ERF115 Antibody

What is ERF115 and why is it important in plant biology research?

ERF115 is a plant-specific transcription factor belonging to the ethylene response factor (ERF) family. It functions as a wound-inducible stem cell organizer that interprets wound-induced auxin maxima . ERF115 is critically important in plant biology research because it plays a central role in regeneration responses, particularly following stem cell damage. The protein works synergistically with auxin accumulation to specify stem cell identity in cells surrounding damaged tissues, driving formative divisions that allow replacement of lost stem cells . This makes ERF115 a key target for studying plant resilience, regeneration, and stem cell dynamics.

How does ERF115 function in plant cell regeneration pathways?

ERF115 functions through multiple interconnected mechanisms in plant cell regeneration:

• Following wound-induced vascular stem cell death, ERF115 expression is rapidly activated in surrounding cells, particularly in the endodermis and quiescent center .

• This activation grants the endodermal cells partial stem cell identity, as evidenced by the ectopic induction of stem cell markers like WOX5 .

• ERF115 interacts with the RBR-SCR signaling network to regulate stem cell division during recovery .

• It works synergistically with auxin, which accumulates around wounds due to disrupted auxin transport pathways .

• ERF115 directly binds to and activates autophagy-related genes like ATG1b and ATG2, promoting cell expansion in response to stress .

• The transcription factor sensitizes cells to auxin by activating ARF5/MONOPTEROS, facilitating auxin response, tissue patterning, and organ formation .

Together, these mechanisms enable ERF115 to orchestrate the replacement of damaged stem cells, contributing to the remarkable regenerative capacity of plants.

What detection methods are most suitable for ERF115 protein in plant tissues?

For effective detection of ERF115 protein in plant tissues, researchers should consider multiple complementary approaches:

• Immunohistochemistry with fluorescent secondary antibodies: This approach allows for spatial visualization of ERF115 within tissue sections, which is particularly important given its cell-type specific expression patterns in the endodermis and quiescent center following wounding .

• Fluorescence reporter fusion proteins: ERF115:GFP-GUS reporter lines have been successfully used to monitor ERF115 expression patterns in response to treatments like bleomycin, auxin, methyl jasmonate, and hydrogen peroxide .

• Western blotting: For quantitative analysis of ERF115 protein levels, particularly when examining changes in expression following treatments that induce cell death or regeneration.

• Flow cytometry: When studying ERF115 in specific cell populations, flow cytometry can be employed using a panel design that matches this relatively rare protein with bright fluorophores .

When designing detection experiments, consider that ERF115 expression is highly context-dependent and may require specific induction conditions such as DNA damage, wounding, or hormone treatment to achieve detectable levels .

How should I design antibody panels for multicolor flow cytometry experiments targeting ERF115?

When designing antibody panels for multicolor flow cytometry experiments targeting ERF115, follow these methodological guidelines:

• Know your instrument limitations: Before starting, understand the capabilities of your flow cytometer in terms of laser configuration and detection channels .

• Prioritize rare antigen detection: Since ERF115 is typically expressed at low levels unless induced by stress or damage , match it with bright fluorophores to enhance detection sensitivity .

• Consider marker co-expression: Avoid placing fluorophores with similar emission spectra on markers that might be co-expressed with ERF115, such as auxin signaling components or cell death markers .

• Manage autofluorescence: Plant tissues often exhibit high autofluorescence; select fluorophores that are spectrally distinct from this background signal .

• Incorporate proper controls: Include:

  • Unstained controls

  • Single-color controls for compensation

  • Fluorescence-minus-one (FMO) controls

  • Isotype controls to assess nonspecific binding

• Include relevant markers in your panel: Consider adding markers for:

  • Cell viability (to exclude dead cells)

  • Auxin signaling (e.g., using DR5 reporters) since auxin works synergistically with ERF115

  • Cell cycle status (to correlate with ERF115's role in promoting formative divisions)

This approach will help ensure reliable detection of ERF115 in complex plant samples while minimizing artifacts from spectral overlap or autofluorescence.

