CYP79F1 Antibody

What is the role of CYP79F1 in glucosinolate biosynthesis and why are antibodies useful for studying it?

CYP79F1 belongs to the cytochrome P450 CYP79 family and catalyzes the conversion of amino acids to oximes in the first committed step of aliphatic glucosinolate biosynthesis. Specifically, CYP79F1 metabolizes mono- to hexahomomethionine, producing both short- and long-chain aliphatic glucosinolates . This distinguishes it from its paralog CYP79F2, which exclusively metabolizes long-chain penta- and hexahomomethionines .

Antibodies against CYP79F1 are invaluable research tools for:

• Tracking protein expression patterns in different tissues and developmental stages

• Confirming knockout or knockdown efficiency in mutant lines

• Investigating protein-protein interactions within the glucosinolate metabolic network

• Determining subcellular localization of the enzyme

Knockout studies have demonstrated that CYP79F1 mutants completely lack short-chain aliphatic glucosinolates while showing elevated levels of long-chain aliphatic glucosinolates, especially in leaves and seeds . Using antibodies to validate these changes at the protein level provides crucial complementary evidence to transcript and metabolite analyses.

What are the expression patterns of CYP79F1 in Arabidopsis tissues and how can antibodies help visualize this distribution?

CYP79F1 shows distinct spatial and developmental regulation in Arabidopsis thaliana. The enzyme is strongly expressed in cotyledons, rosette leaves, stems, and siliques, while its paralog CYP79F2 is primarily expressed in hypocotyl and roots . This differential expression pattern suggests tissue-specific roles in glucosinolate biosynthesis.

Immunohistochemistry using specific CYP79F1 antibodies can:

• Provide high-resolution visualization of protein distribution at the cellular and subcellular levels

• Confirm expression patterns initially identified through transcript analysis

• Reveal potential discrepancies between transcript and protein levels due to post-transcriptional regulation

• Track changes in expression during plant development or stress responses

When designing immunohistochemistry experiments, researchers should include appropriate controls, including CYP79F1 knockout tissues, to validate antibody specificity. Comparing immunostaining patterns with reporter gene constructs (such as promoter-GUS fusions) can provide complementary evidence for expression patterns .

How can researchers confirm the specificity of CYP79F1 antibodies given the high similarity with CYP79F2?

Confirming antibody specificity is crucial when working with CYP79F1 due to its high sequence similarity with CYP79F2. Several methodological approaches can address this challenge:

• Western blot validation using:

  • Recombinant CYP79F1 and CYP79F2 proteins expressed in heterologous systems

  • Protein extracts from single knockout mutants (cyp79F1 and cyp79F2) and double knockout mutants (cyp79F1 cyp79F2)

  • Tissues with differential expression (e.g., leaves for CYP79F1, roots for CYP79F2)

• Immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein

• Peptide competition assays using specific peptides designed from unique regions of CYP79F1

• Cross-reactivity testing against protein extracts from species with varying degrees of conservation in CYP79F homologs

The presence of tissue-specific expression patterns provides an excellent natural system for validation, as antibodies can be tested against tissues known to express predominantly CYP79F1 (leaves) versus CYP79F2 (roots) .

How can CYP79F1 antibodies be used to investigate flux control mechanisms in the glucosinolate pathway?

Flux control analysis in metabolic pathways is crucial for understanding regulatory mechanisms and identifying potential targets for metabolic engineering. Evidence suggests that CYP79F1 exercises majority flux control in the aliphatic glucosinolate pathway, making it a critical target for regulation .

Methodological approaches using CYP79F1 antibodies to investigate flux control include:

• Protein-level quantification in correlation with metabolic flux analysis:

  • Quantitative Western blotting to determine CYP79F1 protein abundance across different conditions

  • Correlation of protein levels with metabolite profiles and flux measurements

  • Analysis of enzyme levels in different genetic backgrounds with altered glucosinolate profiles

• Investigation of post-translational modifications:

  • Immunoprecipitation followed by mass spectrometry to identify PTMs

  • Western blotting with phospho-specific antibodies if phosphorylation sites are known

  • Analysis of how PTMs correlate with enzyme activity and metabolic flux

• Protein complexes and metabolons:

  • Co-immunoprecipitation to identify protein-protein interactions

  • Proximity labeling techniques using antibody-guided approaches

  • Investigation of whether complex formation correlates with flux control

A study by Olson-Manning et al. demonstrated that CYP79F1 has substantial control over pathway flux through enzyme perturbation experiments using gene insertion lines . The table below shows relative substitution rates for enzymes in the glucosinolate pathway, with CYP79F1 showing significant selective pressure:

What methodological approaches can resolve the apparent contradiction between negative DoS values and elevated πN/πS ratios observed for CYP79F1?

