CLE41 Antibody

What is CLE41 and what is its primary function in plants?

CLE41, also known as tracheary element differentiation inhibitory factor (TDIF), is a member of the CLE (CLAVATA3/ESR) family of peptide hormones in plants. It functions primarily as a signaling molecule that promotes stem cell activity specifically in the vascular meristem . Unlike some other CLE family members such as CLV3 that inhibit stem cell proliferation in shoot and root meristems, CLE41 plays a positive regulatory role in vascular stem cell maintenance . CLE41 acts through binding to its receptor TDIF RECEPTOR (TDR, also known as PXY) to regulate vascular development . This peptide hormone is particularly important for preventing the premature differentiation of procambial cells into xylem cells, thereby maintaining the vascular stem cell population .

How does CLE41 differ from other CLE peptides in terms of structure and function?

CLE41 possesses distinct structural features that differentiate it from other CLE peptides such as CLV3. Most notably, CLE41 contains a characteristic serine residue at position 11 (S11th) that is conserved specifically among CLE41-type peptides within the CLE family . This S11th residue is particularly important as it prevents CLE41 from displaying CLV3-like activity, thus ensuring functional specificity .

Unlike CLV3, which requires a histidine residue at position 11 (H11th) for its activity, CLE41 utilizes S11th to maintain its specific function . Molecular dynamics simulations have shown that S11th interacts with the TDR receptor, although this interaction is reduced at room temperature compared to the stable hydrogen bond observed in X-ray crystallography at lower temperatures . Additionally, the N-terminal residue of CLE41 contributes to its specific bioactivity without affecting its receptor specificity, as demonstrated by substitution experiments .

What is the CLE41-TDR signaling pathway and why is it important for plant development?

The CLE41-TDR signaling pathway represents a critical peptide-receptor module that regulates vascular development in plants. CLE41 functions as the ligand while TDR (also known as PXY) serves as its specific transmembrane receptor . This signaling pathway is essential for:

• Promoting vascular stem cell proliferation

• Inhibiting premature differentiation of procambial cells into xylem cells

• Regulating proper vascular patterning

• Maintaining organized cell division in the vascular cambium

Disruption of this pathway, as observed in tdr-1 and cle41-1 mutants, results in reduced stele width due to compromised vascular stem cell maintenance . The application of exogenous CLE41 can rescue the cle41-1 mutant phenotype but not the receptor mutant tdr-1, confirming the specificity of this ligand-receptor interaction . This signaling pathway represents a fundamental mechanism for maintaining the balance between cell proliferation and differentiation in the vascular meristem.

What are the most effective methods for detecting CLE41 expression in plant tissues?

Detection of CLE41 expression in plant tissues can be accomplished through several complementary approaches:

RNA-based detection methods:

• Quantitative real-time PCR (qRT-PCR) to measure CLE41 transcript levels

• In situ hybridization to visualize spatial expression patterns

• RNA-seq for genome-wide expression analysis

Protein-based detection methods:

• Immunohistochemistry using anti-CLE41 antibodies

• GFP reporter fusions to visualize protein localization

• Mass spectrometry for peptide identification

For studying CLE41 activity rather than just expression, researchers can use bioassays that measure:

• Stele thickness in roots (CLE41 increases stele width)

• Xylem differentiation patterns in leaf veins (CLE41 causes discontinued xylem strands)

• Vascular cell proliferation rates

When selecting detection methods, consider that CLE peptides are often present at low concentrations and undergo post-translational modifications, making protein detection challenging. Combining multiple approaches provides more comprehensive insights into CLE41 expression and function.

How can researchers differentiate between the effects of CLE41 and other CLE peptides in experimental settings?

