CYP735A1 Antibody

What is CYP735A1 and what is its primary function in plants?

CYP735A1 is a cytochrome P450 monooxygenase that plays a crucial role in cytokinin (CK) biosynthesis. It specifically catalyzes the hydroxylation of iP (isopentenyladenine) nucleotides to yield tZ-type cytokinins (trans-Zeatin type CKs) . Along with its homolog CYP735A2, these enzymes are responsible for the critical step of converting iP-type cytokinins to tZ-type cytokinins, which have distinct biological activities in plants . This hydroxylation reaction represents a key branching point in cytokinin biosynthesis pathways that determines which class of cytokinins will predominate in specific plant tissues.

Where is CYP735A1 localized within plant cells?

CYP735A1, like other cytochrome P450 monooxygenases, is an integral endoplasmic reticulum (ER)-resident protein . The hydroxylation reaction of cytokinins catalyzed by CYP735A1 takes place on the surface of the ER . This subcellular localization is significant because it indicates that the biosynthetic intermediates of cytokinins must be transported between different cellular compartments for the complete biosynthesis pathway to occur. The spatial organization of cytokinin biosynthesis enzymes across different subcellular compartments suggests that intracellular transport mechanisms are essential for cytokinin homeostasis.

How does CYP735A1 differ from CYP735A2 in terms of expression and function?

While both CYP735A1 and CYP735A2 catalyze the same biochemical reaction, they show distinct tissue-specific expression patterns and potentially different regulatory mechanisms. The functional differences between these two enzymes are still being investigated, but research suggests they may have partially redundant yet distinct roles in different plant tissues or developmental stages . Studies with knockout mutants have revealed that when both CYP735A1 and CYP735A2 genes are disrupted, iP-type cytokinins accumulate in the xylem sap, indicating the essential role of these enzymes in converting iP to tZ for long-distance transport .

What are the optimal methods for detecting CYP735A1 protein in plant tissue samples?

For detecting CYP735A1 protein in plant tissues, immunoblotting (Western blot) is the most widely used approach. For optimal results, researchers should consider:

• Sample preparation: Use microsomal fractions enriched for ER proteins, as CYP735A1 is an integral ER membrane protein

• Protein extraction buffer: Include protease inhibitors and membrane protein solubilization agents (e.g., 1% Triton X-100 or 0.5% SDS)

• SDS-PAGE conditions: 10-12% acrylamide gels provide good resolution for CYP735A1 (predicted molecular weight ~55-60 kDa)

• Transfer conditions: Semi-dry transfer at 15V for 45 minutes or wet transfer at 30V overnight at 4°C for efficient transfer of membrane proteins

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Use purified anti-CYP735A1 at 1:1000 to 1:2000 dilution overnight at 4°C

• Detection method: HRP-conjugated secondary antibodies with ECL detection or fluorescently-labeled secondary antibodies with imaging systems provide sensitive detection

How can researchers differentiate between CYP735A1 and the closely related CYP735A2 using antibodies?

Differentiating between CYP735A1 and CYP735A2 is challenging due to their sequence similarity. Researchers should:

• Use antibodies raised against unique peptide regions of CYP735A1 not present in CYP735A2

• Validate antibody specificity using:

  • Recombinant proteins of both CYP735A1 and CYP735A2 as positive and negative controls

  • Tissue samples from cyp735a1 and cyp735a2 knockout mutants

  • Competitive binding assays with the immunizing peptide

• Perform parallel immunoblots with antibodies specific to each protein

• Consider using epitope-tagged transgenic lines expressing CYP735A1 or CYP735A2 for unambiguous identification

What experimental approaches can be used to study CYP735A1 and cytokinin biosynthesis with antibodies?

Several antibody-based approaches can advance our understanding of CYP735A1 and cytokinin biosynthesis:

• Co-immunoprecipitation (Co-IP) to identify protein interaction partners of CYP735A1 within the cytokinin biosynthetic pathway

• Immunolocalization to confirm ER localization and potential tissue-specific expression patterns

• Chromatin immunoprecipitation (ChIP) to study transcriptional regulation of CYP735A1

• Proximity ligation assays to visualize protein-protein interactions in situ

• Pulse-chase experiments combined with immunoprecipitation to study the stability and turnover of CYP735A1

What are the recommended protocols for immunolocalization of CYP735A1?

