At4g08869 Antibody

What is At4g08869 and what is its biological function?

At4g08869 is an Arabidopsis thaliana gene encoding AtLURE1.7, a cysteine-rich peptide belonging to the LURE family. It functions primarily in reproductive processes, specifically in pollen tube attraction. AtLURE1.7 is expressed in synergid cells and the encoded protein localizes to the filiform apparatus of these cells. Research has demonstrated that AtLURE1.7, along with other AtLURE peptides, plays a critical role in species-specific pollen tube guidance, contributing to reproductive isolation between related plant species . The peptide likely functions through interaction with pollen receptor kinases, similar to other LURE family members, to facilitate precise pollen tube navigation toward ovules.

How does At4g08869 (AtLURE1.7) relate to other AtLURE genes?

At4g08869 (AtLURE1.7) belongs to a family of eight A. thaliana-specific AtLURE1 genes (AtLURE1.1 through AtLURE1.8). These genes form a distinct clade representing rapidly evolving cysteine-rich peptides. Phylogenetic analyses place At4g08869 and At4g08875 (AtLURE1.8) as close relatives within this gene family . The entire AtLURE1 family appears to have evolved recently, with At4g08869 serving as one of the functional members (unlike AtLURE1.6, which is considered a pseudogene). All functional AtLURE peptides share similar expression patterns in synergid cells and contribute collectively to pollen tube attraction, though potentially with varying degrees of specificity and attraction potency .

What cellular localization pattern is expected for the At4g08869 protein?

The At4g08869-encoded protein (AtLURE1.7) primarily localizes to the filiform apparatus of synergid cells within the female gametophyte. This has been confirmed through fluorescent protein fusion studies showing AtLURE1.7-GFP accumulation in this specialized cell wall region . This localization pattern is consistent with the protein's role in pollen tube attraction, as the filiform apparatus serves as a secretory region for attractant molecules that guide growing pollen tubes. Researchers using At4g08869 antibodies should expect immunolocalization signals predominantly in this region of ovules, with the strongest signals observed in mature, unfertilized ovules when the attraction system is fully operational .

What critical controls should be included when using At4g08869 antibody?

When designing experiments with At4g08869 antibody, researchers must implement several essential controls to ensure result validity and interpretability. The following table outlines recommended controls:

Additionally, researchers should consider spatiotemporal controls (tissues/developmental stages where At4g08869 is not expressed) and cross-reactivity controls with other AtLURE family members, particularly the closely related AtLURE1.8 .

How can researchers distinguish At4g08869 (AtLURE1.7) from other AtLURE family members?

Distinguishing At4g08869 (AtLURE1.7) from other AtLURE family members requires careful antibody validation and experimental design due to potential cross-reactivity issues. Researchers should:

• Perform peptide competition assays using each individual LURE peptide to determine specificity profiles

• Validate antibody recognition using recombinant protein panels including all LURE family members

• Test antibody performance in genetic backgrounds where specific AtLURE genes are knocked out (particularly the closely related AtLURE1.8)

• Consider using epitope-tagged transgenic lines expressing individual LURE proteins for validation

Western blot analysis should include size discrimination tests since AtLURE peptides typically possess similar molecular weights due to their conserved structure. Additionally, mass spectrometry validation can provide definitive identification when antibody-based approaches yield ambiguous results .

What sample preparation methods optimize At4g08869 antibody performance in immunolocalization studies?

