PSY (PSY1, At5g17230, UniProt P37271) - research notes

Arabidopsis thaliana phytoene synthase 1, chloroplastic. Single-copy PSY in
Arabidopsis; EC 2.5.1.32; module exemplar in
modules/carotene_backbone_biosynthesis.yaml (part 1, psy_activity,
PANTHER:PTHR31480).

1. Identity and what UniProt says

• Precursor of 422 aa with a predicted chloroplast transit peptide 1-70
  [file:ARATH/PSY/PSY-uniprot.txt "TRANSIT 1..70 /note=\"Chloroplast\""].
• Function: "Catalyzes the reaction from prephytoene diphosphate to phytoene"
  (evidence PubMed:25675505) [file:ARATH/PSY/PSY-uniprot.txt].
• Catalytic activities (all ECO:0000269|PubMed:25675505): overall
  RHEA:34475 (2 GGPP = 15-cis-phytoene + 2 diphosphate, EC 2.5.1.32); half
  reactions RHEA:22296 (2 GGPP -> prephytoene diphosphate + PPi) and
  RHEA:34479 (prephytoene diphosphate -> 15-cis-phytoene + PPi)
  [file:ARATH/PSY/PSY-uniprot.txt "Xref=Rhea:RHEA:34475, ChEBI:CHEBI:27787,"].
• Subunit: monomer (by similarity); interacts with OR (PubMed:25675505,
  PubMed:26224804) and ORLIKE (PubMed:25675505).
• Subcellular location: "Plastid, chloroplast membrane"; peripheral membrane
  protein [file:ARATH/PSY/PSY-uniprot.txt "SUBCELLULAR LOCATION: Plastid,
  chloroplast membrane"].
• Family: "Belongs to the phytoene/squalene synthase family."
  InterPro: IPR002060 Squalene/phytoene synthase (family), IPR019845
  Squalene/phytoene synthase conserved site, IPR033904 Trans-isoprenyl
  diphosphate synthases head-to-head (domain), IPR044843 Trans-isoprenyl
  diphosphate synthases bacterial-type (family), IPR008949 isoprenoid
  synthase domain superfamily. PANTHER PTHR31480 "BIFUNCTIONAL LYCOPENE
  CYCLASE/PHYTOENE SYNTHASE" (family name dominated by the fungal
  bifunctional members; plant PSY is single-domain).
• Original cDNA: Scolnik & Bartley 1994 PMID:8016277 - cached record is title-only
  (no abstract text available), so it can only be cited for the cloning.

2. Cached publications: what each actually shows

PMID:25675505 (Zhou et al. 2015 PNAS) - full text available

The key experimental paper behind the IDA rows (GO:0046905, GO:0016117,
GO:0009507) and the IPI rows (protein binding with OR Q8VYD8 and OR-like
Q9FKF4).

• Pathway position: PMID:25675505.
• Localization: PSY identified as an OR co-IP partner; PMID:25675505; BiFC of OR-PSY: PMID:25675505.
• Direct enzyme assay on isolated Arabidopsis chloroplast membranes:
  PMID:25675505. The assay design is itself informative
  for the InterPro-derived GGPP synthase row: PMID:25675505 - i.e. PSY does not itself make
  GGPP.
• In vivo activity: PMID:25675505.
• Interaction: split-ubiquitin Y2H, co-IP in N. benthamiana, BiFC:
  PMID:25675505; OR/OR-like control PSY protein abundance post-
  transcriptionally: PMID:25675505.
• The paper explicitly declines to call OR a chaperone: PMID:25675505 - so GO:0051087 chaperone binding is NOT supported by this paper
  as a MODIFY target for the protein-binding rows.

PMID:26224804 (Yuan et al. 2015 Plant Physiol) - fetched this session, full text

Cited by UniProt for interaction with OR and subcellular location. Confirms
AtOR(His) variant also interacts with PSY by BiFC: PMID:26224804. Pathway statement: PMID:26224804. Not in GOA; used as a corroborating reference.

