CPK33 Antibody

What is CPK33 and what are its primary functions in plants?

CPK33 is a calcium-dependent protein kinase that functions as an essential Ca²⁺ signal mediator in plants. It primarily mediates strigolactone (SL)-induced stomatal closure in guard cells and is involved in florigen complex formation through phosphorylation of the FD transcription factor. CPK33 has been shown to suppress ABA-induced stomatal closure while stimulating GORK (Guard cell Outward Rectifying K⁺) channel activity to promote stomatal closure under specific conditions. The protein's kinase activity is essential for its functions, as demonstrated by studies using kinase-dead mutants .

Why are CPK33 antibodies important research tools?

CPK33 antibodies are critical tools for studying calcium-dependent signaling pathways in plants, particularly in investigating guard cell responses and flowering regulation. These antibodies enable researchers to detect, localize, and quantify CPK33 expression in different tissues, assess protein modifications such as phosphorylation states, and study protein-protein interactions involving CPK33. They facilitate experiments that help elucidate the mechanisms of calcium signaling cascades that regulate important physiological processes such as stomatal movement and floral transition .

How can I determine the specificity of a CPK33 antibody?

To determine the specificity of a CPK33 antibody, you should:

• Perform Western blot analysis using wild-type plant samples alongside cpk33 mutant samples to confirm absence of signal in the mutant

• Test for cross-reactivity with other CPKs, particularly closely related family members like CPK10

• Conduct immunoprecipitation followed by mass spectrometry to verify that the antibody pulls down CPK33 and not other proteins

• Consider epitope mapping to identify the specific region of CPK33 that the antibody recognizes

• Include appropriate controls such as pre-immune serum or isotype controls in immunostaining experiments

How can I use CPK33 antibodies to study stomatal regulation?

CPK33 antibodies can be employed in multiple ways to study stomatal regulation:

• Immunolocalization studies: Use CPK33 antibodies for immunofluorescence microscopy to examine the spatial distribution of CPK33 in guard cells before and after strigolactone treatment.

• Co-immunoprecipitation assays: Deploy CPK33 antibodies to pull down CPK33 and identify interacting proteins involved in stomatal closure signaling pathways.

• Phosphorylation state analysis: Combine CPK33 antibodies with phospho-specific antibodies to examine changes in CPK33 phosphorylation status during stomatal movement.

• Chromatin immunoprecipitation (ChIP): If CPK33 associates with transcription factors, use CPK33 antibodies to identify target genes regulated during stomatal responses.

• In situ protein activity assays: Apply CPK33 antibodies in combination with activity-based probes to monitor kinase activity changes in guard cells under different environmental conditions .

What methods can be used to detect CPK33 phosphorylation activity?

To detect CPK33 phosphorylation activity, researchers can employ several approaches:

• In vitro kinase assays: Purify CPK33 using CPK33 antibodies and assess its ability to phosphorylate known substrates like FD, measuring activity through techniques such as phos-tag SDS-PAGE to visualize mobility shifts of phosphorylated products.

• Phospho-specific western blotting: Use antibodies that specifically recognize phosphorylated forms of CPK33 substrates.

• Mass spectrometry: Identify phosphorylation sites on target proteins after incubation with immunoprecipitated CPK33.

• Fluorescence-based kinase assays: Utilize fluorescently labeled ATP or substrate peptides to monitor kinase activity in real-time.

• Mobility shift assays: As demonstrated with CPK33KD (kinase-dead) mutants, phos-tag SDS-PAGE can detect both substrate phosphorylation and autophosphorylation of CPK33 .

How can I visualize CPK33 localization in plant cells?

To visualize CPK33 localization in plant cells, consider these methodological approaches:

• Immunofluorescence microscopy: Use primary CPK33 antibodies followed by fluorescently-labeled secondary antibodies on fixed plant tissues or cells.

• Confocal microscopy with fluorescent protein fusions: While not directly using antibodies, this complementary approach involves creating CPK33-GFP fusion constructs and transforming plants to visualize native localization patterns.

• Immunoelectron microscopy: For subcellular resolution, use CPK33 antibodies with gold-conjugated secondary antibodies to precisely localize CPK33 within cell structures.

• Proximity ligation assay (PLA): Detect in situ protein-protein interactions involving CPK33 by combining CPK33 antibodies with antibodies against potential interacting partners.

• Live cell immunostaining: For non-fixed samples, use membrane-permeable CPK33 antibody fragments to track dynamic localization changes during signaling events .

What are common issues with CPK33 antibody specificity and how can they be addressed?

Common specificity issues with CPK33 antibodies include:

• Cross-reactivity with related CPKs: The calcium-dependent protein kinase family has significant sequence homology, particularly in the catalytic domain. To address this:

  • Use antibodies raised against unique regions of CPK33, preferably the variable N-terminal domain

  • Validate with knockout mutants (cpk33-1 and cpk33-2)

  • Pre-absorb antibodies with recombinant proteins of closely related CPKs like CPK10

• Non-specific binding: To reduce background:

  • Optimize blocking conditions using different blockers (BSA, milk, normal serum)

  • Increase washing stringency in immunoblots and immunostaining

  • Use monoclonal antibodies when higher specificity is required

• Poor signal-to-noise ratio:

  • Titrate antibody concentrations

  • Include additional controls such as pre-immune serum

  • Consider signal amplification methods for low-abundance CPK33 detection

How can I optimize immunoprecipitation protocols for CPK33?