For optimal immunolocalization of At4g08869/AtLURE1.7 in plant reproductive tissues, sample preparation requires special consideration due to the protein's localization in the specialized cell wall structures of synergid cells. A recommended protocol includes:

• Harvest pistils at stages 12-14 of flower development (when AtLURE1.7 expression peaks)

• Fix tissues in 4% paraformaldehyde with 0.1% Triton X-100 for 4 hours at 4°C

• Wash thoroughly in PBS and perform careful microdissection to expose ovules

• Perform cell wall permeabilization using enzymatic digestion (1% cellulase, 0.5% macerozyme) for 10-15 minutes

• Block with 3% BSA in PBS with 0.1% Tween-20 for at least 2 hours

• Apply primary At4g08869 antibody at optimized dilution (typically 1:200-1:500) overnight at 4°C

• Perform extensive washing (at least 6×20 minutes) to remove unbound antibody

• Apply appropriate fluorophore-conjugated secondary antibody

This protocol can be adapted for both light and electron microscopy applications. For whole-mount immunolocalization, additional clearing steps may be necessary to improve signal visualization in complex reproductive structures .

How can At4g08869 antibody be used to investigate species-specific pollen tube guidance?

At4g08869 antibody provides a powerful tool for investigating the molecular mechanisms of species-specific pollen tube guidance. Researchers can implement several sophisticated approaches:

• Comparative immunolocalization: Perform simultaneous immunolocalization of AtLURE1.7 (At4g08869) and related LURE peptides from other species (e.g., AlLURE from A. lyrata) in interspecific crosses to visualize protein distributions during compatible versus incompatible pollinations.

• Antibody inhibition assays: Apply purified At4g08869 antibody to in vitro pollen tube guidance systems to selectively block AtLURE1.7 function, allowing assessment of its specific contribution to attraction relative to other family members.

• Co-immunoprecipitation studies: Use At4g08869 antibody to identify interacting receptor proteins in pollen tubes, potentially revealing species-specific receptor-ligand pairs that contribute to reproductive isolation.

• Quantitative immunoassays: Develop ELISA or similar quantitative assays using At4g08869 antibody to measure precise AtLURE1.7 concentrations in the ovule micropylar region during various reproductive scenarios .

These approaches can help determine how AtLURE1.7 contributes to reproductive barriers between closely related plant species, with implications for understanding plant speciation mechanisms and potentially developing tools for controlled hybridization in agricultural contexts.

What insights can At4g08869 antibody provide about evolution of reproductive isolation mechanisms?

At4g08869 antibody enables researchers to study evolutionary aspects of reproductive isolation with unprecedented molecular detail. Since At4g08869 encodes AtLURE1.7, which belongs to a clade of rapidly evolving cysteine-rich peptides, this antibody can help elucidate several key evolutionary questions:

• Divergence patterns: By comparing immunolocalization patterns of AtLURE1.7 across closely related Arabidopsis species, researchers can visualize how expression domains and protein localization have diverged during evolution.

• Functional conservation: Combining antibody detection with functional assays can reveal whether sequence divergence corresponds to functional specialization across species barriers.

• Selection pressure mapping: Correlation of epitope recognition patterns with sequence conservation/divergence data can identify protein regions under strong selection pressure, potentially representing functionally critical domains.

• Gene duplication dynamics: Using At4g08869 antibody alongside antibodies against other LURE family members can illuminate sub-functionalization patterns following gene duplication events .

This evolutionary perspective is particularly valuable given that At4g08869 and At4g08875 appear as outgroups in phylogenetic analyses of related cysteine-rich peptides, suggesting a potentially distinct evolutionary trajectory for these genes compared to other members of the family .

How can researchers use At4g08869 antibody to study CRISPR-generated mutant phenotypes?

CRISPR-Cas9 technology has been successfully employed to generate atlure1 null septuple mutants by simultaneously knocking out multiple AtLURE genes, including At4g08869. At4g08869 antibody offers valuable tools for characterizing these mutants:

• Validation of knockout efficiency: Immunoblotting and immunolocalization with At4g08869 antibody can confirm complete protein elimination in mutant lines.

• Compensation detection: Antibody-based quantification can reveal whether expression of non-targeted related genes increases to compensate for At4g08869 loss.

• Phenotypic correlation: Combining immunolocalization with detailed phenotypic analysis (e.g., pollen tube tracking) can correlate protein levels with functional outcomes.

