GID1A Antibody

What is GID1A and what is its function in plants?

GID1A is one of three homologous gibberellin (GA) receptors in Arabidopsis thaliana, alongside GID1B and GID1C. It functions as the primary GA receptor in plants, playing a crucial role in stem elongation through both proteolytic and non-proteolytic signaling pathways . GID1A belongs to a family of proteins with similarity to hormone-sensitive lipases that bind preferentially to bioactive gibberellins in vitro. The direct physiological role of GID1A involves mediating GA-dependent growth processes by interacting with DELLA proteins, which are growth repressors, in a GA-dependent manner .

How do GID1A, GID1B, and GID1C differ functionally in plant development?

While all three GID1 receptors display some functional redundancy, they exhibit specific developmental roles:

The relative importance of GID1A is evidenced by the severe phenotypes observed in mutants. While single mutants develop normally (suggesting functional redundancy), double mutants show specific defects: gid1a gid1c displays reduced stem height, while gid1a gid1b shows lower male fertility .

How does feedback regulation affect GID1A expression?

GID1A expression is subject to feedback regulation similar to GA biosynthesis genes. GA treatment reduces GID1A transcript abundance to approximately 50% of normal levels, suggesting that plants modulate receptor levels in response to hormone status . This feedback mechanism appears to be mediated through the GA signaling pathway involving DELLA proteins. Mutations in DELLA proteins (such as rga-24 and gai-t6 null mutations) result in slightly decreased GID1A mRNA levels, while gain-of-function DELLA mutations (rga-Δ17) cause an opposite effect on GID1A expression . This regulatory mechanism likely helps maintain hormone homeostasis in plant tissues.

How specific are commercially available GID1A antibodies, and what cross-reactivity concerns should researchers address?

When selecting a GID1A antibody, researchers must carefully evaluate specificity due to the high sequence similarity among the three GID1 homologs in Arabidopsis. Most commercially available antibodies show some degree of cross-reactivity between GID1A, GID1B, and GID1C unless specifically validated for GID1A-exclusive detection . Researchers should review antibody validation data showing tests against all three GID1 proteins, preferably in both wild-type and corresponding knockout mutant backgrounds.

For applications requiring absolute specificity, techniques like epitope-specific antibodies targeting unique regions of GID1A or recombinant antibody fragments may offer improved selectivity . Always confirm specificity through Western blotting against control samples and consider pre-absorption with recombinant GID1B and GID1C proteins when absolute specificity is critical.

What are the optimal antibody formats for different GID1A experimental applications?

The selection of antibody format depends on the specific experimental application:

For co-immunoprecipitation studies investigating GID1A interactions with DELLA proteins or other partners, antibodies recognizing epitopes outside the protein-protein interaction domains are preferable to avoid interference with natural binding events .

What are the recommended protocols for detecting GID1A-DELLA protein interactions using antibody-based approaches?

Detection of GID1A-DELLA protein interactions requires careful experimental design due to the GA-dependency of these interactions. A comprehensive approach includes:

• Co-immunoprecipitation (Co-IP): Conduct experiments in both GA-treated and untreated samples. Use anti-GID1A antibodies to immunoprecipitate protein complexes, then probe for DELLA proteins (RGA, GAI, etc.) with specific antibodies. Include appropriate negative controls such as immunoprecipitation in gid1a knockout backgrounds .

• Pull-down assays: Express recombinant GST-tagged GID1A and incubate with plant extracts containing DELLA proteins in the presence and absence of bioactive GA. Wash thoroughly and detect bound DELLA proteins using anti-DELLA antibodies .

• Bimolecular Fluorescence Complementation (BiFC): For in vivo visualization, create fusion constructs of GID1A and DELLA proteins with split fluorescent protein fragments. Co-express in plant cells with and without GA treatment to observe interaction-dependent fluorescence reconstitution.

The GA-dependency of these interactions is critical - GID1A-DELLA interactions significantly increase in the presence of bioactive GA (except for GID1B which shows some GA-independent interaction) . Include both positive controls (known interactors) and negative controls (non-interacting proteins) in all experimental designs.