What experimental controls are essential when validating ERF115 antibody specificity?

Validating ERF115 antibody specificity requires rigorous controls to ensure reliable research outcomes:

• Genetic controls:

  • Use knockout mutants like erf115-1 as negative controls, though be aware that potential functional redundancy might exist with closely related transcription factors

  • Include ERF115 overexpression lines as positive controls, considering that co-overexpression of ERF115-PAT1 results in hyperproliferation

  • The 35S:ERF115-SRDX dominant negative line can serve as a useful comparison to wild-type samples

• Peptide competition assays: Pre-incubate the antibody with purified ERF115 peptide or recombinant protein before applying to samples; this should abolish specific signals.

• Cross-reactivity assessment:

  • Test against closely related ERF family proteins to ensure specificity

  • Examine potential cross-reactivity with PAT1, a known interaction partner of ERF115

• Treatment validation:

  • Verify increased antibody signal following treatments known to induce ERF115, such as bleomycin-induced DNA damage, auxin application, or mechanical wounding

  • Confirm reduced signal when ERF115 expression is suppressed, such as in tissues treated with auxin biosynthesis inhibitors kynurenine and yucasin

• Subcellular localization consistency: Ensure the detected subcellular localization is consistent with ERF115's role as a transcription factor (primarily nuclear).

Implementing these controls systematically will help establish antibody specificity and validate experimental findings related to ERF115 function.

How can I use ERF115 antibodies to study the temporal dynamics of wound response in plant meristems?

To study temporal dynamics of wound response using ERF115 antibodies, implement this methodological approach:

• Time-course experimental design:

  • Establish a precise wounding protocol (laser ablation, mechanical damage, or chemical induction with bleomycin)

  • Collect samples at multiple timepoints (e.g., 0, 1, 3, 6, 12, 24, 48, and 72 hours post-wounding)

  • Process tissues simultaneously using identical fixation and staining protocols

• Dual immunolabeling technique:

  • Combine ERF115 antibodies with markers for:

    • Auxin signaling/transport (using DR5 reporters or PIN protein antibodies) to track the relationship between auxin accumulation and ERF115 expression

    • Cell death markers to establish the spatial relationship between damaged cells and ERF115 expression

    • Cell cycle proteins to correlate ERF115 expression with subsequent formative divisions

• Quantitative image analysis:

  • Implement computerized image analysis to quantify fluorescence intensity as a proxy for ERF115 protein levels

  • Measure the distance between dead cells and ERF115-expressing cells over time

  • Track changes in cell morphology and division patterns in ERF115-positive cells

• Integration with live-cell imaging:

  • When possible, complement fixed tissue analysis with live-cell imaging using reporter lines

  • Consider using the pERF115:GFP-GUS reporter in conjunction with antibody-based methods to validate findings

• Perturbation experiments:

  • Apply auxin transport inhibitors or biosynthesis inhibitors (kynurenine and yucasin) at different timepoints to determine when auxin signaling is critical for ERF115 expression maintenance

  • Use 35S:ERF115-SRDX or tissue-specific expression of the dominant negative construct (e.g., EN7:ERF115-SRDX) to assess the impact of ERF115 inhibition on regeneration dynamics

This comprehensive approach will reveal the spatiotemporal patterns of ERF115 expression in relation to wound response and regeneration processes.

What are the challenges in detecting post-translational modifications of ERF115 and how can they be overcome?

