CYP81F4 Antibody

What is CYP81F4 and what is its primary function in plants?

CYP81F4 belongs to a small cytochrome P450 monooxygenase family in Arabidopsis that includes CYP81F1, CYP81F2, and CYP81F3. Its primary function is catalyzing the conversion of indole-3-ylmethyl glucosinolate (I3M, also known as glucobrassicin or GBS) to 1-hydroxy-indole-3-ylmethyl glucosinolate (1OH-I3M), which is subsequently converted to 1-methoxy-indole-3-ylmethyl glucosinolate (1MO-I3M, also known as neoglucobrassicin or NGBS) by indole glucosinolate methyltransferase 1 (IGMT1) or IGMT2 .

How does CYP81F4 differ functionally from other members of the CYP81F family?

While all CYP81F family members participate in indole glucosinolate modification, they exhibit distinct substrate specificities and product formation:

• CYP81F4 primarily catalyzes the conversion of I3M to 1OH-I3M, eventually leading to 1MO-I3M (neoglucobrassicin) formation

• CYP81F2 catalyzes the conversion of I3M to 4OH-I3M, which is then converted to 4MO-I3M

• CYP81F3 appears to be involved in the generation of 4OH-I3M and 4MO-I3M, similar to CYP81F2

• CYP81F1 shows some involvement in 4OH-I3M production but has a less pronounced effect

These functional differences have been confirmed through T-DNA insertion mutant analysis and heterologous expression studies in Nicotiana benthamiana .

How is CYP81F4 expression regulated during pathogen infection?

CYP81F4 expression is strongly induced by pathogen infection, particularly by bacteria like Pectobacterium brasiliense. In Brassica rapa, CYP81F4 shows distinct temporal expression patterns compared to other indole glucosinolate biosynthesis genes. While genes like BrMYB51, BrST5a, and BrIGMT1 are strongly induced at 6 hours post-infection and then decrease by 24 hours, BrCYP81F4 is most strongly induced at 24 hours post-inoculation .

This delayed expression pattern suggests CYP81F4 may play a role in later stages of the defense response, potentially involved in the production of specific defense compounds that accumulate as the infection progresses .

What is the relationship between plant hormones and CYP81F4 expression?

Plant hormones, particularly jasmonic acid (JA) and salicylic acid (SA), significantly influence CYP81F4 expression:

• Jasmonic acid (JA) and its derivative methyl jasmonate (MeJA) strongly induce CYP81F4 expression, correlating with increased neoglucobrassicin (NGBS) production

• Salicylic acid (SA) also affects CYP81F4 expression, though the response varies between plant subspecies

• The hormone-mediated regulation of CYP81F4 shows subspecies-specific patterns in Brassica plants

Principal component analysis of gene expression data shows that CYP81F4 homologs (Bol032712, Bol032714, and Bol028918) respond differently to hormone treatments, with some variants showing stronger correlation with MeJA treatment (PC2 loading values of -0.082, 0.326, and 0.349 respectively) .

What are the best approaches to study CYP81F4 function in planta?

To effectively study CYP81F4 function in plants, researchers should consider multiple complementary approaches:

• Genetic analysis: Utilize T-DNA insertion lines (knockouts/knockdowns) to assess phenotypic changes in glucosinolate profiles. For example, the cyp81f4 mutant (SALK_024438) shows nearly undetectable 1MO-I3M levels in leaves and altered glucosinolate profiles in roots .

• Expression analysis: Employ qRT-PCR to analyze CYP81F4 expression patterns after pathogen infection or hormone treatments. Use stable reference genes like eIF4A1 (At3g13920) which shows consistent expression across tissues and developmental stages .

• Metabolite profiling: Combine genetic manipulation with HPLC-based metabolite analysis to directly link CYP81F4 function to specific glucosinolate profiles. This approach revealed CYP81F4's specific role in 1MO-I3M production .

• Heterologous expression: Express CYP81F4 in systems like Nicotiana benthamiana to confirm enzymatic function in a controlled environment .

How can researchers develop reliable immunodetection methods for CYP81F4?

