DI19-3 Antibody

What is Di19-3 and what is its role in plant biology?

Di19-3 (Drought-induced 19-3) is a protein that functions as a positive regulator of auxin signaling pathways in plants. It physically interacts with AtIAA14, an Aux/IAA protein that serves as a negative regulator of auxin signaling. Research has demonstrated that Di19-3 plays a significant role in plant development processes and abiotic stress responses. The protein influences hypocotyl development, lateral root formation, and mediates cross-talk between auxin and ethylene signaling pathways. Studies with Atdi19-3 mutant seedlings reveal altered expression of genes involved in auxin biosynthesis and homeostasis, including NIT2, ILL5, and YUCCA genes, along with auxin-responsive genes like AUX1 and MYB77 .

How does Di19-3 influence auxin signaling pathways?

Di19-3 interacts directly with AtIAA14, an Aux/IAA protein, as demonstrated through multiple interaction assays including yeast two-hybrid (Y2H), bimolecular fluorescence complementation, and in vitro pull-down assays. This interaction affects auxin-induced degradation of AtIAA14, which is typically regulated through the proteasome pathway. In Atdi19-3 mutants, the auxin-induced degradation of AtIAA14 is delayed, suggesting that Di19-3 facilitates proper auxin response by regulating IAA14 stability. Expression studies using pIAA14::mIAA14-GFP in Atdi19-3 mutant backgrounds confirm that both proteins function in the same pathway to influence lateral root development in Arabidopsis .

What phenotypes are observed in Di19-3 mutant plants?

The Atdi19-3 mutant exhibits several distinct phenotypes that reflect disrupted auxin signaling:

• Short hypocotyl development in both light and dark conditions

• Compromised temperature-induced hypocotyl elongation

• Enhanced root growth inhibition when grown in auxin-supplemented medium

• Reduced lateral root formation under normal growth conditions

• Higher accumulation of IAA compared to wild-type plants

• Enhanced sensitivity to ethylene in triple response assays

• Increased tolerance to abiotic stress during seed germination and cotyledon greening phases

These phenotypes collectively indicate Di19-3's multifaceted role in plant development and stress responses .

What are the key considerations when selecting antibodies for Di19-3 research?

When selecting antibodies for Di19-3 research, scientists should consider:

• Specificity: The antibody should exclusively recognize Di19-3 with minimal cross-reactivity with other Di19 family proteins. Validation through knockout/knockdown controls is essential.

• Application compatibility: Determine if the antibody has been validated for your specific application (Western blot, immunoprecipitation, immunohistochemistry, ChIP).

• Species reactivity: Ensure the antibody recognizes Di19-3 from your experimental organism (Arabidopsis thaliana is the most common model for Di19-3 studies).

• Epitope information: Understanding which region of Di19-3 the antibody targets can be important for experimental design, especially if studying protein-protein interactions or protein domains.

• Format appropriateness: Consider whether you need primary antibodies for detection or functional grade antibodies for manipulation of protein function.

Similar to antibody validation protocols used for other research antibodies, Di19-3 antibodies should be tested through multiple assays and controls .

How can researchers validate the specificity of Di19-3 antibodies?

Validating antibody specificity for Di19-3 requires a systematic approach:

• Knockout validation: Test the antibody against wild-type and Di19-3 knockout samples. A specific antibody should show signal in wild-type samples but no signal in knockout samples.

• Overexpression validation: Test samples overexpressing Di19-3 to confirm increased signal intensity.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application to samples. This should block specific binding and eliminate signal.

• Western blot analysis: Confirm the antibody detects a band at the expected molecular weight (~30 kDa for Di19-3) with minimal non-specific bands.

• Comparative analysis with multiple antibodies: When possible, use multiple antibodies targeting different epitopes of Di19-3 to confirm results.

These validation approaches ensure experimental results are attributable to Di19-3 and not to cross-reactivity with other proteins .

What applications are most appropriate for studying Di19-3 protein interactions?

Based on current research methodologies, the following applications are most effective for studying Di19-3 protein interactions:

• Yeast Two-Hybrid (Y2H): Successfully used to demonstrate interaction between Di19-3 and AtIAA14. This approach is particularly useful for initial screening of potential interaction partners.

