CYP82C4 Antibody

What is CYP82C4 and why is it significant in plant research?

CYP82C4 is a cytochrome P450 enzyme belonging to the CYP82C family in Arabidopsis thaliana. It plays a critical role in plant iron deficiency responses and is involved in the biosynthesis of specialized metabolites. CYP82C4 specifically catalyzes the conversion of fraxetin into sideretin (5,7,8-trihydroxy-6-methoxycoumarin), a catecholic coumarin with strong iron chelation properties . This enzyme's expression is tightly regulated under conditions of reduced iron availability, particularly in calcareous soils where plants must activate their "Fe Deficiency Response" mechanisms .

The significance of CYP82C4 is highlighted by studies showing that full suppression of CYP82C4 expression (as observed in atc82c4-1 knockout mutants) results in longer roots at the seedling stage, suggesting its involvement in early iron deficiency responses . Additionally, CYP82C4 is part of a gene cluster that includes paralogs CYP82C2 and CYP82C3, with phylogenetic analyses showing high sequence identity (>88%) among homologs identified in related plant species . Understanding CYP82C4 function helps illuminate plant adaptation mechanisms to nutrient-limited conditions.

How do CYP82C4 antibodies help study iron deficiency responses in plants?

CYP82C4 antibodies serve as invaluable tools for investigating iron deficiency responses in plants by enabling protein detection, localization, and quantification. When combined with techniques like western blotting, immunohistochemistry, and flow cytometry, these antibodies allow researchers to track CYP82C4 expression patterns under varying iron conditions and across different tissues.

For instance, immunolocalization studies using anti-CYP82C4 antibodies can reveal the spatial distribution of CYP82C4 protein in root tissues during early iron deficiency response, providing insights beyond what transcript analysis alone can offer . This is particularly valuable since CYP82C4 expression appears to be regulated through an IDE1-like mediated pathway, a key component of iron deficiency signaling . Antibody-based protein detection complements transcriptomic data, allowing researchers to identify post-transcriptional regulation mechanisms that might affect CYP82C4 protein levels independently of mRNA abundance.

Additionally, CYP82C4 antibodies enable co-immunoprecipitation experiments to identify protein interaction partners, potentially revealing regulatory complexes that control iron homeostasis in plants under stress conditions. Such experiments help establish comprehensive models of iron deficiency signaling networks and their downstream metabolic pathways.

What are the key considerations when selecting a CYP82C4 antibody for plant research?

When selecting CYP82C4 antibodies for plant research, several critical factors must be considered to ensure experimental success. First, antibody specificity is paramount - researchers should verify that the antibody can distinguish CYP82C4 from its close paralogs CYP82C2 and CYP82C3, which share high sequence similarity . This is especially important since CYP82C4 resides in a near-tandem cluster with these paralogs in the Arabidopsis genome.

Second, researchers should consider the antibody format based on intended applications. Polyclonal antibodies generally offer broader epitope recognition but may have higher batch-to-batch variability, while monoclonal antibodies provide consistent results with higher specificity for single epitopes. For co-immunoprecipitation experiments or chromatin immunoprecipitation studies, antibodies must maintain binding affinity under native conditions.

Third, cross-reactivity with orthologs from different plant species should be evaluated when working with non-model organisms. Since CYP82C4 homologs show >88% identity across related species , some antibodies may recognize conserved epitopes, enabling comparative studies. Researchers should verify cross-reactivity through preliminary western blot analyses when extending studies beyond Arabidopsis.

Finally, antibody validation through appropriate controls is essential. This includes testing antibody specificity using protein extracts from cyp82c4 knockout mutants as negative controls, and using recombinant CYP82C4 protein as a positive control to establish detection limits and linear response ranges.

How can ChIP-Seq with CYP82C4 antibodies illuminate transcriptional regulation mechanisms?

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using CYP82C4 antibodies represents an advanced approach to understanding the transcriptional regulation of iron deficiency responses. While CYP82C4 itself is not a transcription factor, ChIP-Seq can be employed to identify proteins that regulate CYP82C4 expression by immunoprecipitating transcription factors known to bind its promoter region.

The search results suggest that CYP82C4 expression may be regulated by mechanisms similar to those controlling CYP82C2, which is directly activated by the transcription factor WRKY33 . In CYP82C2's case, WRKY33 binds to W-box regions containing WRKY33-specific motifs, as demonstrated by ChIP-PCR experiments showing greater than fivefold enrichment at these sites . Similar experimental designs could be employed to identify transcription factors regulating CYP82C4.

