BZIP10 Antibody

What is BZIP10 and why is it significant in plant research?

BZIP10 (AtbZIP10) is a plant-specific transcription factor belonging to the Group C basic leucine zipper (bZIP) family. It contains a conserved bZIP domain enabling DNA binding and dimerization with other bZIP partners. BZIP10 plays critical roles in several fundamental plant processes, making it an important research target:

• It regulates programmed cell death in response to oxidative stress signals

• It functions as a positive mediator of hypersensitive response (HR) during pathogen recognition

• It shuttles dynamically between the nucleus and cytoplasm via exportin-mediated transport

• It interacts with LSD1 (Lesions Simulating Disease Resistance 1), which retains BZIP10 in the cytoplasm to suppress uncontrolled cell death

Understanding BZIP10's function has significant implications for improving plant stress tolerance and pathogen resistance. The protein serves as a molecular hub connecting oxidative stress perception with transcriptional responses, making antibodies against it essential tools for studying these processes.

What DNA sequences does BZIP10 recognize and bind to?

BZIP10 recognizes and binds to specific DNA sequences, including:

• C-box-like motif (5'-TGCTGACGTCA-3')

• G-box-like motif

Importantly, BZIP10 exhibits limited DNA-binding capacity as a homodimer. DAP-seq analysis shows that Group C bZIPs, including BZIP10, demonstrate minimal direct DNA-binding activity unless partnered with Group S bZIPs. BZIP10 forms functional heterodimers with other bZIP proteins (particularly bZIP53 and bZIP25), which significantly enhances its DNA binding affinity and transcriptional activation properties .

This heterodimer formation is crucial for BZIP10's function, as evidenced by in vitro DNA binding assays. For example, experiments using double-stranded oligonucleotides attached to ELISA plates have shown that heterodimerization dramatically increases binding to target sequences .

What are the recommended applications for BZIP10 antibodies in research?

BZIP10 antibodies serve multiple critical applications in plant molecular biology research:

When selecting antibodies for these applications, researchers should consider:

• Epitope accessibility in different experimental contexts

• Potential cross-reactivity with other Group C bZIP proteins

• The need for validation in genetic backgrounds (e.g., atbzip10 mutants as negative controls)

• Whether native BZIP10 or epitope-tagged versions will be detected

How does BZIP10 shuttle between cellular compartments and how can antibodies track this movement?

BZIP10 exhibits dynamic nuclear-cytoplasmic shuttling regulated through several mechanisms:

BZIP10 contains both nuclear localization signals (NLS) and nuclear export signals (NES), with the NES located within the first 105 amino acids based on interaction studies with exportin (XPO1) . This shuttling is actively regulated rather than occurring through passive diffusion, as evidenced by leptomycin B (LMB) experiments. When treated with this nuclear export inhibitor, BZIP10-GFP becomes restricted to the nucleus, confirming active exportin-mediated transport .

Researchers can effectively track BZIP10 localization using several approaches:

• Cell fractionation with immunoblotting:

  • Separate nuclear and cytoplasmic fractions

  • Perform Western blotting with anti-BZIP10 antibodies

  • Studies show that in wild-type plants, BZIP10 is detectable in both soluble and nuclear fractions, while in lsd1 mutants, it localizes predominantly to the nuclear fraction

• Immunofluorescence microscopy:

  • Fix and permeabilize cells/tissues

  • Incubate with anti-BZIP10 primary antibodies followed by fluorescent secondary antibodies

  • Counterstain nuclei with DAPI

  • Quantify nuclear/cytoplasmic signal ratios

• Live-cell imaging with GFP fusions and immunovalidation:

  • Express BZIP10-GFP fusion proteins

  • Track localization in real-time

  • Validate observations with fixed-cell immunofluorescence using BZIP10 antibodies

When conducting these studies, it's important to note that BZIP10 localization is significantly affected by interaction with LSD1. In the absence of LSD1, 82% of cells show mixed cytoplasmic/nuclear distribution of BZIP10-GFP, whereas when co-expressed with LSD1, 34% of cells display exclusively cytoplasmic accumulation .

How does the BZIP10-LSD1 interaction affect subcellular localization?

The interaction between BZIP10 and LSD1 represents a critical regulatory mechanism controlling BZIP10's function:

LSD1 (Lesions Simulating Disease Resistance 1) is a plant-specific zinc-finger protein that functions as a negative regulator of cell death, protecting plant cells from reactive oxygen-induced stress . When BZIP10 interacts with LSD1 in the cytoplasm, it results in partial retention of BZIP10 outside the nucleus, effectively limiting its transcriptional activity .