Detecting post-translational modifications (PTMs) of ERF115 presents several challenges that can be addressed through specialized methodological approaches:

• Challenge: Low abundance of modified protein

  • Solution: Implement enrichment strategies such as immunoprecipitation with ERF115 antibodies followed by detection with modification-specific antibodies (phospho-, ubiquitin-, or SUMO-specific)

  • Method enhancement: Use inducible expression systems to increase baseline ERF115 levels prior to PTM analysis

• Challenge: Transient nature of modifications during signaling

  • Solution: Employ phosphatase or proteasome inhibitors during sample preparation to preserve labile modifications

  • Temporal approach: Design precise time-course experiments following wound induction or auxin treatment to capture dynamic modification states

• Challenge: Identifying specific modified residues

  • Solution: Combine immunoprecipitation with mass spectrometry analysis

  • Validation strategy: Generate site-specific antibodies against predicted modification sites, particularly those in regulatory domains

• Challenge: Determining functional significance of modifications

  • Solution: Create point mutations at modification sites in ERF115 and test their function in complementation studies with erf115 mutants

  • Experimental design: Compare regeneration efficiency and auxin responsiveness between wild-type and modification-site mutants

• Challenge: Cross-talk between different modifications

  • Solution: Employ multiplexed detection methods combining antibodies against different modifications

  • Analysis approach: Use sequential immunoprecipitation to identify proteins carrying multiple modifications

• Challenge: Distinguishing ERF115 from closely related family members

  • Solution: Validate antibody specificity using recombinant proteins representing closely related ERF family members

  • Control implementation: Include the erf115 mutant in all experiments as a negative control, while acknowledging potential functional redundancy

By addressing these challenges systematically, researchers can gain deeper insights into how post-translational regulation influences ERF115's function in regeneration and stress response pathways.

How do I troubleshoot inconsistent ERF115 antibody staining patterns in plant tissue sections?

• Sample preparation variability:

  • Problem: Inconsistent fixation can affect epitope accessibility

  • Solution: Standardize fixation protocols with precisely timed incubations and fresh reagents

  • Validation approach: Compare multiple fixation methods (paraformaldehyde, methanol, acetone) to determine optimal epitope preservation

• Context-dependent expression:

  • Problem: ERF115 expression is highly dependent on wounding, stress, and developmental context

  • Solution: Carefully control induction conditions; consider using bleomycin treatment at standardized concentrations to reliably induce ERF115

  • Verification method: Include pERF115:GFP-GUS reporter lines as positive controls to verify induction conditions

• Tissue penetration issues:

  • Problem: Inadequate antibody penetration, especially in dense plant tissues

  • Solution: Optimize permeabilization steps; consider extending incubation times or implementing vacuum infiltration

  • Comparative approach: Section thickness standardization (10-20 μm typically provides good results)

• Autofluorescence interference:

  • Problem: Plant tissues exhibit significant autofluorescence that may mask specific signals

  • Solution: Include appropriate blocking steps with BSA or normal serum; consider autofluorescence quenching treatments

  • Control implementation: Image untreated sections to establish baseline autofluorescence levels

• Antibody specificity concerns:

  • Problem: Cross-reactivity with related ERF family proteins

  • Solution: Validate with peptide competition assays and genetic controls (use erf115 mutant tissue as negative control)

  • Verification strategy: Confirm staining patterns match known ERF115 expression domains (e.g., endodermis and QC following DNA damage)

Systematic implementation of these troubleshooting strategies will lead to more consistent and reliable ERF115 antibody staining patterns, enhancing data quality and reproducibility.

How can I differentiate between specific and non-specific binding when using ERF115 antibodies in co-immunoprecipitation experiments?

Differentiating between specific and non-specific binding in ERF115 co-immunoprecipitation (co-IP) experiments requires rigorous controls and validation strategies:

• Essential negative controls:

  • Input control: Reserve a portion of pre-IP lysate to confirm target protein presence

  • IgG control: Perform parallel IP with isotype-matched non-specific IgG

  • Genetic control: Use tissue from erf115 mutants to identify non-specific bands

  • Peptide competition: Pre-incubate antibody with excess ERF115 peptide to block specific binding sites

• Stringency optimization:

  • Methodological approach: Perform parallel co-IPs with increasing salt concentrations (150mM, 300mM, 450mM NaCl)