PMID:37966976 (Iglesias-Sanchez et al. 2024 Plant Physiol) - full text available

Behind the IPI (protein binding with FBN6, AT5G19940) and IDA chloroplast rows.

• Interaction: FBN6 pulled down with PSY-GFP (Welsch et al. 2018), confirmed by
  co-IP in N. benthamiana; PMID:37966976.
• Localization: PMID:37966976; PMID:37966976. Importantly the paper notes that FBN6 is NOT a plastoglobule
  protein but is in thylakoid and envelope membranes, and that PSY is found in
  envelope proteomes: PMID:37966976. This bears on the plastoglobule IDA (below).
• FBN6 stimulates plant PSY catalysis without changing PSY amount:
  PMID:37966976; in planta PMID:37966976.
  Conclusion: PMID:37966976.
• The E. coli complementation (pAC-85b lacking crtB, rescued by His-tagged
  Arabidopsis PSY lacking its transit peptide) is an independent demonstration
  that P37271 has phytoene synthase activity.

PMID:23023170 (Shumskaya et al. 2012 Plant Cell) - full text available

Behind the plastoglobule IDA (GO:0010287).

• Method: transient expression of fluorescent-protein fusions in leaf
  protoplasts; At-PSY-RFP was expressed in cowpea (dicot) protoplasts, i.e. a
  heterologous host, not Arabidopsis. PMID:23023170.
• Result: PMID:23023170; speckles identified as plastoglobuli by colocalization with a
  maize fibrillin (shown directly for Zm-PSY2/3): PMID:23023170; the authors extend the conclusion to
  Arabidopsis: PMID:23023170.
• Caveat recorded by the authors themselves: PMID:23023170.
• Assessment: genuine IDA, but from overexpressed fusion protein in a
  heterologous protoplast; the FBN6 paper shows an identical punctate pattern
  for a partner that proteomics places in envelope/thylakoid membranes rather
  than plastoglobules, and envelope proteomics detects PSY. I therefore keep
  the plastoglobule row but as non-core, and treat "chloroplast membrane"
  (envelope / thylakoid-associated, peripheral) as the functional location.
  The sub-plastid site is a genuine open question (see suggested_questions).

PMID:12805607 (Lindgren et al. 2003 Plant Physiol) - full text available

Behind the IMP GO:0016117 and TAS GO:0046905 rows.

• The gene overexpressed is the endogenous Arabidopsis PSY cloned by RT-PCR
  homologous to the Scolnik & Bartley cDNA, i.e. At5g17230/P37271:
  PMID:12805607.
• TAS statement: PMID:12805607.
• Phenotype (napA seed-specific overexpression): 43-fold beta-carotene,
  increased lutein, violaxanthin, lycopene, alpha-carotene, chlorophyll, ABA;
  delayed germination PMID:12805607. Gain-of-function rather than loss-of-function, but a
  clean demonstration that PSY flux drives carotenoid biosynthesis in planta.
  The germination/ABA effects are downstream consequences (xanthophyll ->
  ABA) and are not annotated to PSY; I do not propose them.

PMID:18431481 (Zybailov et al. 2008 PLoS One) - full text available

Behind the HDA chloroplast row. Large-scale chloroplast proteome
PMID:18431481. The cached
text (main body) does not name At5g17230; the identification list is in
supplementary tables not in the cache. The HDA is consistent with all direct
evidence, so ACCEPT, deferring to the TAIR curator for the table entry.

