CCOAOMT Antibody

What is CCOAOMT and why are CCOAOMT antibodies important in plant research?

CCoAOMT (caffeoyl CoA O-methyltransferase) is a critical enzyme in the lignin biosynthesis pathway that catalyzes the methylation of caffeoyl CoA to feruloyl CoA. This enzyme plays essential roles in phenylpropanoid metabolism and participates in the synthesis of various secondary metabolites, including lignin components and anthocyanins .

CCoAOMT antibodies are vital research tools because they allow for:

• Detection and quantification of CCoAOMT protein expression across different tissues

• Monitoring changes in CCoAOMT levels during plant development

• Investigating CCoAOMT's role in plant defense responses through immunoblotting

• Conducting protein-protein interaction studies via co-immunoprecipitation assays

• Analyzing subcellular localization through immunohistochemistry techniques

These antibodies enable researchers to study the complex roles of CCoAOMT in plant metabolism, stress responses, and development .

How are polyclonal CCOAOMT antibodies typically generated for research applications?

The generation of polyclonal CCoAOMT antibodies typically follows this methodological approach:

• Recombinant protein expression: The CCoAOMT gene is cloned into an expression vector (typically pGEX-KG) and expressed in E. coli cells to produce a recombinant CCoAOMT protein .

• Protein purification: The recombinant protein is purified using chromatography on an agarose-glutathione matrix, which allows for isolation of the target protein with high purity .

• Immunization protocol: The purified CCoAOMT protein (approximately 50-100 μg) is emulsified with adjuvant (Freund's complete adjuvant for initial injection, incomplete adjuvant for booster injections) and administered through multiple intramuscular injections at 5-week intervals .

• Serum collection: Serum is collected approximately 10 days after each boost immunization. After clot removal, the serum is clarified by centrifugation and stored in small aliquots at -20°C .

• Antibody validation: The specificity of the antibodies is verified through immunoblotting against both recombinant CCoAOMT proteins and native plant extracts to confirm cross-reactivity with various CCoAOMT isoforms .

For example, antibodies raised against CCoAOMT-1 recombinant protein have been shown to effectively recognize CCoAOMT proteins from multiple classes with similar efficiencies .

What are the different classes of CCOAOMT enzymes and how do antibodies recognize them?

CCoAOMT enzymes are classified into distinct groups based on their sequence homology and evolutionary relationships:

Antibodies raised against CCoAOMT-1 recombinant protein can efficiently detect CCoAOMT proteins from all three classes with similar efficiencies. This cross-reactivity is due to the conservation of key epitopes across CCoAOMT classes, making these antibodies valuable tools for studying multiple CCoAOMT isoforms simultaneously .

In immunoblotting applications, CCoAOMT isoforms typically appear as 27-kD and 32-kD immunoreactive bands, allowing researchers to distinguish between different isoforms based on their molecular weights .

How can CCOAOMT antibodies be used to study protein-protein interactions in plant defense responses?

CCoAOMT antibodies serve as powerful tools for investigating protein-protein interactions in plant defense mechanisms through several advanced methodological approaches:

• Co-immunoprecipitation (Co-IP) assays: CCoAOMT antibodies can be used to capture CCoAOMT proteins along with their interacting partners. This technique has revealed that CCoAOMT2 physically associates with both NLR (nucleotide-binding leucine-rich repeat) proteins like Rp1-dp2 and other enzymes in the lignin biosynthesis pathway such as HCT1806 and HCT4918 .

• Validation of interaction specificity: Through carefully controlled Co-IP experiments, researchers have demonstrated differential interaction specificities among CCoAOMT isoforms. For example, CCoAOMT2 shows strong interaction with Rp1-dp2, while CCoAOMT1 exhibits little to no interaction with the same protein .

• Complex formation analysis: CCoAOMT antibodies have helped establish that CCoAOMT2, HCTs, and Rp1 proteins can form multiprotein complexes in planta, suggesting coordinated roles in defense regulation .

• Domain-specific interaction studies: By using antibodies in combination with protein variants expressing specific domains (such as the coiled-coil domain of Rp1-D21), researchers have identified which regions of proteins are critical for the interaction with CCoAOMT .