  • Buffer composition: Test different detergent types and concentrations to minimize non-specific interactions

  • Validation strategy: True interactors should persist under higher stringency conditions while non-specific binders are eliminated

• Reciprocal co-IP validation:

  • Experimental design: Confirm interactions by performing reverse co-IP using antibodies against putative interacting partners

  • Known interaction verification: Include known ERF115 partners such as PAT1 as positive controls

• Analytical approaches:

  • Quantitative comparison: Compare band intensities between experimental and control samples

  • Mass spectrometry validation: Identify co-precipitated proteins using mass spectrometry and filter against databases of common contaminants

  • Statistical analysis: Implement significance scoring for protein interactions based on peptide counts and reproducibility

• Functional validation of interactions:

  • Reporter assays: Test functional relevance using transactivation assays for putative targets like ATG1b and ATG2

  • Chromatin immunoprecipitation: Validate DNA-binding interactions using ChIP-qPCR for suspected target promoters

This systematic approach will help distinguish bona fide ERF115 interactions from experimental artifacts, leading to more reliable identification of protein complexes involving this important regeneration-promoting transcription factor.

How do results from ERF115 antibody-based methods compare with transgenic reporter systems for studying ERF115 activity?

Understanding the complementarity and limitations of antibody-based detection versus transgenic reporter systems is crucial for comprehensive ERF115 research:

When studying ERF115, an integrated approach combining both methods yields the most comprehensive results:

• Use transgenic reporters like pERF115:GFP-GUS for initial expression pattern analysis and live cell imaging

• Validate key findings with antibody-based detection of endogenous ERF115

• Employ antibodies for detection of post-translational modifications and protein-protein interactions

• Use reporters for lineage tracing experiments, as demonstrated with the pSCR:CRE_GR p35S loxp-tOCS-loxp-CFP system

This complementary approach leverages the strengths of both methods while mitigating their respective limitations.

How should I interpret conflicting results between ERF115 protein levels and gene expression data?

Discrepancies between ERF115 protein levels and gene expression data require systematic analysis and interpretation:

• Mechanistic considerations for divergent results:

  • Post-transcriptional regulation: ERF115 mRNA may be subject to microRNA targeting or RNA-binding protein regulation affecting translation efficiency

  • Protein stability dynamics: ERF115 protein could undergo rapid turnover through ubiquitin-mediated degradation, particularly following wound response

  • Temporal lag effects: Consider that protein accumulation naturally lags behind transcriptional activation

  • Cell type-specific regulation: Protein translation or stability may differ between cell types (e.g., endodermis versus vascular cells)

• Methodological validation approaches:

  • mRNA half-life determination: Use actinomycin D to block transcription and measure ERF115 mRNA decay rates

  • Protein stability assessment: Apply cycloheximide or proteasome inhibitors to determine ERF115 protein turnover rates

  • Cell-specific analysis: Implement cell sorting or single-cell approaches to resolve cell type-specific differences

• Integrated experimental design:

  • Time-course resolution: Sample at finer time intervals to capture the relationship between transcription and translation

  • Parallel measurements: Analyze mRNA and protein from the same samples to enable direct correlation

  • Pathway perturbation: Manipulate key regulatory pathways (auxin, jasmonate, ROS) to identify regulators affecting transcription versus translation

• Statistical analysis framework:

  • Correlation analysis: Calculate Pearson or Spearman correlations between mRNA and protein levels across conditions

  • Principal component analysis: Identify patterns explaining variance between transcript and protein measurements

  • Regression modeling: Develop predictive models that account for time lags between transcription and translation

• Biological interpretation guidelines:

  • Discrepancies may reveal novel regulatory mechanisms specific to stress response transcription factors

  • Consider that different pools of ERF115 protein may exist (active/inactive) that are not distinguished by standard antibody detection

  • Auxin has been shown to sustain ERF115 activity rather than inducing initial expression, which could explain certain discrepancies

This systematic approach transforms conflicting data into opportunities for deeper mechanistic insights into ERF115 regulation.

