CESA9 Antibody

What is CESA9 and why are antibodies against it valuable for plant research?

CESA9 is a cellulose synthase protein that functions as part of a complex with other CESA proteins (notably CESA4 and CESA7) to synthesize cellulose in plant cell walls. Antibodies against CESA9 are valuable research tools for studying cellulose biosynthesis, protein-protein interactions within CESA complexes, and cellular localization of these critical enzymes. These antibodies allow researchers to investigate how mutations in CESA9 affect plant development, cellulose properties, and biomass production .

What approaches should be used to generate specific antibodies against CESA9?

Generation of specific CESA9 antibodies typically involves raising antibodies against the N-terminal predicted cytoplasmic domain of the protein, which contains unique sequences that distinguish it from other CESA family members. The methodology includes expressing recombinant N-terminal fragments of CESA9 in E. coli, immunizing animals (typically rabbits or goats), and employing a two-step immunopurification procedure to enrich for CESA9-specific antibodies. Verification of specificity should be performed using immunoblots with corresponding N-terminal fragments of various CESA proteins to confirm absence of cross-reactivity .

How can co-immunoprecipitation experiments be optimized to study CESA9 interactions with other cellulose synthase complex proteins?

For optimal co-immunoprecipitation of CESA9 with interacting partners:

• Protein extraction should be performed using non-denaturing conditions with mild detergents like Triton X-100 to preserve protein-protein interactions

• Use microsomal membrane extracts that are enriched for cell wall synthesis machinery

• Employ a negative control without primary antibody to verify specificity

• Compare results under both non-denaturing and denaturing conditions to distinguish direct interactions from indirect associations

• Verify antibody specificity prior to co-IP experiments using recombinant protein fragments

Research has shown that CESA proteins interact in Triton X-100-solubilized extracts but these interactions are disrupted under denaturing conditions, indicating the presence of stable but non-covalent associations .

What are the optimal methods for detecting CESA9 protein levels in plant samples?

Detection of CESA9 protein levels requires:

When analyzing CESA9 levels, researchers should be aware that this protein undergoes proteasome-dependent degradation, which can be assessed by treating samples with the proteasome inhibitor MG132 .

How can CESA9 antibodies be used to investigate the impact of mutations on cellulose synthase complex formation?

CESA9 antibodies provide valuable tools for investigating how mutations affect cellulose synthase complex assembly and function:

• Co-immunoprecipitation with antibodies against other CESA proteins (e.g., CESA4 and CESA7) to assess complex formation

• Immunoblotting of microsomal fractions to quantify CESA9 protein levels

• Comparative analysis of wild-type and mutant plants to determine CESA9 stability and association with other complex components

• Proteasome inhibition studies to assess protein degradation rates

Studies have shown that mutations in conserved regions of CESA9 (such as the P-CR region) can significantly reduce protein levels by promoting rapid proteasome degradation. For example, the Osfc16 mutation results in 71% reduction in CESA9 protein levels and also reduces levels of CESA4 and CESA7 by 34% and 22%, respectively .

What controls are essential when using CESA9 antibodies for immunolocalization experiments?

Essential controls for CESA9 immunolocalization include:

• Antibody specificity controls: Pre-immune serum and secondary antibody-only controls

• Genetic controls: CESA9 mutant or knockout plants as negative controls

• Competitive binding controls: Pre-incubation of antibody with immunizing peptide

• Cross-validation: Comparison with other localization methods (e.g., fluorescent protein tagging)

• Co-localization controls: Markers for subcellular compartments (plasma membrane, Golgi, etc.)

These controls help distinguish specific from non-specific binding and validate the subcellular localization patterns observed, which is particularly important when studying the dynamic trafficking of CESA complexes between the Golgi apparatus and the plasma membrane .

Why might Western blot analysis with CESA9 antibodies show multiple bands, and how should this be interpreted?

Multiple bands in Western blots using CESA9 antibodies may result from:

• Post-translational modifications (phosphorylation, glycosylation)

• Proteolytic degradation during sample preparation

• Cross-reactivity with closely related CESA family members (CESA2, CESA5)

• Alternative splicing variants

This phenomenon has been observed with other CESA antibodies, such as CESA6, where researchers detected a second band on immunoblots . To address this issue:

• Include recombinant CESA9 protein as a positive control

• Optimize extraction buffers with various protease inhibitor combinations

• Perform peptide competition assays to verify specificity

• Compare patterns between wild-type and CESA9 mutant plants

How can researchers distinguish between effects on CESA9 expression versus protein stability when studying mutants?