• Bimolecular Fluorescence Complementation (BiFC): Effective for visualizing protein-protein interactions in plant cells. This technique has confirmed the Di19-3 and AtIAA14 interaction in vivo.

• Pull-down assays: In vitro pull-down assays provide biochemical confirmation of direct protein-protein interactions. This has been used to validate the interaction between Di19-3 and auxin signaling components.

• Co-immunoprecipitation (Co-IP): While not explicitly mentioned in the search results for Di19-3, Co-IP is a standard technique that would be appropriate for studying endogenous protein interactions in plant tissues.

• Fluorescence Resonance Energy Transfer (FRET): This technique could provide quantitative information about interaction dynamics and proximity between Di19-3 and other proteins in living cells .

How can researchers investigate the role of Di19-3 in abiotic stress responses?

Investigating Di19-3's role in abiotic stress responses requires a multi-faceted experimental approach:

• Stress tolerance assays: Compare wild-type and di19-3 mutant plants under various stress conditions (drought, salt, heat, cold). Measure parameters such as:

  • Germination rate

  • Survival percentage

  • Biomass accumulation

  • Root/shoot growth

  • Chlorophyll content

  • Electrolyte leakage

• Gene expression analysis: Perform RNA-seq or qRT-PCR to identify differential expression patterns of stress-responsive genes in wild-type versus di19-3 mutant plants under normal and stress conditions.

• Protein accumulation studies: Use Di19-3 specific antibodies to monitor protein accumulation patterns during stress exposure through Western blotting or immunolocalization.

• Promoter analysis: Investigate the regulation of Di19-3 expression using promoter-reporter constructs (GUS, LUC) under various stress conditions.

• Complementation studies: Express Di19-3 under constitutive or inducible promoters in di19-3 mutant backgrounds to confirm functional relevance to observed phenotypes.

• Biochemical assays: Measure stress-related metabolites (proline, soluble sugars, malondialdehyde) and enzyme activities (SOD, CAT, APX) to assess stress response mechanisms .

What approaches can be used to study Di19-3 involvement in auxin-ethylene crosstalk?

The crosstalk between auxin and ethylene signaling pathways involving Di19-3 can be investigated through these methodological approaches:

• Hormone sensitivity assays:

  • Perform triple response assays with various concentrations of ethylene precursors (ACC) on di19-3 mutants

  • Test auxin response using DR5::GUS reporter lines crossed with di19-3 mutants

  • Analyze root growth inhibition in response to exogenous auxins and ethylene in combination

• Genetic interaction studies:

  • Generate double mutants between di19-3 and key components of auxin (tir1, arf7/19) and ethylene (ein2, etr1) signaling

  • Compare phenotypes of single and double mutants to establish epistatic relationships

• Biochemical interaction studies:

  • Investigate whether Di19-3 directly interacts with ethylene signaling components using Y2H, BiFC, and pull-down assays

  • Examine if ethylene affects the interaction between Di19-3 and Aux/IAA proteins

• Hormone quantification:

  • Measure endogenous auxin and ethylene levels in di19-3 mutants under normal and stress conditions using GC-MS or LC-MS/MS

• Pharmacological approaches:

  • Apply ethylene inhibitors (AgNO₃, AVG) to di19-3 mutants and monitor auxin-related phenotypes

  • Use auxin transport inhibitors (NPA) to determine if Di19-3 functions upstream or downstream of auxin transport

Data from these approaches would comprehensively map Di19-3's position in the auxin-ethylene signaling network .

What are the recommended protocols for analyzing Di19-3 protein localization and dynamics?

For analyzing Di19-3 protein localization and dynamics, researchers should consider these methodological approaches:

• Fluorescent protein fusion constructs:

  • Generate N- and C-terminal GFP/YFP/mCherry fusions with Di19-3 under native promoter

  • Express in di19-3 mutant background to confirm functionality through complementation

  • Use confocal microscopy to visualize subcellular localization under different conditions

• Immunolocalization:

  • Use validated Di19-3 antibodies for immunofluorescence microscopy

  • Apply various fixation and permeabilization protocols to preserve protein localization

  • Use colabeling with organelle markers to confirm subcellular compartmentalization

• Protein dynamics studies:

