BHLH121 Antibody

What is BHLH121 and why is it significant in plant iron homeostasis research?

BHLH121 is a transcription factor belonging to the basic-helix-loop-helix (bHLH) family in Arabidopsis thaliana. It plays a crucial role in regulating iron (Fe) homeostasis by acting upstream of the iron homeostasis regulatory network. BHLH121 directly regulates the expression of the majority of genes encoding proteins and peptides involved in the Fe regulatory cascade . This transcription factor acts as a direct transcriptional activator of key genes in the Fe regulatory network, including bHLH38, bHLH39, bHLH100, bHLH101, POPEYE (PYE), BRUTUS (BTS), and BRUTUS LIKE1 (BTSL1), as well as IRONMAN1 (IMA1) and IRONMAN2 (IMA2) . Additionally, BHLH121 is necessary for activating the expression of the transcription factor gene FIT in response to Fe deficiency, though this occurs through an indirect mechanism . Understanding BHLH121 is essential for comprehending plant adaptation to Fe availability and developing strategies to improve Fe efficiency in crops.

How does BHLH121 function within the transcriptional network regulating iron homeostasis?

BHLH121 operates as a master regulator within the iron homeostasis transcriptional network. It interacts with several other bHLH transcription factors, particularly ILR3 (bHLH105) and its three closest homologs—bHLH34, bHLH104, and bHLH115 . Interaction studies confirm that BHLH121 forms heterodimers with these transcription factors but cannot form homodimers and does not interact with PYE, bHLH11, or FIT .

The regulatory activity of BHLH121 is positioned upstream of several key components in the Fe homeostasis network. Expression analyses revealed that BHLH121 activates the expression of bHLH38, bHLH39, bHLH100, bHLH101, and PYE in response to Fe deficiency . Furthermore, it is required for the induction of FIT expression under Fe-deficient conditions. Notably, BHLH121 expression itself is not affected by Fe availability, but its cellular localization in roots is influenced by Fe status . This suggests a post-translational regulatory mechanism controlling BHLH121 activity.

Recent studies have also identified that BHLH121, PYE, and ILR3 form a chain of antagonistic switches that regulate the expression of ferritin genes, which are crucial for iron storage in plants . This positioning makes BHLH121 antibodies valuable tools for investigating the hierarchical organization of the Fe deficiency response network.

What are the recommended applications for BHLH121 antibodies in iron homeostasis research?

BHLH121 antibodies serve multiple critical applications in iron homeostasis research:

• Chromatin Immunoprecipitation (ChIP) Assays: BHLH121 antibodies can be used in ChIP experiments to identify direct binding targets of BHLH121. Previous research has successfully employed ChIP assays to demonstrate that BHLH121 directly binds to the promoters of key genes in the Fe regulatory network, including bHLH38, bHLH39, bHLH100, bHLH101, PYE, BTS, and BTSL1 .

• Co-Immunoprecipitation (Co-IP): BHLH121 antibodies are effective for co-IP experiments to study protein-protein interactions. This approach has been used to identify BHLH121 as an ILR3-interacting transcription factor and to confirm its interactions with other bHLH transcription factors .

• Immunolocalization Studies: These antibodies can be employed to determine the subcellular localization of BHLH121 under different Fe conditions, as the protein's cellular localization in roots is affected by Fe availability .

• Western Blot Analysis: For detecting and quantifying BHLH121 protein levels in different plant tissues and under various experimental conditions.

• Immunoprecipitation-Mass Spectrometry (IP-MS): To identify novel protein interactions and post-translational modifications of BHLH121.

For optimal results in each application, researchers should validate antibody specificity using appropriate controls, including bhlh121 mutant lines as negative controls.

How can researchers optimize ChIP protocols for BHLH121 in Arabidopsis?

Optimizing ChIP protocols for BHLH121 in Arabidopsis requires careful consideration of several factors:

Recommended ChIP Protocol Optimization:

• Crosslinking Conditions: Use 1% formaldehyde for 10-15 minutes at room temperature. For BHLH121, which functions as part of transcriptional complexes with other bHLH proteins, dual crosslinking with disuccinimidyl glutarate (DSG) followed by formaldehyde may improve capture of protein complexes.

• Chromatin Fragmentation: Sonicate chromatin to achieve fragments of 200-500 bp. Test sonication conditions (amplitude, cycle number, duration) on your specific tissue type, as root and leaf tissues may require different conditions.

