Chloroplastic fructose-1,6-bisphosphatase (cpFBPase, EC 3.1.3.11) from Miscanthus lutarioriparius, a C4 perennial grass in the Poaceae family (Andropogoneae tribe). This enzyme catalyzes the irreversible hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate within the Calvin-Benson-Bassham cycle in the chloroplast stroma. In C4 grasses of the NADP-malic enzyme subtype, the Calvin cycle operates primarily in bundle sheath cell chloroplasts. cpFBPase is a key regulatory enzyme activated by the ferredoxin/thioredoxin system in light and inactivated by oxidation in the dark, preventing futile cycling with phosphofructokinase. It functions as a homotetramer in its inactive form and as a dimer in its active state at the alkaline pH of illuminated stroma. The enzyme requires Mg2+ as a cofactor and belongs to the FBPase class 1 family (HAMAP: MF_01855). This is an unreviewed TrEMBL entry derived from whole genome shotgun sequencing. No direct experimental studies exist for this specific protein; function is inferred from well-characterized orthologs in related C4 grasses (maize, sorghum, sugarcane) and model plants (Arabidopsis, pea, spinach).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0042132
fructose 1,6-bisphosphate 1-phosphatase activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: This is the defining enzymatic activity of cpFBPase - hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate (EC 3.1.3.11). Supported by InterPro domain matches (IPR028343), PANTHER family assignment (PTHR11556:SF1), and HAMAP rule MF_01855. The catalytic reaction is well-characterized in orthologous cpFBPases from multiple plant species.
Reason: Core enzymatic activity. Strong domain-based evidence from multiple sources (InterPro, PANTHER, HAMAP, Pfam PF00316). This is the primary molecular function of the protein.
Supporting Evidence:
PMID:10581254
structure of the oxidized, low-activity form of chloroplastic fructose-1, 6-bisphosphate phosphatase (FBPase), one of the four enzymes of the Calvin cycle whose activity is redox-regulated by light
PMID:16415064
FBPase contributes to the partitioning of the fixed carbon for RuBP regeneration or starch synthesis
|
|
GO:0016791
phosphatase activity
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: This is a parent term of the more specific GO:0042132 (fructose 1,6-bisphosphate 1-phosphatase activity). While technically correct, it is redundant with the more specific annotation already present.
Reason: Redundant with the more specific GO:0042132. The InterPro match (IPR000146, FBPase_class-1) supports the specific FBPase activity, not just generic phosphatase activity. Retaining the general term alongside the specific one adds no information.
|
|
GO:0042578
phosphoric ester hydrolase activity
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: Another parent term of GO:0042132. This InterPro-derived annotation (from IPR020548, Fructose_bisphosphatase_AS active site) is redundant with the more specific FBPase activity annotation.
Reason: Redundant with GO:0042132. The active site match (IPR020548) is specifically the FBPase active site, warranting the specific term, not this general parent.
|
|
GO:0005975
carbohydrate metabolic process
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: Very broad biological process term. While cpFBPase is involved in carbohydrate metabolism, the specific biological process is the reductive pentose-phosphate cycle (Calvin cycle, GO:0019253), which is already annotated in UniProt.
Reason: Too general. The Calvin cycle annotation (GO:0019253) provides the specific biological context. This broad term adds no useful information beyond what the specific term conveys.
|
|
GO:0005985
sucrose metabolic process
|
IEA
GO_REF:0000117 |
MODIFY |
Summary: Sucrose metabolism in plants involves both biosynthesis and degradation. The chloroplastic FBPase contributes indirectly to sucrose synthesis by producing triose-phosphates that are exported to the cytosol, but the direct enzymatic steps of sucrose metabolism occur in the cytosol and are catalyzed by cytosolic FBPase, sucrose-phosphate synthase, and sucrose-phosphate phosphatase. This ARBA-derived annotation does not adequately distinguish between the chloroplastic and cytosolic isoforms.
Reason: The chloroplastic FBPase operates within the Calvin cycle in the chloroplast stroma. While its activity indirectly supports sucrose synthesis by providing carbon skeletons, the enzyme is not directly involved in sucrose metabolic process. The cytosolic FBPase is the isoform directly involved in sucrose biosynthesis. Loss of cytosolic (not chloroplastic) FBPase in rice abolished sucrose synthesis.
Proposed replacements:
reductive pentose-phosphate cycle
Supporting Evidence:
PMID:18811733
OscFBP1 plays a major role in the cytosolic conversion of trioseP to sucrose in leaves during the day
PMID:24317825
Cytosolic FBPase is relatively conserved among various organisms both at amino acid and nucleotide sequence levels. There is slightly higher similarity between mammalian FBPase and plant cytosolic FBPase than there is between the two plant FBPases
|
|
GO:0005986
sucrose biosynthetic process
|
IEA
GO_REF:0000118 |
REMOVE |
Summary: Sucrose biosynthesis is a cytosolic process. The pathway requires export of triose-phosphates from the chloroplast, followed by cytosolic aldolase, cytosolic FBPase, and sucrose-phosphate synthase. The chloroplastic FBPase operates exclusively within the Calvin cycle in the chloroplast stroma and is not directly involved in sucrose biosynthesis.
