BHLH11 Antibody

What is BHLH11 and what is its primary function in plants?

BHLH11 is a basic helix-loop-helix transcription factor that functions as a negative regulator of iron (Fe) homeostasis in plants, particularly in Arabidopsis thaliana. The protein plays a pivotal role in maintaining iron balance by repressing the expression of genes involved in iron uptake and transport .

Functionally, BHLH11:

• Contains two ethylene response factor-associated amphiphilic repression (EAR) motifs in its C-terminal region

• Interacts with and inhibits the activity of bHLH subgroup IVc transcription factors

• Recruits TOPLESS/TOPLESS-RELATED (TPL/TPRs) corepressors to repress gene expression

• Displays dynamic subcellular localization between the cytoplasm and nucleus, which is influenced by iron status and interaction with other bHLH proteins

Research using loss-of-function mutants has demonstrated that when BHLH11 is disrupted, plants show enhanced sensitivity to excess iron, increased iron accumulation, and elevated expression of iron deficiency-responsive genes .

How does BHLH11 differ from other bHLH family members involved in iron homeostasis?

BHLH11 differs from other iron homeostasis-related bHLH proteins in several key aspects:

BHLH11 is most closely related to BHLH121 (65% identity), but unlike BHLH121, BHLH11 functions specifically as a repressor by recruiting TPL/TPR corepressors through its EAR motifs .

What are the most effective methods for detecting endogenous BHLH11 in plant tissues?

For detecting endogenous BHLH11 in plant tissues, researchers should consider multiple complementary approaches:

Immunological detection:

• Western blotting using anti-BHLH11 antibodies is effective for protein quantification and determining subcellular localization

• Perform nuclear and cytoplasmic fractionation before immunoblotting to assess distribution between compartments

• Use appropriate controls, including bhlh11 mutants as negative controls

Transcript analysis:

• Quantitative real-time PCR (qRT-PCR) using BHLH11-specific primers

• RNA-seq for global expression patterns in different tissues or conditions

Subcellular localization:

• Immunohistochemistry with anti-BHLH11 antibodies

• For validating antibody specificity, complement with fluorescent protein tagging approaches

When using antibodies for BHLH11 detection, researchers should be aware that protein levels are responsive to iron status, with both nuclear and cytoplasmic forms showing changes in response to iron deficiency conditions .

What are the critical considerations when selecting or generating antibodies against BHLH11?

When selecting or generating antibodies against BHLH11 for research applications, consider these critical factors:

Epitope selection:

• Target unique regions of BHLH11 that don't share homology with close family members (especially BHLH121)

• Consider targeting the C-terminal region containing the EAR motifs for specificity

• Avoid the conserved bHLH domain if distinguishing from other bHLH family members is important

Antibody validation strategies:

• Use bhlh11 mutant or knockout lines as negative controls

• Perform peptide competition assays to confirm specificity

• Test cross-reactivity with recombinant BHLH121 protein

• Validate using both immunoblotting and immunoprecipitation applications

Technical specifications:

• For subcellular localization studies, ensure antibodies work in both native and fixed conditions

• For co-immunoprecipitation studies, select antibodies that don't interfere with protein-protein interactions

• For chromatin immunoprecipitation (ChIP) applications, verify the antibody can recognize formaldehyde-fixed epitopes

Developing antibodies that can distinguish between phosphorylated and non-phosphorylated forms of BHLH11 may also be valuable, as many transcription factors are regulated by post-translational modifications, though specific information about BHLH11 phosphorylation status is not provided in the search results .

How can antibodies be effectively employed to study BHLH11 interactions with bHLH IVc transcription factors?

