CYP78A6 Antibody

What is CYP78A6 and what role does it play in plant development?

CYP78A6 (also known as EOD3, ENHANCER OF DA1-1) is a cytochrome P450 protein belonging to the CYP78A family in Arabidopsis thaliana. It functions as a key regulator of seed and organ size through maternal control mechanisms.

CYP78A6 is primarily involved in:

The protein is expressed in multiple plant tissues including leaves, sepals, petals, stamens, carpels and developing ovules. It localizes to membranes as a single-pass membrane protein, consistent with its function as a cytochrome P450 enzyme.

How does CYP78A6 function in relation to other CYP78A family members?

CYP78A6 functions redundantly with other CYP78A family members, particularly CYP78A9, in regulating plant development:

Experimental evidence from double and triple mutant analyses demonstrates that while single mutants often show mild phenotypes, disruption of multiple CYP78A genes reveals their collective importance in developmental processes . For example, the cyp78a6 cyp78a9 double mutant exhibits significant reduction in seed size compared to either single mutant .

What are the recommended methods for validating CYP78A6 antibody specificity?

When validating CYP78A6 antibody specificity, researchers should consider:

• Genetic controls:

  • Use cyp78a6 knockout mutants as negative controls to confirm absence of signal

  • Compare antibody reactivity in wild-type versus overexpression lines

  • Test cross-reactivity with closely related CYP78A family members, particularly CYP78A9

• Biochemical validation:

  • Perform Western blot analysis at the expected molecular weight (approximately 57-59 kDa)

  • Include pre-adsorption controls with recombinant CYP78A6 protein

  • Conduct immunoprecipitation followed by mass spectrometry to confirm target identity

• Tissue-specific validation:

  • Compare antibody staining patterns with established expression patterns from reporters (pKLU::YFP, pKLU::GUS)

  • Focus validation in tissues with known expression: developing seeds, leaf primordia, and shoot meristems

The high sequence similarity between CYP78A family members necessitates careful validation to ensure specificity for CYP78A6 versus other family members, particularly CYP78A9 and CYP78A8 .

How can CYP78A6 antibodies be effectively employed to investigate seed development processes?

For investigating CYP78A6's role in seed development, researchers should:

• Implement tissue-specific immunolocalization:

  • Use thin sections (3-5 μm) of developing seeds at critical developmental stages (globular to mature)

  • Apply dual immunolabeling with markers for specific integument cell layers

  • Compare CYP78A6 localization between wild-type and mutant backgrounds (e.g., da1-1 or ttg2)

• Design developmental timecourse experiments:

  • Sample developing seeds at 2-day intervals from fertilization to maturity

  • Quantify CYP78A6 protein levels relative to cell proliferation rates in integuments

  • Correlate protein expression with critical developmental transitions

• Combine with cellular measurements:

  • Pair antibody detection with quantitative analysis of integument cell number and size

  • Track maternal tissue growth parameters alongside CYP78A6 protein abundance

  • Analyze data considering that eod3-1D (CYP78A6 overexpression) forms more and larger cells in integuments, while loss-of-function mutants show reduced cell numbers

This approach enables precise correlation between CYP78A6 protein levels and developmental processes controlling seed size, providing insight beyond transcriptional data alone.

What experimental designs best elucidate the maternal effects of CYP78A6 in seed development?

To investigate maternal effects of CYP78A6, experimental designs should:

• Implement reciprocal crossing strategies:

  • Perform systematic crosses: wild-type (♀) × cyp78a6 (♂) and cyp78a6 (♀) × wild-type (♂)

  • Include cyp78a6 overexpression lines in reciprocal crosses

  • Analyze F1 seed phenotypes (size, weight, morphology) and compare with self-pollinated controls

• Conduct tissue-specific complementation:

  • Generate constructs driving CYP78A6 expression under integument-specific promoters (e.g., INNER NO OUTER)

  • Transform cyp78a6 mutants and assess rescue of seed size phenotypes

  • Compare effectiveness of different tissue-specific promoters in restoring normal seed development

• Analyze cell proliferation dynamics in maternal tissues:

  • Use EdU incorporation assays to monitor cell division rates in integuments

  • Compare division patterns between wild-type and cyp78a6 mutants at defined developmental stages

  • Quantify integument cell numbers using the established range (wild-type: 29-33 cells; mutants: 15-20 cells)

These approaches can definitively establish the maternal role of CYP78A6, as previous studies have demonstrated that EOD3/CYP78A6 acts maternally to promote seed growth through effects on cell proliferation and elongation in the integuments .

How should researchers design experiments to dissect the functional redundancy between CYP78A6 and other family members?