Reason: Incorrect annotation. Sucrose biosynthesis is catalyzed by the cytosolic FBPase isoform, not the chloroplastic one. This TreeGrafter annotation (PANTHER:PTN004269459) likely resulted from failure to distinguish between chloroplastic and cytosolic FBPase paralogs within the PTHR11556 family. Loss of cytosolic FBPase in rice caused dramatically decreased sucrose levels, confirming the cytosolic isoform is responsible.
Supporting Evidence:
PMID:18811733
OscFBP1 plays a major role in the cytosolic conversion of trioseP to sucrose in leaves during the day...The isolated OscFBP1 mutants exhibited markedly decreased photosynthetic rates and severe growth retardation
PMID:24317825
The cytosolic FBPase is one of the regulatory enzymes in the sucrose biosynthetic pathway
|
|
GO:0006000
fructose metabolic process
|
IEA
GO_REF:0000118 |
MARK AS OVER ANNOTATED |
Summary: While cpFBPase does act on a fructose bisphosphate substrate, this broad term is misleading. The biological context of this reaction is the Calvin cycle, not generic fructose metabolism. The fructose-6-phosphate produced is immediately isomerized to glucose-6-phosphate for RuBP regeneration or starch synthesis within the chloroplast.
Reason: Overly vague and misleading. The enzyme's biological role is in the Calvin cycle (GO:0019253), not in fructose metabolism per se. TreeGrafter annotation that fails to capture the specific biological context.
|
|
GO:0006002
fructose 6-phosphate metabolic process
|
IEA
GO_REF:0000118 |
MARK AS OVER ANNOTATED |
Summary: cpFBPase produces fructose-6-phosphate as its reaction product, so it is technically involved in F6P metabolism. However, this annotation without the Calvin cycle context is uninformative and potentially misleading, as it could imply cytosolic fructose phosphate metabolism.
Reason: Technically true but uninformative without Calvin cycle context. The reductive pentose-phosphate cycle (GO:0019253) annotation captures the specific biological process. This TreeGrafter annotation adds no useful information beyond what GO:0019253 already conveys.
|
|
GO:0006094
gluconeogenesis
|
IEA
GO_REF:0000118 |
REMOVE |
Summary: Gluconeogenesis is a cytosolic/mitochondrial pathway that converts non-carbohydrate precursors to glucose. In plants, the cytosolic FBPase participates in gluconeogenesis, analogous to the mammalian liver enzyme. The chloroplastic FBPase functions exclusively in the Calvin-Benson cycle within the chloroplast and does not participate in gluconeogenesis.
Reason: Incorrect annotation. Gluconeogenesis involves the cytosolic FBPase, not the chloroplastic isoform. The regulation of cpFBPase (thioredoxin-mediated redox regulation) differs fundamentally from the allosteric regulation (by fructose-2,6-bisphosphate and AMP) of the cytosolic/gluconeogenic FBPase. This TreeGrafter error stems from failure to distinguish the two paralogs.
Supporting Evidence:
PMID:24317825
In non-photosynthetic tissues, it regulates the rate of gluconeogenesis
PMID:10581254
regulation of plant FBPases by thiol-disulfide interchange differs in every respect from the regulation of mammalian gluconeogenic FBPases by AMP
|
|
GO:0030388
fructose 1,6-bisphosphate metabolic process
|
IEA
GO_REF:0000120 |
KEEP AS NON CORE |
Summary: cpFBPase hydrolyzes fructose-1,6-bisphosphate as its substrate, so this annotation is technically accurate. However, the specific biological context is the Calvin cycle, and the reductive pentose-phosphate cycle annotation (GO:0019253) is more informative.
Reason: Technically correct as the enzyme acts on FBP, but the Calvin cycle annotation (GO:0019253) captures the biological context more accurately. This is an acceptable secondary annotation but should not be considered a core function.
|
|
GO:0005737
cytoplasm
|
IEA
GO_REF:0000118 |
REMOVE |
Summary: This TreeGrafter annotation incorrectly assigns cytoplasm localization to a chloroplastic protein. The N-terminal transit peptide (~60 residues) directs this protein to the chloroplast stroma.
Reason: Incorrect localization. This protein has a chloroplast transit peptide and is classified as chloroplastic by UniProt (ARBA), HAMAP (MF_01855), and FunFam (3.30.540.10:FF:000014 "Fructose-1,6-bisphosphatase, chloroplastic"). The cytoplasm annotation is a TreeGrafter error from the PANTHER family that includes both chloroplastic and cytosolic isoforms.
Supporting Evidence:
PMID:8980497
Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution
|
|
GO:0005829
cytosol
|
IEA
GO_REF:0000118 |
REMOVE |
Summary: Cytosol localization is incorrect for this chloroplastic FBPase. The cytosolic localization applies to the cytosolic FBPase paralog, not to this chloroplast-targeted enzyme.
Reason: Incorrect localization. Same TreeGrafter isoform confusion as the cytoplasm annotation. In maize (a closely related C4 grass), the chloroplastic FBPase is primarily in bundle sheath cells, while cytosolic FBPase is in mesophyll cytoplasm for sucrose synthesis. The PANTHER family PTHR11556 encompasses both isoforms, leading to incorrect annotation propagation.