3. GO_REF / electronic rows

• GO_REF:0000002 InterPro2GO:
• IPR044843 "Trans-isoprenyl diphosphate synthases, bacterial-type" ->
  GO:0004311 GGPP synthase activity. Verified on the InterPro API this
  session: the entry's GO mapping is GO:0004311. PSY consumes GGPP
  (head-to-head condensation of two C20 units); it does not perform the
  head-to-tail FPP + IPP -> GGPP elongation. Direct experimental argument:
  the Arabidopsis PSY assay required exogenous GGPP synthase
  PMID:25675505. REMOVE.
• IPR033904 "Trans-isoprenyl diphosphate synthases, head-to-head" ->
  GO:0051996 squalene synthase [NAD(P)H] activity. The domain is shared by
  squalene synthase and phytoene synthase (same fold, same head-to-head
  chemistry), but squalene synthase condenses two FPP and reduces the
  presqualene intermediate with NAD(P)H; PSY condenses two GGPP and releases
  phytoene without a reductive step (RHEA:34475 has no NAD(P)H). The mapping
  assigns the paralog's activity. REMOVE.
• IPR019845 "Squalene/phytoene synthase, conserved site" -> GO:0016765
  transferase activity, transferring alkyl or aryl (other than methyl)
  groups. Correct parent of EC 2.5.1.32 (GO:0046905). KEEP_AS_NON_CORE as a
  generic grandparent.
• GO_REF:0000033 IBA (PANTHER:PTN000770577): GO:0046905 and GO:0016117.
  The target AT5G17230 appears in its own WITH/FROM, which is expected (its
  IDA is one of the descendant evidences for the IBD). Donors include the
  Neurospora al-2 P37295, Synechococcus/cyanobacterial and plant PSYs. Node
  placement is sound: phytoene synthase activity is the ancestral function of
  the CrtB/PSY family. ACCEPT both.
• GO_REF:0000108 inter-ontology inference: GO:0046905 -> GO:0016120 carotene
  biosynthetic process. Phytoene is a C40 hydrocarbon carotene, so this is
  correct. Note (per brief): GO:0016120 is not currently a descendant of
  GO:0016117; GO:1901174 phytoene biosynthetic process IS a descendant of
  GO:0016120 (checked via QuickGO ancestors this session) and is the most
  precise process for core_functions. Not proposed as NEW because it is a
  descendant of a carried term (redundancy rule).
• GO_REF:0000120 (ARBA00027384 / RHEA:34475 / EC 2.5.1.32) -> GO:0046905:
  correct. ACCEPT.
• GO_REF:0000044 UniProt SubCell SL-0053 -> GO:0031969 chloroplast membrane:
  matches UniProt "Plastid, chloroplast membrane; Peripheral membrane
  protein" and the membrane-bound activity in PMID:25675505. ACCEPT.
• GO_REF:0000122 AtSubP -> GO:0009507 chloroplast: sequence-based prediction
  consistent with the transit peptide and with all IDA evidence. ACCEPT.

4. Protein-binding rows (GO:0005515)

Three IPI rows (OR, OR-like, FBN6). All three interactions are real and
biologically important, but on the PSY side they describe PSY being
regulated (stabilised by OR/OR-like; catalytically stimulated by FBN6). No
more informative MF for PSY is supported: the OR paper explicitly says OR is
not a molecular chaperone, so "chaperone binding" is not licensed, and there
is no GO MF for "being an activated enzyme". Per policy: REMOVE as
uninformative; the informative annotations belong on OR/OR-like and FBN6
(enzyme regulator / activator side). Removal does not deny the interactions.

5. Deep research report (retrieval support only)

PSY-deep-research-falcon.md points to additional non-GOA literature I did
not cache: Alvarez et al. 2016 (5'UTR splice variants controlling PSY
translation), Welsch et al. 2018 (Clp protease and OR control PSY
proteostasis; PSY-GFP co-IP that first found FBN6), Maass et al. 2009
(constitutive AtPSY overexpression: leaves unchanged, roots/callus with
carotenoid crystals), Zhou et al. 2022 review, and a 2023 preprint on
hypomorphic PSY variants and a cis-carotene-derived signal. None of these
are used for identifiers or quotes here; they inform the description and the
suggested questions.

6. Decisions summary

No NEW annotations proposed. GO:1901174 phytoene biosynthetic process (a
descendant of the carried GO:0016120) is the most precise process term, but per
the redundancy rule it is neither proposed as NEW nor placed in core_functions;
core_functions uses the two carried process terms GO:0016117 and GO:0016120 and
names phytoene biosynthesis in its description.