Antibodies can be strategically employed to investigate BHLH11 interactions with bHLH IVc transcription factors through several methodological approaches:

Co-immunoprecipitation (Co-IP):

• Use anti-BHLH11 antibodies to pull down protein complexes from plant extracts

• Detect bHLH IVc proteins (bHLH34, bHLH104, bHLH105, bHLH115) in the immunoprecipitate using specific antibodies

• Alternatively, tag bHLH IVc proteins with epitope tags (MYC, HA) for easier detection

• This approach has successfully demonstrated that BHLH11 and bHLH IVc TFs are present in the same protein complex

Proximity-based labeling:

• Fuse BHLH11 to a proximity-labeling enzyme (BioID or TurboID)

• Identify interacting proteins through streptavidin pulldown and mass spectrometry

• Validate interactions using antibodies against specific bHLH IVc proteins

Subcellular co-localization:

• Use fluorescently labeled antibodies for immunofluorescence microscopy

• Track changes in localization upon co-expression of interaction partners

• This can confirm findings from split-GFP assays showing that bHLH IVc proteins affect the subcellular localization of BHLH11

In vitro binding assays:

• Express and purify recombinant BHLH11 and bHLH IVc proteins

• Perform pull-down assays using antibodies to detect interactions

• Quantify binding affinities using surface plasmon resonance or similar techniques

Research has shown that when any of the four bHLH IVc proteins are co-expressed with BHLH11, they facilitate BHLH11 accumulation exclusively in the nucleus, which can be detected using proper antibody-based localization techniques .

What protocols have proven most effective for immunoprecipitation of BHLH11 from plant tissues?

Based on the research methodologies described in the literature, the following protocol outlines an effective approach for immunoprecipitation of BHLH11 from plant tissues:

Sample preparation:

• Harvest plant tissues (preferably young seedlings) and flash-freeze in liquid nitrogen

• Grind tissue to fine powder while maintaining frozen state

• Extract proteins in buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail

  • Phosphatase inhibitors if interested in phosphorylation status

• Clarify lysate by centrifugation at 14,000 × g for 15 minutes at 4°C

Immunoprecipitation procedure:

• Pre-clear lysate with protein A/G agarose beads for 1 hour at 4°C

• Incubate pre-cleared lysate with anti-BHLH11 antibody (or anti-tag antibody for tagged versions) overnight at 4°C with gentle rotation

• Add protein A/G agarose beads and incubate for 2-3 hours at 4°C

• Wash beads 4-5 times with washing buffer (extraction buffer with reduced detergent)

• Elute bound proteins by boiling in SDS sample buffer

• Analyze by SDS-PAGE and western blotting

Key considerations:

• When studying interactions with bHLH IVc TFs, consider crosslinking before extraction

• For nuclear interactions, perform nuclear extraction before immunoprecipitation

• Include appropriate controls (IgG control, bhlh11 mutant tissues)

• For detecting interactions in different iron conditions, grow plants under iron-sufficient and iron-deficient conditions before extraction

This protocol has been successfully adapted from the Co-IP assays conducted to confirm interactions between bHLH IVc TFs and BHLH11 in Nicotiana benthamiana leaves, where MYC-tagged bHLH IVc TFs and HA-tagged BHLH11 were co-expressed and immunoprecipitated .

How can researchers effectively investigate the mechanism by which BHLH11 recruits TPL/TPR corepressors?

Investigating the mechanism of BHLH11 recruitment of TPL/TPR corepressors requires a multi-faceted approach focusing on the EAR motifs and their functional significance:

Structure-function analysis:

• Generate EAR motif mutants of BHLH11:

  • Single mutations in each EAR motif (bHLH11m1, bHLH11m2)

  • Double mutations affecting both EAR motifs (bHLH11dm)

• Assess interaction with TPL/TPRs using:

  • Yeast two-hybrid assays

  • Co-immunoprecipitation with antibodies against BHLH11 and TPL/TPRs

  • Bimolecular fluorescence complementation (BiFC)

Functional repressor assays:

• Conduct reporter-effector transient expression assays:

  • Use promoters of known BHLH11 target genes (e.g., bHLH38) fused to a luciferase reporter

  • Compare effects of wild-type BHLH11 versus EAR motif mutants

  • Create fusion proteins with activation domains (e.g., VP16) to convert the repressor to an activator

In vivo functional validation:

• Generate transgenic plants expressing:

  • Wild-type BHLH11

  • EAR motif mutants (bHLH11dm)

  • Dominant activator versions (bHLH11dm-VP16)

• Assess phenotypes related to iron homeostasis

• Measure expression of target genes using RT-qPCR

Research has demonstrated that mutation of both EAR motifs abolishes the interaction between BHLH11 and TPL/TPRs, confirming these motifs are essential for recruitment of the corepressor complex. Further, replacing the repression domain with an activation domain (VP16) not only eliminates the repressive function but can convert BHLH11 into an activator .