Supporting Evidence:
PMID:16663806
There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts...The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells
|
|
GO:0009507
chloroplast
|
IEA
GO_REF:0000044 |
MODIFY |
Summary: Chloroplast localization is correct. The protein has a predicted chloroplast transit peptide and is classified as chloroplastic by multiple domain databases. More specifically, cpFBPase resides in the chloroplast stroma where it operates as part of the Calvin cycle.
Reason: Correct but could be more specific. The enzyme operates in the chloroplast stroma (GO:0009570), which is the site of the Calvin cycle reactions. Stromal localization is supported by immunolocalization of the related cpFBPaseII and by the enzyme's pH-dependent regulation (active at stromal alkaline pH ~8.0 in light).
Proposed replacements:
chloroplast stroma
Supporting Evidence:
PMID:19220782
Immunolocalization experiments and chloroplast isolation confirmed that the new isoenzyme is located in the stroma
PMID:40485148
active cFBP1 is strictly dimeric at pH values occurring in illuminated chloroplasts and...Cys95 is an important determinant of the stromal pH-driven structure and activity of cFBP1
|
Q: Does the Miscanthus lutarioriparius cpFBPase have dual FBPase/SBPase activity like some cyanobacterial homologs, or is it strictly specific for fructose-1,6-bisphosphate?
Q: Is the NCGR_LOCUS1270 locus the only cpFBPase gene in M. lutarioriparius, or are there additional paralogs (e.g., a cpFBPaseII-like redox-independent isoform as described in Arabidopsis)?
Experiment: Express recombinant NCGR_LOCUS1270 protein, confirm FBPase activity in vitro, and test for thioredoxin f-dependent activation. Compare kinetic parameters (Km for FBP, metal ion requirements, pH optimum) with characterized cpFBPases from maize and Arabidopsis.
Hypothesis: NCGR_LOCUS1270 encodes a functional chloroplastic FBPase with thioredoxin-dependent redox regulation.
Experiment: Perform in situ hybridization or cell-type-specific transcriptomic analysis (e.g., laser capture microdissection) of M. lutarioriparius leaves to determine whether NCGR_LOCUS1270 is preferentially expressed in bundle sheath versus mesophyll cells.
Hypothesis: The cpFBPase in Miscanthus is preferentially expressed in bundle sheath cells, consistent with its role in C4 Calvin cycle operation.
Chloroplastic FBPase (cpFBPase, also called cFBP1) catalyzes the irreversible hydrolysis of fructose-1,6-bisphosphate (FBP) to fructose-6-phosphate (F6P) and inorganic phosphate within the Calvin-Benson-Bassham (CBB) cycle. This is one of two irreversible phosphatase reactions in the CBB cycle (the other being catalyzed by sedoheptulose-1,7-bisphosphatase, SBPase).
cpFBPase occupies a critical branch point in the CBB cycle: it controls the partitioning of carbon between RuBP regeneration and output of fixed carbon (as starch within the chloroplast or as triose-phosphate exported for sucrose synthesis in the cytosol) PMID:16415064. Overexpression of cyanobacterial FBPase/SBPase in tobacco chloroplasts increased photosynthetic CO2 fixation 1.24-fold and dry matter 1.5-fold PMID:11581664. Transgenic plants with enhanced chloroplastic FBPase showed increased starch in source leaves and higher RuBP levels PMID:16415064.
The enzyme is essential for photoautotrophic growth. In cyanobacteria, mutants lacking the combined SBP/FBPase protein cannot grow photoautotrophically PMID:36518499. Antisense reduction of chloroplastic FBPase in potato resulted in reduced photosynthetic assimilation rates, with saturation at much lower light intensities PMID:10467036.
The chloroplastic and cytosolic FBPase isoforms are encoded by distinct genes that arose through an ancient gene duplication early in eukaryotic evolution PMID:8980497.
| Feature | Chloroplastic FBPase (cpFBPase/cFBP1) | Cytosolic FBPase (cyFBPase) |
|---|---|---|
| Pathway | Calvin-Benson cycle (CO2 fixation) | Sucrose biosynthesis; gluconeogenesis |
| Localization | Chloroplast stroma | Cytosol |
| Regulation | Redox-regulated by thioredoxin f; light-activated | Allosteric inhibition by fructose-2,6-bisphosphate and AMP |
| Key regulatory feature | Contains "loop 170" with regulatory Cys residues forming disulfide bridge | Lacks redox regulatory domain; regulated like mammalian/yeast FBPase |
| pH optimum | Alkaline (pH ~8.0-8.3, matching illuminated stroma) | Near-neutral |
| Metal requirement | Mg2+ (high concentration, reflecting stromal Mg2+ in light) | Mg2+ (lower concentration) |
The cytosolic FBPase shares higher sequence similarity with mammalian gluconeogenic FBPase than with the chloroplastic isoform from the same plant PMID:24317825. The kinetic and allosteric properties of the plant cytosolic FBPase "are remarkably similar to the mammalian and yeast FBPase, but differ greatly from those of the chloroplastic FBPase" PMID:24317825.