This approach has provided significant insights into how CCoAOMT enzymes can modulate plant immune responses through protein-protein interactions that are independent of their enzymatic activity, suggesting additional regulatory roles beyond their metabolic functions .

What methodologies are recommended for analyzing CCOAOMT protein expression during pathogen-induced hypersensitive response?

When analyzing CCoAOMT protein expression during pathogen-induced hypersensitive response (HR), researchers should implement the following comprehensive methodology:

• Time-course sampling: Collect plant tissue samples at multiple time points before and after pathogen inoculation (e.g., 0, 24, 48, 72, and 96 hours post-inoculation) to capture the dynamic changes in CCoAOMT expression .

• Protein extraction optimization: Use optimized extraction buffers containing protease inhibitors to ensure preservation of CCoAOMT proteins, particularly when working with tissues undergoing HR which may have elevated protease activity .

• Quantitative immunoblotting:

  • Separate proteins using SDS-PAGE

  • Transfer to membranes for immunoblotting with CCoAOMT-specific antibodies

  • Include multiple biological replicates

  • Use imaging software to quantify band intensities for statistical analysis

• Parallel enzyme activity assays: Complement immunoblotting data with CCoAOMT enzyme activity measurements using appropriate substrates (preferably CoA esters) to correlate protein abundance with functional activity .

• Comparative analysis with other defense-related proteins: Analyze CCoAOMT expression alongside other defense-related enzymes like COMTs (caffeic acid O-methyltransferases) to establish temporal relationships in the defense response network .

• RNA expression correlation: Perform RT-PCR or qRT-PCR analysis of CCoAOMT transcript levels to determine whether protein accumulation correlates with gene expression, helping to distinguish between transcriptional and post-transcriptional regulation .

This methodology has revealed that CCoAOMT accumulation follows a pattern similar to COMT I and II during HR, with significant induction occurring around 48 hours after inoculation, coinciding with the appearance of necrotic lesions in tobacco leaves infected with TMV .

How can CCOAOMT antibodies be optimized for cross-species immunodetection in comparative plant studies?

Optimizing CCoAOMT antibodies for cross-species immunodetection requires a strategic approach to maximize detection efficiency while maintaining specificity:

• Epitope selection for antibody generation:

  • Target highly conserved regions of CCoAOMT proteins based on multi-species sequence alignments

  • Avoid regions with high sequence variability between species

  • Select epitopes that maintain structural integrity across phylogenetic distances

• Validation across multiple species:

  • Test antibody reactivity against recombinant CCoAOMT proteins from various plant species

  • Create a reactivity profile documenting detection efficiency across different taxonomic groups

  • Determine minimum detectable protein amounts for each species

• Optimizing immunoblotting conditions:

  • Adjust blocking reagents to minimize background in different plant extracts

  • Optimize primary antibody concentration for each plant species

  • Determine optimal incubation times and temperatures for different plant materials

  • Consider using enhanced chemiluminescence or fluorescent detection systems for improved sensitivity

• Addressing species-specific challenges:

  • For species with lower antibody affinity, increase protein loading or antibody concentration

  • For species with high background, incorporate additional washing steps or alternative blocking agents

  • Consider using protein A/G conjugated secondary antibodies for improved binding across species

Existing evidence shows that antibodies raised against tobacco CCoAOMT-1 can effectively detect CCoAOMT proteins from different classes within the same species . This suggests that targeting conserved epitopes can yield antibodies with broad detection capabilities across CCoAOMT isoforms and potentially across related plant species.

What are common technical challenges when using CCOAOMT antibodies in immunoprecipitation experiments?

When using CCoAOMT antibodies for immunoprecipitation (IP) experiments, researchers frequently encounter several technical challenges that require methodological solutions:

• Low abundance of native CCoAOMT:

  • Challenge: Native CCoAOMT proteins may be expressed at low levels in certain tissues or under certain conditions.

  • Solution: Scale up starting material, use tissues with known high expression (such as stems for lignification studies or pathogen-induced tissues where CCoAOMT is upregulated) .

  • Validation approach: Include positive controls with known expression patterns.

• Transient or weak protein-protein interactions:

  • Challenge: Interactions between CCoAOMT and partner proteins (like Rp1 proteins) may be transient or condition-dependent.

  • Solution: Use chemical crosslinking agents before cell lysis to stabilize protein complexes; optimize buffer conditions to preserve interactions .