What are the most informative experimental approaches to study the dynamic subcellular localization of BHLH11 in response to iron conditions?

To investigate the dynamic subcellular localization of BHLH11 in response to iron conditions, researchers should implement these complementary experimental approaches:

Live-cell imaging with fluorescent fusion proteins:

• Generate BHLH11-fluorescent protein fusions (e.g., BHLH11-GFP)

• Transform plants and observe localization under:

  • Iron-sufficient conditions

  • Iron-deficient conditions

  • Iron resupply after deficiency

• Perform time-course experiments to track relocalization dynamics

• Co-express with fluorescently tagged bHLH IVc proteins to observe interaction effects

Biochemical fractionation with antibody detection:

• Grow plants under varying iron conditions

• Isolate nuclear and cytoplasmic fractions

• Perform immunoblotting with anti-BHLH11 antibodies

• Quantify the nuclear/cytoplasmic ratio under different conditions

• Include markers for nuclear (histone H3) and cytoplasmic (GAPDH) fractions as controls

Immunohistochemistry:

• Fix plant tissues from different iron treatments

• Perform immunostaining with anti-BHLH11 antibodies

• Counterstain nuclei with DAPI

• Quantify nuclear signal intensity across conditions

Factors affecting localization to investigate:

• The role of bHLH IVc proteins in facilitating nuclear accumulation

• Potential post-translational modifications affecting localization

• Iron-sensing mechanisms linking iron status to BHLH11 localization

Research has shown that BHLH11 protein is localized in both the cytoplasm and nucleus, and both its nuclear and cytoplasmic counterparts respond to iron status. Additionally, co-expression with bHLH IVc TFs causes BHLH11 to accumulate exclusively in the nucleus .

How can ChIP-seq approaches with BHLH11 antibodies advance our understanding of its gene regulatory networks?

ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) using BHLH11 antibodies offers powerful insights into the gene regulatory networks controlled by this transcription factor. Here's a methodological framework:

Experimental design considerations:

• Generate highly specific antibodies against BHLH11 or use epitope-tagged BHLH11 in complemented mutants

• Include appropriate controls:

  • Input DNA

  • IgG control immunoprecipitation

  • bhlh11 mutant negative control

• Test multiple growth conditions:

  • Iron-sufficient versus iron-deficient

  • Different developmental stages

  • Various tissues (roots vs. shoots)

ChIP-seq protocol optimization:

• Crosslinking: Optimize formaldehyde concentration (1-2%) and duration (10-15 minutes)

• Sonication: Adjust conditions to yield 200-500 bp DNA fragments

• Immunoprecipitation: Use affinity-purified antibodies against BHLH11

• Library preparation: Generate sequencing libraries from immunoprecipitated DNA

• Sequencing: Aim for >20 million reads per sample for good coverage

Data analysis approaches:

• Peak calling to identify BHLH11 binding sites genome-wide

• Motif analysis to define the BHLH11 binding motif

• Integration with RNA-seq data to correlate binding with gene expression changes

• Comparison of binding sites in different conditions to identify context-dependent regulation

Validation experiments:

• ChIP-qPCR for selected target genes

• Reporter gene assays using identified binding regions

• EMSA (Electrophoretic Mobility Shift Assay) to confirm direct binding

While not explicitly demonstrated in the provided search results, ChIP-seq could reveal whether BHLH11 directly represses iron uptake genes like IRT1 and FRO2 as suggested in the literature, and could identify the broader set of genes regulated by the BHLH11-TPL/TPR repressor complex .

What are common technical challenges when working with BHLH11 antibodies and how can they be overcome?