The review by Serrato et al. (2009) provides a comprehensive overview of both isoforms in sugar partitioning, emphasizing their distinct regulatory mechanisms and the consequences of compartmentalization for plant metabolism PMID:19325167.
Sucrose biosynthesis is a cytosolic process. The pathway for sucrose synthesis involves:
1. Export of triose-phosphates from the chloroplast via the triose-phosphate/phosphate translocator
2. Conversion to fructose-1,6-bisphosphate (by aldolase) in the cytosol
3. Dephosphorylation by cytosolic FBPase to fructose-6-phosphate
4. Conversion to glucose-6-phosphate, then glucose-1-phosphate, then UDP-glucose
5. Sucrose-phosphate synthase + sucrose-phosphate phosphatase produce sucrose
The chloroplastic FBPase operates within the chloroplast stroma and is NOT directly involved in sucrose biosynthesis. Loss of cytosolic FBPase in rice caused dramatically decreased sucrose levels and severe growth retardation PMID:18811733. This demonstrates that sucrose biosynthesis depends on the cytosolic isoform, not the chloroplastic one.
The GO annotation GO:0005986 (sucrose biosynthetic process) assigned by TreeGrafter to this chloroplastic FBPase is therefore incorrect -- this annotation likely results from the automated pipeline failing to distinguish between the two isoforms.
cpFBPase is localized to the chloroplast stroma. This has been demonstrated by:
The most specific GO CC term should be GO:0009570 (chloroplast stroma), not just GO:0009507 (chloroplast).
The GO:0005829 (cytosol) annotation from TreeGrafter is INCORRECT for this protein. The cytosolic localization applies to the cytosolic FBPase isoform, not the chloroplastic one. TreeGrafter likely propagated this from an ancestor node that included both isoforms.
Gluconeogenesis is a cytosolic/mitochondrial pathway that converts non-carbohydrate precursors (pyruvate, amino acids, etc.) to glucose. In plants, the cytosolic FBPase participates in gluconeogenesis, analogous to the mammalian liver enzyme PMID:24317825.
The chloroplastic FBPase functions exclusively in the Calvin-Benson cycle within the chloroplast. The structural study of oxidized pea cpFBPase explicitly noted that "regulation of plant FBPases by thiol-disulfide interchange differs in every respect from the regulation of mammalian gluconeogenic FBPases by AMP" PMID:10581254.
The GO annotation GO:0006094 (gluconeogenesis) assigned by TreeGrafter is therefore incorrect for this chloroplastic isoform -- it should only apply to the cytosolic FBPase.
GO:0006000 (fructose metabolic process): This is a very broad term. While cpFBPase does catalyze a reaction involving a fructose derivative (FBP to F6P), the biological process is the Calvin cycle, not "fructose metabolism" per se. The F6P produced by cpFBPase in the Calvin cycle is isomerized to glucose-6-phosphate and then used for RuBP regeneration or starch synthesis. This annotation is overly vague and not informative for a Calvin cycle enzyme.
GO:0006002 (fructose 6-phosphate metabolic process): Technically accurate in that the enzyme produces F6P, but again, using this term without the Calvin cycle context is misleading. The reductive pentose-phosphate cycle (GO:0019253) is the appropriate biological process term.
GO:0030388 (fructose 1,6-bisphosphate metabolic process): Similar -- technically the enzyme acts on FBP, but this is in the context of the Calvin cycle. This is an acceptable secondary annotation but less informative than the primary Calvin cycle annotation.
GO:0005829 (cytosol) is NOT appropriate for this chloroplastic protein. The protein has a clear chloroplast transit peptide (N-terminal ~60 residues), is annotated by UniProt as chloroplast-localized (ARBA), classified in FunFam 3.30.540.10:FF:000014 as "Fructose-1,6-bisphosphatase, chloroplastic", and the HAMAP family MF_01855 (FBPase_class1) covers chloroplastic forms.
The cytosol annotation from TreeGrafter (GO_REF:0000118) is an error from automated phylogenetic propagation. The PANTHER family PTHR11556 includes both chloroplastic and cytosolic FBPases, and the annotation was likely inherited from an ancestral node without proper isoform distinction.
cpFBPase is one of the best-characterized targets of the ferredoxin/thioredoxin (Fd/Trx) system in chloroplasts PMID:18377232.
The regulatory mechanism:
1. Light drives photosynthetic electron transport
2. Reduced ferredoxin is produced by PSI
3. Ferredoxin:thioredoxin reductase (FTR) reduces thioredoxin f
4. Thioredoxin f reduces the regulatory disulfide bridge in cpFBPase (Cys153-Cys173 in pea)
5. Reduction activates the enzyme
In darkness, cpFBPase is oxidized (disulfide formed) and inactive, preventing futile cycling with phosphofructokinase PMID:6260483.
The type-f thioredoxins are the primary reductants for cpFBPase. In Arabidopsis trxf1f2 double mutants, FBPase showed "retarded and incomplete reduction... upon illumination" and carbon fixation activation was delayed PMID:26842981.