This interaction has been confirmed through multiple experimental approaches:

• Yeast two-hybrid assays: LSD1 was identified as an interaction partner using the nuclear transportation trap (NTT) system

• Co-immunoprecipitation: In planta studies confirm the interaction occurs in plant cells

• Cell fractionation studies: In lsd1 mutants, BZIP10 localizes exclusively to the nuclear-enriched microsomal fraction, whereas in wild-type plants, it distributes between soluble and nuclear fractions

• Quantitative localization analysis: When co-expressed with LSD1, the percentage of cells showing exclusively cytoplasmic BZIP10-GFP nearly doubles (from 18% to 34%)

The functional significance of this interaction is demonstrated by genetic studies showing that BZIP10 and LSD1 act antagonistically in both pathogen-induced hypersensitive response and basal defense responses . LSD1 appears to serve as a cellular hub, where its interaction with BZIP10 (and likely other proteins) contributes significantly to plant oxidative stress responses by controlling the cell death-related transcriptional activity of BZIP10 via altering its intracellular partitioning .

What are the optimal immunoprecipitation conditions for studying BZIP10 protein complexes?

Optimizing immunoprecipitation (IP) protocols for BZIP10 requires careful consideration of several factors:

• Protein expression challenges:
  BZIP10 is typically expressed at relatively low levels when driven by its native promoter. Studies show that conditionally overexpressed BZIP10 accumulates to approximately 10-fold higher levels than constitutively expressed BZIP10 . This presents challenges for detection and requires careful optimization of antibody amounts and incubation conditions.

• Buffer composition optimization:
  For effective IP of BZIP10 complexes, buffer composition is critical:

  • Use mild detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions

  • Include protease inhibitors to prevent degradation

  • Add reducing agents (5-10 mM DTT) to maintain proper folding of cysteine-rich proteins like LSD1

• Stabilizing heterodimer interactions:
  Research shows that co-expression of BZIP10 and bZIP53 leads to enhanced protein levels, suggesting heterodimer formation might stabilize the proteins from degradation . This has implications for IP protocols:

  • Consider cross-linking approaches to capture transient interactions

  • Optimize salt concentration to preserve heterodimeric complexes

  • Avoid harsh washing conditions that might disrupt important interactions

• Controls and validation strategies:

  • Include appropriate negative controls (IgG, unrelated antibody)

  • Validate detected interactions using reciprocal IP (e.g., IP with anti-bZIP53, detect with anti-BZIP10)

  • Include known non-interacting proteins as specificity controls (e.g., bZIP63, which does not interact with LSD1)

When studying BZIP10-LSD1 interactions specifically, cell fractionation may be necessary to enrich cytoplasmic compartments where this interaction predominantly occurs .

How can I optimize chromatin immunoprecipitation (ChIP) protocols for BZIP10?

ChIP experiments with BZIP10 antibodies present several specific challenges that require optimization:

• Antibody specificity considerations:
  BZIP10 shares sequence similarity with other Group C bZIPs, necessitating careful validation:

  • Validate antibody specificity using atbzip10 mutants as negative controls

  • Consider using epitope-tagged BZIP10 and ChIP with anti-tag antibodies if specificity issues arise

• DNA binding heterodimer complexes:
  BZIP10 cannot bind DNA alone but requires heterodimerization with other bZIPs, particularly from Group S:

  • This heterodimer requirement affects ChIP efficiency compared to transcription factors that bind DNA as homodimers

  • Optimize crosslinking conditions (test 1% formaldehyde for 5-15 minutes) to capture heterodimeric complexes

  • Consider dual ChIP approaches targeting both BZIP10 and its heterodimerization partners

• Target sequence considerations:
  BZIP10 binds specific DNA motifs when in heterodimeric complexes:

  • Design positive control primers for regions containing C-box-like (5'-TGCTGACGTCA-3') and G-box-like motifs

  • Include negative control regions lacking these motifs

  • Use DNA binding assays (like those described in the research literature with ELISA plate-bound oligonucleotides) to validate binding specificity

• Accounting for nuclear-cytoplasmic shuttling:
  BZIP10's dynamic localization affects ChIP efficiency:

  • LSD1 interaction retains BZIP10 in the cytoplasm, reducing nuclear availability

  • Compare ChIP efficiency in wild-type vs. lsd1 mutants (where nuclear accumulation is enhanced)

  • Consider treatments that affect localization (e.g., BTH treatment which induces oxidative stress responses)

• Validation approaches:

  • Perform qPCR of candidate target regions

  • Compare results with published binding data for related bZIP proteins

  • Correlate binding with gene expression changes to establish functional relevance

When analyzing ChIP-seq data, account for the fact that BZIP10 acts as part of heterodimeric complexes and analyze enrichment of specific DNA motifs in peak regions that match the known binding preferences of BZIP10 heterodimers .