To effectively analyze functional redundancy between CYP78A family members:

• Generate and characterize comprehensive mutant series:

  • Create single, double, and higher-order mutants using CRISPR/Cas9 to ensure complete knockout

  • Include key combinations: cyp78a6 cyp78a9, cyp78a6 cyp78a8, cyp78a8 cyp78a9, and triple mutants

  • Systematically phenotype each combination across multiple developmental stages and conditions

• Perform protein-level complementation studies:

  • Express each CYP78A family member under the CYP78A6 promoter in cyp78a6 background

  • Quantify the degree of phenotypic rescue for each construct

  • Use Western blot with anti-CYP78A6 antibody to confirm expression levels

• Conduct domain-swap experiments:

  • Generate chimeric proteins between CYP78A6 and related family members

  • Express these under native promoters in respective mutant backgrounds

  • Use immunodetection to confirm protein expression and localization

This approach has revealed that while cyp78a6 or cyp78a9 single mutants show minimal phenotypes, the double mutant exhibits significantly enhanced defects in seed development and other processes, supporting their functional redundancy .

What methodologies can detect post-translational modifications or protein-protein interactions involving CYP78A6?

For investigating CYP78A6 post-translational modifications and interactions:

• Analyze post-translational modifications:

  • Immunoprecipitate CYP78A6 using validated antibodies followed by mass spectrometry

  • Employ phospho-specific antibodies to detect potential phosphorylation events

  • Use native gel electrophoresis to identify potential complex formation

• Identify protein interaction partners:

  • Perform co-immunoprecipitation with anti-CYP78A6 antibodies followed by mass spectrometry

  • Consider potential interactions with ubiquitin pathway components (DA1, UBP15) implicated in seed size control

  • Validate interactions using yeast two-hybrid and bimolecular fluorescence complementation

• Investigate membrane complex formation:

  • Apply blue native PAGE for membrane protein complex analysis

  • Consider potential interactions with components of COPⅡ pathway as observed with rice CYP78As

  • Use proximity labeling approaches (BioID) with CYP78A6 as bait

Research has demonstrated that CYP78A proteins function in complex pathways with other proteins. For example, in rice, SMG4 interacts with CYP78As and COPⅡ components to regulate grain size , suggesting similar interactions may exist for Arabidopsis CYP78A6.

What are the key considerations for immunolocalization studies using CYP78A6 antibodies?

When performing immunolocalization with CYP78A6 antibodies:

• Optimize tissue fixation and processing:

  • For developing seeds and meristems, use 4% paraformaldehyde with careful vacuum infiltration

  • Consider shorter fixation times (2-4 hours) to preserve epitope accessibility

  • Test both paraffin embedding and cryosectioning to determine optimal tissue preservation

• Implement rigorous controls:

  • Include parallel experiments with pre-immune serum

  • Process cyp78a6 mutant tissues alongside wild-type samples

  • Consider the expression pattern overlap with CYP78A9 when interpreting results

• Target specific developmental stages:

  • Focus on tissues with known expression: developing seeds, integuments, leaf primordia

  • Pay particular attention to the "horseshoe-like pattern" in cotyledons reported for CYP78A5

  • Examine vegetative shoots where CYP78A6 expression overlaps with AMP1 expression

Studies have shown that CYP78A5 (related to CYP78A6) shows strong expression in specific patterns: "at the lateral bases encompassing the developing SAM" and "in the suspensor and the hypophysis from the globular stage onward" . Similar patterns may be expected for CYP78A6.

How can researchers effectively quantify CYP78A6 protein levels across different developmental stages?

For quantitative analysis of CYP78A6 protein levels:

• Establish stage-specific extraction protocols:

  • Develop micro-extraction methods for developing seeds at defined stages

  • Use specialized buffers optimized for membrane-bound cytochrome P450 proteins

  • Consider detergent selection carefully (CHAPS or Triton X-100) to maintain native structure

• Implement quantitative Western blot analysis:

  • Utilize recombinant CYP78A6 protein standards for absolute quantification

  • Apply fluorescent secondary antibodies for wider linear range

  • Include loading controls specific to membrane proteins (e.g., H⁺-ATPase)

• Couple with morphological measurements:

  • Correlate protein levels with developmental parameters (seed size, integument cell number)

  • Track protein abundance in relation to expression of known markers (WUS, CLV3)

  • Compare with transcript levels to identify post-transcriptional regulation

Research has shown that CYP78A6/EOD3 influences both cell proliferation and cell elongation in integuments , suggesting protein levels may correlate with these cellular processes during development.

What approaches should be used to investigate the molecular mechanisms of CYP78A6 in relation to the ubiquitin pathway?