The redox states of FBPase and SBPase are linearly correlated with photosynthetic electron transport rates PMID:38305687.
cpFBPase is also regulated by S-nitrosylation in pea, specifically at Cys153. This occurs during the light period and triggers formation of the regulatory disulfide, providing an additional layer of redox regulation PMID:29059554.
Native 2-Cys peroxiredoxin stimulates cpFBPase activity through a non-reductive mechanism requiring both FBP and Ca2+, distinct from the thioredoxin-mediated activation PMID:17307139.
Illumination raises stromal pH from ~7 to ~8, which promotes cpFBPase activation through a conformational transition PMID:6096140. A recent study demonstrated that Cys95 mediates pH-driven structural changes: at pH 8.3 (illuminated stroma), cFBP1 is active as a dimer; at pH 7.0, it forms inactive tetramers PMID:40485148.
CBSX2 participates in the oxidation (inactivation) of cpFBPase during dark transitions PMID:38028645.
In C4 plants (NADP-ME subtype, including Miscanthus, maize, sugarcane, sorghum), the Calvin-Benson cycle operates primarily in the bundle sheath cell chloroplasts. CO2 is initially fixed in mesophyll cells by PEP carboxylase, transported as C4 acids to bundle sheath cells, decarboxylated to release CO2, and this concentrated CO2 is then fixed by Rubisco in the bundle sheath chloroplast CBB cycle.
In maize, chloroplastic FBPase is primarily located in bundle sheath cells, with large (5-fold) light activation observed in isolated bundle sheath strands versus little light activation in mesophyll protoplasts PMID:16663806. In mesophyll protoplasts, FBPase was largely cytoplasmic (cytosolic isoform for sucrose synthesis).
Miscanthus x giganteus and related species (including M. lutarioriparius) are NADP-malic enzyme subtype C4 grasses PMID:20693355. The C4 photosynthesis model has been parameterized for this group PMID:34173821.
Photosynthetic efficiency in Miscanthus has been studied in the context of chilling tolerance, light responses, and bundle sheath conductance [PMID:26714623, PMID:22812384], though these studies focused on PEPCase, Rubisco, and PPDK rather than cpFBPase specifically.
No publications were found specifically about chloroplastic FBPase in M. lutarioriparius. The protein A0A811M8A5 derives from the whole genome shotgun sequence of M. lutarioriparius submitted by Han et al. (2020) to EMBL/GenBank/DDBJ databases.
Transcriptomic studies of M. lutarioriparius exist but focus on rhizome development PMID:28446913 and secondary cell wall biosynthesis PMID:28831170, not on Calvin cycle enzymes.
Given the close phylogenetic relationship of Miscanthus to maize, sorghum, and sugarcane (all Andropogoneae), the functional properties of cpFBPase can be reasonably inferred from studies in these related C4 grasses.
Based on articles retrieved from PubMed:
id: A0A811M8A5
gene_symbol: NCGR_LOCUS1270
taxon:
id: NCBITaxon:422564
label: Miscanthus lutarioriparius
description: >-
Chloroplastic fructose-1,6-bisphosphatase (cpFBPase, EC 3.1.3.11) from Miscanthus
lutarioriparius, a C4 perennial grass in the Poaceae family (Andropogoneae tribe).
This enzyme catalyzes the irreversible hydrolysis of fructose-1,6-bisphosphate to
fructose-6-phosphate and inorganic phosphate within the Calvin-Benson-Bassham cycle
in the chloroplast stroma. In C4 grasses of the NADP-malic enzyme subtype, the Calvin
cycle operates primarily in bundle sheath cell chloroplasts. cpFBPase is a key
regulatory enzyme activated by the ferredoxin/thioredoxin system in light and
inactivated by oxidation in the dark, preventing futile cycling with
phosphofructokinase. It functions as a homotetramer in its inactive form and as a
dimer in its active state at the alkaline pH of illuminated stroma. The enzyme
requires Mg2+ as a cofactor and belongs to the FBPase class 1 family (HAMAP:
MF_01855). This is an unreviewed TrEMBL entry derived from whole genome shotgun
sequencing. No direct experimental studies exist for this specific protein; function
is inferred from well-characterized orthologs in related C4 grasses (maize, sorghum,
sugarcane) and model plants (Arabidopsis, pea, spinach).
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through InterPro
- id: GO_REF:0000044
title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular
Location vocabulary mapping
- id: GO_REF:0000117
title: Electronic Gene Ontology annotations created by ARBA machine learning
models
- id: GO_REF:0000118
title: Gene Ontology annotation by TreeGrafter
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
- id: PMID:18377232
title: The ferredoxin/thioredoxin system of oxygenic photosynthesis
- id: PMID:8980497
title: Higher-plant chloroplast and cytosolic fructose-1,6-bisphosphatase
isoenzymes origins via duplication rather than prokaryote-eukaryote divergence
- id: PMID:16415064
title: Contribution of fructose-1,6-bisphosphatase and
sedoheptulose-1,7-bisphosphatase to the photosynthetic rate and carbon flow
in the Calvin cycle in transgenic plants
- id: PMID:16663806
title: Activation of NADP-Malate Dehydrogenase, Pyruvate Pi Dikinase, and
Fructose 1,6-Bisphosphatase in Relation to Photosynthetic Rate in Maize
- id: PMID:18811733
title: "Loss of cytosolic fructose-1,6-bisphosphatase limits photosynthetic sucrose synthesis and causes severe growth retardations in rice (Oryza sativa)."
