CLE42 Antibody

What is CLE42 and why are antibodies against it valuable for plant research?

CLE42 is a plant-specific peptide hormone belonging to the CLE (CLAVATA3/EMBRYO SURROUNDING REGION-RELATED) family in Arabidopsis thaliana. It functions as a negative regulator of leaf senescence by antagonizing the ethylene pathway . The mature CLE42 peptide consists of 12 amino acids (HGVPSGPNPISN) derived from the CLE domain of the CLE42 precursor .

Antibodies against CLE42 are valuable research tools because they enable:

• Precise detection of CLE42 protein levels in plant tissues

• Investigation of spatial and temporal CLE42 distribution patterns

• Study of protein-protein interactions involving CLE42

• Analysis of how CLE42 regulates the ethylene signaling pathway

• Examination of CLE42's role in delaying leaf senescence

How does CLE42 function in plant development and senescence?

CLE42 serves as a negative regulator of leaf senescence through antagonizing the ethylene pathway . The evidence for this includes:

• The cle42 mutant displays earlier senescence phenotypes compared to wild-type plants

• Overexpression of CLE42 delays both age-dependent and dark-induced leaf senescence

• Application of synthetic CLE42 peptide (CLE42p) delays leaf senescence under both natural and dark conditions

At the molecular level, CLE42 suppresses ethylene biosynthesis and promotes protein accumulation of EBF, which in turn decreases the function of EIN3 . This regulation creates a communication pathway between CLE peptide hormones and ethylene signaling during leaf senescence .

What distinguishes CLE42 from other closely related CLE peptides?

CLE42 shares functional redundancy with CLE41/44 in regulating leaf senescence, but with some notable distinctions:

The cle41 cle42 cle44 triple mutant exhibits more pronounced earlier senescence phenotypes than any single mutant, confirming functional redundancy .

What strategies are most effective for generating specific antibodies against CLE42?

Developing antibodies against small peptides like CLE42 requires careful immunogen design strategies:

• Peptide-based approaches:

  • Use the synthetic 12-amino acid CLE42p (HGVPSGPNPISN) conjugated to carrier proteins like KLH

  • Focus on N-terminal regions where CLE42 differs from CLE41/44 (position 2: G vs E)

  • Employ multiple antigenic determinants to increase specificity

• Recombinant protein strategies:

  • Express the full CLE42 precursor protein with fusion tags

  • Design protein fragments that enhance CLE domain exposure

  • Use display technologies (phage/yeast) for antibody selection

• Screening considerations:

  • Implement competitive ELISAs with related peptides to identify specific clones

  • Test antibody binding to both synthetic peptides and native protein

  • Validate against plant extracts from wild-type and cle42 mutant plants

The epitope binning approach described in search result can help identify antibodies that bind distinct regions, improving specificity for CLE42 over related peptides.

What validation methods confirm CLE42 antibody specificity?

Rigorous validation is critical for CLE42 antibodies due to potential cross-reactivity with related CLE peptides:

• Genetic validation:

  • Compare immunoblot signals between wild-type and cle42 mutant plants

  • Include cle41 cle42 cle44 triple mutants to address redundancy

  • Test CLE42 overexpression lines for enhanced signal

• Biochemical validation:

  • Perform peptide competition assays with synthetic CLE42p

  • Compare reactivity with CLE41/44p to assess cross-reactivity

  • Use mass spectrometry to confirm antibody-captured proteins

• Functional validation:

  • Verify if antibody-mediated neutralization mimics cle42 mutant phenotypes

  • Test antibody specificity in receptor binding inhibition assays

  • Correlate antibody binding with biological activity

Similar to the antibody validation approach used for CLEC-2 in search result , antibody specificity should be assessed by multiple complementary methods.

How can researchers optimize sample preparation for CLE42 detection?

Detecting low-abundance peptide hormones like CLE42 in plant tissues requires optimized sample preparation:

• Extraction considerations:

  • Use buffers containing protease inhibitors to prevent degradation

  • Consider tissue-specific extraction protocols (CLE42 may have tissue-specific expression)

  • Include reducing agents to maintain peptide structure

• Enrichment strategies:

  • Implement size-exclusion chromatography to isolate small peptides

  • Use immunoaffinity purification with validated antibodies

  • Consider subcellular fractionation to target compartments with highest CLE42 content

• Detection optimization:

  • Adjust antibody concentration through titration experiments

  • Employ signal amplification methods for low-abundance detection

  • Use appropriate blocking reagents to minimize background

These approaches maximize the signal-to-noise ratio for reliable CLE42 detection in complex plant samples.

How can CLE42 antibodies elucidate the relationship between CLE42 and the ethylene pathway?