What are the most promising applications for ERF115 antibodies in studying plant regeneration and stress response mechanisms?

ERF115 antibodies offer several promising research avenues for advancing our understanding of plant regeneration and stress responses:

• Cellular reprogramming studies:

  • Use ERF115 antibodies to track acquisition of stem cell identity in differentiated cells responding to damage

  • Combine with cell fate markers to map the precise trajectory of cellular reprogramming during regeneration

  • Investigate how ERF115 binding to ATG promoters promotes autophagy-dependent cell expansion in response to stress

• Stress resilience mechanism elucidation:

  • Apply ERF115 antibodies to compare regeneration responses across different stress types (heat, drought, pathogen attack)

  • Investigate whether ERF115 expression patterns correlate with regeneration capacity differences between stress-tolerant and sensitive plant varieties

  • Study how ERF115 activation might be involved in priming responses that enhance future stress resilience

• Hormone crosstalk visualization:

  • Use dual immunolabeling to map the spatial relationships between ERF115 expression and hormone signaling components

  • Investigate the relationship between auxin transport disruption, auxin accumulation, and ERF115 activation

  • Explore how ERF115 sensitizes cells to auxin by activating ARF5/MONOPTEROS

• Synthetic biology applications:

  • Develop engineered plants with modified ERF115 expression to enhance regeneration capacity

  • Design synthetic circuits incorporating ERF115 elements for improved tissue engineering applications

  • Investigate whether controlled expression of ERF115 could enhance recovery from agricultural stresses

• Comparative developmental biology:

  • Apply ERF115 antibodies across plant species to determine conservation of regeneration mechanisms

  • Compare ERF115 activation patterns between plants with different regenerative capacities

  • Investigate ERF115 expression during natural developmental processes that involve controlled cell death and regeneration

These research directions leverage ERF115 antibodies as powerful tools for understanding fundamental aspects of plant resilience while potentially developing applications for agriculture and biotechnology.

How can multi-omics approaches incorporating ERF115 antibody-based techniques advance our understanding of plant regeneration networks?

Multi-omics approaches incorporating ERF115 antibody-based techniques can revolutionize our understanding of plant regeneration networks through systematic data integration:

• Integrated experimental design strategies:

  • Sequential sampling: Collect tissues at defined intervals post-wounding for parallel multi-omic analysis

  • Cell-type resolution: Combine antibody-based cell sorting with transcriptomics and proteomics

  • Perturbation series: Apply treatments that modify ERF115 function (auxin inhibitors, DNA damage agents) for network response mapping

• Complementary omics integration:

  • ChIP-seq with ERF115 antibodies: Map global ERF115 binding sites beyond known targets like ATG1b and ATG2

  • Proteomics of ERF115 complexes: Identify interacting proteins through immunoprecipitation followed by mass spectrometry

  • Transcriptomics of ERF115-expressing cells: Profile gene expression in FACS-sorted ERF115-positive cells

  • Metabolomics correlation: Associate metabolite changes with ERF115 activation patterns

• Advanced computational integration approaches:

  • Network inference algorithms: Construct gene regulatory networks centered on ERF115

  • Multi-modal data visualization: Develop tools to visualize ERF115-dependent processes across omics layers

  • Causal modeling: Implement causal inference methods to identify directional relationships in ERF115 signaling

• Validation experimental designs:

  • CRISPR-based perturbation: Target predicted network nodes and measure effects on ERF115-dependent regeneration

  • Synthetic promoter analysis: Test predicted ERF115 binding motifs in reporter constructs

  • Protein-fragment complementation: Validate predicted protein interactions in vivo

• Data representation model:

This integrated multi-omics approach would reveal the comprehensive molecular landscape of ERF115-mediated regeneration, identifying key intervention points for enhancing plant resilience and regenerative capacity.