- id: PMID:24317825
title: Cytosolic fructose-1,6-bisphosphatase A key enzyme in the sucrose
biosynthetic pathway
- id: PMID:10581254
title: Redox signalling in the chloroplast structure of oxidized pea
fructose-1,6-bisphosphate phosphatase
- id: PMID:19325167
title: 'Changing sugar partitioning in FBPase-manipulated plants.'
- id: PMID:19220782
title: cpFBPaseII a novel redox-independent chloroplastic isoform of
fructose-1,6-bisphosphatase
- id: PMID:40485148
title: The Highly Conserved Cys95 Residue of Fructose-1,6-Bisphosphatase 1
Mediates the pH-Driven Structure and Activity of the Enzyme and Photosynthesis
- id: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-notes.md
title: Research notes for NCGR_LOCUS1270 cpFBPase
existing_annotations:
- term:
id: GO:0042132
label: fructose 1,6-bisphosphate 1-phosphatase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
This is the defining enzymatic activity of cpFBPase - hydrolysis of
fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate
(EC 3.1.3.11). Supported by InterPro domain matches (IPR028343), PANTHER
family assignment (PTHR11556:SF1), and HAMAP rule MF_01855. The catalytic
reaction is well-characterized in orthologous cpFBPases from multiple plant
species.
action: ACCEPT
reason: >-
Core enzymatic activity. Strong domain-based evidence from multiple sources
(InterPro, PANTHER, HAMAP, Pfam PF00316). This is the primary molecular
function of the protein.
supported_by:
- reference_id: PMID:10581254
supporting_text: >-
structure of the oxidized, low-activity form of chloroplastic
fructose-1, 6-bisphosphate phosphatase (FBPase), one of the four
enzymes of the Calvin cycle whose activity is redox-regulated by light
- reference_id: PMID:16415064
supporting_text: >-
FBPase contributes to the partitioning of the fixed carbon for RuBP
regeneration or starch synthesis
- term:
id: GO:0016791
label: phosphatase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This is a parent term of the more specific GO:0042132 (fructose 1,6-bisphosphate
1-phosphatase activity). While technically correct, it is redundant with the more
specific annotation already present.
action: MARK_AS_OVER_ANNOTATED
reason: >-
Redundant with the more specific GO:0042132. The InterPro match (IPR000146,
FBPase_class-1) supports the specific FBPase activity, not just generic
phosphatase activity. Retaining the general term alongside the specific one
adds no information.
- term:
id: GO:0042578
label: phosphoric ester hydrolase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
Another parent term of GO:0042132. This InterPro-derived annotation (from
IPR020548, Fructose_bisphosphatase_AS active site) is redundant with the
more specific FBPase activity annotation.
action: MARK_AS_OVER_ANNOTATED
reason: >-
Redundant with GO:0042132. The active site match (IPR020548) is specifically
the FBPase active site, warranting the specific term, not this general parent.
- term:
id: GO:0005975
label: carbohydrate metabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
Very broad biological process term. While cpFBPase is involved in carbohydrate
metabolism, the specific biological process is the reductive pentose-phosphate
cycle (Calvin cycle, GO:0019253), which is already annotated in UniProt.
action: MARK_AS_OVER_ANNOTATED
reason: >-
Too general. The Calvin cycle annotation (GO:0019253) provides the specific
biological context. This broad term adds no useful information beyond what
the specific term conveys.
- term:
id: GO:0005985
label: sucrose metabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000117
review:
summary: >-
Sucrose metabolism in plants involves both biosynthesis and degradation.
The chloroplastic FBPase contributes indirectly to sucrose synthesis by
producing triose-phosphates that are exported to the cytosol, but the direct
enzymatic steps of sucrose metabolism occur in the cytosol and are catalyzed
by cytosolic FBPase, sucrose-phosphate synthase, and sucrose-phosphate
phosphatase. This ARBA-derived annotation does not adequately distinguish
between the chloroplastic and cytosolic isoforms.
action: MODIFY
reason: >-
The chloroplastic FBPase operates within the Calvin cycle in the chloroplast
stroma. While its activity indirectly supports sucrose synthesis by providing
carbon skeletons, the enzyme is not directly involved in sucrose metabolic
process. The cytosolic FBPase is the isoform directly involved in sucrose
biosynthesis. Loss of cytosolic (not chloroplastic) FBPase in rice abolished
sucrose synthesis.
proposed_replacement_terms:
- id: GO:0019253
label: reductive pentose-phosphate cycle
supported_by:
- reference_id: PMID:18811733
supporting_text: >-
OscFBP1 plays a major role in the cytosolic conversion of trioseP to
sucrose in leaves during the day
- reference_id: PMID:24317825
supporting_text: >-
Cytosolic FBPase is relatively conserved among various organisms both
at amino acid and nucleotide sequence levels. There is slightly higher
similarity between mammalian FBPase and plant cytosolic FBPase than
there is between the two plant FBPases
- term:
id: GO:0005986
label: sucrose biosynthetic process
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: >-
Sucrose biosynthesis is a cytosolic process. The pathway requires export of
triose-phosphates from the chloroplast, followed by cytosolic aldolase,
cytosolic FBPase, and sucrose-phosphate synthase. The chloroplastic FBPase
operates exclusively within the Calvin cycle in the chloroplast stroma and
is not directly involved in sucrose biosynthesis.