CLE42 antibodies provide powerful tools for investigating how CLE42 antagonizes the ethylene pathway:

• Protein interaction studies:

  • Co-immunoprecipitation to identify direct interactors of CLE42

  • Analyze protein complexes involving CLE42, PXY receptor, and ethylene signaling components

  • Evaluate how CLE42 influences EBF protein accumulation and EIN3 function

• Pathway analysis approaches:

  • Monitor ethylene biosynthesis enzyme levels in response to CLE42

  • Track EBF protein accumulation using immunoblotting in various genetic backgrounds

  • Investigate how CLE42 application affects downstream signaling components

• Genetic interaction studies:

  • Compare antibody-detected protein levels in wild-type, ein3/eil1 mutants, and ebf1 overexpression lines

  • Analyze how CLE42 levels correlate with senescence marker expression

  • Investigate the rescue of cle42 mutant phenotypes by ein3/eil1 mutation or ebf1 overexpression

RNA-Seq analysis revealed that the ethylene pathway was suppressed when CLE42 was overexpressed, providing a foundation for antibody-based validation studies .

What methods can determine if CLE42 antibodies have neutralizing activity?

Assessing neutralizing activity of CLE42 antibodies requires functional assays:

• In vitro assays:

  • Receptor binding inhibition tests to determine if antibodies block CLE42-PXY interaction

  • Competitive binding assays with labeled synthetic peptides

  • Structure-function studies to identify binding epitopes

• Ex vivo approaches:

  • Test antibody effects on leaf disc senescence assays

  • Measure chlorophyll content and photosystem II efficiency (Fv/Fm) in antibody-treated samples

  • Compare with phenotypes observed in cle42 mutants

• In planta studies:

  • Apply antibodies to growing plants and assess senescence timing

  • Combine antibody treatment with dark-induced senescence assays

  • Compare antibody effects with those of synthetic CLE42p application

Similar to the antibody-mediated depletion approach used for CLEC-2 in search result , neutralizing antibodies against CLE42 should induce phenotypes resembling genetic knockouts.

How can CLE42 antibodies investigate receptor complex formation?

CLE42 antibodies can reveal insights into receptor complex formation and signaling:

• Receptor identification:

  • Immunoprecipitate CLE42 to identify bound receptors

  • Compare receptor binding in wild-type versus pxy mutant backgrounds

  • Investigate potential co-receptors similar to the CLV1/BAM1/BAM2 complexes described in search result

• Complex visualization:

  • Use antibodies in proximity ligation assays to visualize CLE42-receptor interactions in situ

  • Employ co-localization studies using fluorescently-labeled antibodies

  • Analyze the subcellular localization of CLE42-receptor complexes

• Functional analysis:

  • Evaluate how antibody binding affects receptor phosphorylation

  • Test if antibodies disrupt receptor oligomerization

  • Investigate downstream signaling effects of antibody-mediated receptor disruption

Research indicates CLE42 regulates leaf senescence primarily through the PXY receptor, as CLE42p treatment shows reduced effect in pxy mutant backgrounds .

What controls are essential when using CLE42 antibodies in research?

Proper experimental design requires comprehensive controls:

• Genetic controls:

  • Wild-type plants (positive control)

  • cle42 knockout mutants (negative/specificity control)

  • cle41 cle42 cle44 triple mutants (to address redundancy)

  • CLE42 overexpression lines (enhanced signal control)

• Peptide controls:

  • Synthetic CLE42p (HGVPSGPNPISN) for competition assays

  • CLE41/44p (HEVPSGPNPISN) to assess cross-reactivity

  • Unrelated CLE peptides as negative controls

• Technical controls:

  • Isotype control antibodies

  • Pre-immune serum controls for polyclonal antibodies

  • Secondary antibody-only controls

  • Peptide competition/blocking controls

These controls help validate antibody specificity and experimental outcomes.

How can researchers address challenges in detecting low-abundance CLE peptides?

Detecting low-abundance peptides like CLE42 requires specialized approaches:

• Sample enrichment:

  • Use immunoaffinity purification with validated antibodies

  • Implement peptide extraction methods optimized for small signaling molecules

  • Consider concentrating samples through precipitation or lyophilization

• Signal enhancement:

  • Employ high-sensitivity detection systems (e.g., chemiluminescence, fluorescence)

  • Use signal amplification methods (tyramide signal amplification, polymer detection)

  • Optimize antibody concentration and incubation conditions

• Alternative detection approaches:

  • Consider mass spectrometry-based targeted peptide detection

  • Use highly sensitive ELISA formats with optimized antibody pairs

  • Implement digital ELISA or single-molecule detection platforms

These strategies can help overcome the challenges of detecting physiologically relevant levels of CLE42 in complex plant samples.

What methodologies can distinguish between CLE42 precursor and mature peptide forms?

Understanding CLE42 processing requires antibodies that differentiate between precursor and mature forms:

• Antibody design strategy:

  • Generate antibodies against the precursor-specific regions outside the CLE domain

  • Develop antibodies specific to the mature 12-aa CLE42p

  • Create antibodies recognizing processing intermediates or cleavage sites

• Analytical approaches:

  • Use immunoblotting with appropriate gel systems to resolve size differences

  • Employ immunoprecipitation followed by mass spectrometry to identify forms

  • Implement subcellular fractionation to track processing through cellular compartments

• Application of antibody panels:

  • Use combinations of antibodies recognizing different regions in multiplexed assays

  • Track processing kinetics with time-course analyses

  • Correlate detection of different forms with biological activity

This approach allows researchers to investigate the processing, transport, and function of different CLE42 forms.