• Antibody Selection and Validation:

  • Validate antibody specificity using Western blot against wild-type and bhlh121 mutant tissues

  • Test antibody performance in preliminary ChIP-qPCR experiments using known BHLH121 target genes (bHLH38, bHLH39, PYE, etc.)

  • Consider using epitope-tagged BHLH121 (in bhlh121 mutant background) if native antibodies show limited specificity

• Controls:

  • Input DNA (pre-immunoprecipitation sample)

  • IgG negative control

  • Positive control (antibody against a known transcription factor)

  • ChIP in bhlh121 mutant background as biological negative control

• Fe Conditions: Since BHLH121 regulates genes responding to Fe availability, perform ChIP under both Fe-sufficient and Fe-deficient conditions to capture condition-specific binding events .

• Data Analysis: For ChIP-seq applications, use appropriate peak calling algorithms and motif analysis tools to identify BHLH121 binding motifs.

Previous studies have successfully used ChIP assays to demonstrate BHLH121 binding to promoters of key Fe homeostasis genes , confirming the feasibility of this approach with proper optimization.

What controls are essential for validating BHLH121 antibody specificity?

Validating BHLH121 antibody specificity is crucial for obtaining reliable experimental results. The following controls are essential:

• Genetic Controls:

  • CRISPR-generated bhlh121 knockout mutants serve as ideal negative controls. The literature describes multiple characterized bhlh121 loss-of-function alleles .

  • Complementation lines where BHLH121 is reintroduced into the mutant background (e.g., ProUBI:bHLH121 overexpression lines) .

  • Comparisons between wild-type and mutant samples in Western blot should show absence or significant reduction of signal in the mutant.

• Recombinant Protein Controls:

  • Purified recombinant BHLH121 protein can serve as a positive control.

  • Pre-adsorption of the antibody with the recombinant protein should abolish specific signals in immunodetection experiments.

• Cross-reactivity Assessment:

  • Test against closely related proteins, particularly BHLH11 (65% identity with BHLH121) , to ensure specificity.

  • Include protein extracts from tissues overexpressing BHLH11 to verify absence of cross-reactivity.

• Epitope Competition:

  • If using a peptide-derived antibody, pre-incubation with the immunizing peptide should block specific binding.

• Cellular Localization Consistency:

  • Immunolocalization results should be consistent with known BHLH121 nuclear localization patterns and its Fe-dependent distribution .

• Molecular Weight Verification:

  • The detected protein band should match the predicted molecular weight of BHLH121.

  • Post-translational modifications may alter apparent molecular weight and should be considered.

The table below summarizes validation approaches and their expected outcomes:

How can BHLH121 antibodies be used to study protein-protein interactions in the iron regulatory network?

BHLH121 antibodies are powerful tools for investigating protein-protein interactions within the iron regulatory network. The following methodologies leveraging these antibodies can provide valuable insights:

• Co-Immunoprecipitation (Co-IP):

  • BHLH121 antibodies can be used to pull down BHLH121 and its interacting partners from plant extracts.

  • This approach has successfully identified interactions between BHLH121 and bHLH IVc transcription factors (bHLH34, bHLH104, bHLH115, and ILR3) .

  • For optimal results, perform Co-IP under both Fe-sufficient and Fe-deficient conditions, as these interactions may be Fe-dependent.

• Immunoprecipitation Coupled with Mass Spectrometry (IP-MS):

  • This approach can identify novel BHLH121 interaction partners beyond those already described.

  • Previous studies using ILR3:GFP as bait in Co-IP LC-MS/MS analyses successfully identified BHLH121 as an interacting protein .

  • When using BHLH121 antibodies for IP-MS, include appropriate controls such as IgG pull-downs and samples from bhlh121 mutants.

• Proximity-dependent Biotin Identification (BioID) or TurboID:

  • Though not directly using antibodies, these techniques can complement antibody-based approaches.

  • BHLH121 fused to a biotin ligase can biotinylate proteins in close proximity, which can then be purified and identified.

  • BHLH121 antibodies can validate the expression and localization of the BHLH121-biotin ligase fusion protein.

• Sequential ChIP (ChIP-reChIP):

  • This technique can determine if BHLH121 and other transcription factors (like ILR3) co-occupy the same genomic regions.

  • Perform initial ChIP with BHLH121 antibody, followed by a second ChIP with antibodies against potential partner proteins.