For ChIP-Seq experiments targeting putative transcription factors binding to the CYP82C4 promoter, researchers should:

• Cross-link proteins to DNA in plant tissues under iron-deficient conditions

• Lyse cells and shear chromatin to appropriate fragment sizes (200-500 bp)

• Immunoprecipitate chromatin using antibodies against candidate transcription factors

• Reverse cross-links, isolate DNA, and prepare libraries for next-generation sequencing

• Map reads to the reference genome and identify enriched binding regions

• Validate binding sites through reporter gene assays and DNA binding studies

This approach has successfully revealed that the IMA1/IMA2 peptides may act as transcriptional coactivators that modulate CYP82C4 expression, as demonstrated in luciferase reporter assays in transiently transformed protoplasts . Such studies provide crucial insights into the complex regulatory networks controlling iron homeostasis in plants.

What methodologies are most effective for quantifying CYP82C4 protein expression across different experimental conditions?

Quantifying CYP82C4 protein expression across different experimental conditions requires robust methodologies that provide accurate, reproducible results. Several complementary approaches can be employed, each with specific advantages for particular research questions.

Western blotting with CYP82C4 antibodies remains a fundamental technique for semi-quantitative protein analysis. For rigorous quantification, researchers should include standard curves using recombinant CYP82C4 protein and normalize to appropriate loading controls. Digital image analysis software can enhance quantification accuracy. When comparing protein levels across multiple conditions (such as varying iron concentrations or pH levels), consistent sample preparation and loading are essential .

For more precise quantification, enzyme-linked immunosorbent assays (ELISAs) using CYP82C4 antibodies offer higher sensitivity and better quantitative capacity. Sandwich ELISA formats provide particularly robust results when two different CYP82C4 antibodies recognizing distinct epitopes are available.

Mass spectrometry-based approaches represent the gold standard for absolute quantification of CYP82C4. Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) with isotopically labeled peptide standards allows for highly accurate protein quantification across multiple samples. While these methods don't directly utilize antibodies, they can be combined with immunoprecipitation steps (IP-MS) to enrich for CYP82C4 before analysis.

The choice of methodology should align with experimental objectives. For example, when studying pH-dependent regulation of CYP82C4, as suggested by recent work showing differential expression at variable pH , western blotting might be sufficient for identifying broad expression patterns, while mass spectrometry would provide more precise quantification needed for mathematical modeling of regulatory networks.

How can dose-response modeling approaches be applied to CYP82C4 antibody-based assays?

Dose-response modeling provides a powerful framework for quantifying relationships between experimental variables and biological responses in CYP82C4 antibody-based assays. Although the search results don't specifically describe dose-response models for CYP82C4 antibodies, we can adapt modeling approaches similar to those used for monoclonal antibody functional activity assessments .

For antibody-based quantification of CYP82C4 in response to varying iron concentrations or pH levels, researchers can employ four-parameter logistic (4PL) models of the form:

$y = L+(U − L)/(1 + (x/IC50)^h)$

Where:

• L represents the minimum CYP82C4 expression (lower limit)

• U represents the maximum CYP82C4 expression (upper limit)

• IC50 is the iron concentration where CYP82C4 expression is 50% of maximum

• h is the Hill slope determining curve steepness

This modeling approach allows for robust comparison of CYP82C4 expression patterns across different experimental conditions, genotypes, or treatments. For example, when comparing CYP82C4 expression in wild-type versus mutant plants under varying iron concentrations, the IC50 values can provide a quantitative measure of sensitivity to iron deficiency.

To implement this approach effectively, researchers should:

• Design experiments with sufficient concentration points (minimum 7-8) to accurately define the curve

• Include appropriate controls for upper and lower limits

• Perform technical and biological replicates to assess variability

• Use statistical methods to compare parameter estimates between conditions

Such modeling approaches provide statistical rigor when analyzing complex relationships between environmental conditions and CYP82C4 expression, facilitating hypothesis testing and experimental design optimization .

How can researchers troubleshoot low signal or high background issues when using CYP82C4 antibodies?

When researchers encounter low signal or high background issues using CYP82C4 antibodies, systematic troubleshooting and optimization are essential. For low signal problems, several factors may be responsible. First, CYP82C4 protein expression is highly dependent on iron availability and pH conditions, so researchers should verify that their experimental conditions indeed induce CYP82C4 expression . Studies have shown that CYP82C4 is part of the early Fe deficiency response, and its expression patterns may change rapidly under varying conditions .

Antibody concentration and incubation conditions should be systematically optimized. A titration experiment using different antibody dilutions (1:250, 1:500, 1:1000, 1:2000) can help identify the optimal concentration. Extended incubation times at 4°C (overnight versus 1-2 hours) may improve signal without increasing background. Signal amplification systems like biotin-streptavidin or tyramide signal amplification can enhance detection sensitivity for low-abundance proteins.

For high background issues, several strategies can be implemented. More stringent blocking conditions using 5% BSA or 5% non-fat dry milk in TBST, with longer blocking times (2-3 hours), can reduce non-specific binding. Increasing wash stringency by using higher salt concentrations (up to 500 mM NaCl) or adding 0.1% SDS to wash buffers can help reduce background. For immunohistochemistry applications, autofluorescence can be quenched using treatments like sodium borohydride or Sudan Black B.