To optimize immunoprecipitation (IP) protocols for CPK33:

• Buffer optimization:

  • Include calcium chelators (EGTA) when studying non-calcium-bound forms

  • Add calcium when studying active forms

  • Use phosphatase inhibitors to preserve phosphorylation states

  • Include protease inhibitors to prevent degradation

• Antibody coupling strategies:

  • Covalently couple CPK33 antibodies to beads to prevent co-elution

  • Optimize antibody-to-bead ratios for maximum capture efficiency

• Extraction conditions:

  • Test different detergents (NP-40, Triton X-100, digitonin) for optimal solubilization

  • Adjust salt concentrations to maintain protein-protein interactions

• Elution methods:

  • Use gentle elution with peptide competition for native protein recovery

  • Consider on-bead digestion for downstream mass spectrometry analysis

• Validation:

  • Confirm successful IP by western blot analysis

  • Verify kinase activity of immunoprecipitated CPK33

What controls should be included when using CPK33 antibodies for Western blotting?

When performing Western blotting with CPK33 antibodies, include these essential controls:

• Negative controls:

  • cpk33 mutant tissues (cpk33-1 and cpk33-2) to confirm antibody specificity

  • Secondary antibody only (no primary antibody) to identify non-specific binding

  • Pre-immune serum to detect background binding

• Positive controls:

  • Recombinant CPK33 protein as a size reference

  • Tissues known to express high levels of CPK33

  • Immunoprecipitated CPK33 protein

• Loading controls:

  • Housekeeping proteins (actin, tubulin) to normalize protein loading

  • Total protein staining (Ponceau S, SYPRO Ruby) as an alternative normalization method

• Specificity controls:

  • Peptide competition assay to confirm epitope-specific binding

  • Comparison with CPK33-GFP transgenic plants using both anti-CPK33 and anti-GFP antibodies

• Treatment controls:

  • Samples treated with phosphatase to identify phosphorylation-dependent signals

  • Calcium-dependent binding evaluation using samples with varying calcium concentrations

How does CPK33 interact with the strigolactone signaling pathway in guard cells?

CPK33 serves as a critical mediator in the strigolactone (SL) signaling pathway in guard cells through several mechanisms:

• Calcium dependency: Pharmacological studies with calcium chelators and channel blockers demonstrate that CPK33 functions downstream of calcium influx in the SL-induced stomatal closure pathway. The cpk33 mutant is impaired in both SL- and Ca²⁺-induced stomatal closure, positioning CPK33 as an essential calcium transducer in this pathway.

• Relationship with ROS signaling: While the cpk33 mutant shows impaired H₂O₂-induced stomatal closure, SL-mediated H₂O₂ production remains intact in these mutants. This suggests CPK33 functions downstream of H₂O₂ in the SL signaling cascade rather than regulating ROS production.

• Substrate specificity: CPK33 likely phosphorylates specific targets in guard cells that regulate ion channel activity and membrane transport, though these substrates remain to be fully characterized in the context of SL signaling.

• Kinase activity requirement: Studies with kinase-dead CPK33 mutants (D197N) confirm that the catalytic activity of CPK33 is essential for SL-induced stomatal closure, not just its physical presence in protein complexes .

What is known about the interplay between CPK33 and other calcium-dependent kinases in flowering regulation?

The interplay between CPK33 and other calcium-dependent kinases in flowering regulation reveals complex redundancy and specialization patterns:

• Functional redundancy: The relatively limited impact of cpk33 single mutants on flowering time under standard conditions suggests functional redundancy with other calcium-dependent protein kinases. The stronger phenotype observed with dominant-negative approaches (expressing kinase-dead CPK33) supports this redundancy hypothesis.

• FD phosphorylation: CPK33 phosphorylates threonine 282 of FD, a basic leucine zipper transcription factor, which is critical for the formation of the florigen complex with FLOWERING LOCUS T (FT) protein and 14-3-3 proteins. Other CDPKs may also contribute to this phosphorylation in the absence of CPK33.

• Calcium signaling integration: The involvement of CPK33 in flowering indicates that calcium signaling integrates with photoperiodic flowering pathways. How calcium oscillations or signatures are decoded by different CDPKs remains an important area for investigation.

• Tissue-specific functions: CPK33's role in the shoot apical meristem for flowering regulation contrasts with its function in guard cells for stomatal regulation, suggesting tissue-specific roles for this kinase that may overlap with different sets of related kinases in each tissue .

How can kinase-dead CPK33 mutants be utilized to study calcium signaling networks?

Kinase-dead CPK33 mutants (such as CPK33D197N) offer valuable tools for dissecting calcium signaling networks:

• Dominant-negative approach: When expressed in wild-type plants, kinase-dead CPK33 can interfere with endogenous CDPK activity against shared substrates, revealing functions that might be masked by redundancy in knockout studies. This approach produced a clear delayed-flowering phenotype when CPK33KD was expressed in the shoot apical meristem.