How should researchers optimize immunohistochemical detection of GID1A in plant tissues?

Successful immunohistochemical detection of GID1A in plant tissues requires addressing several challenges specific to plant materials:

• Tissue fixation: Use a mixture of 4% paraformaldehyde with 0.1-0.5% glutaraldehyde for optimal protein preservation while maintaining antigenicity. Vacuum infiltration is essential for complete penetration into plant tissues.

• Cell wall considerations: Include cell wall digesting enzymes (pectolyase, cellulase) in a controlled partial digestion step to improve antibody accessibility without compromising tissue integrity.

• Antigen retrieval: Perform heat-mediated antigen retrieval in citrate buffer (pH 6.0) to expose GID1A epitopes that may be masked during fixation, but optimize conditions to avoid tissue damage.

• Background reduction: Pre-block with 5% normal serum from the secondary antibody host species plus 3% BSA and 0.3% Triton X-100. Include 0.1% Tween-20 in all wash steps to reduce non-specific binding common in plant tissues.

• Signal amplification: For low-abundance detection, implement tyramide signal amplification (TSA) or quantum dot-conjugated secondary antibodies to enhance sensitivity while maintaining specificity.

When examining tissue-specific expression patterns, parallel staining of gid1a mutant tissues is essential to validate signal specificity. Additionally, consider complementary RNA in situ hybridization to correlate protein localization with transcript distribution .

How can researchers quantitatively analyze GID1A expression levels across different developmental stages?

Quantitative analysis of GID1A expression requires a multi-method approach to overcome the challenges of plant tissue heterogeneity and developmental variation:

• Quantitative Western Blotting: Implement a standardized protocol using recombinant GID1A protein standards at known concentrations to create a calibration curve. Normalize GID1A signals to stable reference proteins (preferably multiple references like actin, tubulin, and GAPDH) using digital image analysis software.

• qRT-PCR correlation: Measure both GID1A protein (via quantitative immunoblotting) and transcript levels (via qRT-PCR) to establish the relationship between mRNA and protein abundance across developmental stages. This helps identify stages with post-transcriptional regulation .

• Statistical considerations: Apply appropriate statistical methods for time-series data analysis. Using a minimum of 4-5 biological replicates per developmental stage is recommended to account for natural variation.

Developmental expression analysis has revealed that GID1A transcripts are subject to feedback regulation, with GA treatment reducing transcript levels to approximately 50% of normal abundance . This feedback control must be considered when interpreting expression data from different developmental contexts where endogenous GA levels fluctuate naturally.

What control experiments are essential when interpreting GID1A antibody data in the context of GA signaling studies?

Rigorous controls are crucial for accurate interpretation of GID1A antibody data in GA signaling research:

• Genetic controls:

  • gid1a single mutant tissues (negative control)

  • gid1a/b/c triple mutant tissues (complete negative control)

  • Tissues with GID1A overexpression (positive control)

  • GA-deficient mutant backgrounds (e.g., ga1-3) to assess GA-dependency

• Treatment controls:

  • GA treatment time course (analyzing changes in GID1A-DELLA interactions)

  • GA biosynthesis inhibitor treatments (paclobutrazol)

  • Comparison between bioactive and inactive GA forms

• Antibody controls:

  • Pre-immune serum controls

  • Epitope blocking with purified recombinant GID1A

  • Secondary antibody-only controls

• Protein interaction controls:

  • For co-IP experiments, include known non-interacting proteins

  • When studying GID1A-DELLA interactions, include GA-insensitive DELLA mutants lacking the DELLA domain, which prevents interaction with GID1 proteins

One key aspect often overlooked is the feedback regulation of GID1A expression by GA treatment. Studies should account for the approximately 50% reduction in GID1A transcript levels following GA treatment when interpreting protein-level data .

How can GID1A antibodies be applied in studying non-proteolytic GA signaling mechanisms?

Non-proteolytic GA signaling represents an important research frontier where GID1A antibodies can provide crucial insights:

• Immunoprecipitation-mass spectrometry (IP-MS): Use anti-GID1A antibodies to capture native GID1A-DELLA complexes from sly1 mutant backgrounds where DELLA degradation is impaired. This approach allows identification of additional complex components specific to non-proteolytic signaling .