Differentiating between the effects of CLE41 and other CLE peptides requires strategic experimental design:

Genetic approaches:

• Use specific mutants: cle41 mutants show reduced stele width while clv3 mutants exhibit enlarged shoot meristems

• Employ receptor mutants: tdr-1 (CLE41 receptor) versus clv1-101 (CLV3 receptor) to distinguish pathway-specific effects

• Create double mutants to analyze potential interactions between different CLE signaling pathways

Biochemical approaches:

• Conduct competitive binding assays using labeled peptides like [125I]ASA-KIN to determine receptor specificity

• Perform dose-response experiments - CLE41 promotes stele thickening at higher concentrations (>1μM), while CLV3 inhibits stele growth at lower concentrations (≥30nM)

Tissue-specific phenotypic analysis:

• Examine tissue-specific responses - CLE41 affects vascular tissue while CLV3 affects shoot and root meristems

• Analyze discontinued xylem strands in leaf veins (CLE41-specific phenotype)

• Measure stele width in roots (differentially affected by CLE41 and CLV3)

The table below summarizes key distinguishing features between CLE41 and CLV3 peptides:

What controls and validation steps are essential when using antibodies to study CLE41 in plant tissues?

When using antibodies to study CLE41 in plant tissues, several critical controls and validation steps must be implemented:

Essential controls:

• Negative controls:

  • cle41 mutant tissues to confirm antibody specificity

  • Pre-immune serum control to assess background staining

  • Secondary antibody-only control to detect non-specific binding

• Positive controls:

  • Tissues with confirmed CLE41 expression (e.g., vascular cambium)

  • CLE41 overexpression lines

  • Purified CLE41 peptide for Western blot standardization

Validation approaches:

• Cross-reactivity assessment:

  • Test antibody against other CLE peptides, particularly those with similar sequences

  • Perform peptide competition assays to confirm specificity

• Multiple detection methods:

  • Confirm antibody results with orthogonal techniques (e.g., RNA expression, reporter lines)

  • Use different antibodies targeting different epitopes of CLE41

• Functional validation:

  • Correlate antibody staining patterns with known CLE41-dependent phenotypes

  • Verify detection in tissues where CLE41 signaling is active

Technical considerations:

• Optimize fixation methods to preserve peptide epitopes

• Consider synthetic peptide immunization strategies that account for post-translational modifications

• Validate antibody performance under various experimental conditions (fixation, embedding, antigen retrieval)

How can synthetic peptide analogs of CLE41 be designed for enhanced research applications?

Designing synthetic CLE41 peptide analogs requires strategic modification approaches based on structure-function relationships:

Structural optimization strategies:

• Residue substitution:

  • Replace S11th with H11th to create dual-function peptides that interact with both TDR and CLV1 receptors

  • Modify N-terminal residues to alter peptide stability while maintaining specificity

  • Substitute non-critical residues with non-natural amino acids to enhance stability

• Hybrid peptide design:

  • Create chimeric peptides like KIN (combining properties of different CLE peptides) that exhibit both CLV3 and CLE41 activities

  • Develop peptides with specific amino acid swaps to target multiple receptors

• Chemical modifications:

  • Add fluorescent tags at non-critical positions for tracking peptide movement

  • Incorporate photoactivatable groups for temporal control of peptide activity

  • Design biotinylated versions for pull-down experiments

Functional considerations:

• Test dose-response relationships to determine optimal concentrations for desired activities

• Verify receptor binding using competitive displacement assays with labeled peptides

• Assess both inhibitory and promotional activities on stele development

The table below presents examples of synthetic CLE peptide variants and their bioactivities:

What approaches can be used to investigate the interaction between CLE41 and its receptor TDR at the molecular level?

