CRTISO (At1g06820, CCR2; UniProt Q9M9Y8) — research notes

Journal for the de-novo review of Arabidopsis thaliana prolycopene isomerase /
carotenoid isomerase. Append, do not rewrite.

1. Identity check

• UniProt Q9M9Y8, CRTSO_ARATH, "Prolycopene isomerase, chloroplastic", EC 5.2.1.13,
  synonyms CCR2 (Carotenoid and chloroplast regulation protein 2), CRTISO; locus
  At1g06820, ORF F4H5.10; 595 aa precursor with a 56-residue chloroplast transit
  peptide [file:ARATH/CRTISO/CRTISO-uniprot.txt "TRANSIT 1..56 /note=\"Chloroplast\"
  /evidence=\"ECO:0007744|PubMed:22223895\""].
• Family: carotenoid/retinoid oxidoreductase family, CrtISO subfamily; PANTHER
  PTHR46313:SF3 "PROLYCOPENE ISOMERASE, CHLOROPLASTIC" (verified in
  interpro/panther/panther.obo; Q9M9Y8 is a listed member of PTHR46313:SF3 in
  interpro/panther/panther-members.tsv). InterPro IPR014101 (CrtISO), IPR045892
  (CrtISO-like), IPR002937 (amine oxidase domain), IPR036188 (FAD/NAD-binding
  superfamily); NCBIfam TIGR02730 carot_isom.
• The GOA rows, the UniProt record and the deep-research report all agree on the
  same locus. There is a distinct Arabidopsis paralog (At1g57770, "CRTISO2") that is
  NOT this gene; nothing in this review should be transferred to or from it.

2. What the cached publications actually contain

PMIDs for the non-GOA papers were resolved by exact-title PubMed esearch and title
confirmed by esummary before fetching with uv run ai-gene-review fetch-pmid.

3. Molecular function

• Genetic and enzymatic identification in Arabidopsis: "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. After positional cloning, enzymatic analysis of CRTISO expressed in
  Escherichia coli confirmed that the enzyme catalyzes the isomerization of
  poly-cis-carotenoids to all-trans-carotenoids." PMID:11884677. This is exactly the
  GO:0046608 definition ("Catalysis of the isomerization of poly-cis-carotenoids to
  all-trans-carotenoids", QuickGO, verified 2026-09-26).
• Reaction: Rhea RHEA:30971 "7,7',9,9'-tetra-cis-lycopene = all-trans-lycopene",
  EC 5.2.1.13 (verified at rhea-db.org). UniProt CATALYTIC ACTIVITY cites
  PMID:11884677 for it [file:ARATH/CRTISO/CRTISO-uniprot.txt].
• Regiospecificity (tomato ortholog, in vitro): "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 C9′" and it converts
  "7,9,9′-tri-cis-neurosporene to 9′-cis-neurosporene and 7′9′-di-cis-lycopene into
  all-trans-lycopene" PMID:15557094. cis-zeta-carotenes are NOT substrates
  ("demonstrating that cis- ζ -carotenes are not substrates of CRTISO"
  PMID:15557094) — that step belongs to Z-ISO.
• Cofactor and mechanism (tomato ortholog, purified enzyme): "FAD is the cofactor
  required by CRTISO" and "it is the reduced form of FAD, partially replaceable by FMN
  red , which drives enzymatic activity of CRTISO" PMID:21209101. Crucially, the
  reaction is NOT a redox reaction: "The reduced form of this cofactor catalyzes a
  reaction not involving net redox changes" PMID:21209101. UniProt lists FAD, NAD(+)
  and NADP(+) as cofactors "by similarity" (ECO:0000250); only FAD is experimentally
  established, and only in the tomato ortholog.
• Membrane requirement: purified 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" unless a membrane-containing lysate was present
  PMID:15557094; Yu et al. assayed it with substrate "embedded into phosphatidylcholine
  liposomal membranes" PMID:21209101.