• Substrate trapping: Kinase-dead mutants can still bind substrates but cannot phosphorylate them, effectively "trapping" the substrates. This property can be exploited to identify physiological substrates through co-immunoprecipitation followed by mass spectrometry.

• Structure-function analysis: By comparing the interaction profiles of wild-type and kinase-dead CPK33, researchers can distinguish between interactions that depend on phosphorylation versus those that rely on physical docking.

• Calcium dependency studies: CPK33KD mutants can help elucidate which aspects of CPK33 function are dependent on its kinase activity versus its calcium-binding or scaffold properties.

• Cross-talk analysis: By expressing CPK33KD in different genetic backgrounds (with mutations in other CDPKs or calcium decoders), researchers can uncover hierarchical relationships and redundancies in calcium signaling networks .

What are the best methods to analyze CPK33 gene expression patterns?

To analyze CPK33 gene expression patterns effectively, consider these methodological approaches:

• Quantitative RT-PCR (qRT-PCR):

  • Design primers specific to CPK33, avoiding cross-amplification of related CPKs

  • Use reference genes with stable expression across experimental conditions

  • Include technical and biological replicates for statistical validity

• RNA-seq analysis:

  • For genome-wide expression context, analyze CPK33 expression patterns across tissues and conditions

  • Apply careful bioinformatic analysis to distinguish CPK33 from close homologs

  • Validate RNA-seq findings with qRT-PCR for key conditions

• In situ hybridization:

  • Design probes specific to CPK33 mRNA

  • Use sense probes as negative controls

  • Apply to tissue sections to visualize spatial expression patterns

• Reporter gene constructs:

  • Generate CPK33 promoter-GUS or CPK33 promoter-GFP fusions

  • Create transgenic plants to visualize tissue-specific expression patterns

  • Examine expression under various treatments and developmental stages

• Single-cell RNA-seq:

  • For cell-type specific expression patterns in complex tissues like the shoot apex or leaf

What techniques can reveal CPK33 protein-protein interactions in planta?

To investigate CPK33 protein-protein interactions in planta, researchers can employ these techniques:

• Co-immunoprecipitation (Co-IP):

  • Use CPK33-specific antibodies to pull down native protein complexes

  • Analyze interacting partners by Western blot or mass spectrometry

  • Include calcium in buffers to maintain calcium-dependent interactions

• Bimolecular Fluorescence Complementation (BiFC):

  • Split YFP system with CPK33 and potential interactors

  • Allows visualization of interactions in living plant cells

  • Can reveal subcellular locations of interactions

• Förster Resonance Energy Transfer (FRET):

  • Create CPK33 fusions with donor fluorophores and potential interactors with acceptor fluorophores

  • Provides dynamic information about interactions in living cells

  • Can detect calcium-dependent interaction changes

• Proximity-dependent biotin identification (BioID):

  • Fuse CPK33 to a biotin ligase

  • Identify proteins in close proximity through biotinylation and streptavidin pulldown

  • Less affected by interaction strength than Co-IP

• Yeast two-hybrid (Y2H) and split-ubiquitin systems:

  • For initial screening of potential interactors

  • Results should be confirmed with in planta methods

  • Consider using calcium-independent mutants for consistent results

The interaction between CPK33 and FD, as demonstrated by biochemical assays, shows that CPK33 can still interact with substrate proteins even when its kinase activity is abolished (CPK33KD) .

How should I design experiments to analyze CPK33 function across different environmental conditions?

When designing experiments to analyze CPK33 function across environmental conditions, consider these methodological approaches:

• Genetic resources preparation:

  • Use multiple cpk33 mutant alleles (cpk33-1, cpk33-2)

  • Create complementation lines with native CPK33 promoter

  • Generate kinase-dead (CPK33D197N) lines for dominant-negative studies

  • Develop CPK33-overexpression lines for gain-of-function analysis

  • Consider tissue-specific expression systems (like the FD promoter for shoot apex studies)

• Environmental variables to test:

  • Light conditions (intensity, quality, photoperiod)

  • Temperature regimes (normal, heat stress, cold stress)

  • Water availability (drought, flooding)

  • Hormone treatments (strigolactones, ABA, other phytohormones)

  • Calcium availability (altered [Ca²⁺] in growth media)

• Phenotypic analyses:

  • For stomatal regulation: measure stomatal apertures, water loss rates, thermal imaging

  • For flowering time: count leaf numbers, days to bolting, floral meristem development

  • Physiological measurements: photosynthetic rates, transpiration, ABA sensitivity

• Biochemical analyses:

  • Monitor CPK33 expression and protein levels across conditions

  • Assess phosphorylation status of CPK33 and its substrates

  • Measure kinase activity with substrate phosphorylation assays

  • Analyze calcium binding properties under different conditions

• Data integration approaches:

  • Use statistical methods appropriate for multi-factorial experimental designs

  • Consider principal component analysis for complex datasets

  • Develop mathematical models of CPK33 function in signaling networks