What role does BZIP10 play in pathogen defense, and how can antibodies help elucidate this function?

BZIP10 serves as a positive regulator of both pathogen-induced hypersensitive response (HR) and basal defense responses, with these activities antagonized by LSD1 . This makes it a crucial target for studying plant immunity.

Key findings on BZIP10's role in pathogen defense:

• Genetic evidence:

  • lsd1-2 atbzip10 double mutants display significant reduction of ion leakage compared to lsd1-2 single mutants after BTH treatment, indicating BZIP10's role in cell death regulation

  • Conversely, overexpression of AtbZIP10 in lsd1-2 dramatically enhances BTH-induced ion leakage

  • BZIP10 function is required for superoxide-induced runaway cell death in lsd1

• Pathogen interactions:

  • BZIP10 positively regulates basal defense against Hyaloperonospora parasitica (Hp), an obligate biotrophic oomycete parasite

  • It contributes to both RPP2-mediated HR and basal defense responses

Antibody-based approaches to study BZIP10 in pathogen defense:

• Protein expression dynamics:

  • Use Western blotting with anti-BZIP10 antibodies to track expression levels during infection

  • Compare expression in infected vs. adjacent non-infected tissues

  • Analyze time-course samples to capture expression dynamics throughout the infection process

• Subcellular localization changes:

  • Employ immunolocalization to track BZIP10 translocation during pathogen challenge

  • Compare nuclear vs. cytoplasmic distribution in resistant and susceptible interactions

  • Correlate localization patterns with defense outcomes

• Protein complex dynamics:

  • Use co-immunoprecipitation with anti-BZIP10 antibodies to identify changing interaction partners during infection

  • Focus on BZIP10-LSD1 interaction dynamics during different phases of pathogen response

  • Identify novel defense-related interactors through mass spectrometry of immunoprecipitated complexes

• Target gene identification:

  • Employ ChIP with anti-BZIP10 antibodies to map binding sites during infection

  • Compare target profiles in compatible vs. incompatible interactions

  • Correlate with transcriptional changes of defense genes

These approaches can be applied across different genetic backgrounds (wild-type, atbzip10, lsd1, lsd1 atbzip10, BZIP10 overexpression lines) to comprehensively map BZIP10's role in pathogen defense networks .

How does heterodimerization affect BZIP10 function and how can it be studied?

Heterodimerization is central to BZIP10's functionality, dramatically enhancing its activity and stability:

• Enhanced transcriptional activity:

  • Research demonstrates that bZIP53 heterodimerization with bZIP10 or bZIP25 significantly increases its transactivation properties

  • This enhanced activity is essential for effective regulation of target genes

• Increased protein stability:

  • Co-expression of bZIP10 and bZIP53 leads to enhanced protein levels, suggesting that heterodimer formation might protect the proteins from degradation

  • This stabilization effect has significant implications for protein half-life and activity duration

• Improved DNA binding:

  • BZIP10 alone shows limited DNA-binding capacity

  • Heterodimerization, particularly with Group S bZIPs like bZIP53, dramatically enhances binding to target sequences

  • This is demonstrated through DNA binding assays using double-stranded oligonucleotides attached to ELISA plates

Methods to study BZIP10 heterodimer formation using antibodies:

• Co-immunoprecipitation approaches:

  • Use anti-BZIP10 antibodies to pull down complexes and detect partners

  • Perform reciprocal IP with antibodies against partner proteins

  • Quantify relative amounts of heterodimers under different conditions

• Protein stability assessment:

  • Express BZIP10 with or without dimerization partners

  • Track protein levels over time using Western blotting

  • Quantify degradation rates to confirm stabilization effects

• DNA binding analysis with heterodimers:

  • Perform DNA binding assays with purified proteins or cell extracts

  • Compare binding efficiency of BZIP10 alone versus heterodimeric complexes

  • Use competition experiments with wild-type or mutated oligonucleotides to confirm binding specificity, as demonstrated in published research

• Functional validation in mutant backgrounds:

  • Assess target gene expression in plants lacking specific bZIP proteins

  • Complement with various combinations to determine which heterodimers are functionally relevant

  • Correlate with phenotypic outcomes in stress response and development

These approaches provide complementary insights into how BZIP10 heterodimers form, their binding preferences, and their functional significance in plant biology.