Investigating CLE41-TDR interactions at the molecular level requires sophisticated biochemical, structural, and computational approaches:

Structural biology techniques:

• X-ray crystallography:

  • Co-crystallize CLE41 peptide with TDR receptor domain

  • Determine atomic resolution structure of the complex

  • Identify key interaction residues at the binding interface

• Cryo-electron microscopy:

  • Visualize larger receptor complexes in near-native conditions

  • Study conformational changes upon ligand binding

• NMR spectroscopy:

  • Analyze dynamic interactions between CLE41 and TDR

  • Map binding interfaces through chemical shift perturbations

Biochemical interaction analyses:

• Binding affinity measurements:

  • Surface plasmon resonance (SPR) to determine kon and koff rates

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Radiolabeled peptide binding assays using [125I]ASA-labeled peptides

• Cross-linking approaches:

  • Photo-affinity labeling to capture transient interactions

  • Chemical cross-linking followed by mass spectrometry (XL-MS)

Computational methods:

• Molecular dynamics simulations:

  • Model hydrogen bond formation between CLE41 and TDR at different temperatures

  • Analyze flexibility and stability of critical residues (e.g., S11th, N12th)

  • Simulate conformational changes upon binding

• Structure-based virtual screening:

  • Identify potential small-molecule modulators of CLE41-TDR interaction

  • Design peptide mimetics with enhanced properties

Research has shown that the interaction between S11th of CLE41 and TDR is reduced at room temperature compared to lower temperatures, suggesting temperature-dependent binding dynamics . Additionally, N12th forms stable hydrogen bonds with TDR receptor residues more than 95% of the time despite increased flexibility at physiological temperatures .

How does the CLE41/TDR signaling module integrate with other signaling pathways to regulate vascular development?

The CLE41/TDR signaling module interacts with multiple pathways to orchestrate vascular development through complex signaling networks:

Pathway interactions:

• Relationship with CLV3 signaling:

  • CLV3 and CLE41 pathways can exhibit synergistic effects when co-applied

  • The clv2-101 mutation affects the sensitivity to both CLV3 and CLE41, suggesting pathway convergence

  • Synthetic peptides like KIN can activate both pathways simultaneously

• Interaction with hormonal pathways:

  • Potential crosstalk with auxin transport and signaling for coordinated vascular patterning

  • Integration with cytokinin signaling, which also regulates procambial cell proliferation

  • Possible interaction with gibberellin pathways affecting cell elongation in vascular tissues

• Downstream transcriptional networks:

  • Regulation of WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors

  • Modulation of cell cycle regulators controlling vascular stem cell division

  • Potential influence on differentiation-promoting transcription factors

Genetic evidence for pathway integration:

• The clv2-101 mutant shows enhanced CLE41 responses and resistance to CLV3 inhibitory effects

• Simultaneous treatment with 10μM CLV3 and 10μM CLE41 produces a strong stele-thickening effect in clv2-101 mutants but not in wild-type plants

• Synthetic peptides like KIN exhibit concentration-dependent activation of different pathways

Understanding these pathway integrations provides opportunities for precise manipulation of vascular development through targeted interventions in specific signaling components or their interactions.

What are common challenges in CLE41 peptide preparation and how can they be addressed?

Researchers face several challenges when preparing and working with CLE41 peptides:

Peptide synthesis and stability issues:

• Chemical synthesis challenges:

  • Achieving high purity due to hydrophobic regions in the sequence

  • Ensuring proper disulfide bond formation if present

  • Maintaining correct post-translational modifications

• Stability concerns:

  • Peptide degradation during storage

  • Aggregation in aqueous solutions

  • Variable activity across different batches

Practical solutions:

• Synthesis optimization:

  • Use solid-phase peptide synthesis with optimized protection schemes

  • Consider native chemical ligation for challenging sequences

  • Verify peptide identity via mass spectrometry and HPLC

• Storage and handling:

  • Store lyophilized peptides at -20°C or -80°C

  • Prepare single-use aliquots to avoid freeze-thaw cycles

  • Use low-binding tubes to prevent adsorption to surfaces

• Quality control:

  • Test each batch in bioassays measuring stele thickening

  • Include positive controls (commercial peptides) in experiments

  • Determine optimal working concentrations through dose-response curves

Formulation recommendations:

• Dissolve peptides in DMSO before diluting to working concentration

• Include carrier proteins (0.1% BSA) for very dilute solutions

• Use buffers with physiological pH and defined ionic strength

• Consider cyclization strategies to enhance peptide stability

How can researchers address contradictory results when studying CLE41 function across different plant species or tissues?