4. Pathway position

• Plant poly-cis desaturation route: PDS -> Z-ISO -> ZDS -> CRTISO, giving
  all-trans-lycopene for the cyclases. "CRTISO isomerizes all cis double bonds formed by
  the action of PDS and ZDS ( 14 ) yielding 7,9,9′,7′-tetra- cis -lycopene 3 (commonly
  referred to as prolycopene; 15 ) to finally form all- trans -lycopene. The latter is a
  substrate for the subsequent introduction of cyclic β- and/or ϵ-ionone end groups"
  PMID:21209101. Matches modules/carotene_backbone_biosynthesis.yaml (crtiso_step,
  concept GO:1901177 lycopene biosynthetic process; representative member Q9M9Y8).
• Light can substitute: "In the dark, the isomerisation of tri-cis-ζ-carotene to
  di-cis-ζ-carotene and tetra-cis-lycopene to all-trans-lycopene has a strict
  requirement for ZISO and CRTISO activity respectively" but "light-mediated
  photoisomerisation in the presence of a photosensitiser can substitute for a lack of
  isomerase activity" PMID:32003746. Hence ccr2 leaves are lutein-deficient but
  green, while etiolated tissue accumulates prolycopene.
• Tomato ortholog phenotype: "Fruit of tangerine are orange and accumulate prolycopene
  (7Z,9Z,7'Z,9'Z-tetra-cis-lycopene) instead of the all-trans-lycopene" PMID:11884678.
• Ontology note (from the brief, verified by QuickGO ancestors 2026-09-26): GO:1901177
  is_a GO:0016120 carotene biosynthetic process, which is NOT a descendant of GO:0016117
  carotenoid biosynthetic process. This is an ontology structure issue, not something
  to fix in this review; both GO:0016117 (existing) and GO:1901177 (proposed) are
  therefore retained side by side.

5. Localization

• Transit peptide 1-56, cleavage after Ser-56 with N-acetyl-Val-57, from the
  large-scale N-terminal acetylome (PMID:22223895; reflected in UniProt FT lines). The
  body text of that paper does not name CRTISO; the datum is in its supplementary data.
• UniProt SUBCELLULAR LOCATION: "Plastid, chloroplast membrane {ECO:0000250};
  Peripheral membrane protein {ECO:0000250}" — inferred by similarity, not measured for
  Arabidopsis CRTISO. GO:0031969 "chloroplast membrane" is defined as either envelope
  bilayer. The deep-research report cites AT_CHLORO proteomics for an envelope
  assignment; the cached Joyard 2009 record is abstract-only and does not mention
  CRTISO, so I cannot quote it. Membrane association per se is well supported by the
  in vitro requirement for membranes (section 3), and plastid localization by the
  transit peptide and the etioplast phenotype.

6. The etioplast / prolamellar body phenotype (GO:0009662)

• "Etioplasts of dark-grown crtISO mutants accumulate acyclic poly-cis-carotenoids in
  place of cyclic all-trans-xanthophylls and also lack prolamellar bodies (PLBs)... This
  demonstrates a requirement for carotenoid biosynthesis to form the PLB."
  PMID:11884677.
• Mechanism resolved later: "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" and blocking upstream cis-carotene formation (ziso-155) or
  carotenoid cleavage dioxygenase activity "restored PLB formation in ccr2 etioplasts"
  PMID:32003746. So the PLB defect is caused by a cis-carotene-derived apocarotenoid
  signal produced when CRTISO is absent, not by CRTISO doing any of the work of
  etioplast assembly.
• Reasoning on the annotation: the phenotype is genuine, reproducible and mechanistically
  understood, and TAIR curated it with acts_upstream_of_or_within, which is the
  right relationship for an enzyme whose loss perturbs a downstream structure. CRTISO
  does not catalyse or scaffold any step of PLB assembly; it is upstream of it. I keep
  both the IMP and the PAINT IBA as KEEP_AS_NON_CORE (not REMOVE: the assertion is
  correct as an upstream requirement; not ACCEPT: it is not the enzyme's function).
  Comparator check (QuickGO 2026-09-26): among Arabidopsis proteins GO:0009662 is
  carried only by CRTISO, FLN2 and TOC75-4; the ~500 other rows are IBA/IEA
  propagations of the CRTISO clade to plant orthologs. That propagation is a considered
  PAINT judgement (PTN005296160) seeded by the Arabidopsis IMP, and Q9M9Y8 appearing in
  its own WITH/FROM is expected, not circular.