• Förster Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC) Validation:

  • Antibodies can validate the expression of fusion proteins used in these interaction assays.

  • Previous studies have successfully used BiFC to confirm BHLH121 interactions with other bHLH transcription factors .

The literature indicates that BHLH121 interacts with bHLH34, bHLH104, bHLH115, and ILR3, but not with PYE, FIT, BTS, BTSL1, BTSL2, or itself . In contrast, its close homolog BHLH11 can form homodimers and interacts with the same bHLH IVc proteins . These differences highlight the importance of specificity when using antibodies to study these interactions.

What role does BHLH121 play in regulating ferritin gene expression, and how can antibodies help investigate this function?

BHLH121 plays a significant role in regulating ferritin gene expression, with ferritins being crucial proteins for iron storage in plants. Recent research has revealed the following:

• Impact on Ferritin Gene Expression:

  • Under iron-sufficient conditions, the expression of ferritin genes (FER1, FER3, and FER4) is lower in bhlh121 mutants compared to wild-type plants, indicating that BHLH121 is necessary to maintain ferritin gene expression when iron availability is not limiting .

  • Under iron-deficient conditions, FER3 mRNA accumulation is higher in wild-type plants than in bhlh121 mutants, suggesting that BHLH121 has a positive effect on FER3 expression specifically when iron availability is low .

  • These findings contrast with previous microarray-based studies that suggested ferritin gene expression is not affected in bhlh121 mutants under iron-replete conditions, highlighting the higher sensitivity of qRT-PCR methods in detecting these differences .

• Regulatory Mechanism:

  • BHLH121, PYE, and ILR3 form a chain of antagonistic switches that regulate the expression of ferritin genes .

  • This regulatory network ensures appropriate iron storage responses under varying iron availability conditions.

BHLH121 antibodies can help investigate this function through several approaches:

• Chromatin Immunoprecipitation (ChIP):

  • ChIP assays using BHLH121 antibodies can determine whether BHLH121 directly binds to ferritin gene promoters or if the regulation occurs through intermediate factors.

  • qPCR following ChIP can quantify binding to specific regions of ferritin gene promoters under different iron conditions.

• Protein Complex Analysis:

  • Co-IP with BHLH121 antibodies followed by Western blot for other transcription factors can reveal which protein complexes form under different iron conditions to regulate ferritin gene expression.

  • This can help elucidate whether BHLH121 interacts with specific co-factors when regulating ferritin genes versus other iron homeostasis genes.

• Temporal Dynamics:

  • Time-course experiments using BHLH121 antibodies can track changes in BHLH121 localization, abundance, and interaction partners during iron status transitions.

  • This can reveal how quickly BHLH121-mediated regulation of ferritin genes responds to changing iron conditions.

A proposed experimental workflow for investigating BHLH121's role in ferritin gene regulation would include:

What are common challenges when working with BHLH121 antibodies and how can they be addressed?

Researchers working with BHLH121 antibodies may encounter several challenges. Here are common issues and recommended solutions:

• Limited Antibody Specificity:

  • Challenge: Cross-reactivity with closely related proteins, particularly BHLH11 (65% identity) .

  • Solution:

    • Pre-adsorb antibodies with recombinant BHLH11 protein

    • Use peptide-derived antibodies targeting unique regions of BHLH121

    • Validate specificity using bhlh121 mutant tissues as negative controls

    • Consider using epitope-tagged BHLH121 expression systems

• Protein Abundance Issues:

  • Challenge: BHLH121 may be expressed at low levels in certain tissues or conditions.

  • Solution:

    • Increase starting material amount

    • Optimize extraction buffers to improve protein solubility

    • Use more sensitive detection methods (e.g., enhanced chemiluminescence systems)

    • Consider tissue-specific or cell-type-specific approaches when appropriate

• Iron-Dependent Localization Effects:

  • Challenge: BHLH121 cellular localization varies with Fe availability , potentially affecting antibody accessibility.

  • Solution:

    • Use different fixation protocols depending on growth conditions

    • Include multiple extraction fractions (nuclear, cytoplasmic) in analyses

    • Track and normalize to Fe status markers in the same samples

• Protein Complex Integrity:

  • Challenge: BHLH121 functions in protein complexes that may be disrupted during extraction.

  • Solution:

    • Use gentle extraction conditions

    • Include protein crosslinking steps before extraction

    • Add protease inhibitors and phosphatase inhibitors to preserve post-translational modifications

    • Consider native versus denaturing conditions based on experimental goals

• Reproducibility Issues:

  • Challenge: Variable results between experiments or antibody batches.

