activity, not under oxidoreductase activity. Purified CRTISO was shown to be an FAD(red)-dependent flavoprotein catalysing a non-redox reaction and to lack desaturase activity. The earlier 'reversible redox' wording in the 2004 in vitro study and in UniProt describes a proposed transient-hydride mechanism, not an oxidoreductase activity on a substrate. Removing this IEA does not contradict any experimental annotation. Supporting Evidence: PMID:21209101 The reduced form of this cofactor catalyzes a reaction not involving net redox changes PMID:21209101 the findings presented place CRTISO into a novel group of flavoproteins catalyzing non-redox reactions with the help of reduced flavins PMID:15557094 However, CRTISO does not possess any desaturase activity file:ARATH/CRTISO/CRTISO-uniprot.txt EC=5.2.1.13; |
| GO:0031969 chloroplast membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Mapped from the UniProt subcellular location 'Plastid, chloroplast membrane; peripheral membrane protein', which UniProt itself records by similarity (ECO:0000250). Membrane association is strongly supported biochemically: purified CRTISO is inactive on micellar substrate and requires a membrane environment (bacterial membranes or liposomes) for catalysis, and its substrates are membrane-embedded hydrocarbons. GO:0031969 denotes the envelope bilayers, the usual site of the plant carotene desaturation enzymes; direct sub-plastid localization data for the Arabidopsis protein are not in the cached literature. Reason: Consistent with UniProt, with the transit peptide, and with the membrane requirement of the enzyme. Accepted as the core site of action while noting that the envelope versus thylakoid assignment for Arabidopsis CRTISO rests on similarity and proteomics rather than on direct imaging. Supporting Evidence: file:ARATH/CRTISO/CRTISO-uniprot.txt SUBCELLULAR LOCATION: Plastid, chloroplast membrane {ECO:0000250}; PMID:15557094 A purified His-tagged form of CRTISO was enzymatically inactive with poly-cis-carotenes when present in Triton X-100 micelles or in liposomes prepared from E. coli lipids or DMPC |
| GO:0046608 carotenoid isomerase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference at node PTN008706876 for the CRTISO/CrtH clade; the Arabidopsis gene's own IMP is one of the descendant evidences and appears in WITH/FROM as expected. The activity is the defining function of the clade (plant and cyanobacterial prolycopene isomerases). Reason: Correct at the right level of specificity; GO:0046608 is defined as catalysis of the isomerisation of poly-cis-carotenoids to all-trans-carotenoids, exactly what Park et al. measured for recombinant Arabidopsis CRTISO and what Rhea 30971 / EC 5.2.1.13 describe. Supporting Evidence: PMID:11884677 enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids |
| GO:0046608 carotenoid isomerase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from IPR014101, the prolycopene isomerase family defined on Arabidopsis CRTISO. Correct activity. Reason: Domain-level inference that coincides with the experimentally demonstrated activity of this very protein. Supporting Evidence: file:ARATH/CRTISO/CRTISO-uniprot.txt Reaction=7,7',9,9'-tetra-cis-lycopene = all-trans-lycopene; |
| GO:0046608 carotenoid isomerase activity | IMP PMID:11884677 Identification of the carotenoid isomerase provides insight ... | ACCEPT | Summary: TAIR annotation from the identification paper: ccr2 mutants accumulate poly-cis carotene precursors in the dark and are lutein-deficient in the light, and CRTISO expressed in E. coli isomerises poly-cis carotenoids to all-trans carotenoids. Later in vitro work on the tomato ortholog defined the regiospecificity (adjacent 7,9 cis pairs of neurosporene and lycopene; not cis-zeta-carotenes) and the FAD(red) requirement. Reason: The core molecular function, established by mutant analysis and recombinant enzyme assay in Arabidopsis and corroborated by purified-enzyme kinetics on the tomato ortholog. Supporting Evidence: PMID:11884677 enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids PMID:15557094 CRTISO isomerizes adjacent cis-double bonds at C7 and C9 pairwise into the trans-configuration, but is incapable of isomerizing single cis-double bonds at C9 and C9β² |
| GO:0046608 carotenoid isomerase activity | IMP PMID:11884677 Identification of the carotenoid isomerase provides insight ... | ACCEPT | Summary: TIGR annotation of the same evidence as the TAIR IMP row above (duplicate row from a second annotating group). Reason: Same evidence and same conclusion as the TAIR row; duplicates with identical term and evidence are acceptable. Supporting Evidence: PMID:11884677 enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids |
| GO:1901177 lycopene biosynthetic process | IMP PMID:11884677 Identification of the carotenoid isomerase provides insight ... | NEW | Summary: Proposed specific process term. CRTISO catalyses the final reaction of lycopene formation in plants, the isomerisation of 7,7',9,9'-tetra-cis-lycopene (prolycopene) to all-trans-lycopene (Rhea 30971; UniProt pathway 'Carotenoid biosynthesis; lycopene biosynthesis'). ccr2 mutants accumulate prolycopene and its poly-cis precursors in the dark, and the recombinant enzyme converts them to the all-trans products. Reason: Participation test passed: CRTISO itself performs a catalytic step of lycopene biosynthesis, not merely a requirement for it. Comparator check passed: Arabidopsis ZDS1, which performs the preceding step of the same route, carries GO:1901177 by IDA, and the carotene backbone module uses GO:1901177 as the concept for the PDS/Z-ISO/ZDS/CRTISO node. GO:1901177 is not an ancestor or descendant of the existing GO:0016117 annotation (in the current ontology it descends from GO:0016120 carotene biosynthetic process, which is not under carotenoid biosynthetic process), so it adds pathway-level precision rather than redundancy. Supporting Evidence: PMID:11884677 The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants PMID:21209101 CRTISO isomerizes all cis double bonds formed by the action of PDS and ZDS ( 14 ) yielding 7,9,9β²,7β²-tetra- cis -lycopene 3 (commonly referred to as prolycopene; 15 ) to finally form all- trans -lycopene PMID:15557094 converting 7,9,9β²-tri-cis-neurosporene to 9β²-cis-neurosporene and 7β²9β²-di-cis-lycopene into all-trans-lycopene file:ARATH/CRTISO/CRTISO-uniprot.txt PATHWAY: Carotenoid biosynthesis; lycopene biosynthesis. |
Loading supporting contentβ¦
Download this section (compressed HTML)Q: What is the physiological reductant that keeps CRTISO-bound FAD reduced inside plastids? In vitro the enzyme depended on the E. coli respiratory chain or on chemical reductants under nitrogen; the plastid electron donor (plastoquinol, NAD(P)H via a membrane dehydrogenase, or another carrier) has not been identified.
Suggested experts: Peter Beyer, Joseph Hirschberg
Q: Where within the plastid is Arabidopsis CRTISO located (envelope inner membrane versus thylakoid), and does it associate physically with ZDS or the cyclases? The current 'chloroplast membrane' annotation rests on similarity and on proteomic fractionation rather than on direct localization of the Arabidopsis protein.
Suggested experts: Barry J. Pogson, Christopher I. Cazzonelli
Q: Do the substrate specificity (strict requirement for adjacent 7,9 cis pairs, no action on cis-zeta-carotenes) and the FAD(red) dependence shown for the tomato enzyme hold quantitatively for Arabidopsis CRTISO, and does the Arabidopsis paralog At1g57770 (CRTISO2) have any isomerase activity in planta?
Suggested experts: Barry J. Pogson, Peter Beyer
Experiment: Express and purify His-tagged mature Arabidopsis CRTISO (residues 57-595), confirm bound FAD by HPLC-MS, and assay it under N2 with dithionite-reduced FAD in phosphatidylcholine liposomes loaded with prolycopene, 7,9,9'-tri-cis-neurosporene and 9,9'-di-cis-zeta-carotene; follow products by C30 reversed-phase HPLC with photodiode-array detection. Include apo-enzyme and oxidized-FAD controls.
Hypothesis: Arabidopsis CRTISO, like the tomato enzyme, is an FAD(red)-dependent non-redox isomerase that acts only on adjacent 7,9 cis pairs.
Type: in vitro enzyme assay with purified recombinant protein
Experiment: Combine confocal imaging of a CRTISO-GFP fusion driven by the native promoter in the ccr2 background (checking complementation of the lutein phenotype) with immunoblotting of purified envelope, stroma and thylakoid fractions from Arabidopsis chloroplasts and of etioplast membranes, using salt and carbonate washes to test peripheral versus integral association.
Hypothesis: CRTISO is a peripheral protein of the chloroplast inner envelope membrane.
Type: subcellular localization by fluorescence microscopy and fractionation
Experiment: Compare etioplast ultrastructure and POR/PChlide levels in ccr2, ccr2 ziso, ccr2 treated with carotenoid cleavage dioxygenase inhibitors, and ccr2 complemented with a catalytically dead CRTISO (dinucleotide-binding-motif mutant); identify the candidate apocarotenoid by LC-MS of etiolated tissue.
Hypothesis: The prolamellar-body defect of ccr2 is caused by a cleavage product of accumulated cis-carotenes rather than by the absence of CRTISO protein or of all-trans carotenoids.
Type: genetic epistasis with electron microscopy and metabolite profiling
Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)