Based on established protocols for similar proteins, an effective immunodetection strategy for CYP81F4 would involve:

• Antibody generation: Develop polyclonal antibodies against a unique region of CYP81F4, similar to approaches used for other plant proteins such as ETR1, where antibodies were generated against amino acids 401-738 .

• Microsomal fraction isolation:

  • Homogenize plant tissue in buffer containing 30 mM Tris (pH 8.3 at 4°C), 150 mM NaCl, 1 mM EDTA, and 20% (v/v) glycerol with protease inhibitors

  • Centrifuge at 8,000g for 15 min followed by 100,000g for 30 min

  • Resuspend the membrane pellet in 10 mM Tris (pH 7.6), 150 mM NaCl, 0.1 mM EDTA, and 10% (v/v) glycerol with protease inhibitors

• Protein quantification: Determine protein concentration using bicinchoninic acid reagent after solubilizing membrane proteins with 0.1 mL of 0.5% (w/v) SDS .

• Specificity validation: Verify antibody specificity using tissue from CYP81F4 knockout/knockdown plants as negative controls.

How can researchers distinguish between CYP81F family members in experimental settings?

Distinguishing between highly similar CYP81F family members requires a multi-faceted approach:

• Gene-specific primers: Design primers targeting unique regions in each CYP81F gene for qRT-PCR analysis. This approach was successfully used to differentiate CYP81F1, CYP81F2, CYP81F3, and CYP81F4 expression patterns .

• Metabolite profiling of mutants: Analyze glucosinolate profiles in single gene knockouts. For example:

  • cyp81f4 mutants show dramatically reduced 1MO-I3M levels

  • cyp81f2 mutants show reduced 4OH-I3M and 4MO-I3M levels

  • cyp81f1 mutants show minimal changes in glucosinolate profiles

• Tissue-specific analysis: Examine both shoots and roots separately, as the effects of CYP81F mutations differ between these tissues. In roots, cyp81f4 mutants show elevated I3M, 4OH-I3M, and 4MO-I3M levels, indicating metabolic rerouting when 1MO-I3M production is blocked .

What is the significance of CYP81F4 in plant pathogen resistance?

CYP81F4 plays a critical role in plant defense through several mechanisms:

• Indole glucosinolate modification: CYP81F4's production of 1MO-I3M (neoglucobrassicin) provides plants with specialized defense compounds against pathogens. In Brassica rapa, pathogen-induced NGBS accumulation correlates with increased resistance to Pectobacterium brasiliense .

• Jasmonate-mediated defense: CYP81F4 functions within the jasmonate (JA) signaling pathway, which generally protects against necrotrophic pathogens. Upregulation of CYP81F4 by JA treatment leads to increased NGBS production and enhanced pathogen resistance .

• Integration with broader defense networks: CYP81F4 expression correlates with other defense-related genes, including transcription factors like MYB51, which regulate indole glucosinolate biosynthesis in response to pathogen attack .

The importance of CYP81F4 in pathogen resistance is particularly evident when comparing wild-type plants with mutant lines. While not as severely affected as plants with mutations in upstream indole glucosinolate biosynthesis genes (like cyp79B2/B3 or myb34/51/122), cyp81F mutants still show increased susceptibility to pathogens like Alternaria brassicicola .

How should researchers address data inconsistencies when studying CYP81F4 expression?

When encountering inconsistent data on CYP81F4 expression, researchers should implement the following strategies:

• Control for temporal dynamics: CYP81F4 shows distinct temporal expression patterns, with peak expression at different time points compared to other glucosinolate-related genes. Sample at multiple time points (e.g., 0, 6, and 24 hours post-treatment) to capture the complete expression profile .

• Consider tissue-specific differences: CYP81F4 functions differently in leaves versus roots. In roots, blocking 1MO-I3M production in cyp81f4 mutants results in metabolic rerouting, with elevated levels of I3M, 4OH-I3M, and 4MO-I3M. Always analyze tissues separately .

• Account for subspecies variation: Different plant subspecies (e.g., cabbage, broccoli, and kale) show distinct CYP81F4 expression patterns in response to the same treatments. Principal component analysis revealed significant subspecies × treatment interactions (p<0.01) affecting CYP81F4 expression .