How can A-ZIP inhibitors be used to study BZIP10 function?

A-ZIP inhibitors represent powerful tools for studying BZIP10 function through specific disruption of bZIP dimerization:

A-ZIP inhibitors are engineered dominant-negative proteins designed to specifically disrupt bZIP dimerization. They contain the leucine zipper domain but lack the basic DNA-binding region, allowing them to form non-functional heterodimers with endogenous bZIP proteins .

Experimental applications of A-ZIP inhibitors:

• Transient expression assays:

  • Research shows that co-transfection with A-ZIP53 plasmid reduces GUS signals from reporter constructs in a dose-dependent manner

  • A-ZIP53 inhibits the DNA binding of bZIP53, bZIP10, and bZIP25, demonstrating its effectiveness in disrupting functional heterodimers

• Validation of heterodimer-specific functions:

  • A-ZIP inhibitors can distinguish functions requiring specific heterodimer combinations

  • By selectively disrupting particular bZIP interactions, researchers can identify which heterodimer partners are essential for specific biological processes

• Complementary approach to genetic studies:

  • While genetic knockouts eliminate entire proteins, A-ZIP inhibitors specifically target dimerization

  • This allows differentiation between dimerization-dependent and independent functions

Methodological considerations when using A-ZIP inhibitors:

• Controls for specificity:

  • Include unrelated transcription factors to confirm specificity

  • Test multiple concentrations to establish dose-dependent effects

  • Use mutated versions of A-ZIP inhibitors as negative controls

• Validation of inhibition mechanism:

  • Confirm that A-ZIP inhibitors actually form heterodimers with target bZIPs using co-IP

  • Verify reduced DNA binding using techniques like EMSA or DNA-binding ELISAs

• Combination with antibody-based detection:

  • Use BZIP10 antibodies to confirm expression levels are not affected

  • Employ co-IP with BZIP10 antibodies to verify A-ZIP association

  • Perform ChIP to confirm reduced chromatin association

These approaches provide powerful tools for dissecting the specific contributions of heterodimer formation to BZIP10 function in various biological contexts .

What are the most promising future directions for BZIP10 antibody-based research?

Several promising research directions emerge at the intersection of BZIP10 biology and antibody-based methodologies:

• Single-cell analysis of BZIP10 dynamics:

  • Developing immunofluorescence approaches for tracking BZIP10 in individual cells within tissues

  • Correlating cellular heterogeneity in BZIP10 localization with cell-specific responses to pathogens

  • This would address the finding that BZIP10 localization varies significantly between cells, with some showing exclusively nuclear, others cytoplasmic, and many mixed distributions

• Protein modification mapping:

  • Generating modification-specific antibodies to detect phosphorylated, ubiquitinated, or otherwise modified BZIP10

  • Mapping how these modifications affect BZIP10-LSD1 interaction and nuclear-cytoplasmic shuttling

  • Correlating modifications with changes in heterodimer preferences and transcriptional activity

• Comprehensive interactome analysis:

  • Using BZIP10 antibodies to pull down interaction networks under various stress conditions

  • Building a dynamic map of how BZIP10's protein partnerships change during pathogen attack

  • Identifying novel components of the LSD1-BZIP10 regulatory pathway

• Heterodimer-specific antibodies:

  • Developing antibodies that specifically recognize BZIP10 in complex with particular partners

  • This would enable tracking of specific heterodimer combinations in different cell types and conditions

  • Such tools would complement the A-ZIP inhibitor approaches already being employed

• Translational applications to crop species:

  • Developing antibodies against BZIP10 homologs in major crop species

  • Using these to study how BZIP10-related pathways might be harnessed to enhance disease resistance

  • Creating diagnostic tools to monitor stress responses in agricultural settings

These future directions build upon the solid foundation of BZIP10 research, potentially opening new avenues for understanding and manipulating plant stress responses and disease resistance .