  • Example: Co-IP experiments have successfully demonstrated that CCoAOMT2 interacts with Rp1-dp2, while CCoAOMT1 does not show detectable interaction .

• Specificity issues between CCoAOMT isoforms:

  • Challenge: Antibodies may recognize multiple CCoAOMT isoforms with different affinities.

  • Solution: Pre-clear lysates with control antibodies; use peptide competition assays to confirm specificity; complement IP results with alternative interaction methods .

  • Data validation: Always include controls for non-specific binding.

• Buffer compatibility issues:

  • Challenge: Different extraction buffers may affect CCoAOMT solubility and interaction stability.

  • Solution: Test multiple lysis buffers with varying detergent types and concentrations; include appropriate protease inhibitors to prevent degradation during IP procedures.

• Quantification challenges:

  • Challenge: Accurate quantification of immunoprecipitated proteins can be difficult.

  • Solution: Use standardized loading controls, consistent antibody batches, and quantitative western blotting techniques with appropriate imaging software .

Successful Co-IP experiments have been performed by coexpressing tagged versions of proteins (e.g., HA-tagged Rp1 proteins and EGFP-tagged CCoAOMT) in heterologous systems like Nicotiana benthamiana, demonstrating the feasibility of studying CCoAOMT interactions despite these challenges .

How can researchers distinguish between CCOAOMT isoforms using antibody-based techniques?

Distinguishing between CCoAOMT isoforms presents a significant challenge due to their high sequence similarity. Researchers can employ these methodological approaches:

• Isoform-specific antibody development:

  • Target unique epitopes in non-conserved regions of different CCoAOMT isoforms

  • Develop antibodies against synthetic peptides corresponding to variable regions

  • Validate specificity against recombinant proteins of each isoform

• Two-dimensional gel electrophoresis coupled with immunoblotting:

  • Separate proteins based on both isoelectric point and molecular weight

  • Perform immunoblotting with general CCoAOMT antibodies

  • Identify isoform-specific spots based on their unique migration patterns

  • This approach can separate CCoAOMT isoforms that have similar molecular weights but different pI values

• Sequential immunoprecipitation:

  • Perform initial immunoprecipitation with pan-CCoAOMT antibodies

  • Subject the precipitate to isoform-specific antibodies or analyze by mass spectrometry

  • Quantify relative abundances of different isoforms

• Differential interaction patterns:

  • Exploit known differences in protein-protein interactions between isoforms

  • For example, CCoAOMT2 strongly interacts with Rp1-dp2 and HCT proteins, while CCoAOMT1 shows weak or no interaction

  • Use co-immunoprecipitation followed by specific antibody detection to identify isoforms based on their binding partners

• Correlation with transcript analysis:

  • Combine protein detection with isoform-specific RT-PCR

  • Design primers that specifically amplify each CCoAOMT class transcript

  • Verify transcript identity through RFLP analysis of PCR products

  • Correlate transcript abundance with protein detection

Research has shown that different CCoAOMT isoforms can exhibit distinct molecular weights (e.g., 27-kD and 32-kD bands) on immunoblots, which can be used as an initial distinguishing characteristic . Additionally, differences in expression patterns during development and stress responses can provide contextual information to aid in isoform identification .

What are the best methodological approaches for analyzing CCOAOMT protein subcellular localization using antibodies?

Analyzing CCoAOMT protein subcellular localization using antibodies requires a multi-faceted methodological approach to ensure accurate results:

• Immunofluorescence microscopy protocol optimization:

  • Tissue fixation: Use 4% paraformaldehyde for protein cross-linking while preserving cellular architecture

  • Permeabilization: Optimize detergent concentration (0.1-0.5% Triton X-100) to allow antibody access while maintaining cellular structures

  • Blocking: Use 2-5% bovine serum albumin or normal serum to reduce non-specific binding

  • Primary antibody incubation: Determine optimal concentration through titration experiments

  • Secondary antibody selection: Use fluorophore-conjugated antibodies compatible with available microscopy equipment

  • Controls: Include secondary-only controls and pre-immune serum controls to verify specificity

• Subcellular fractionation combined with immunoblotting:

  • Isolate distinct cellular compartments (cytosol, nucleus, mitochondria, chloroplasts, etc.) using differential centrifugation

