GRF4 Antibody

What is GRF4 and why are antibodies against it valuable for plant research?

GRF4 (GROWTH-REGULATING FACTOR4) is a transcription factor that functions as a crucial regulator of nitrogen use efficiency (NUE) in plants. It serves as an integrative regulator of multiple nitrogen metabolism genes and a coordinator of carbon metabolism. Antibodies against GRF4 are valuable research tools for studying protein-DNA interactions, protein localization, and regulatory mechanisms in plant development. These antibodies enable researchers to investigate how GRF4 directly regulates the transcription of genes like MYB61, which is involved in cellulose synthesis and biomass production .

What experimental evidence demonstrates GRF4's regulatory function in plants?

Multiple experimental approaches have confirmed GRF4's regulatory role. In loss-of-function mutants (grf4-1 and grf4-2), researchers observed reduced flag leaf size, decreased internode diameter, thinner cell walls, and lower cellulose content. Conversely, gain-of-function mutants (GS2) exhibited larger flag leaves, thicker internodes, enhanced cell wall thickness, and increased cellulose levels. These phenotypic changes correspond with altered MYB61 expression levels - upregulated in gain-of-function mutants and significantly repressed in loss-of-function mutants .

How does GRF4 regulate target gene expression at the molecular level?

GRF4 functions through direct binding to specific DNA motifs in the promoter regions of target genes. Chromatin-immunoprecipitation (ChIP) analysis with GRF4-cMyc in grf4 mutant backgrounds has revealed associations between GRF4 and multiple promoter segments (specifically P1, P2, and P8 regions in the MYB61 promoter). The binding at the P8 segment containing motif 2 has been further confirmed through electrophoretic mobility shift assays (EMSA) .

What protocols yield optimal results for GRF4 ChIP experiments?

For optimal GRF4 ChIP experiments, researchers should:

• Express epitope-tagged GRF4 (e.g., GRF4-cMyc) in appropriate genetic backgrounds (preferably grf4 mutants to avoid competition with endogenous protein)

• Use crosslinking conditions optimized for transcription factors (typically 1% formaldehyde for 10-15 minutes)

• Employ sonication parameters that generate DNA fragments of 200-500 bp

• Include appropriate controls: input DNA (pre-immunoprecipitation), IgG control, and negative control regions

• Validate findings with quantitative PCR targeting multiple regions of interest

The ChIP protocol has successfully identified three association points between GRF4 and the MYB61 promoter at segments P1, P2, and P8, providing a validated experimental framework .

What are the critical parameters for successful EMSA experiments with GRF4?

Based on published methodologies, successful EMSA experiments with GRF4 require:

This approach successfully demonstrated specific binding of GST-GRF4 to the MYB61 promoter fragment at P8, while no binding was detected with fragments harboring motif 1 or at P1 and P2 regions .

How can researchers validate the specificity of GRF4 antibodies?

To validate GRF4 antibody specificity, implement the following multi-step approach:

• Genetic validation: Compare antibody signal between wild-type and grf4 knockout lines (signal should be absent or significantly reduced in knockouts)

• Protein validation: Perform Western blot analysis to confirm detection at the expected molecular weight

• Epitope competition: Pre-incubate antibody with purified GRF4 peptide/protein before application (should reduce or eliminate specific signal)

• Cross-reactivity assessment: Test antibody against related GRF family members to ensure specificity

• Functional validation: Confirm antibody can immunoprecipitate GRF4 by validating pulled-down protein with mass spectrometry

This comprehensive validation approach ensures reliable results in subsequent experimental applications.

How can GRF4 antibodies be utilized to study protein-protein interactions within transcriptional complexes?

GRF4 antibodies can reveal protein-protein interactions through these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use GRF4 antibodies to precipitate the protein complex, followed by Western blot analysis with antibodies against suspected interaction partners

• Proximity ligation assay (PLA): Combine GRF4 antibody with antibodies against potential interacting proteins to visualize interactions in situ

• ChIP-re-ChIP: Perform sequential immunoprecipitations with GRF4 antibody followed by antibodies against other transcription factors to identify co-occupancy at specific genomic loci

• Yeast two-hybrid validation: Use antibodies to confirm interactions identified through Y2H screens in plant tissues

• Mass spectrometry following IP: Identify novel interaction partners by immunoprecipitating GRF4 complexes and analyzing by mass spectrometry

These approaches can help decipher how GRF4 functions within larger regulatory networks coordinating carbon and nitrogen metabolism .

What strategies can resolve inconsistent results when using GRF4 antibodies in different experimental contexts?

When troubleshooting inconsistent GRF4 antibody results:

• Assess epitope accessibility: GRF4 conformation may differ between applications (native vs. denatured conditions)

• Optimize fixation protocols: Crosslinking conditions may affect epitope recognition in immunohistochemistry

• Consider tissue-specific post-translational modifications: GRF4 may be differently modified in various tissues or under different nitrogen conditions

• Evaluate antibody batch variation: Validate each new lot against a known positive control

• Test multiple antibodies: Use antibodies targeting different GRF4 epitopes to confirm findings

• Examine buffer compatibility: Adjust buffers to optimize antibody performance for specific applications

Additionally, consider that GRF4 shows inducible performance under limited nitrogen conditions, which may affect detection levels in different experimental setups .