action: REMOVE
reason: >-
Incorrect annotation. Sucrose biosynthesis is catalyzed by the cytosolic
FBPase isoform, not the chloroplastic one. This TreeGrafter annotation
(PANTHER:PTN004269459) likely resulted from failure to distinguish between
chloroplastic and cytosolic FBPase paralogs within the PTHR11556 family.
Loss of cytosolic FBPase in rice caused dramatically decreased sucrose
levels, confirming the cytosolic isoform is responsible.
supported_by:
- reference_id: PMID:18811733
supporting_text: >-
OscFBP1 plays a major role in the cytosolic conversion of trioseP to
sucrose in leaves during the day...The isolated OscFBP1 mutants
exhibited markedly decreased photosynthetic rates and severe growth
retardation
- reference_id: PMID:24317825
supporting_text: >-
The cytosolic FBPase is one of the regulatory enzymes in the sucrose
biosynthetic pathway
- term:
id: GO:0006000
label: fructose metabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: >-
While cpFBPase does act on a fructose bisphosphate substrate, this broad
term is misleading. The biological context of this reaction is the Calvin
cycle, not generic fructose metabolism. The fructose-6-phosphate produced
is immediately isomerized to glucose-6-phosphate for RuBP regeneration or
starch synthesis within the chloroplast.
action: MARK_AS_OVER_ANNOTATED
reason: >-
Overly vague and misleading. The enzyme's biological role is in the Calvin
cycle (GO:0019253), not in fructose metabolism per se. TreeGrafter
annotation that fails to capture the specific biological context.
- term:
id: GO:0006002
label: fructose 6-phosphate metabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: >-
cpFBPase produces fructose-6-phosphate as its reaction product, so it is
technically involved in F6P metabolism. However, this annotation without
the Calvin cycle context is uninformative and potentially misleading, as
it could imply cytosolic fructose phosphate metabolism.
action: MARK_AS_OVER_ANNOTATED
reason: >-
Technically true but uninformative without Calvin cycle context. The
reductive pentose-phosphate cycle (GO:0019253) annotation captures the
specific biological process. This TreeGrafter annotation adds no useful
information beyond what GO:0019253 already conveys.
- term:
id: GO:0006094
label: gluconeogenesis
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: >-
Gluconeogenesis is a cytosolic/mitochondrial pathway that converts
non-carbohydrate precursors to glucose. In plants, the cytosolic FBPase
participates in gluconeogenesis, analogous to the mammalian liver enzyme.
The chloroplastic FBPase functions exclusively in the Calvin-Benson cycle
within the chloroplast and does not participate in gluconeogenesis.
action: REMOVE
reason: >-
Incorrect annotation. Gluconeogenesis involves the cytosolic FBPase, not
the chloroplastic isoform. The regulation of cpFBPase (thioredoxin-mediated
redox regulation) differs fundamentally from the allosteric regulation
(by fructose-2,6-bisphosphate and AMP) of the cytosolic/gluconeogenic
FBPase. This TreeGrafter error stems from failure to distinguish the two
paralogs.
supported_by:
- reference_id: PMID:24317825
supporting_text: >-
In non-photosynthetic tissues, it regulates the rate of
gluconeogenesis
- reference_id: PMID:10581254
supporting_text: >-
regulation of plant FBPases by thiol-disulfide interchange differs in
every respect from the regulation of mammalian gluconeogenic FBPases
by AMP
- term:
id: GO:0030388
label: fructose 1,6-bisphosphate metabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
cpFBPase hydrolyzes fructose-1,6-bisphosphate as its substrate, so this
annotation is technically accurate. However, the specific biological context
is the Calvin cycle, and the reductive pentose-phosphate cycle annotation
(GO:0019253) is more informative.
action: KEEP_AS_NON_CORE
reason: >-
Technically correct as the enzyme acts on FBP, but the Calvin cycle annotation
(GO:0019253) captures the biological context more accurately. This is an
acceptable secondary annotation but should not be considered a core function.