To differentiate between effects on CESA9 expression versus protein stability:

• Compare transcript levels using RT-qPCR with protein levels from Western blots

• Conduct pulse-chase experiments to measure protein turnover rates

• Treat samples with proteasome inhibitors (e.g., MG132) to block degradation

• Perform co-immunoprecipitation experiments under various conditions

Research has demonstrated that the CESA9 conserved-site mutation in the Osfc16 mutant affects protein stability rather than expression. When treated with MG132, the mutant showed a 100% increase in CESA9 biosynthesis rates compared to only a 15% increase in wild-type plants, indicating rapid proteasome degradation of the mutated protein .

How can CESA9 antibodies be used to study cellulose synthase complex dynamics during environmental stress?

CESA9 antibodies enable investigation of cellulose synthase complex responses to environmental stressors:

• Quantitative Western blot analysis to track changes in protein abundance

• Co-immunoprecipitation to assess complex stability under stress conditions

• Immunolocalization to observe changes in subcellular distribution

• Phospho-specific antibodies to monitor stress-induced post-translational modifications

These approaches can reveal how stresses like drought, temperature extremes, or pathogen exposure affect CESA9 incorporation into functional complexes and subsequent cellulose synthesis.

What considerations are important when using CESA9 antibodies for studying engineered plants with modified cell wall properties?

When studying engineered plants with modified cell wall properties:

• Epitope conservation: Verify that genetic modifications haven't altered the antibody recognition site

• Expression level variations: Use dilution series to ensure quantification within the linear range

• Background genetics: Include appropriate wild-type controls with matching genetic background

• Developmental staging: Compare tissues at equivalent developmental stages

• Cross-validation: Complement antibody-based approaches with direct cellulose measurements

These considerations are particularly important when evaluating CRISPR/Cas9-engineered plants with conserved-site mutations in CESA9, which have shown enhanced biomass enzymatic saccharification and improved lodging resistance while maintaining normal growth patterns .

How do antibody-based approaches complement other methods for studying CESA9 function?

CESA9 antibody applications provide unique insights when integrated with other research approaches:

This integrated approach has revealed that CESA9 mutations can affect the association of cellulose synthase complexes, leading to modified β-1,4-glucan chain synthesis and altered cellulose degree of polymerization .

What are the methodological differences when using CESA9 antibodies across different plant species?

When applying CESA9 antibodies across different plant species, researchers should consider:

• Epitope conservation: Sequence alignment analysis to predict cross-reactivity

• Extraction protocols: Optimization for different tissue types and cell wall compositions

• Detection sensitivity: Adjustment of antibody concentrations for species-specific expression levels

• Background reactivity: More extensive blocking procedures may be needed for some species

• Validation requirements: Independent confirmation with species-specific controls

These considerations ensure robust cross-species comparisons when investigating the conservation of CESA9 function and cellulose synthase complex organization across plant taxa.

How might phospho-specific antibodies advance our understanding of CESA9 regulation?

Phospho-specific antibodies targeting CESA9 could revolutionize our understanding of cellulose synthase regulation by:

• Identifying specific phosphorylation sites that regulate CESA9 activity

• Monitoring phosphorylation status changes during development and stress responses

• Determining how phosphorylation affects protein-protein interactions within cellulose synthase complexes

• Revealing kinase-phosphatase signaling networks that control cellulose synthesis

Development of such antibodies requires identification of phosphorylation sites through mass spectrometry, generation of phospho-specific antibodies, and careful validation using phosphatase treatments and phospho-null mutants.

What novel applications of CESA9 antibodies might emerge from combining them with advanced imaging techniques?

Integration of CESA9 antibodies with cutting-edge imaging approaches offers exciting research possibilities:

• Super-resolution microscopy: Nanoscale visualization of CESA complex organization at the plasma membrane

• Single-molecule tracking: Following individual CESA9 molecules during cellulose synthesis

• Expansion microscopy: Enhanced visualization of cell wall-plasma membrane interfaces

• Correlative light and electron microscopy: Connecting protein localization with ultrastructural features

• Proximity labeling: Identifying transient interaction partners in specific subcellular compartments

These applications could provide unprecedented insights into the dynamic assembly and function of cellulose synthase complexes containing CESA9, advancing our understanding of plant cell wall biosynthesis .