7. Review of the IEA rows

• IPR014101 (CrtISO family) -> GO:0046608 and GO:0016117: entry description is
  specifically the Arabidopsis prolycopene isomerase; both mappings correct. ACCEPT.
• IPR045892 (CrtISO-like family) -> GO:0016116 carotenoid metabolic process: the
  broader family also contains cyanobacterial CrtD (myxoxanthophyll C-3',4'
  desaturase), so InterPro chose the metabolic parent. For CRTISO itself the process is
  biosynthetic; MODIFY to GO:0016117 (already carried by the gene) — not wrong, just
  less specific than the evidence allows.
• IPR002937 (amine oxidase domain) -> GO:0016491 oxidoreductase activity: a fold-level
  mapping. CRTISO shares the FAD-binding amine-oxidase fold with CRTI-type desaturases
  and monoamine oxidases but catalyses a cis-trans isomerization with no net redox
  change PMID:21209101; EC 5.2.1.13 is an isomerase
  class, and GO:0046608 sits under GO:0016859 cis-trans isomerase activity, not under
  GO:0016491. REMOVE as a paralog/fold activity the protein does not have (the brief's
  explicit rule for InterPro2GO rows). Note the older Isaacson 2004 model ("reversible
  redox reaction acting at specific double bonds") is a transient-hydride mechanism
  hypothesis, not evidence of an oxidoreductase activity; UniProt still echoes that
  sentence.
• SL-0053 -> GO:0031969 chloroplast membrane: consistent with UniProt (by similarity)
  and with membrane-dependent catalysis. ACCEPT, flagging that direct sub-plastid
  localization data for Arabidopsis CRTISO are not in the cached literature.

8. NEW proposal

• GO:1901177 lycopene biosynthetic process ("The chemical reactions and pathways
  resulting in the formation of lycopene"; not obsolete, QuickGO 2026-09-26).
  Participation test: CRTISO catalyses the terminal step (prolycopene ->
  all-trans-lycopene; RHEA:30971). Comparator check: Arabidopsis ZDS1 (Q38893) carries
  GO:1901177 by IDA (PMID:9914519) and the module uses GO:1901177 as the concept for the
  whole poly-cis desaturation/isomerization node. UniProt PATHWAY: "Carotenoid
  biosynthesis; lycopene biosynthesis." Not redundant with GO:0016117 because of the
  ontology structure noted in section 4. Evidence: IMP/IDA from PMID:11884677 (mutant
  accumulates poly-cis precursors; recombinant enzyme isomerizes them), corroborated by
  the tomato ortholog in vitro [PMID:15557094, PMID:21209101].
• Considered and NOT proposed: FAD binding (GO:0050660/GO:0071949) — experimentally
  shown only for the tomato enzyme PMID:21209101; UniProt lists it by similarity. Left
  as a suggested experiment rather than an annotation. GO:0016120 carotene biosynthetic
  process — ancestor of GO:1901177, redundant.

9. Regulation (context only, not annotated)

• SDG8/CCR1 histone methyltransferase is required for CRTISO transcription: "The level
  of CRTISO transcripts in ccr1-4 leaf tissue was only 10% that of wild-type leaves"
  and a CRTISO genomic fragment "completely restoring lutein levels by 96% in ccr2-1"
  PMID:19174535. Confirms At1g06820 is the ccr2 gene and that CRTISO can be
  rate-limiting for lutein.

10. Open questions carried into the review

• Physiological reductant that keeps CRTISO-bound FAD reduced in plastids (Yu et al.
  used dithionite/N2; Isaacson et al. used E. coli respiratory chain).
• Sub-plastid membrane (envelope vs thylakoid) for the Arabidopsis protein.
• Whether Arabidopsis CRTISO shares the tomato enzyme's strict 7,9-pair requirement and
  FAD dependence (all in vitro work used the tomato ortholog).