Researchers should also consider tissue-specific extraction protocols, as CYP82C4 is a membrane-associated cytochrome P450 enzyme that may require specialized extraction methods to maintain protein integrity and antibody accessibility. Detergent selection and concentration are particularly important for solubilizing membrane proteins while preserving epitope recognition.

What are the optimal experimental designs for studying CYP82C4 protein-protein interactions?

Optimal experimental designs for studying CYP82C4 protein-protein interactions require careful consideration of both the membrane-associated nature of this cytochrome P450 enzyme and the conditions under which its interactions are likely to occur. Co-immunoprecipitation (Co-IP) using CYP82C4 antibodies represents a primary approach, but must be optimized for membrane proteins.

For effective Co-IP experiments, researchers should use mild detergents like digitonin (0.5-1%), CHAPS (0.5-1%), or NP-40 (0.1-0.5%) that solubilize membranes while preserving protein-protein interactions. Crosslinking with membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) before cell lysis can stabilize transient interactions. To ensure specificity, parallel Co-IPs should be performed using samples from cyp82c4 knockout plants as negative controls .

Bimolecular Fluorescence Complementation (BiFC) offers a powerful in vivo approach to visualize CYP82C4 interactions. This technique involves fusing potential interacting partners with complementary fragments of a fluorescent protein (such as YFP). When CYP82C4 and its partner interact, the fluorescent protein fragments reconstitute, producing a detectable signal. This approach is particularly valuable for confirming interactions in living plant cells and determining their subcellular localization.

Proximity-dependent biotin identification (BioID) presents an emerging technique well-suited for studying CYP82C4 interactions. By fusing CYP82C4 to a biotin ligase, proteins in close proximity become biotinylated and can be purified and identified by mass spectrometry. This approach is especially valuable for identifying weak or transient interactions that might be missed by Co-IP.

For all protein interaction studies, researchers should design experiments that account for iron availability conditions, as CYP82C4 functions in iron deficiency responses . Interaction profiles should be compared between iron-sufficient and iron-deficient conditions to identify condition-specific interactions that might reveal regulatory mechanisms.

How can researchers effectively compare data from CYP82C4 antibody-based assays with transcriptomic data?

Integrating CYP82C4 antibody-based protein data with transcriptomic datasets requires careful methodological approaches to account for the different regulatory levels and temporal dynamics of mRNA versus protein expression. This integration is particularly important for CYP82C4, as its expression appears to be regulated at multiple levels including transcriptional control by factors like IMA1/IMA2 peptides and possibly post-transcriptional mechanisms.

To effectively compare these datasets, researchers should first ensure temporal alignment by collecting protein and RNA samples at multiple matched time points following iron deficiency treatment. This time-course approach can reveal important regulatory dynamics, such as delays between transcriptional induction and protein accumulation, or post-transcriptional regulation that may result in different mRNA and protein profiles.

Statistical approaches for integrating these data types include:

• Correlation analysis: Computing Pearson or Spearman correlation coefficients between CYP82C4 mRNA and protein levels across conditions and time points.

• Dynamic time warping: This algorithm aligns time-series data that may be out of phase, accommodating differences in the temporal dynamics of mRNA versus protein expression.

• Regression analysis: Models that predict protein levels based on current and past mRNA levels can help quantify the relationship between transcription and translation.

When discrepancies between mRNA and protein levels are observed, researchers should investigate potential mechanisms, including:

• mRNA stability differences under varying iron conditions

• Translational efficiency changes mediated by RNA-binding proteins

• Post-translational modifications affecting protein stability

• Protein compartmentalization or trafficking changes

For example, when studying the IDE1-like mediated pathway that appears to regulate CYP82C4 in early Fe deficiency responses , researchers might observe that transcriptional changes precede detectable protein changes by several hours. Such observations can guide mechanistic studies into the regulatory layers controlling CYP82C4 expression and function.

How can CYP82C4 antibodies be employed in studying cross-talk between iron deficiency and pathogen defense pathways?

CYP82C4 antibodies provide valuable tools for investigating the cross-talk between iron deficiency responses and pathogen defense pathways in plants. This research direction has emerged from findings that members of the CYP82C family play roles in both nutrient homeostasis and pathogen resistance. While CYP82C4 is primarily involved in iron deficiency responses, its paralog CYP82C2 is regulated by WRKY33 and participates in pathogen-induced metabolic responses .

Immunoprecipitation experiments using CYP82C4 antibodies coupled with mass spectrometry (IP-MS) can identify protein interaction partners that bridge these two response pathways. By comparing protein complexes formed under iron deficiency alone, pathogen challenge alone, and combined stress conditions, researchers can map the signaling networks that integrate these responses. This approach might reveal whether transcription factors like WRKY33, which directly regulates CYP82C2 in pathogen responses , also influence CYP82C4 under certain conditions.