  • Solution:

    • Standardize plant growth conditions, especially iron availability

    • Include internal controls in each experiment

    • Validate new antibody batches against previous ones

    • Document detailed protocols with specific conditions

Recommended extraction buffer for BHLH121 immunoprecipitation:

• 50 mM Tris-HCl (pH 7.5)

• 150 mM NaCl

• 1% Triton X-100

• 0.5% Sodium deoxycholate

• 5 mM EDTA

• 1 mM DTT

• Protease inhibitor cocktail

• Phosphatase inhibitor cocktail

• 10% glycerol

How can researchers optimize detection of nuclear versus cytoplasmic BHLH121 in immunolocalization studies?

Optimizing detection of nuclear versus cytoplasmic BHLH121 is crucial for understanding its subcellular dynamics, especially since its localization is affected by iron availability . Here are detailed recommendations:

• Sample Preparation Considerations:

  • Fixation Protocol: Use 4% paraformaldehyde for 20-30 minutes at room temperature. For better preservation of nuclear and cytoplasmic structures, consider a dual fixation approach with 0.5% glutaraldehyde and 3% paraformaldehyde.

  • Permeabilization: Different permeabilization approaches may favor detection in specific compartments:

    • For balanced detection: 0.1% Triton X-100 for 10 minutes

    • For enhanced nuclear detection: 0.5% Triton X-100 for 15 minutes

    • For enhanced cytoplasmic detection: 0.05% Tween-20 for 5 minutes

• Subcellular Fractionation Approach:

  • For quantitative assessment, perform subcellular fractionation followed by Western blot analysis.

  • This approach has been successfully used to demonstrate that BHLH11 protein was detected in both nuclear and cytoplasmic fractions, with levels responsive to iron status .

  • Include appropriate markers for fractionation quality:

    • Nuclear markers: Histone H3, PARP-1

    • Cytoplasmic markers: GAPDH, tubulin

• Confocal Microscopy Optimization:

  • Z-stack imaging to capture the full cell volume

  • High-resolution imaging (at least 100x objective with oil immersion)

  • Appropriate filters to distinguish BHLH121 signal from autofluorescence

  • Co-staining with DAPI for nuclear visualization

  • Consider using organelle-specific markers as counterstains

• Fe-Status Considerations:

  • Always include both Fe-sufficient and Fe-deficient conditions in parallel experiments

  • Document Fe status with established markers (ferritin levels, IRT1/FRO2 expression)

  • Consider time-course experiments during Fe status transitions

• Quantification Methods:

  • For accurate comparison of nuclear versus cytoplasmic distribution, use:

    • Nuclear/cytoplasmic signal intensity ratios

    • Colocalization coefficients with compartment markers

    • Fluorescence intensity line profiles across cells

• Controls and Validation:

  • bhlh121 mutants as negative controls

  • Competition with immunizing peptide

  • Comparison with GFP-tagged BHLH121 localization patterns

  • Testing multiple antibodies (if available) targeting different epitopes

Previous research has shown that when BHLH IVc transcription factors are co-expressed with BHLH11-mCherry, the latter accumulates exclusively in the nucleus . Similar dynamics may occur with BHLH121, and should be considered when interpreting localization results.

How might BHLH121 antibodies contribute to understanding iron homeostasis in crop species beyond Arabidopsis?

BHLH121 antibodies have significant potential to advance our understanding of iron homeostasis in economically important crop species, bridging fundamental research in Arabidopsis to applied agricultural improvements. Here's how these antibodies can contribute:

• Comparative Functional Studies:

  • BHLH121 orthologs exist in various crop species with high sequence conservation.

  • Antibodies raised against Arabidopsis BHLH121 may cross-react with orthologs in closely related species, enabling comparative studies.

  • For crops with more divergent sequences, development of species-specific antibodies targeting conserved epitopes would be valuable.

  • Such studies could reveal both conserved and species-specific aspects of BHLH121 function across plant lineages.

• Stress Response Integration:

  • Iron homeostasis intersects with multiple abiotic stress responses in crops.

  • BHLH121 antibodies can help examine how this transcription factor's abundance, localization, and interactions change under combined stresses (drought, salinity, heat) that are particularly relevant to crop production.