To explore CYP78A6's relationship with the ubiquitin pathway:

• Examine potential ubiquitination of CYP78A6:

  • Immunoprecipitate CYP78A6 followed by ubiquitin-specific Western blotting

  • Test protein stability in the presence of proteasome inhibitors

  • Generate lysine-to-arginine mutants of potential ubiquitination sites

• Investigate interactions with known ubiquitin pathway components:

  • Test direct interactions with DA1 (ubiquitin receptor) and UBP15 (deubiquitinating enzyme)

  • Examine CYP78A6 protein levels in da1-1 and ubp15 mutant backgrounds

  • Consider the genetic interactions that affect seed size

• Analyze effects on downstream targets:

  • Compare protein levels of miRNA targets in wild-type versus cyp78a mutants

  • Investigate whether CYP78A6 affects the stability of proteins involved in cell proliferation

  • Track changes in ubiquitination patterns of development-related proteins

Research has established connections between the ubiquitin pathway and seed size regulation. The ubiquitin receptor DA1 and the deubiquitinating enzyme UBP15 antagonistically regulate seed size , and there may be functional connections with CYP78A6's role in the same developmental process.

How can researchers integrate CYP78A6 antibody studies with metabolomic approaches to identify potential substrates?

To identify potential CYP78A6 substrates through integrated approaches:

• Combine immunoprecipitation with metabolite analysis:

  • Use CYP78A6 antibodies for affinity purification of protein complexes

  • Analyze co-purified metabolites using LC-MS/MS

  • Compare metabolite profiles between wild-type and cyp78a6 mutants

• Investigate predicted biochemical pathways:

  • Focus on phenylpropanoid pathway metabolites, as bioinformatic predictions place CYP78A9 in this pathway

  • Examine potential roles in flavonol biosynthesis, particularly conversion of dihydrokaempferol to dihydroquercetin

  • Consider luteolin biosynthesis and eriodictyol formation from naringenin

• Analyze seed coat composition differences:

  • Examine changes in seed coat color observed in cyp78a8 cyp78a9 mutants (pale brown)

  • Quantify phenylpropanoid pathway components in seed coats

  • Correlate metabolite changes with protein expression patterns

Bioinformatic predictions have positioned CYP78A9 (closely related to CYP78A6) in the phenylpropanoid pathway, specifically in flavonol biosynthesis . Similar substrate specificity might be expected for CYP78A6, making this pathway a prime target for investigation.

What experimental strategies can link CYP78A6 function to hormone signaling pathways in seed development?

To investigate connections between CYP78A6 and hormone signaling:

• Analyze hormone-responsive elements in the CYP78A6 promoter:

  • Conduct detailed promoter analysis for hormone-responsive elements

  • Generate reporter constructs with mutations in key elements

  • Monitor expression changes under various hormone treatments

• Examine protein-level responses to hormone treatments:

  • Treat plants with various hormones (auxin, cytokinin, brassinosteroids)

  • Quantify CYP78A6 protein levels using validated antibodies

  • Compare protein stability and post-translational modifications across treatments

• Investigate genetic interactions with hormone mutants:

  • Create double mutants between cyp78a6 and hormone biosynthesis/signaling mutants

  • Focus on auxin (ARF2) and brassinosteroid pathways implicated in seed size control

  • Analyze epistatic relationships through detailed phenotyping

Microarray data has shown that CYP78A9 overexpression affects genes involved in various hormone pathways, including CYP724A1 (brassinosteroid C-22 hydroxylase) , suggesting potential crosstalk between CYP78A family members and hormone signaling.

How can researchers distinguish between the mechanistic roles of CYP78A6 in cell proliferation versus cell expansion in integuments?

To differentiate between effects on cell proliferation and expansion:

• Implement stage-specific cellular analyses:

  • Track integument cell numbers throughout seed development in wild-type and mutants

  • Measure both cell number and cell size in the same samples at defined intervals

  • Compare findings with the established parameters (wild-type: 29-33 cells; mutants: 15-20 cells)

• Utilize cell cycle and expansion markers:

  • Monitor cell cycle markers (CYCB1;1, KRP proteins) alongside CYP78A6 protein

  • Track expression of cell expansion regulators (expansins, XTHs) in parallel

  • Correlate protein levels with cellular measurements

• Apply inducible expression systems:

  • Generate inducible CYP78A6 lines for temporal control of expression

  • Induce expression at specific developmental time points

  • Analyze differential effects on proliferation versus expansion phases

What approaches can determine if CYP78A6 and related family members produce mobile signals regulating development?

To investigate potential mobile signals produced by CYP78A6:

• Design grafting experiments:

  • Perform reciprocal grafting between wild-type and cyp78a6 mutants

  • Include overexpression lines as scions or rootstocks

  • Analyze developmental phenotypes in tissues distant from graft junctions

• Implement tissue-specific complementation:

  • Express CYP78A6 under tissue-specific promoters in mutant background

  • Examine rescue effects in adjacent non-expressing tissues

  • Quantify mobile metabolites in expressing versus non-expressing regions

• Analyze enzymatic products in phloem and transport tissues:

  • Collect phloem exudates from wild-type and mutant plants

  • Compare metabolite profiles using targeted and untargeted approaches

  • Test candidate mobile signals in feeding experiments

This hypothesis follows research on CYP78A5/KLUH, which has been proposed to generate a mobile growth signal affecting organ size , suggesting similar mechanisms may exist for CYP78A6.