  • Perform Fluorescence Recovery After Photobleaching (FRAP) with fluorescently tagged Di19-3

  • Use photoactivatable or photoconvertible tags to track protein movement

  • Apply hormone treatments or stress conditions to monitor changes in localization patterns

• Cell fractionation and biochemical verification:

  • Isolate nuclear, cytoplasmic, and membrane fractions

  • Perform Western blot analysis with Di19-3 antibodies on each fraction

  • Compare fractionation patterns under normal and stress conditions

• Time-lapse imaging:

  • Monitor Di19-3-GFP localization in response to hormones and stresses in real-time

  • Quantify changes in fluorescence intensity across cellular compartments

These approaches provide complementary data on protein localization and dynamics that are essential for understanding Di19-3 function .

How can researchers overcome challenges in detecting low abundance Di19-3 protein?

Detecting low abundance proteins like Di19-3 can be challenging. Here are recommended strategies to enhance detection:

• Sample enrichment techniques:

  • Immunoprecipitation to concentrate Di19-3 before analysis

  • Subcellular fractionation to reduce sample complexity

  • TCA precipitation to concentrate proteins from dilute samples

• Signal amplification methods:

  • Use high-sensitivity ECL substrates for Western blotting

  • Apply tyramide signal amplification for immunohistochemistry

  • Consider biotin-streptavidin detection systems for enhanced sensitivity

• Optimized extraction procedures:

  • Test multiple extraction buffers to improve Di19-3 solubilization

  • Include appropriate protease inhibitors to prevent degradation

  • Optimize extraction conditions based on Di19-3's predicted properties

• Alternative detection methods:

  • Consider targeted mass spectrometry approaches (MRM/PRM)

  • Use antibody arrays for multiplexed detection of low-abundance proteins

  • Apply proximity ligation assays for in situ detection with enhanced sensitivity

• Expression enhancement:

  • Use stress conditions known to induce Di19-3 expression

  • Consider tissues/developmental stages with higher Di19-3 expression

These approaches can significantly improve detection of low-abundance Di19-3 in experimental samples .

What strategies can resolve contradictory results in Di19-3 functional studies?

When confronted with contradictory results in Di19-3 functional studies, consider these systematic troubleshooting approaches:

• Genetic material verification:

  • Reconfirm genotypes of all plant lines through PCR and sequencing

  • Ensure multiple independent mutant/transgenic lines are tested

  • Check for background mutations through whole-genome sequencing

• Experimental condition standardization:

  • Standardize growth conditions (light, temperature, humidity, medium composition)

  • Control for plant developmental stage during experiments

  • Document and report all experimental parameters in detail

• Methodological triangulation:

  • Apply multiple independent techniques to address the same question

  • Combine genetic, biochemical, and imaging approaches

  • Use both gain-of-function and loss-of-function strategies

• Inter-laboratory validation:

  • Collaborate with other research groups to independently confirm results

  • Compare protocols and identify variables that might explain differences

  • Consider blind experimental design to eliminate unconscious bias

• Context-dependent function analysis:

  • Test whether Di19-3 function varies with developmental stage

  • Investigate tissue-specific differences in Di19-3 activity

  • Examine potential redundancy with other Di19 family members

This systematic approach can help resolve contradictory findings and establish a more robust understanding of Di19-3 function .

What are the best controls for specificity when using Di19-3 antibodies in different applications?

These controls ensure experimental results accurately reflect Di19-3 biology rather than technical artifacts or cross-reactivity. For all applications, include both wild-type and di19-3 mutant samples processed identically to confirm antibody specificity .

How does Di19-3 function relate to other drought-responsive transcription factors?

Di19-3 functions within a complex network of drought-responsive transcription factors, with several important relationships and distinctions:

• Comparative signaling mechanisms:

  • Unlike DREB/CBF transcription factors that primarily function through ABA-independent pathways, Di19-3 appears to intersect with hormone signaling pathways, particularly auxin and ethylene.

  • While many drought-responsive transcription factors directly bind to stress-responsive elements, Di19-3 seems to function through protein-protein interactions, particularly with Aux/IAA proteins like AtIAA14.

• Functional overlap and distinctiveness:

  • Di19-3 shares functional similarity with other Di19 family members in stress response but has unique roles in auxin signaling.