• Verify gene copy numbers: Some plant species contain multiple copies of CYP81F4 (e.g., Bol032712, Bol032714, and Bol028918 in Brassica), each potentially responding differently to treatments. When working with non-model organisms, identify and monitor all relevant homologs .

What experimental design considerations are crucial when analyzing CYP81F4 function in different plant subspecies?

When designing experiments to study CYP81F4 across plant subspecies, researchers should consider:

• Factorial experimental design: Implement a design that accounts for both subspecies and treatment effects, as well as their interactions. This approach revealed significant subspecies × treatment effects on CYP81F4 expression and glucosinolate profiles in Brassica subspecies .

• Multivariate statistical analysis: Use principal component analysis (PCA) to identify patterns in complex datasets involving multiple CYP81F4 homologs and glucosinolate metabolites. The PCA approach successfully identified that:

  • PC1 (explaining 41.5% of variation) showed significant effects of subspecies, treatment, and their interaction

  • PC2 (explaining 15.0% of variation) showed significant effects of treatment and subspecies × treatment interaction

  • Different CYP81F4 homologs contributed differently to these principal components

• Adequate biological replication: Use at least three biological replicates per treatment combination to account for natural variation, as demonstrated in studies comparing fold increases in CYP81F4 expression across cabbage, broccoli, and kale .

• Comprehensive gene family analysis: Analyze all CYP81F family members simultaneously to understand functional redundancy and specialization. For example, research on Arabidopsis revealed distinct but overlapping functions among CYP81F1-4 .

How might CYP81F4 research contribute to developing enhanced crop protection strategies?

CYP81F4 research has several promising applications for crop protection strategies:

• Transgenic approaches: Overexpression of CYP81F4 could enhance plant resistance to pathogens by increasing production of protective neoglucobrassicin (NGBS). Previous studies have shown that altering glucosinolate profiles through overexpression of related genes (e.g., CYP79) enhanced resistance to Pectobacterium brasiliense .

• Marker-assisted breeding: Identification of natural CYP81F4 variants with enhanced expression or activity could guide breeding programs for pathogen-resistant crop varieties. The significant variation in CYP81F4 response among Brassica subspecies provides a foundation for such approaches .

• Priming treatments: Exogenous application of jasmonic acid or methyl jasmonate could prime CYP81F4 expression and boost plant immunity prior to pathogen exposure. MeJA treatment significantly upregulated CYP81F4 expression in Brassica subspecies, particularly in broccoli (PC2 score of 2.52±0.38) .

• Integration with other defense pathways: Combining CYP81F4 enhancement with activation of complementary defense pathways could provide more durable resistance. Research has shown that CYP81F4 functions within a network including PAMP-triggered immunity and jasmonate signaling .

What technological advancements would facilitate deeper understanding of CYP81F4 structure-function relationships?

Several emerging technologies could advance our understanding of CYP81F4:

• Cryo-electron microscopy: Determining the 3D structure of CYP81F4 would enable structure-based design of enhanced variants with improved catalytic efficiency or substrate specificity.

• CRISPR-based approaches: Precise genome editing could generate series of CYP81F4 variants to map critical functional domains. This could help distinguish the unique features that allow CYP81F4 to produce 1OH-I3M while related enzymes like CYP81F2 produce 4OH-I3M .

• Single-cell transcriptomics: Analyzing CYP81F4 expression at cellular resolution would reveal tissue-specific and cell-type-specific regulation patterns, potentially explaining the differential responses observed in various plant tissues.

• Protein interaction studies: Identifying proteins that physically interact with CYP81F4 would clarify its integration into broader metabolic and signaling networks. Expression correlation analysis has already suggested functional relationships between CYP81F4 and several O-methyltransferases, which could be confirmed through protein interaction studies .

• Metabolic flux analysis: Tracing the fate of isotope-labeled glucosinolate precursors in wild-type versus CYP81F4 mutant plants would provide dynamic insights into how CYP81F4 influences metabolic flow through the glucosinolate pathway under different conditions. : https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.964092/full : https://pmc.ncbi.nlm.nih.gov/articles/PMC3077789/ : https://www.mdpi.com/1420-3049/21/10/1417 : https://www.biorxiv.org/content/10.1101/2021.04.22.440953v1.full-text : https://academic.oup.com/plphys/article/159/2/682/6109258