  • Understanding these interactions could lead to breeding strategies for multi-stress-resistant crops with improved iron utilization.

• Developmental Regulation in Crop-Specific Tissues:

  • Crops have specialized tissues not present in Arabidopsis (e.g., endosperm in cereals).

  • BHLH121 antibodies can reveal tissue-specific expression patterns in these crop-specific structures.

  • This could be particularly important for biofortification efforts targeting edible tissues.

• Methodology Transfer:

  • ChIP protocols optimized with BHLH121 antibodies in Arabidopsis can be adapted for crops.

  • This would allow identification of species-specific regulatory targets that might contribute to differences in iron efficiency between species.

• Potential Applications in Crop Improvement:

  • Screening germplasm collections for natural variation in BHLH121 protein abundance or post-translational modifications.

  • Monitoring BHLH121 status during breeding programs targeting improved iron nutrition.

  • Developing diagnostic tools for iron status in crops based on BHLH121 regulatory network components.

The translation of BHLH121 research to crops is particularly relevant given that iron deficiency is a major constraint for crop productivity worldwide and a significant human nutritional problem. Understanding the conservation and divergence of this key regulator across species could inform both breeding and biotechnological approaches to developing more iron-efficient and iron-rich crops.

What emerging technologies might enhance the utility of BHLH121 antibodies in plant iron homeostasis research?

Several emerging technologies have the potential to significantly enhance the utility of BHLH121 antibodies in advancing plant iron homeostasis research:

• Single-Cell Proteomics:

  • Application: Detecting BHLH121 abundance and modifications at the single-cell level.

  • Advantage: Would reveal cell-type-specific regulation that may be masked in whole-tissue analyses.

  • Implementation: BHLH121 antibodies could be used for single-cell Western blotting or mass cytometry (CyTOF) with metal-conjugated antibodies.

  • Relevance: The iron deficiency response varies dramatically between cell types in roots, making this approach particularly valuable.

• Proximity Labeling Technologies:

  • Application: BHLH121 antibodies can validate expression of BHLH121 fusion proteins with proximity labeling enzymes (BioID, TurboID, APEX).

  • Advantage: Enables identification of proteins that transiently interact with BHLH121 or are part of the same complex without direct binding.

  • Implementation: Generate transgenic plants expressing BHLH121-TurboID fusions and validate with antibodies.

  • Relevance: Could reveal novel components of the iron regulatory network that interact with BHLH121 under specific conditions.

• Live-Cell Imaging with Nanobodies:

  • Application: Development of anti-BHLH121 nanobodies (single-domain antibodies) for live-cell imaging.

  • Advantage: Would allow tracking of BHLH121 dynamics in real-time in living cells.

  • Implementation: Express fluorescently tagged nanobodies in plants to visualize endogenous BHLH121.

  • Relevance: Could reveal rapid changes in BHLH121 localization in response to iron availability fluctuations.

• Proteomics of Isolated Crosslinked Chromatin Segments (PICh-MS):

  • Application: BHLH121 antibodies can help validate results from this technique that identifies proteins associated with specific genomic regions.

  • Advantage: Would reveal the complete protein complex assembled at BHLH121 target genes.

  • Implementation: Use oligonucleotides targeting known BHLH121-regulated promoters combined with mass spectrometry.

  • Relevance: Could identify additional co-factors involved in BHLH121-mediated transcriptional regulation.

• CUT&Tag or CUT&RUN Technologies:

  • Application: More sensitive alternatives to traditional ChIP for mapping BHLH121 binding sites.

  • Advantage: Requires fewer cells and provides higher resolution than conventional ChIP.

  • Implementation: Conjugate BHLH121 antibodies to Protein A-Tn5 transposase fusion protein.

  • Relevance: Could more precisely map BHLH121 binding sites, especially in specific cell types or under conditions where BHLH121 is less abundant.

• Antibody-Based Biosensors:

  • Application: Development of BHLH121 antibody-based biosensors to monitor protein levels in real-time.

  • Advantage: Would allow continuous monitoring of BHLH121 abundance or modifications.

  • Implementation: Immobilize antibodies on field-effect transistors or optical sensors.

  • Relevance: Could provide insights into the temporal dynamics of BHLH121 regulation during iron status changes.

These emerging technologies, when combined with high-quality BHLH121 antibodies, have the potential to reveal new dimensions of iron homeostasis regulation in plants, leading to both fundamental insights and practical applications in crop improvement.