  • Verify fraction purity using compartment-specific marker proteins

  • Perform immunoblotting of each fraction with CCoAOMT antibodies

  • Quantify relative distribution across cellular compartments

• Immuno-electron microscopy for high-resolution localization:

  • Process tissues for transmission electron microscopy

  • Perform immunogold labeling using CCoAOMT antibodies

  • Analyze gold particle distribution quantitatively across cellular compartments

  • This approach provides nanometer-scale resolution of protein localization

• Comparative analysis with fluorescent protein fusions:

  • Complement antibody-based localization with expressed fluorescent protein-tagged CCoAOMT

  • Compare localization patterns between both approaches

  • Use this dual approach to validate findings and rule out artifacts

• Dynamic localization studies:

  • Analyze potential changes in CCoAOMT localization during development or stress responses

  • Sample tissues at multiple time points after treatment

  • Quantify changes in compartmental distribution

Research on StCCoAOMT10 in Solanum tuberosum has demonstrated that this protein is primarily localized to the cytoplasm and nucleus . This finding illustrates the importance of comprehensive localization studies, as the subcellular distribution may provide insights into the diverse functions of CCoAOMT beyond its canonical enzymatic role in the cytoplasm.

How do CCOAOMT proteins contribute to plant defense responses and how can antibodies help study this mechanism?

CCoAOMT proteins play multifaceted roles in plant defense responses that extend beyond their conventional metabolic functions. CCoAOMT antibodies provide critical tools for elucidating these mechanisms:

• Induction during pathogen response:

  • CCoAOMT protein levels increase significantly following pathogen infection or during hypersensitive response (HR)

  • Immunoblotting with CCoAOMT antibodies has demonstrated that enzyme accumulation patterns mirror those of other defense-related enzymes like COMT I and II

  • Protein accumulation is particularly pronounced around 48 hours post-infection, coinciding with the appearance of necrotic lesions

• Protein complex formation in immune regulation:

  • CCoAOMT2 physically interacts with nucleotide-binding leucine-rich repeat (NLR) immune receptors like Rp1-D21

  • Co-immunoprecipitation using CCoAOMT antibodies has revealed that CCoAOMT2 can suppress Rp1-D21-induced HR

  • This suppression occurs through direct protein-protein interaction rather than through CCoAOMT's enzymatic activity

• Multi-protein defense complexes:

  • CCoAOMT antibodies have helped demonstrate that CCoAOMT2, HCT enzymes, and Rp1 proteins can form complexes in planta

  • These complexes appear to function in regulating defense responses, potentially by fine-tuning the hypersensitive response

• Allelic variation affecting defense function:

  • Different alleles of CCoAOMT2 show varying abilities to suppress HR

  • Immunodetection has helped characterize these functional differences by confirming comparable protein expression levels despite different functional outcomes

• Research methodology applications:

  • Monitor CCoAOMT induction kinetics during infection using quantitative immunoblotting

  • Track protein-protein interactions via co-immunoprecipitation with CCoAOMT antibodies

  • Localize CCoAOMT during defense responses using immunohistochemistry

  • Compare CCoAOMT isoform contributions to defense through differential detection

The research demonstrates that CCoAOMT proteins function at the intersection of metabolism and immunity, with CCoAOMT2 specifically acting as a negative regulator of the hypersensitive response through its physical interaction with NLR immune receptors . This represents a novel mechanism linking lignin biosynthesis enzymes with immune regulation in plants.

What methodological approaches can resolve contradictory data when studying CCOAOMT's dual roles in metabolism and defense?

When investigating the seemingly contradictory roles of CCoAOMT in both primary metabolism (lignin biosynthesis) and defense responses, researchers should employ these methodological approaches to resolve discrepancies:

• Separation of enzymatic activity from protein interaction effects:

  • Generate and express catalytically inactive CCoAOMT mutants while preserving protein structure

  • Test both wild-type and mutant proteins for their ability to suppress HR

  • Measure enzymatic activity in parallel with defense phenotypes

  • This approach has revealed that CCoAOMT2's role in suppressing HR is likely independent of its metabolic activity

• Domain-specific interaction studies:

  • Create truncated or chimeric CCoAOMT proteins to identify domains responsible for defense functions