  • Unlike NAC and WRKY transcription factors that often act as master regulators of large gene sets, Di19-3 appears to have more specific regulatory functions in hormone homeostasis.

• Evolutionary conservation:

  • Analysis of Di19 family proteins across plant species reveals conservation of key functional domains, suggesting fundamental roles in plant stress adaptation throughout evolutionary history.

  • The specific interaction between Di19-3 and auxin signaling components represents a specialized adaptation that may vary across species.

• Hierarchical positioning:

  • Evidence suggests Di19-3 functions downstream of initial stress perception but upstream of physiological responses, potentially serving as an integration node between stress sensing and hormone-mediated growth adjustment.

These relationships position Di19-3 as a unique component in drought response networks, particularly in linking environmental stress perception to growth regulation through hormone signaling .

What methodologies can establish causality between Di19-3 and observed phenotypes?

Establishing causality between Di19-3 and observed phenotypes requires multiple complementary approaches:

• Genetic complementation:

  • Introduce wild-type Di19-3 under native promoter into di19-3 mutant background

  • Verify restoration of wild-type phenotypes to confirm causal relationship

  • Use domain-specific mutants to identify functional regions responsible for specific phenotypes

• Dosage-dependent analysis:

  • Generate multiple independent transgenic lines with varying Di19-3 expression levels

  • Establish quantitative correlation between expression level and phenotype intensity

  • Develop inducible expression systems to temporally control Di19-3 activity

• Site-directed mutagenesis:

  • Introduce specific mutations in Di19-3 functional domains

  • Test mutated versions for ability to complement di19-3 mutant phenotypes

  • Map critical amino acids required for interaction with partners like AtIAA14

• Tissue-specific manipulation:

  • Express Di19-3 under tissue-specific promoters in di19-3 background

  • Determine which tissues require Di19-3 for normal development

  • Use cell-type specific promoters to refine understanding of where Di19-3 functions

• Temporal regulation analysis:

  • Apply temporally controlled gene silencing or activation

  • Identify critical developmental windows for Di19-3 function

  • Use heat-shock or chemical-inducible systems for precise temporal control

These approaches collectively provide robust evidence for causality between Di19-3 and the observed developmental and stress response phenotypes .

How can researchers integrate Di19-3 findings into systems biology frameworks?

Integrating Di19-3 research into systems biology frameworks requires multidimensional data collection and analysis approaches:

• Multi-omics integration:

  • Combine transcriptomics data from di19-3 mutants with proteomics and metabolomics

  • Identify key regulated pathways through enrichment analysis

  • Construct regulatory networks centered on Di19-3 function

• Interactome mapping:

  • Perform proteome-wide interaction screens (Y2H, AP-MS) with Di19-3 as bait

  • Validate key interactions through orthogonal methods

  • Map Di19-3 position within larger protein-protein interaction networks

• Computational modeling:

  • Develop mathematical models of auxin signaling incorporating Di19-3

  • Simulate system behavior under various perturbations

  • Test model predictions experimentally to refine understanding

• Cross-species comparative analysis:

  • Identify Di19-3 homologs across plant species

  • Compare function, regulation, and interaction partners

  • Determine conserved and divergent aspects of Di19-3 biology

• Phenomics approaches:

  • Apply high-throughput phenotyping to monitor multiple parameters simultaneously

  • Quantify growth, development, and stress responses in an unbiased manner

  • Correlate phenotypic clusters with molecular signatures

• Network analysis visualization:

These approaches collectively position Di19-3 research within broader biological contexts, enabling systems-level understanding of its function .

How might CRISPR/Cas9 genome editing advance Di19-3 functional studies?

