NCGR_LOCUS1270 is a predicted chloroplast fructose-1,6-bisphosphatase from the C4 grass Miscanthus lutarioriparius. It hydrolyzes fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate, supporting carbon regeneration and partitioning in the Calvin-Benson-Bassham cycle. Conserved sequence features and close grass homologs support stromal localization and light-dependent regulation through the ferredoxin/thioredoxin system. The enzyme belongs to the class1 FBPase family and uses magnesium as a cofactor.
| 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 | ACCEPT | Summary: The FBPase reaction is a phosphoric monoester hydrolysis, so phosphatase activity is correct. Reason: The specific FBPase activity does not invalidate this independently sourced broad InterPro annotation. ACCEPT reflects the same core catalytic function at broader granularity. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-notes.md The defining FBPase reaction hydrolyzes fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. |
| GO:0042578 phosphoric ester hydrolase activity | IEA GO_REF:0000002 | ACCEPT | Summary: Hydrolysis of fructose 1,6-bisphosphate is phosphoric ester hydrolysis. Reason: The broad InterPro activity is entailed by the defining enzymatic reaction. Coexistence with the more specific GO:0042132 is not over-annotation. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-notes.md The defining FBPase reaction hydrolyzes fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. |
| GO:0005975 carbohydrate metabolic process | IEA GO_REF:0000002 | MODIFY | Summary: Refine carbohydrate metabolism to the reductive pentose-phosphate cycle supported by the chloroplast FBPase identity. Reason: The chloroplast FBPase itself performs the fructose-1,6-bisphosphate hydrolysis step during Calvin-cycle carbon regeneration, rather than being a substrate or an indirect requirement. The close chloroplast-associated sorghum homolog, conserved catalytic architecture and comparative cpFBPase studies support this inherited pathway role. GO:0019253 is a descendant of GO:0005975 in live QuickGO; refining this broad row makes the already synthesized core process explicit without proposing a redundant NEW annotation. Conditional additional carbohydrate pathways remain separately assessed. Proposed replacements: reductive pentose-phosphate cycle Supporting Evidence: PMID:16415064 FBPase contributes to the partitioning of the fixed carbon for RuBP regeneration or starch synthesis PMID:18377232 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." PMID:16663806 The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells |
| GO:0005985 sucrose metabolic process | IEA GO_REF:0000117 | UNDECIDED | Summary: The chloroplast role does not resolve whether the broader sucrose-process annotation is warranted. Reason: Chloroplast FBPase supplies and regulates carbon partitioning, whereas the best-established sucrose-synthesis reactions use the cytosolic paralog. Supplying precursors alone is not sufficient participation, but a different main compartment is not a negative functional assay. The named TreeGrafter reference is itself a chloroplast-associated sorghum protein, so the previous automatic cytosolic-paralog explanation is unproven. Preserve uncertainty about the actual physiological process boundary. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-bioinformatics/RESULTS.md The named TreeGrafter reference is a chloroplast-associated sorghum homolog, not an established cytosolic-paralog mismatch. |
| GO:0005986 sucrose biosynthetic process | IEA GO_REF:0000118 | UNDECIDED | Summary: Direct participation in sucrose biosynthesis is uncertain for the chloroplast enzyme. Reason: The dominant Calvin-cycle role and separation from cytosolic FBPase are supported, but the previous absolute exclusion and claimed paralog misgraft were not established. Current PTN004269459 is the sorghum A0A1B6QP66 leaf, annotated to chloroplast/Calvin cycle and 96.85% identical across the full global alignment. Rice cytosolic-FBPase perturbation shows that paralog contributes to sucrose synthesis; it does not prove that all conditional contributions of this chloroplast homolog are absent. Direct pathway participation still needs clarification rather than being inferred simply from exported carbon. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-bioinformatics/RESULTS.md The named TreeGrafter reference is a chloroplast-associated sorghum homolog, not an established cytosolic-paralog mismatch. |