  • Use co-immunoprecipitation with specific antibodies to assess how these modifications affect protein interactions

  • Compare interaction patterns across CCoAOMT isoforms that differ in their defense functions

  • For example, studies have shown that CCoAOMT2 interacts with the coiled-coil domain of Rp1-D21, while CCoAOMT1 does not

• Temporal and spatial resolution of activities:

  • Employ tissue-specific and inducible expression systems

  • Monitor both enzymatic activity and defense phenotypes across different tissues and timepoints

  • Use immunolocalization to determine whether CCoAOMT relocates during defense responses

  • Correlate CCoAOMT subcellular localization with different functions

• Comparative analysis across alleles with differential effects:

  • Analyze allelic variants with different suppressive effects on HR

  • Identify key amino acid differences (e.g., in "Patch 1" near the N-terminus in CCoAOMT2 alleles)

  • Test chimeric proteins incorporating these variable regions to pinpoint defense-modulating domains

• Systems biology approach to resolve contextual functions:

  • Use antibodies to track CCoAOMT in different protein complexes

  • Perform immunoprecipitation followed by mass spectrometry to identify all interaction partners

  • Create interaction network maps to visualize context-dependent functions

  • Compare these networks between metabolic and defense contexts

This integrated approach has helped researchers develop a model where CCoAOMT2 can participate both in lignin biosynthesis through its enzymatic activity and in defense regulation through protein-protein interactions with immunity complexes, demonstrating how a single protein can serve distinct functions depending on cellular context .

How can CCOAOMT antibodies be used to investigate the relationship between lignin biosynthesis and immunity in plants?

CCoAOMT antibodies provide powerful tools for investigating the complex relationship between lignin biosynthesis and plant immunity through these advanced methodological approaches:

• Dual pathway monitoring during defense responses:

  • Track changes in both lignin biosynthesis enzymes and immune components simultaneously

  • Use CCoAOMT antibodies alongside antibodies against NLR proteins (like Rp1) and defense signaling components

  • Quantify protein abundance changes in both pathways during infection timecourses

  • This approach has revealed that CCoAOMT2 can physically associate with both HCT enzymes (involved in lignin biosynthesis) and Rp1 proteins (immune receptors)

• Protein complex composition analysis:

  • Perform sequential immunoprecipitations targeting different components

  • Use CCoAOMT antibodies for initial pulldown, followed by detection of associated proteins

  • Alternatively, use NLR protein antibodies for pulldown and detect associated CCoAOMT

  • Characterize the composition and stoichiometry of these multi-protein complexes

  • Research has demonstrated that CCoAOMT2, HCTs, and Rp1 proteins can form complexes in planta

• Subcellular localization studies during defense activation:

  • Track potential relocalization of CCoAOMT during immune activation

  • Use immunofluorescence microscopy at different timepoints after pathogen challenge

  • Determine whether CCoAOMT co-localizes with defense signaling components during infection

  • Compare localization patterns between CCoAOMT isoforms with different defense functions

• Genetic manipulation validation:

  • Generate plants with altered CCoAOMT levels or mutated CCoAOMT proteins

  • Use antibodies to confirm protein expression or absence in these lines

  • Correlate CCoAOMT protein levels with both lignin content and defense phenotypes

  • This approach can distinguish between effects due to protein abundance versus protein activity

• Differential phosphorylation and post-translational modification analysis:

  • Use phospho-specific antibodies or general CCoAOMT antibodies followed by phosphorylation detection

  • Compare modification patterns between metabolic and defense contexts

  • Identify potential regulatory modifications that switch CCoAOMT between its dual functions

These approaches have contributed to a model where lignin biosynthesis enzymes like CCoAOMT2 serve dual roles: their canonical function in phenylpropanoid metabolism and an unexpected role in modulating defense responses through direct interaction with immune components . This research highlights the intricate relationship between primary metabolism and immunity in plants, suggesting that metabolic enzymes can serve as regulators of defense responses.

What emerging techniques might enhance the utility of CCOAOMT antibodies in plant research?