CRISPR/Cas9 genome editing offers several transformative approaches for Di19-3 research:

• Precise mutation generation:

  • Create domain-specific mutations to dissect Di19-3 function with nucleotide precision

  • Generate allelic series of mutations to establish structure-function relationships

  • Introduce reporter tags at endogenous loci for physiological expression studies

• Multiplexed mutant generation:

  • Simultaneously target Di19-3 and related family members to address functional redundancy

  • Create higher-order mutants with auxin signaling components to dissect genetic interactions

  • Target multiple domains within Di19-3 in parallel to identify critical functional regions

• Base editing applications:

  • Introduce specific amino acid changes without double-strand breaks

  • Create phosphorylation site mutants to study post-translational regulation

  • Modify promoter elements to alter expression patterns while maintaining genomic context

• Prime editing opportunities:

  • Make precise nucleotide substitutions without donor templates

  • Introduce or remove specific regulatory elements with minimal off-target effects

  • Create conditional alleles through strategic sequence modifications

• CRISPR activation/interference:

  • Modulate Di19-3 expression through CRISPRa (activation) or CRISPRi (interference)

  • Target Di19-3 promoter to achieve temporal and spatial expression control

  • Apply multiplexed CRISPRa/i to study regulatory networks involving Di19-3

These approaches would significantly advance understanding of Di19-3 function beyond what conventional mutant analysis allows .

What high-throughput screening approaches could identify novel Di19-3 interaction partners or regulators?

Several high-throughput screening approaches can efficiently identify novel Di19-3 interaction partners and regulators:

• Protein-protein interaction screens:

  • Split-ubiquitin yeast two-hybrid using Di19-3 as bait against cDNA libraries

  • BiFC-based screens in plant protoplasts with arrayed candidate proteins

  • Proximity-dependent biotin identification (BioID) with Di19-3 fusion proteins

  • Affinity purification-mass spectrometry (AP-MS) with tagged Di19-3

• Genetic interaction screens:

  • CRISPR-based screens in plant cells expressing Di19-3 reporters

  • Suppressor/enhancer screens in di19-3 mutant backgrounds

  • Synthetic genetic array analysis to identify genetic interactions

• Small molecule screens:

  • Chemical genomics approaches to identify compounds affecting Di19-3 function

  • Small molecule perturbation of Di19-3-IAA14 interaction using fluorescence-based assays

  • Identification of compounds that modify Di19-3 stress response phenotypes

• Transcriptional regulation screens:

  • Yeast one-hybrid screens to identify transcription factors binding Di19-3 promoter

  • CRISPR activation/interference screens targeting transcriptional regulators

  • Chromatin immunoprecipitation sequencing (ChIP-seq) to identify Di19-3 binding sites

• Post-translational modification screens:

  • Protein arrays to identify kinases/phosphatases acting on Di19-3

  • Mass spectrometry-based identification of Di19-3 modifications under different conditions

  • In vitro enzymatic assays with protein modification enzyme libraries

These screening approaches would significantly expand our understanding of Di19-3 regulatory networks and functional interactions .

How can computational modeling predict Di19-3 function across environmental conditions?

Computational modeling offers powerful approaches to predict Di19-3 function across diverse environmental conditions:

• Structural modeling approaches:

  • Predict Di19-3 protein structure through homology modeling and AlphaFold2

  • Simulate binding interactions with known partners like AtIAA14

  • Identify potential binding pockets for small molecule modulators

  • Predict effects of mutations on protein stability and interaction interfaces

• Gene regulatory network modeling:

  • Integrate transcriptomic data from di19-3 mutants under various conditions

  • Build Boolean or ordinary differential equation (ODE) models of Di19-3 regulatory circuits

  • Simulate network behavior under different environmental perturbations

  • Identify critical nodes and feedback loops in Di19-3-dependent pathways

• Multiscale modeling integration:

  • Link molecular interactions to cellular responses and whole-plant phenotypes

  • Predict emergent properties from Di19-3 molecular function

  • Integrate hormone transport and signaling models with Di19-3 function

  • Model cross-talk between stress response and developmental pathways

• Machine learning applications:

  • Train models on phenotypic and molecular data from di19-3 and wild-type plants

  • Develop predictive algorithms for plant responses under novel stress combinations

  • Identify previously unrecognized patterns in Di19-3-dependent responses

  • Predict optimal environmental conditions for studying specific Di19-3 functions

• Evolutionary modeling:

  • Analyze selection pressures on Di19-3 across plant species

  • Predict functional divergence of Di19-3 orthologs

  • Model coevolution of Di19-3 with interacting partners

  • Reconstruct ancestral Di19-3 sequences and predicted functions

These computational approaches complement experimental work and generate testable hypotheses about Di19-3 function across environmental conditions that would be impractical to test experimentally .