- term:
id: GO:0005737
label: cytoplasm
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: >-
This TreeGrafter annotation incorrectly assigns cytoplasm localization to
a chloroplastic protein. The N-terminal transit peptide (~60 residues)
directs this protein to the chloroplast stroma.
action: REMOVE
reason: >-
Incorrect localization. This protein has a chloroplast transit peptide
and is classified as chloroplastic by UniProt (ARBA), HAMAP (MF_01855),
and FunFam (3.30.540.10:FF:000014 "Fructose-1,6-bisphosphatase,
chloroplastic"). The cytoplasm annotation is a TreeGrafter error from the
PANTHER family that includes both chloroplastic and cytosolic isoforms.
supported_by:
- reference_id: PMID:8980497
supporting_text: >-
Chloroplast and cytosolic FBP isoenzymes of higher plants arose
through a gene duplication event which occurred early in eukaryotic
evolution
- term:
id: GO:0005829
label: cytosol
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: >-
Cytosol localization is incorrect for this chloroplastic FBPase. The
cytosolic localization applies to the cytosolic FBPase paralog, not to
this chloroplast-targeted enzyme.
action: REMOVE
reason: >-
Incorrect localization. Same TreeGrafter isoform confusion as the cytoplasm
annotation. In maize (a closely related C4 grass), the chloroplastic FBPase
is primarily in bundle sheath cells, while cytosolic FBPase is in mesophyll
cytoplasm for sucrose synthesis. The PANTHER family PTHR11556 encompasses
both isoforms, leading to incorrect annotation propagation.
supported_by:
- reference_id: PMID:16663806
supporting_text: >-
There was a large (5-fold) light activation of FBPase in isolated
bundle sheath strands of maize, whereas there was little light
activation of the enzyme in isolated mesophyll protoplasts...The
results suggest the chloroplastic FBPase in maize is primarily located
in the bundle sheath cells
- term:
id: GO:0009507
label: chloroplast
evidence_type: IEA
original_reference_id: GO_REF:0000044
review:
summary: >-
Chloroplast localization is correct. The protein has a predicted
chloroplast transit peptide and is classified as chloroplastic by multiple
domain databases. More specifically, cpFBPase resides in the chloroplast
stroma where it operates as part of the Calvin cycle.
action: MODIFY
reason: >-
Correct but could be more specific. The enzyme operates in the chloroplast
stroma (GO:0009570), which is the site of the Calvin cycle reactions.
Stromal localization is supported by immunolocalization of the related
cpFBPaseII and by the enzyme's pH-dependent regulation (active at stromal
alkaline pH ~8.0 in light).
proposed_replacement_terms:
- id: GO:0009570
label: chloroplast stroma
supported_by:
- reference_id: PMID:19220782
supporting_text: >-
Immunolocalization experiments and chloroplast isolation confirmed
that the new isoenzyme is located in the stroma
- reference_id: PMID:40485148
supporting_text: >-
active cFBP1 is strictly dimeric at pH values occurring in
illuminated chloroplasts and...Cys95 is an important determinant of
the stromal pH-driven structure and activity of cFBP1
core_functions:
- description: >-
Chloroplastic fructose-1,6-bisphosphatase catalyzes the hydrolysis of
fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate
as part of the Calvin-Benson-Bassham cycle in the chloroplast stroma. This
is the primary evolved function of this enzyme, operating at a critical
branch point that controls partitioning of fixed carbon between RuBP
regeneration and carbon output for starch synthesis. The enzyme is
light-activated via the ferredoxin/thioredoxin system and requires Mg2+.
In C4 grasses like Miscanthus, this activity occurs primarily in bundle
sheath cell chloroplasts.
molecular_function:
id: GO:0042132
label: fructose 1,6-bisphosphate 1-phosphatase activity
directly_involved_in:
- id: GO:0019253
label: reductive pentose-phosphate cycle
locations:
- id: GO:0009570
label: chloroplast stroma
supported_by:
- reference_id: PMID:16415064
supporting_text: >-
FBPase contributes to the partitioning of the fixed carbon for RuBP
regeneration or starch synthesis
- reference_id: PMID:18377232
supporting_text: >-
ferredoxin (Fdx) was shown to activate fructose 1,6-bisphosphatase in
illuminated chloroplast preparations, thereby laying the foundation for
the field now known as "redox biology."
- reference_id: PMID:16663806
supporting_text: >-
The results suggest the chloroplastic FBPase in maize is primarily
located in the bundle sheath cells
suggested_questions:
- question: >-
Does the Miscanthus lutarioriparius cpFBPase have dual FBPase/SBPase
activity like some cyanobacterial homologs, or is it strictly specific
for fructose-1,6-bisphosphate?
experts: []
- question: >-
Is the NCGR_LOCUS1270 locus the only cpFBPase gene in M. lutarioriparius,
or are there additional paralogs (e.g., a cpFBPaseII-like redox-independent
isoform as described in Arabidopsis)?
experts: []
suggested_experiments:
- hypothesis: >-
NCGR_LOCUS1270 encodes a functional chloroplastic FBPase with
thioredoxin-dependent redox regulation.
description: >-
Express recombinant NCGR_LOCUS1270 protein, confirm FBPase activity in
vitro, and test for thioredoxin f-dependent activation. Compare kinetic
parameters (Km for FBP, metal ion requirements, pH optimum) with
characterized cpFBPases from maize and Arabidopsis.
- hypothesis: >-
The cpFBPase in Miscanthus is preferentially expressed in bundle sheath
cells, consistent with its role in C4 Calvin cycle operation.
description: >-
Perform in situ hybridization or cell-type-specific transcriptomic analysis
(e.g., laser capture microdissection) of M. lutarioriparius leaves to
determine whether NCGR_LOCUS1270 is preferentially expressed in bundle
sheath versus mesophyll cells.
status: COMPLETE