Several cutting-edge techniques are poised to significantly enhance the utility of CCoAOMT antibodies in plant research:

• Proximity labeling coupled with immunoprecipitation:

  • Fuse CCoAOMT with proximity labeling enzymes (BioID, TurboID, or APEX2)

  • Allow in vivo biotinylation of proteins in close proximity to CCoAOMT

  • Use CCoAOMT antibodies to immunoprecipitate the protein complex

  • Identify biotinylated proteins through mass spectrometry

  • This approach would provide a comprehensive view of the CCoAOMT interactome under various conditions

• Single-cell proteomics with CCoAOMT immunodetection:

  • Apply antibody-based detection methods at single-cell resolution

  • Combine with single-cell transcriptomics to correlate protein and mRNA levels

  • Map CCoAOMT distribution across different cell types in plant tissues

  • This would reveal cell-type-specific roles of CCoAOMT that may be masked in whole-tissue analyses

• Super-resolution microscopy for precise localization:

  • Apply techniques like STORM or PALM using fluorophore-conjugated CCoAOMT antibodies

  • Achieve nanometer-scale resolution of CCoAOMT localization

  • Visualize dynamic protein complex formation during defense responses

  • Combine with other labeled proteins to observe direct interactions in situ

• In planta protein dynamics using antibody-based biosensors:

  • Develop FRET-based biosensors incorporating CCoAOMT antibody fragments

  • Monitor real-time changes in CCoAOMT conformation or interactions

  • Track protein dynamics during pathogen infection or environmental stresses

  • This would provide temporal resolution currently missing from static analyses

• CCoAOMT complex cryo-EM structural studies:

  • Use antibodies to purify native CCoAOMT-containing complexes

  • Apply cryo-electron microscopy to determine complex structures

  • Resolve the structural basis for CCoAOMT's dual functions in metabolism and immunity

  • This approach could reveal how CCoAOMT2 physically interacts with both HCT enzymes and NLR proteins

These emerging techniques would address current knowledge gaps regarding the spatiotemporal dynamics of CCoAOMT function, the structural basis of its protein-protein interactions, and its cell-type-specific roles in both metabolism and immunity, potentially revealing new therapeutic targets for enhancing plant disease resistance.

How might research on CCOAOMT expand our understanding of the evolution of plant defense mechanisms?

Research on CCoAOMT using antibody-based approaches can significantly expand our understanding of plant defense evolution through these methodological investigations:

• Comparative immunological analysis across plant lineages:

  • Apply CCoAOMT antibodies to detect homologous proteins across evolutionary diverse plant species

  • Compare protein abundance, localization, and complex formation patterns

  • Determine when the dual roles of CCoAOMT in metabolism and immunity emerged

  • This approach could reveal whether the defense function of CCoAOMT is ancestral or derived

• Structural and functional analysis of CCoAOMT variants:

  • Compare CCoAOMT variants across plant species using antibody-based detection

  • Correlate structural differences with functional divergence in immunity roles

  • Research has already identified critical sequence regions (like "Patch 1" near the N-terminus) that differ between CCoAOMT alleles with varying effects on immunity

  • Phylogenetic analysis has shown that monocot CCoAOMTs form a distinct clade

• Co-evolutionary studies with pathogen effectors:

  • Use antibodies to investigate whether pathogens directly target CCoAOMT

  • Perform co-immunoprecipitation between CCoAOMT and known pathogen effectors

  • Determine if pathogen pressure has driven CCoAOMT evolution

  • This could reveal whether CCoAOMT is a direct target of pathogen manipulation

• Allelic diversity characterization across populations:

  • Apply antibodies to detect variant-specific CCoAOMT proteins

  • Quantify expression levels of different alleles in diverse germplasm

  • Correlate allelic variants with disease resistance phenotypes

  • Studies have already identified variable suppressive effects of different CCoAOMT2 alleles on hypersensitive response

• Multi-level analysis of defense network evolution:

  • Use CCoAOMT antibodies alongside antibodies against other defense components

  • Compare defense complex composition across evolutionary diverse plant species

  • Trace the evolutionary history of the interconnection between phenylpropanoid metabolism and immunity

This research could reveal how plants have repurposed metabolic enzymes for defense functions throughout evolutionary history. The discovery that CCoAOMT2 physically interacts with NLR immune receptors represents a novel connection between primary metabolism and immunity , suggesting that such dual-function proteins might be more common in plant defense systems than previously recognized. Understanding this evolutionary process could provide new strategies for engineering disease resistance in crops.