Contradictory results when studying CLE41 function may arise from various sources:

Common sources of contradiction:

• Species-specific differences:

  • Variations in receptor-ligand affinities across species

  • Different downstream signaling components

  • Evolutionary divergence in CLE peptide functions

• Experimental variables:

  • Differences in peptide concentrations used (CLE41 shows dose-dependent effects)

  • Variations in application methods (soil drenching vs. direct application)

  • Different plant ages or developmental stages

  • Growth conditions affecting sensitivity to peptides

• Genetic background effects:

  • Presence of compensatory mechanisms in different backgrounds

  • Modifier genes affecting signaling outcomes

  • Varying receptor expression levels

Systematic resolution approaches:

• Standardized experimental design:

  • Use consistent peptide concentrations across experiments

  • Control plant age and growth conditions rigorously

  • Include appropriate genetic controls (wild-type, receptor mutants)

• Multi-layered validation:

  • Combine genetic, biochemical, and phenotypic analyses

  • Test multiple independent lines or accessions

  • Verify results with both loss-of-function and gain-of-function approaches

• Context-specific interpretation:

  • Consider tissue-specific effects (e.g., stele vs. xylem differentiation)

  • Account for differential receptor expression patterns

  • Acknowledge potential cross-talk with other signaling pathways

Research has shown that sensitivity to CLE peptides can differ by plant age, and specific mutations like clv2-101 can dramatically alter response patterns to both CLV3 and CLE41 , highlighting the importance of genetic background in experimental interpretation.

What strategies can improve the specificity and sensitivity of CLE41 detection in complex plant tissues?

Enhancing CLE41 detection in complex tissues requires optimized strategies:

Advanced immunological approaches:

• Antibody optimization:

  • Develop peptide-specific antibodies targeting unique regions of CLE41

  • Use monoclonal antibodies for increased specificity

  • Consider nanobodies for improved tissue penetration

• Signal amplification techniques:

  • Employ tyramide signal amplification (TSA) for low-abundance detection

  • Use proximity ligation assays (PLA) to detect CLE41-receptor interactions

  • Apply RNAscope technology for coupled RNA-protein detection

Genetic reporter systems:

• Transcriptional reporters:

  • Create promoter:GUS or promoter:GFP fusions to visualize expression domains

  • Use destabilized fluorescent proteins for dynamic expression studies

  • Develop split reporters to track both CLE41 and TDR expression simultaneously

• Translational fusions:

  • Generate CLE41-GFP fusions with minimal functional interference

  • Use epitope tags (HA, FLAG, Myc) for detection with commercial antibodies

  • Employ CRISPR/Cas9 to tag endogenous CLE41 loci

Tissue preparation techniques:

• Cellular resolution methods:

  • Use high-resolution confocal microscopy with clearing techniques

  • Apply expansion microscopy for subcellular localization

  • Employ laser capture microdissection for tissue-specific analysis

• Preservation strategies:

  • Optimize fixation protocols to maintain peptide antigenicity

  • Use cryosectioning to prevent antigenic loss

  • Consider hydrogel embedding techniques for structure preservation

The combination of these approaches allows for more sensitive and specific detection of CLE41 in complex plant tissues, enabling detailed analysis of its spatial distribution and functional dynamics.

What are promising areas for developing engineered CLE41 variants with novel functions?