Sequence analysis of CYP79F1 presents an intriguing evolutionary signature that appears contradictory: a significantly negative Direction of Selection (DoS) value (-4.46, P=0.0029) alongside a πN/πS ratio substantially greater than 1 (bootstrap 99% confidence interval: 1.15–8.02) . This apparent contradiction requires sophisticated methodological approaches to resolve.

Researchers can use CYP79F1 antibodies in conjunction with population genetics to investigate:

• Geographic variation in protein function and abundance:

  • Quantitative Western blotting across diverse Arabidopsis accessions

  • Correlation of protein variants with ecological conditions

  • Functional characterization of protein variants through in vitro enzyme assays

• Haplotype-specific protein analysis:

  • Haplotype-specific antibodies targeting polymorphic regions

  • Analysis of expression levels across different natural variants

  • Investigation of whether distinct protein variants show differential subcellular localization

• Experimental validation of selective pressures:

  • Reciprocal transplant experiments with protein quantification

  • Analysis of protein stability and function under varying environmental conditions

  • Investigation of herbivore response to different CYP79F1 variants

The high πN/πS ratio for CYP79F1 may indicate balancing selection, while site-frequency spectrum analysis reveals high-frequency nonsynonymous polymorphisms with widespread geographic distribution . These patterns suggest that CYP79F1 has undergone complex selection processes, possibly involving adaptation to different environments or pathogen pressures across Arabidopsis populations.

How can researchers utilize CYP79F1 antibodies to understand the relationship between glucosinolate biosynthesis and plant hormone signaling?

Mutations in CYP79F1 (also known as SPS/CYP79F1) affect not only glucosinolate profiles but also plant development through changes in hormone levels, particularly cytokinin and auxin . CYP79F1 antibodies can help elucidate these complex interactions.

Methodological approaches include:

• Spatial correlation of protein expression with hormone response:

  • Co-immunolocalization of CYP79F1 with hormone reporters or signaling components

  • Comparison of CYP79F1 protein levels in specific tissues with hormone measurements

  • Analysis of hormone reporter expression in wild-type versus cyp79f1 mutant backgrounds

• Temporal dynamics of protein expression and hormone signaling:

  • Time-course analysis of CYP79F1 protein levels during developmental transitions

  • Correlation with hormone-responsive reporter expression

  • Inducible suppression or overexpression of CYP79F1 followed by monitoring hormone responses

• Protein interaction networks linking glucosinolate metabolism and hormone signaling:

  • Immunoprecipitation coupled with mass spectrometry

  • Yeast two-hybrid or BiFC validation of potential interactors

  • Investigation of whether CYP79F1 physically interacts with hormone biosynthesis or signaling components

Research has shown that cyp79f1 mutants display increased expression of both cytokinin and auxin-responsive reporters, but at different sites in the plant: cytokinin reporter expression is elevated at the leaf axil of the mutant, while auxin reporter expression is higher in the leaf blade . Understanding these spatial differences requires precise localization of CYP79F1 protein in relation to hormone biosynthesis and response components.

What techniques can be employed to investigate potential cross-talk between CYP79F1 and CYP79F2 in planta using antibody-based approaches?

CYP79F1 and CYP79F2 are tandem-duplicated genes with overlapping but distinct substrate specificities and expression patterns . Investigating their potential cross-talk requires sophisticated antibody-based approaches.

Methodological strategies include:

• Co-expression analysis in tissues with overlapping expression:

  • Dual immunofluorescence with specific antibodies against each enzyme

  • Super-resolution microscopy to determine subcellular co-localization

  • Quantitative Western blotting to determine relative abundance in different tissues

• Compensatory mechanisms in single mutants:

  • Analysis of CYP79F2 protein levels in cyp79f1 mutants and vice versa

  • Investigation of changes in subcellular localization in single mutants

  • Correlation with altered glucosinolate profiles

• Protein-protein interaction analysis:

  • Co-immunoprecipitation to test direct interaction

  • Proximity labeling to identify closely associated proteins

  • FRET analysis with labeled antibodies or fluorescent protein fusions