• Proximity-dependent labeling: Implement BioID or TurboID-GID1A fusion proteins combined with specific antibodies to identify proteins in the vicinity of GID1A-DELLA complexes in living cells, capturing transient interactions in non-proteolytic contexts.

• Chromatin studies: Apply GID1A antibodies in ChIP-seq workflows to investigate whether GID1A-DELLA complexes associate with chromatin in non-proteolytic signaling, potentially identifying direct transcriptional regulation mechanisms.

Research has established that GID1A is the primary GA receptor mediating stem elongation in both proteolytic and non-proteolytic signaling pathways, while GID1B has a stronger role in fertility during non-proteolytic signaling . This differential involvement may be related to GID1B's higher affinity for DELLA proteins, as it shows some interaction even in the absence of GA . Antibody-based approaches can help elucidate these mechanistic differences by enabling precise tracking of protein complex formation in various genetic backgrounds.

What methodological adaptations are needed when using GID1A antibodies to study GA-GID1-DELLA-SLY1 complex formation?

Studying the quaternary GA-GID1-DELLA-SLY1 complex formation requires specialized methodological adaptations:

• Sequential co-immunoprecipitation: Implement a two-step immunoprecipitation protocol where first GID1A is immunoprecipitated with anti-GID1A antibodies, followed by a second immunoprecipitation targeting DELLA proteins or SLY1 from the first eluate. This approach helps isolate complete quaternary complexes.

• Yeast three-hybrid assays: While not directly using antibodies, these assays have demonstrated that the GA-GID1 complex promotes the interaction between DELLA proteins (like RGA) and the F-box protein SLY1, a component of the SCF^SLY1 E3 ubiquitin ligase that targets DELLA proteins for degradation .

• Protein crosslinking: Apply membrane-permeable crosslinkers before immunoprecipitation to stabilize transient quaternary complexes. MS/MS analysis of crosslinked peptides can reveal interaction interfaces.

• Competitive binding studies: Use purified proteins and antibodies in in vitro binding assays to determine how GA concentration affects the relative affinity of GID1A for DELLA proteins versus SLY1 in quaternary complex formation.

Research has shown that the N-terminal sequence containing the DELLA domain is necessary for GID1 binding . Additionally, the formation of the GA-GID1-DELLA complex enhances the interaction with the SLY1 F-box component, presumably promoting polyubiquitination by the SCF ubiquitin E3 ligase . These complex interactions can be precisely mapped using antibody-based approaches combined with structural biology techniques.

What are common pitfalls when working with GID1A antibodies and how can researchers address them?

Researchers frequently encounter several challenges when working with GID1A antibodies:

• Cross-reactivity issues: Due to the high sequence similarity between GID1A, GID1B, and GID1C (66-82% amino acid identity), antibodies may not distinguish between these homologs .

  • Solution: Validate specificity using tissues from single, double, and triple gid1 mutants. Consider using peptide antibodies targeting unique regions of GID1A or performing pre-absorption with recombinant GID1B and GID1C proteins.

• Low signal strength: GID1A may be expressed at relatively low levels in some tissues.

  • Solution: Implement signal amplification methods such as tyramide signal amplification for immunohistochemistry or use more sensitive detection systems for immunoblotting (e.g., chemiluminescent substrates with extended exposure times).

• GA-dependent conformational changes: GA binding induces conformational changes in GID1A that may mask or expose different epitopes.

  • Solution: Test antibody recognition under both GA-treated and untreated conditions. Consider using multiple antibodies targeting different regions of the protein.

• Feedback regulation affecting expression levels: GA treatment reduces GID1A transcript levels by approximately 50% .

  • Solution: Account for this feedback regulation when comparing samples with different GA status by normalizing to appropriate reference proteins and including proper controls.

• Fixation-sensitive epitopes: Plant-specific fixatives may interfere with antibody recognition.

  • Solution: Test multiple fixation protocols and include antigen retrieval steps to optimize epitope accessibility without compromising tissue structure.

How can researchers validate the specificity of their GID1A antibody results in plant experimental systems?