Several promising research directions exist for engineering CLE41 variants with enhanced or novel functions:

Peptide engineering opportunities:

• Receptor specificity modification:

  • Develop bifunctional peptides like KIN that interact with multiple receptors

  • Create peptides with altered receptor preferences through targeted amino acid substitutions

  • Design variants with modified binding kinetics for sustained or pulsed signaling

• Biophysical property enhancement:

  • Improve peptide stability through cyclization or backbone modifications

  • Engineer membrane-permeable variants for enhanced tissue penetration

  • Develop pH-responsive peptides for environment-specific activation

• Functional expansions:

  • Create inducible CLE41 variants controlled by light or small molecules

  • Design peptides with altered dose-response characteristics

  • Develop CLE41 antagonists to block specific signaling events

Applications of engineered variants:

• Research tools:

  • Peptide biosensors to visualize receptor activation in real-time

  • Labeled variants for tracking receptor-ligand dynamics

  • Controlled perturbation of vascular development processes

• Biotechnological applications:

  • Modulation of wood formation in forestry species

  • Engineering of vascular development for improved stress resistance

  • Enhancement of secondary cell wall formation for biomass applications

The successful engineering of KIN, a synthetic hybrid peptide that exhibits both CLV3 and CLE41 activities through systematic swapping of amino acid residues , demonstrates the feasibility of creating novel peptides with expanded functions through rational design approaches.

How might systems biology approaches advance our understanding of CLE41 signaling networks?

Systems biology offers powerful approaches to unravel the complexity of CLE41 signaling:

Integrative methodologies:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from CLE41-treated tissues

  • Correlate phosphoproteomics with receptor activation dynamics

  • Map epigenetic changes associated with long-term CLE41 exposure

• Network modeling approaches:

  • Develop mathematical models of CLE41-TDR signal transduction

  • Simulate cross-talk between CLE41 and other signaling pathways

  • Predict emergent properties of vascular development regulation

• Single-cell technologies:

  • Apply single-cell RNA-seq to identify cell-type-specific responses to CLE41

  • Use spatial transcriptomics to map signaling gradients

  • Develop single-cell proteomics methods to track receptor activation

Anticipated research impacts:

• Mechanistic insights:

  • Identification of previously unknown pathway components

  • Discovery of feedback mechanisms regulating signal strength and duration

  • Elucidation of tissue-specific response differences

• Translational applications:

  • Rational design of interventions to modify vascular development

  • Identification of critical nodes for biotechnological manipulation

  • Development of predictive models for plant growth under varying conditions

The observation that CLE41 and CLV3 can act synergistically in certain genetic backgrounds suggests complex pathway interactions that would benefit from systems-level analysis to fully understand the regulatory networks governing plant vascular development.

What potential applications exist for manipulating CLE41 signaling in agricultural and biotechnological contexts?

Manipulation of CLE41 signaling offers diverse applications in agriculture and biotechnology:

Agricultural applications:

• Crop improvement strategies:

  • Enhance vascular development for improved nutrient and water transport

  • Modify wood properties in timber species through CLE41 signaling modulation

  • Develop stress-resistant varieties with optimized vascular architecture

• Yield enhancement approaches:

  • Improve photoassimilate transport through vascular system optimization

  • Enhance fruit development through modulated vascular bundle formation

  • Increase biomass production by promoting cambial activity

Biotechnological applications:

• Bioenergy sector:

  • Engineer plants with enhanced secondary cell wall development for biofuel production

  • Modify lignin content and composition through targeted CLE41 pathway manipulation

  • Develop rapid-growing woody biomass crops with enhanced vascular cambium activity

• Pharmaceutical applications:

  • Use CLE41-based peptide engineering as a model for developing synthetic peptide drugs

  • Apply receptor-peptide interaction principles to drug design

  • Develop plant-based production systems for therapeutic peptides

Technological approaches:

• Gene editing strategies:

  • CRISPR/Cas9 modification of CLE41 or TDR to alter signaling properties

  • Fine-tuning of promoter activity for spatial and temporal control

  • Creation of synthetic regulatory circuits incorporating CLE41 signaling components

• Peptide application technologies:

  • Develop slow-release formulations of synthetic CLE peptides

  • Create seed treatments to modulate early vascular development

  • Design targeted delivery systems for tissue-specific effects

The synthetic biology approach demonstrated in the creation of hybrid CLE peptides with novel activities provides a conceptual framework for rationally designing peptide variants with specific agricultural or biotechnological applications.