How can researchers distinguish direct from indirect effects of GRF4 in gene regulatory networks?

To differentiate direct from indirect GRF4 regulatory effects:

• Integrate ChIP-seq with RNA-seq: Compare GRF4 binding sites with transcriptional changes to identify direct targets

• Use time-course experiments: Direct targets typically show more rapid expression changes following GRF4 induction

• Analyze cis-regulatory elements: Confirm the presence of validated GRF4 binding motifs in promoters of putative target genes

• Employ transactivation assays: Test GRF4's ability to activate transcription from target promoters (as demonstrated with MYB61 promoter)

• Perform motif mutation studies: Mutate predicted binding sites and measure effects on GRF4 binding and transactivation

This multi-faceted approach revealed that GRF4 directly regulates MYB61 transcription, with the 9311 allele of MYB61 showing higher activation levels than the NP allele in transactivation assays .

How should researchers analyze ChIP-seq data to identify genuine GRF4 binding sites?

For robust analysis of GRF4 ChIP-seq data:

This approach can expand understanding beyond the three validated GRF4 binding regions (P1, P2, and P8) in the MYB61 promoter to a genome-wide perspective .

What considerations are important when using GRF4 antibodies to study nitrogen-responsive regulation?

When investigating nitrogen-responsive GRF4 activity:

• Implement precise nitrogen treatment protocols: Use defined media with controlled nitrogen sources and concentrations

• Include time-course sampling: GRF4 activity shows dynamic responses to nitrogen availability

• Compare multiple tissue types: GRF4 regulation may differ between roots, leaves, and reproductive tissues

• Account for developmental stage: Nitrogen responses often interact with developmental programming

• Consider genotype differences: As observed with differential regulation of indica and japonica MYB61 alleles under varied nitrogen availability

• Quantify both total and phosphorylated GRF4: Post-translational modifications may affect GRF4 activity

GRF4 has been shown to accumulate under low nitrogen availability, making controlled nitrogen conditions crucial for experimental reproducibility .

How can transactivation activity analyses be optimized to study GRF4 function?

For optimal transactivation assays with GRF4:

• Clone the full-length coding sequence of GRF4 into appropriate expression vectors (such as p2GW7)

• Generate reporter constructs containing target gene promoters (like ProMYB61:LUC) in vectors with luciferase genes

• Use protoplast systems from appropriate species (Arabidopsis rosette leaves have been successful)

• Include internal controls (such as Renilla reniformis luciferase driven by CaMV 35S)

• Allow adequate incubation time (overnight incubation has proven effective)

• Implement appropriate normalization strategies to account for transformation efficiency

This methodology successfully demonstrated that GRF4 activates the MYB61 promoter, with the 9311 allele showing approximately one-fold higher activation than the NP allele .

How might GRF4 antibodies contribute to understanding plant responses to climate change?

GRF4 antibodies can advance climate change adaptation research through:

• Monitoring GRF4 protein levels across diverse environmental conditions (drought, temperature stress, CO₂ enrichment)

• Identifying changes in GRF4 binding patterns under stress conditions via ChIP-seq

• Investigating how altered nitrogen availability (due to climate change) affects GRF4-mediated regulation

• Comparing GRF4 dynamics between climate-resilient and susceptible varieties

• Studying how GRF4's integration of carbon and nitrogen metabolism responds to changing climate variables

Since GRF4 constitutes a regulatory cascade governing NUE and cellulosic biomass production, understanding its behavior under climate stress could inform breeding strategies for climate-resilient crops .

What methodological advances are needed to study tissue-specific GRF4 activity?

To advance tissue-specific GRF4 research:

• Develop cell-type-specific antibody-based techniques: Adapt RNAscope-like approaches for protein detection

• Implement tissue-clearing protocols compatible with immunohistochemistry: Enable whole-organ imaging of GRF4 localization

• Create GRF4 biosensors: Design fusion proteins that report on GRF4 activity in living tissues

• Establish cell-type-specific ChIP protocols: Adapt INTACT or FACS-based methods for GRF4 binding studies

• Apply spatial transcriptomics: Correlate GRF4 protein levels with transcriptional outputs at cellular resolution

These methodological advances would help resolve how GRF4 functions differently in specific cell types, such as between periportal hepatocytes or in proliferating versus non-proliferating intestinal epithelia .

How can researchers integrate GRF4 antibody data with multi-omics approaches?

For comprehensive multi-omics integration:

• Correlate ChIP-seq binding profiles with:

  • RNA-seq: Identify direct transcriptional effects

  • ATAC-seq: Map chromatin accessibility changes influenced by GRF4

  • Proteomics: Link transcriptional changes to protein abundance

  • Metabolomics: Connect regulatory events to metabolic outcomes

• Implement computational integration approaches:

  • Network analysis to identify GRF4-centered regulatory hubs

  • Machine learning to predict GRF4 binding under various conditions

  • Bayesian modeling to infer causal relationships in GRF4 regulatory networks

This integrated approach would provide a systems-level understanding of how GRF4 coordinates carbon and nitrogen metabolism to govern NUE and biomass production, potentially leading to applications in crop improvement .