| GO:0006000 fructose metabolic process | IEA GO_REF:0000118 | UNDECIDED | Summary: Fructose-process scope remains unresolved; the report does not establish a chemical exclusion. Reason: The report correctly separates fructose from its phosphorylated derivatives in the is_a hierarchy, but absence of an ancestor relation does not make pathways disjoint or require direct free-fructose turnover by every enzyme. QuickGO explicitly assigns this term to human FBP1/FBP2 with TAS and Dictyostelium fbp with IDA, and the F6P-process definition names fructose metabolism. Those comparators refute a blanket exclusion while not proving this chloroplast enzyme contributes to every fructose pathway. Retain UNDECIDED pending physiological and curator-scope clarification. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-bioinformatics/RESULTS.md The named TreeGrafter reference is a chloroplast-associated sorghum homolog, not an established cytosolic-paralog mismatch. |
| GO:0006002 fructose 6-phosphate metabolic process | IEA GO_REF:0000118 | ACCEPT | Summary: FBPase directly produces fructose 6-phosphate by hydrolysis of fructose 1,6-bisphosphate. Reason: GO:0006002 covers chemical reactions involving F6P without requiring a cytosolic location. Chloroplast Calvin-cycle chemistry therefore satisfies the term. The previous review incorrectly treated absent pathway specificity as over-annotation. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-notes.md The defining FBPase reaction hydrolyzes fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. |
| GO:0006094 gluconeogenesis | IEA GO_REF:0000118 | UNDECIDED | Summary: A secondary gluconeogenic contribution is unresolved despite a well-supported Calvin-cycle specialization. Reason: GO:0006094 describes glucose formation from noncarbohydrate precursors and does not impose the prior asserted cytosolic-only location. Chloroplast targeting and redox regulation distinguish this enzyme from the canonical cytosolic paralog, but neither is a direct demonstration of loss of all gluconeogenic participation. Shared FBPase chemistry alone also does not establish the complete pathway role. The named TreeGrafter reference is a close chloroplast-associated sorghum homolog; the actual query insertion and lineage-specific loss have not been established. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-bioinformatics/RESULTS.md The named TreeGrafter reference is a chloroplast-associated sorghum homolog, not an established cytosolic-paralog mismatch. |
| GO:0030388 fructose 1,6-bisphosphate metabolic process | IEA GO_REF:0000120 | ACCEPT | Summary: FBPase directly consumes fructose 1,6-bisphosphate in its core catalytic reaction. Reason: This substrate-centered process directly describes the work performed by the enzyme and is core. Calvin-cycle context is complementary; it does not relegate FBP metabolism to a non-core function. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-notes.md The defining FBPase reaction hydrolyzes fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. |
| GO:0005737 cytoplasm | IEA GO_REF:0000118 | ACCEPT | Summary: Chloroplast localization is compatible with the GO definition of cytoplasm. Reason: GO:0005737 includes subcellular structures outside the nucleus and plasma membrane, including plastids. The previous REMOVE conflated cytoplasm with cytosol. Retain this broad localization while retaining the separate challenge to GO:0005829 cytosol. Supporting Evidence: PMID:8980497 Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-notes.md GO:0005737 cytoplasm includes other subcellular structures; a chloroplast is therefore compatible with cytoplasm, although it is distinct from cytosol. |
| GO:0005829 cytosol | IEA GO_REF:0000118 | UNDECIDED | Summary: The principal chloroplast assignment is supported, while a functional cytosolic pool remains unresolved. Reason: The predicted transit sequence, conserved redox-regulatory insertion and close chloroplast homologs strongly support stromal localization. They do not by themselves establish exclusive import or exclude a conditional cytosolic pool. The source PTN leaf maps to the closely matching chloroplast-associated sorghum homolog, contradicting the assumption that TreeGrafter necessarily selected a cytosolic paralog. Retain the compartment conflict as UNDECIDED rather than asserting a demonstrated negative localization. Supporting Evidence: file:9POAL/NCGR_LOCUS1270/NCGR_LOCUS1270-bioinformatics/RESULTS.md The named TreeGrafter reference is a chloroplast-associated sorghum homolog, not an established cytosolic-paralog mismatch. |
| 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 |
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Download this section (compressed HTML)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)?
Q: Which physiological process boundaries justify fructose/sucrose metabolism or gluconeogenesis for this chloroplast FBPase, and is there a functional conditional cytosolic pool? The named TreeGrafter reference is a closely matching chloroplast-associated sorghum homolog; distinguish an ancestral assertion needing refinement from a demonstrated wrong-paralog query insertion.
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.
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