Researchers working with BHLH11 antibodies may encounter several technical challenges. Here are the most common issues and recommended solutions:

Challenge 1: Cross-reactivity with related bHLH proteins

• Solution: Pre-absorb antibodies with recombinant BHLH121 protein (65% identity to BHLH11)

• Alternative: Target antibodies to unique regions outside the conserved bHLH domain

• Validation: Always confirm specificity using bhlh11 mutant tissues as negative controls

Challenge 2: Low signal-to-noise ratio in immunoprecipitation

• Solution: Increase stringency of washing steps gradually

• Alternative: Use tandem affinity purification with dual-tagged BHLH11

• Optimization: Test different detergent concentrations in wash buffers

Challenge 3: Poor nuclear extraction efficiency

• Solution: Optimize nuclear isolation protocol with proper buffer composition

• Alternative: Use cell fractionation approaches with appropriate controls

• Validation: Include markers for nuclear (histone H3) and cytoplasmic (GAPDH) fractions

Challenge 4: Detecting dynamic changes in response to iron status

• Solution: Carefully control iron conditions in growth media

• Time course: Collect samples at multiple timepoints after iron status changes

• Quantification: Use standardized loading controls and quantitative western blotting

Challenge 5: Antibody performance in fixed tissues

• Solution: Test multiple fixation protocols (paraformaldehyde concentrations and times)

• Alternative: Use epitope retrieval techniques if formaldehyde fixation masks epitopes

• Control: Include positive controls with overexpressed tagged BHLH11

Based on the research methodologies described, successful detection of BHLH11 has been achieved in both plant tissues and in transient expression systems like Nicotiana benthamiana, suggesting that with proper optimization, antibody-based detection of this protein is feasible for multiple applications .

How can researchers effectively use antibodies to distinguish between the active and inactive forms of BHLH11?

Distinguishing between active and inactive forms of BHLH11 using antibodies requires focusing on its subcellular localization, protein-protein interactions, and potential post-translational modifications:

Subcellular localization-based approaches:

• Generate antibodies that work in immunofluorescence microscopy

• Quantify nuclear versus cytoplasmic distribution, as BHLH11 appears to be active as a repressor in the nucleus

• Track changes in localization under different iron conditions or when co-expressed with bHLH IVc proteins

• Consider subcellular fractionation followed by western blotting as a complementary approach

Protein complex-specific antibodies:

• Develop antibodies that specifically recognize BHLH11 when bound to TPL/TPR corepressors

• Use proximity ligation assays (PLA) to detect BHLH11-TPL/TPR complexes in situ

• Perform sequential immunoprecipitation to isolate specific protein complexes

Post-translational modification detection:

• Generate phospho-specific antibodies if phosphorylation sites are identified

• Use 2D gel electrophoresis followed by western blotting to separate differently modified forms

• Combine with mass spectrometry to identify specific modifications

Functional readouts:

• Correlate BHLH11 binding to chromatin (via ChIP) with repression activity

• Assess recruitment of TPL/TPR corepressors as a proxy for active repression

• Monitor target gene expression in parallel with BHLH11 status

Research has shown that BHLH11 functions as an active repressor when it:

• Is localized in the nucleus

• Interacts with bHLH IVc transcription factors

• Recruits TPL/TPR corepressors through its EAR motifs

The nuclear accumulation of BHLH11 is facilitated by bHLH IVc proteins, suggesting that interaction with these proteins is a key step in activating BHLH11's repressive function .

What protocols are most effective for studying the protein-protein interactions between BHLH11, bHLH IVc proteins, and TPL/TPR corepressors?