Immunolocalization studies can determine whether CYP82C4 protein localization or abundance changes during pathogen infection, particularly when plants are simultaneously experiencing iron limitation. Confocal microscopy with fluorescently labeled CYP82C4 antibodies can track protein redistribution in response to these combined stresses. This is particularly relevant since both iron deficiency and pathogen defense involve specialized metabolites like coumarins, which CYP82C4 helps synthesize by converting fraxetin to sideretin .

To fully explore this cross-talk, researchers should design factorial experiments that systematically vary both iron availability and pathogen exposure, using CYP82C4 antibodies to track protein expression, localization, and interaction partners across all conditions. This comprehensive approach can reveal how plants prioritize and integrate responses to multiple simultaneous stresses, a crucial adaptation for survival in complex natural environments.

What role might CYP82C4 play in pH-dependent iron acquisition strategies, and how can antibodies help elucidate this?

CYP82C4 appears to play a significant role in pH-dependent iron acquisition strategies in plants, particularly in the context of calcareous soils where high pH reduces iron availability. Recent research indicates that IMA peptides regulate CYP82C4 expression in a pH-dependent manner, with different regulatory patterns observed under acidic versus alkaline conditions . CYP82C4 antibodies can help elucidate these complex regulatory mechanisms through several experimental approaches.

Immunoblotting with CYP82C4 antibodies can quantify protein expression across pH gradients (pH 5.0-8.0) and in different genetic backgrounds (wild-type versus ima mutants). This approach can confirm whether transcriptional changes in CYP82C4 observed under different pH conditions translate to corresponding protein level changes. A comprehensive experimental design should include time-course sampling after pH shifts to capture the dynamics of this response.

Immunolocalization studies can determine whether CYP82C4 protein shows altered subcellular or tissue distribution under different pH conditions. This is particularly relevant since iron acquisition strategies may involve specialized cells or tissues depending on soil pH. For example, under high pH conditions where iron solubility is severely limited, plants might increase CYP82C4 expression in specific root tissues to enhance sideretin production for iron chelation .

Co-immunoprecipitation experiments using CYP82C4 antibodies can identify pH-dependent protein interaction partners, potentially revealing how pH sensing mechanisms connect to iron acquisition pathways. These experiments should be performed with protein extracts from plants grown at different pH levels to capture condition-specific interactions.

The research findings suggest that IMA peptides play a crucial role as transcriptional coactivators modulating CYP82C4 expression in response to pH changes . Antibody-based approaches can help validate these regulatory relationships by tracking protein complex formation between IMA peptides, transcription factors, and the CYP82C4 promoter region under varying pH conditions.

How can advanced microscopy techniques enhance CYP82C4 antibody applications in subcellular localization studies?

Advanced microscopy techniques can significantly enhance CYP82C4 antibody applications for subcellular localization studies, providing unprecedented insights into the spatial organization of iron deficiency responses. As a cytochrome P450 enzyme, CYP82C4 is likely associated with the endoplasmic reticulum membrane, but its precise localization and potential redistribution under stress conditions remain to be fully characterized.

Super-resolution microscopy techniques overcome the diffraction limit of conventional light microscopy, enabling visualization of protein localization at nanometer-scale resolution. Specifically, Structured Illumination Microscopy (SIM) provides 2-fold improvement in resolution and is compatible with standard immunofluorescence protocols using CYP82C4 antibodies. For even higher resolution, Stochastic Optical Reconstruction Microscopy (STORM) or Photoactivated Localization Microscopy (PALM) can achieve 10-20 nm resolution, though these techniques require specialized fluorophore-conjugated antibodies.

Multi-color immunofluorescence microscopy using CYP82C4 antibodies in combination with markers for different subcellular compartments can precisely map the enzyme's location relative to organelles involved in iron homeostasis. Co-localization analyses with markers for the endoplasmic reticulum, Golgi apparatus, and secretory vesicles can illuminate the trafficking pathway of CYP82C4 and its metabolic products like sideretin.

Live-cell imaging approaches using genetically encoded tags (like GFP) can complement antibody-based fixed-cell microscopy to capture dynamic changes in CYP82C4 localization during iron deficiency responses. While these approaches don't directly use antibodies, validation of GFP fusion protein localization patterns with antibody-based immunofluorescence in fixed cells ensures that the fusion proteins faithfully represent endogenous protein distribution.

For plants experiencing iron deficiency, researchers should examine whether CYP82C4 shows altered localization patterns, particularly in root tissues where iron acquisition occurs. Changes in subcellular distribution could reflect adaptive responses to enhance coumarin biosynthesis and secretion for iron mobilization in the rhizosphere.