• Rescue experiment verification: When performing genetic complementation, At4g08869 antibody can confirm proper expression and localization of the reintroduced protein .

Additionally, researchers should consider combining At4g08869 antibody studies with fluorescent protein reporters to simultaneously track multiple molecular players in these mutant backgrounds, providing a more comprehensive view of the molecular consequences of gene deletion .

What are common challenges when using At4g08869 antibody in plant reproductive tissues?

Researchers commonly encounter several technical challenges when using At4g08869 antibody in plant reproductive tissues:

• Tissue accessibility limitations: The synergid cells where AtLURE1.7 localizes are embedded within ovule tissues, creating physical barriers to antibody penetration. This may require optimization of fixation and permeabilization protocols, including extended incubation times or alternative fixatives.

• High background in reproductive tissues: Female reproductive tissues often exhibit autofluorescence and non-specific antibody binding. Researchers should implement additional blocking steps (e.g., with normal serum matching the secondary antibody host species) and consider tissue-specific autofluorescence quenching methods.

• Low protein abundance issues: AtLURE1.7 may be expressed at relatively low levels compared to structural proteins, necessitating signal amplification techniques such as tyramide signal amplification or high-sensitivity detection systems.

• Developmental timing variability: AtLURE1.7 expression peaks at specific developmental stages, requiring precise staging of flowers for consistent results. Researchers should establish clear morphological markers for standardizing collection times .

To address these challenges, preliminary studies should include a comprehensive optimization matrix varying fixation conditions, antibody concentrations, incubation times, and detection methods to establish reliable protocols for the specific experimental context.

How should researchers interpret discrepancies between At4g08869 antibody signals and gene expression data?

When researchers encounter discrepancies between At4g08869 antibody detection and gene expression data, systematic investigation is required to determine the cause:

• Post-transcriptional regulation: Differences between mRNA and protein levels may reflect authentic biological regulation. Researchers should examine whether microRNAs or RNA-binding proteins target At4g08869 transcripts, potentially explaining translation efficiency differences.

• Protein stability variations: AtLURE1.7 may undergo developmental or condition-dependent degradation. Pulse-chase experiments with protein synthesis inhibitors can help determine protein half-life under different conditions.

• Protein translocation effects: The secreted nature of AtLURE1.7 means that protein may accumulate at sites distant from synthesis. Combined in situ hybridization and immunolocalization can distinguish between synthesis and accumulation locations.

• Technical artifacts: Antibody sensitivity limitations or mRNA detection problems may cause apparent discrepancies. Researchers should verify results using alternative detection methods such as epitope-tagged transgenic lines or mass spectrometry .

When reporting such discrepancies, researchers should clearly differentiate between technical limitations and potentially interesting biological phenomena that might reveal novel regulatory mechanisms affecting this important signaling molecule.

What quantitative approaches can accurately measure At4g08869 protein levels in experimental samples?

Several quantitative approaches can be employed to accurately measure At4g08869 protein levels:

• Quantitative Western blotting: Using infrared fluorescence-based detection systems with At4g08869 antibody alongside internal loading controls allows reliable relative quantification. Standard curves generated with recombinant AtLURE1.7 enable absolute quantification.

• ELISA development: Sandwich ELISA using capture and detection antibodies against different At4g08869 epitopes provides high sensitivity for protein quantification in complex samples.

• Selected Reaction Monitoring (SRM) mass spectrometry: Combining immunoprecipitation using At4g08869 antibody with targeted mass spectrometry offers highly specific and sensitive quantification, even distinguishing between closely related LURE family members.

• Capillary immunoelectrophoresis: This technique combines the separation power of capillary electrophoresis with immunodetection, offering high resolution for quantifying AtLURE1.7 in minimal sample volumes .

For all quantitative approaches, researchers should establish the linear dynamic range of the assay, determine limits of detection and quantification, and validate reproducibility across technical and biological replicates. Additionally, spike-in controls with known quantities of recombinant protein can correct for matrix effects in complex plant tissue samples.