For comprehensive analysis of the protein-protein interactions within the BHLH11-bHLH IVc-TPL/TPR regulatory complex, researchers should employ multiple complementary techniques:

In vitro interaction assays:

• GST pull-down assays:

  • Express GST-tagged BHLH11 and test binding to in vitro translated bHLH IVc proteins and TPL/TPRs

  • Use mutations in EAR motifs to confirm specificity

• Surface Plasmon Resonance (SPR):

  • Measure binding kinetics and affinities between purified proteins

  • Compare wild-type versus mutant protein interactions

Yeast-based interaction assays:

• Yeast two-hybrid (Y2H):

  • Test direct interactions between BHLH11 and both bHLH IVc and TPL/TPR proteins

  • Map interaction domains using truncated proteins

  • This method has successfully demonstrated that BHLH11 interacts with all four bHLH IVc TFs and that this interaction depends on the EAR motifs

Plant cell-based interaction assays:

• Split-fluorescent protein complementation:

  • Use tripartite split-GFP system as demonstrated in the literature

  • GFP10-bHLH IVc + bHLH11-GFP11 + GFP1-9 showed interactions in the nucleus

  • Include appropriate negative controls

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of proteins in Nicotiana benthamiana

  • Immunoprecipitate with antibodies against one protein (e.g., anti-MYC for MYC-bHLH IVc)

  • Detect co-precipitated proteins with antibodies against the other protein (e.g., anti-HA for HA-BHLH11)

  • This approach confirmed that bHLH IVc and BHLH11 form protein complexes in plant cells

Functional interaction assays:

• Transient expression reporter assays:

  • Use promoters of target genes (e.g., bHLH38) fused to luciferase

  • Test effects of co-expressing BHLH11, bHLH IVc, and TPL/TPR proteins

  • This method demonstrated that BHLH11 inhibits the transactivity of bHLH IVc TFs

The research has established that BHLH11 interacts with bHLH IVc TFs in the nucleus and represses their transactivation function by recruiting TPL/TPR corepressors through its EAR motifs. These interactions are crucial for BHLH11's role in negatively regulating iron homeostasis in plants .

How can genome editing approaches be used to study BHLH11 function and potentially create iron-efficient crop varieties?

Genome editing approaches offer powerful tools for both fundamental research on BHLH11 and potential agricultural applications in developing iron-efficient crops:

CRISPR/Cas9-based strategies for functional characterization:

• Knockout studies:

  • Generate precise bhlh11 knockout mutants to study iron uptake and homeostasis

  • Create multiple knockouts of bHLH family members to assess functional redundancy

  • Analyze iron accumulation phenotypes under various growth conditions

• Domain-specific mutations:

  • Edit EAR motifs to disrupt TPL/TPR interaction without affecting protein expression

  • Modify interaction domains with bHLH IVc proteins

  • Create targeted mutations in DNA-binding regions

Base editing and prime editing applications:

• Precise modifications:

  • Introduce specific amino acid changes without double-strand breaks

  • Create allelic series with varying levels of BHLH11 activity

• Promoter modifications:

  • Alter BHLH11 expression patterns by modifying promoter elements

  • Engineer iron-responsive expression through promoter editing

Agricultural applications:

• Iron efficiency in crops:

  • Translate findings from Arabidopsis to crop species by targeting orthologs

  • Fine-tune BHLH11 expression to optimize iron uptake without toxicity

  • Create varieties with enhanced performance in iron-limiting soils

• Biofortification strategies:

  • Modulate BHLH11 activity to increase iron content in edible tissues

  • Balance increased iron accumulation with plant growth and yield

Experimental validation approaches:

• Use antibodies against BHLH11 to confirm knockout or altered expression

• Employ RNA-seq to assess global transcriptional changes

• Measure iron content in various tissues using ICP-MS

• Assess plant performance under field conditions with varying iron availability

While the search results don't explicitly discuss genome editing of BHLH11, the detailed understanding of its function as a negative regulator of iron homeostasis suggests that precise modification of this gene could be a valuable approach for both research and crop improvement strategies .

What is the current understanding of how BHLH11 interacts with other iron homeostasis regulatory networks beyond the bHLH IVc pathway?

