PHO1 Antibody
Description
PHO1 Antibody: Structure and Function
PHO1 (Phosphate 1) is a membrane-spanning protein with three distinct domains:
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N-terminal SPX domain: Binds phosphoinositol pyrophosphate (PP-InsP) molecules, regulating transport activity .
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EXS domain: Facilitates Pi translocation via residues Trp719 and Tyr610, acting as a gate for phosphate movement .
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C-terminal tail: Partially disordered, contributes to dimer formation and PP-InsP binding .
PHO1 is expressed in root pericycle and xylem parenchyma cells, mediating Pi transfer from roots to shoots . Mutations in PHO1 cause reduced shoot growth and seed production .
Applications in Research
PHO1 antibodies enable diverse experimental approaches:
3.1. Immunoprecipitation (IP)
PHO1 antibodies co-precipitate interacting proteins, revealing functional complexes:
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PHO1-Dpe1 interaction: In sweet potato roots, PHO1 co-IPs with 4-α-glucantransferase (Dpe1), forming a metabolon to regulate starch metabolism .
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PHO1-PsaC interaction: In rice, PHO1 binds photosystem I subunit C (PsaC), suggesting a role in plastidial Pi homeostasis .
3.2. Western Blot (WB)
Detects PHO1 abundance and post-translational modifications:
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Phosphorylation studies: Antibodies track PHO1 phosphorylation at Ser566 in maize, critical for enzyme activity .
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Tissue-specific expression: PHO1 is detected in roots (high expression) and shoots (low expression) .
3.3. Immunohistochemistry (IHC-P)
Localizes PHO1 in plant tissues:
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Human PHO1 antibodies (e.g., Abcepta) detect nuclear/cytoplasmic localization in leukocytes and cancer cells .
Phosphorylation and Regulatory Mechanisms
Phosphorylation modulates PHO1 activity:
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Maize PHO1: Mutations at Ser566 (S566A) reduce Pi release from Glc-1-P by ~50%, indicating phosphorylation enhances activity .
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PHO2 interaction: PHO2 ubiquitinates PHO1, promoting its degradation in endomembranes to maintain Pi homeostasis .
Cross-Reactivity and Specificity
Challenges and Considerations
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SDS-PAGE artifacts: PHO1’s hydrophobic nature may cause faster migration (~68 kDa vs. predicted ~90 kDa) .
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Overexpression effects: Shoot-specific PHO1 overexpression in Arabidopsis triggers Pi efflux, reducing vacuolar Pi pools .
Research Implications
PHO1 antibodies are pivotal for:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research has revealed that PHO1 and PHOH1, the Arabidopsis Pi exporters, are expressed in the chalazal seed coat (CZSC) of developing seeds. Additionally, PHO1 is found in developing ovules. Studies have analyzed phosphorus (P) content and Pi flux between the seed coat and embryo in seeds from grafts between wild-type roots and scions from either pho1, phoh1, or the pho1 phoh1 double mutant. PMID: 28943092
- Genome-wide association mapping has identified PHOSPHATE 1 (PHO1) and genes like Root System Architecture 1 (RSA1) as being associated with variations in root allometry, a highly plastic trait related to the distribution of lateral roots along the primary axis. PMID: 23980140
- The EXS domain of PHO1 plays a critical role in a root-to-shoot signaling pathway involved in the phosphate deficiency response. PMID: 26546667
- AtWRKY42 has been shown to modulate Pi homeostasis by regulating the expression of PHO1 and PHT1;1, enabling plants to adapt to changes in inorganic phosphate (Pi) availability in the environment. PMID: 25733771
- PHR1 and PHO1 are intricately involved in the co-regulation of Zn and Pi homeostasis. PMID: 24420568
- The expression of PHO1 in guard cells of Arabidopsis thaliana is essential for complete stomatal responses to abscisic acid. PMID: 22612335
- PHO1 has the ability to trigger Pi export in ectopic plant cells, providing strong evidence that PHO1 itself functions as a Pi exporter. PMID: 22449068
- Overexpression of PHO1 in the shoot of Arabidopsis thaliana resulted in a substantial increase (two- to threefold) in shoot inorganic phosphate content, leading to a significant reduction in shoot growth. PMID: 21309867
- Reducing PHO1 expression enables plants to exhibit wild-type growth in phosphate-deficient media, despite the Pi deficiency. PMID: 21288266
- Studies have demonstrated that WRKY6 and WRKY42 play crucial roles in Arabidopsis responses to low Pi stress through the regulation of PHO1 expression. PMID: 19934380
Q&A
Basic Research Questions
What is the functional role of PHO1 in phosphate homeostasis, and how is its antibody used to study this process?
PHO1 facilitates xylem loading of inorganic phosphate (Pi) in plant roots and regulates systemic Pi distribution. Its antibody enables:
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Localization studies: Immunohistochemistry to track PHO1 expression in root vascular tissues .
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Protein stability assays: Western blotting under varying Pi conditions (e.g., ±Pi treatments) to quantify PHO1 degradation kinetics .
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Mutant validation: Confirming PHO1 absence in pho1 knockout lines (e.g., pho1-2 vs. wild-type controls) .
Methodological Tip:
"For Western blotting, use a Tris-Tricine gel system to resolve PHO1’s hydrophobic domains, which migrate anomalously at ~68 kDa despite a predicted 90 kDa mass ."
Advanced Research Questions
How do conflicting data on PHO1 stability arise in pho2 mutants, and how can experimental design mitigate this?
PHO1 accumulates in pho2 mutants due to impaired ubiquitination-mediated degradation. Contradictions arise from:
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Temporal dynamics: PHO1 degradation slows after 8–72 hr of Pi resupply in pho2 (vs. wild-type degradation within 48 hr) .
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Post-translational regulation: Cycloheximide chase assays reveal a PHO1 half-life of 21.7 min in wild-type roots under +Pi conditions, but delayed degradation in pho2 .
Experimental Design Table:
| Variable | Wild-Type Protocol | pho2 Protocol | Key Consideration |
|---|---|---|---|
| Pi Treatment | 0–72 hr resupply after -Pi | 0–72 hr resupply after -Pi | Synchronize growth stages |
| Protein Extraction | EMs (endomembranes) lysis | EMs lysis + protease inhibitors | Prevent artifactual degradation |
| Antibody Validation | pho1-2 mutant as negative control | pho1-5 pho2 suppressor line | Confirm specificity |
What technical challenges occur when characterizing PHO1-protein interactions, and how are they addressed?
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Membrane association: PHO1’s localization in endomembranes necessitates detergent-based extraction (e.g., Triton X-114) to retain solubility .
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Ubiquitination assays: Co-immunoprecipitation (Co-IP) with PHO2 (E2 ubiquitin-conjugase) requires transient expression in protoplasts and MG132 treatment to inhibit proteasomal decay .
Data Interpretation Guidance
How should researchers reconcile discrepancies in PHO1 antibody specificity across plant species?
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Cross-reactivity testing: Validate antibodies against PHO1 orthologs (e.g., Arabidopsis vs. rice PHO1) via peptide-blocking assays.
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Epitope mapping: Use truncated PHO1 variants (e.g., ΔSPX domain) to identify antibody-binding regions .