Rigorous validation is essential for ensuring the reliability of GID1A antibody results:

• Genetic validation approach:

  • Test antibody reactivity in wild-type plants versus gid1a single, gid1a/b, gid1a/c double, and gid1a/b/c triple mutants

  • Include GID1A overexpression lines as positive controls

  • Compare signal patterns with known expression domains from promoter-reporter studies

• Molecular validation:

  • Perform peptide competition assays using the immunizing peptide to confirm specificity

  • Compare immunoblot patterns with expected molecular weight (MW ≈ 38 kDa for GID1A)

  • Confirm detection of recombinant GID1A protein at known concentrations

• Functional validation:

  • Correlate antibody signals with known GA responses

  • Compare patterns with RNA expression data from transcriptomic studies

  • Validate interaction partners (e.g., DELLA proteins) in co-IP experiments

• Technical controls:

  • Include secondary antibody-only controls to identify non-specific binding

  • Use pre-immune serum comparisons to establish baseline reactivity

  • Implement isotype controls matching the GID1A antibody class

• Cross-species validation: If the antibody is claimed to recognize GID1A orthologs in multiple species, confirm specificity across species using the appropriate genetic resources in each system.

These validation approaches should be systematically documented to establish confidence in experimental findings, particularly for publications where antibody specificity is critical to data interpretation.

How do the binding affinities of different GA receptors affect experimental design when using GID1A antibodies?

The differential binding affinities among GID1 receptors have significant implications for experimental design:

These differential affinities necessitate careful experimental consideration:

• Binding assays: When studying GA-dependent interactions, researchers should use physiologically relevant GA concentrations that distinguish between the differing affinities of GID1 receptors. GID1B's higher affinity means it will form complexes at lower GA concentrations than GID1A or GID1C .

• Immunoprecipitation conditions: The stronger affinity of GID1B for DELLA proteins means that more stringent washing conditions may be needed when using GID1A antibodies to avoid detecting indirect interactions via GID1B in complex samples.

• Genetic background considerations: When characterizing GID1A antibody specificity, test not only against gid1a mutants but also against gid1b knockout backgrounds, as GID1B's higher affinity for DELLA may create competing interactions that affect experimental outcomes .

• GA dose-response experiments: Include carefully calibrated GA dose-response experiments to distinguish receptor-specific effects, particularly when studying tissues where multiple GID1 receptors are expressed.

Understanding these differential binding properties is critical when interpreting results from antibody-based studies of GA signaling complexes .

What methodological approaches can distinguish between proteolytic and non-proteolytic functions of GID1A in GA signaling?

Distinguishing between these two signaling modes requires specialized experimental approaches:

• Genetic-biochemical combined approach:

  • Use sly1 mutant backgrounds where DELLA proteins cannot be degraded via the conventional proteolytic pathway

  • Compare with sly1 gid1a double mutants to isolate GID1A-specific effects in non-proteolytic signaling

  • Monitor DELLA protein levels via immunoblotting with anti-DELLA antibodies to confirm lack of degradation

• Temporal resolution experiments:

  • Implement time-course studies with anti-GID1A and anti-DELLA antibodies

  • Non-proteolytic responses typically occur more rapidly than proteolytic responses that require protein degradation

  • Use protein synthesis inhibitors (cycloheximide) to distinguish primary signaling events from secondary responses

• Protein modification analysis:

  • Employ phospho-specific antibodies against DELLA proteins to detect post-translational modifications

  • Use immunoprecipitation with GID1A antibodies followed by mass spectrometry to identify modifications associated with non-proteolytic signaling

• Target gene expression:

  • Compare transcriptional responses in wild-type versus sly1 and sly1 gid1a backgrounds

  • Identify genes responding to GA in sly1 (non-proteolytic) but not in sly1 gid1a (neither pathway)

Research has demonstrated that GID1A is the primary GA receptor stimulating stem elongation in both proteolytic and non-proteolytic signaling pathways, while GID1B plays a stronger role in fertility during non-proteolytic GA signaling . These functional differences provide a framework for designing experiments that can distinguish between these parallel signaling mechanisms.