How should researchers interpret discrepancies between antibody detection and genetic data?

When antibody results conflict with genetic data, consider these potential explanations:

• Technical considerations:

  • Antibody specificity limitations (cross-reactivity with CLE41/44)

  • Differences in detection sensitivity between methods

  • Post-translational modifications affecting antibody recognition

• Biological considerations:

  • Functional redundancy between CLE42 and related peptides

  • Compensatory mechanisms in genetic mutants

  • Tissue-specific or developmental stage-dependent effects

• Resolution strategies:

  • Use multiple independent antibodies targeting different epitopes

  • Combine antibody detection with orthogonal methods (e.g., mass spectrometry)

  • Design experiments that directly compare genetic and immunological approaches

Recognizing that CLE42, CLE41, and CLE44 display functional redundancy in leaf senescence helps explain potential discrepancies .

What approaches help resolve cross-reactivity issues with CLE41/44?

Addressing cross-reactivity with closely related peptides requires specialized approaches:

• Antibody refinement:

  • Perform negative selection against cross-reactive peptides

  • Use affinity purification with specific peptides

  • Develop monoclonal antibodies with enhanced specificity

• Experimental design modifications:

  • Implement peptide competition controls with both CLE42p and CLE41/44p

  • Use genetic backgrounds lacking potential cross-reactive proteins

  • Employ parallel detection with different antibody clones

• Analytical considerations:

  • Apply quantitative standards to measure cross-reactivity

  • Use statistical methods to account for background signal

  • Consider advanced computational approaches to deconvolute signals

The functional redundancy between CLE42 and CLE41/44 highlights the importance of addressing cross-reactivity in antibody development .

How can researchers assess if their CLE42 antibodies detect biologically active forms?

Correlating antibody detection with biological activity requires these approaches:

• Functional bioassays:

  • Compare antibody reactivity with samples having known biological activity

  • Correlate antibody signal intensity with phenotypic outcomes

  • Use antibody-mediated depletion to assess functional consequences

• Structure-function analysis:

  • Determine if antibodies recognize epitopes critical for receptor binding

  • Compare detection of native versus denatured forms

  • Assess antibody binding to synthetic peptides with known activity

• Receptor interaction studies:

  • Evaluate if antibody-bound CLE42 can still interact with PXY receptor

  • Determine if antibody binding alters downstream signaling

  • Compare receptor activation in the presence/absence of antibodies

Synthetic CLE42p demonstrates biological activity by delaying leaf senescence , providing a reference standard for evaluating whether antibodies detect the biologically relevant form.

How might CLE42 antibodies contribute to understanding peptide hormone crosstalk?

CLE42 antibodies can advance our understanding of hormone crosstalk in several ways:

• Pathway integration analysis:

  • Investigate how CLE42 levels change in response to other hormone treatments

  • Examine how CLE42 antagonizes ethylene signaling at the protein level

  • Study potential interactions with additional hormone pathways

• Spatial regulation studies:

  • Map CLE42 distribution in relation to hormone response domains

  • Analyze CLE42 transport in relation to hormone movement

  • Investigate tissue-specific crosstalk mechanisms

• Temporal dynamics:

  • Track CLE42 levels during development and stress responses

  • Monitor real-time changes in CLE42 abundance during hormone treatments

  • Correlate CLE42 dynamics with changes in ethylene pathway components

Understanding how CLE42 communicates with the ethylene pathway provides a model for studying peptide-hormone crosstalk in plants .

What emerging technologies might enhance CLE42 antibody applications?

Several emerging technologies could advance CLE42 antibody research:

• Advanced imaging approaches:

  • Super-resolution microscopy for subcellular localization

  • Live-cell imaging with minimally disruptive antibody fragments

  • Correlative light and electron microscopy for structural context

• Single-cell analysis:

  • Single-cell proteomics to analyze cell-specific CLE42 levels

  • Spatial transcriptomics correlated with protein localization

  • High-throughput image-based phenotyping of cellular responses

• Systems biology integration:

  • Antibody-based interactome mapping

  • Multi-omics approaches incorporating antibody-validated protein data

  • Mathematical modeling of CLE42 signaling networks

These technologies could provide unprecedented insights into CLE42 function at cellular and molecular levels.

How might CLE42 antibodies contribute to understanding plant stress adaptation?

CLE42 antibodies can help elucidate stress adaptation mechanisms:

• Stress response analysis:

  • Monitor CLE42 levels during various abiotic stresses

  • Investigate how stress affects CLE42-ethylene pathway antagonism

  • Examine if CLE42 contributes to stress tolerance through delayed senescence

• Developmental adaptation:

  • Track CLE42 abundance during developmental transitions

  • Analyze how environmental factors influence CLE42 levels

  • Investigate if CLE42 contributes to developmental plasticity

• Translational applications:

  • Use antibodies to screen for genetic variants with altered CLE42 levels

  • Identify conditions that optimize CLE42 signaling for improved stress tolerance

  • Develop diagnostic tools to predict plant stress responses

The role of CLE42 in delaying leaf senescence suggests it may contribute to stress adaptation by extending photosynthetic capacity under challenging conditions .