The interaction of BHLH11 with broader iron homeostasis regulatory networks represents an emerging research area with several key aspects to explore:

Interaction with FIT-dependent pathways:

• Research indicates BHLH11 functions independently of FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), suggesting parallel regulatory mechanisms

• This independence creates a complex regulatory landscape where BHLH11 and FIT control distinct but overlapping sets of iron homeostasis genes

• Future research should investigate potential cross-talk between these pathways

Connection to iron sensing mechanisms:

• BHLH11 protein levels decrease in response to iron deficiency, indicating integration with iron sensing pathways

• The mechanisms by which iron status influences BHLH11 expression or stability remain to be fully elucidated

• Potential involvement of BHLH11 in feedback loops regulating iron uptake and distribution

Integration with hormone signaling networks:

• Many plant hormones (ethylene, auxin, jasmonic acid) influence iron homeostasis

• BHLH11 contains EAR motifs that are common in hormone-responsive transcriptional regulators

• Research should explore whether hormones modulate BHLH11 function during stress responses

Potential interaction with BTSL-FEP3 regulatory module:

• The search results mention a BTSL-bHLH-FEP3 interactome

• FEP3/IRON MAN1 is described as a small effector protein inhibiting BTSL1/BTSL2-mediated degradation of bHLH subgroup IVb and IVc proteins

• This suggests potential cross-regulation between BHLH11 and the BTSL-FEP3 module that warrants further investigation

Research approaches to explore these networks:

• Protein-protein interaction mapping using proteomics approaches

• Genetic analysis with higher-order mutants combining bhlh11 with mutations in other iron regulatory genes

• Transcriptomics under various iron conditions comparing wild-type and mutant backgrounds

• ChIP-seq to identify genome-wide binding sites of BHLH11 and other iron homeostasis transcription factors

This complex regulatory landscape highlights the need for systems biology approaches to fully understand how BHLH11 functions within the broader context of iron homeostasis regulation in plants .

How might post-translational modifications regulate BHLH11 function and what methodologies can detect these modifications?

Post-translational modifications (PTMs) likely play crucial roles in regulating BHLH11 function, though specific modifications are not directly described in the search results. Here's an exploration of potential PTMs and methodologies to detect them:

Potential PTMs regulating BHLH11:

• Phosphorylation:

  • May regulate nuclear-cytoplasmic shuttling

  • Could affect interaction with bHLH IVc proteins or TPL/TPRs

  • Potentially responsive to iron status signaling

• Ubiquitination:

  • Might control protein stability in response to iron conditions

  • The search results show that BHLH11 protein levels decrease during iron deficiency

  • Could regulate nuclear-cytoplasmic distribution

• SUMOylation:

  • Often regulates transcription factor activity

  • May affect repressor function or protein-protein interactions

  • Could modulate nuclear retention

• Acetylation/Methylation:

  • Potential regulation of DNA binding activity

  • May influence interaction with corepressors

Advanced methodologies to detect and characterize PTMs:

• Mass spectrometry-based approaches:

  • Immunoprecipitate BHLH11 using specific antibodies

  • Analyze by LC-MS/MS to identify and map modifications

  • Use SILAC or TMT labeling to compare PTM profiles under different iron conditions

  • Employ enrichment strategies for specific modifications (phosphopeptide enrichment, ubiquitin remnant antibodies)

• Modification-specific antibodies:

  • Develop antibodies against predicted phosphorylation sites

  • Use in western blotting to detect changes in modification status

  • Apply in ChIP to correlate modifications with chromatin binding

• Protein mobility analysis:

  • Use Phos-tag gels to detect phosphorylated forms

  • Apply 2D gel electrophoresis to separate differentially modified forms

  • Perform western blotting with anti-BHLH11 antibodies

• Functional validation approaches:

  • Generate site-specific mutants of predicted modification sites

  • Assess impact on localization, protein interactions, and gene regulation

  • Create phosphomimetic and phospho-deficient variants

• In vivo dynamics:

  • Use split-luciferase complementation to monitor protein interactions

  • Apply FRET-based sensors to detect conformational changes upon modification

  • Employ real-time imaging to track localization changes

Understanding the PTM landscape of BHLH11 would provide crucial insights into how this repressor is regulated in response to changing iron conditions and how it interfaces with broader signaling networks controlling iron homeostasis in plants .