For successful immunolocalization of CYP735A1 in plant tissues:

• Fixation: Use 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours, as it preserves protein antigenicity better than glutaraldehyde

• Embedding: Low-melting-point paraffin or LR White resin work well for maintaining antigen accessibility

• Section thickness: 5-10 μm sections provide good resolution for subcellular localization

• Antigen retrieval: Mild citrate buffer (10 mM, pH 6.0) heating can improve antibody binding

• Blocking: 2-5% BSA with 0.1% Triton X-100 in PBS for 1-2 hours

• Primary antibody: Anti-CYP735A1 at 1:50 to 1:200 dilution, incubate overnight at 4°C

• Secondary antibody: Fluorescently-labeled secondary antibody at 1:200 to 1:500

• Controls: Include sections from cyp735a1 knockout plants and peptide competition controls

• Co-staining: Use ER markers (e.g., anti-BiP or anti-calnexin) to confirm ER localization

• Imaging: Confocal microscopy with appropriate excitation/emission settings

How can researchers troubleshoot non-specific binding when using CYP735A1 antibodies?

Non-specific binding is a common challenge with plant protein antibodies. To minimize this issue:

• Increase blocking time and concentration (use 5% BSA or milk in TBST for 2 hours)

• Add 0.1-0.3% Tween-20 to washing buffers

• Pre-absorb antibody with plant extract from knockout mutants

• Increase salt concentration in washing buffers (up to 500 mM NaCl)

• Use monoclonal antibodies when available, as they typically show higher specificity

• Include competing peptide controls to confirm binding specificity

• Optimize antibody dilution through titration experiments

• Consider using protein A/G-purified IgG fractions rather than whole serum

• Reduce primary antibody incubation time or increase dilution if background remains high

What validation methods should be used to confirm CYP735A1 antibody specificity?

Rigorous validation of CYP735A1 antibodies is critical for research reliability:

• Western blot with recombinant CYP735A1 protein as a positive control

• Parallel testing with samples from wild-type and cyp735a1 knockout plants

• Competitive inhibition using the immunizing peptide

• Cross-reactivity testing against closely related proteins (especially CYP735A2)

• Mass spectrometry analysis of immunoprecipitated proteins

• RNA interference or CRISPR knockout validation showing corresponding reduction in detected protein

• Testing across multiple tissue types with known expression patterns based on transcriptomic data

• Verification with epitope-tagged CYP735A1 expression systems

How do CYP735A1 and CYP735A2 contribute to cytokinin transport and distribution in plants?

CYP735A1 and CYP735A2 play critical roles in determining which cytokinin species are transported throughout the plant:

• The conversion of iP-type to tZ-type cytokinins by CYP735A1/A2 occurs primarily in the roots, creating a pool of tZ-type cytokinins for long-distance transport via the xylem

• In plants where both CYP735A1 and CYP735A2 are knocked out, iP-type cytokinins accumulate in the xylem sap instead of tZ-type cytokinins

• This suggests that the hydroxylation step catalyzed by these enzymes is crucial for establishing the characteristic cytokinin profile found in xylem sap (predominantly tZ and tZR)

• The spatial expression patterns of CYP735A1 and CYP735A2 in root vascular tissues supports their role in generating xylem-mobile cytokinins

• The localization of these enzymes at the ER creates a requirement for intracellular transport mechanisms to move cytokinin biosynthetic intermediates between different subcellular compartments

What is known about the regulatory mechanisms controlling CYP735A1 expression and activity?

The regulation of CYP735A1 involves multiple layers of control:

• Transcriptional regulation: CYP735A1 expression is differentially regulated across tissues and developmental stages

• Hormonal regulation: Cytokinin levels themselves may feedback-regulate CYP735A1 expression

• Environmental responses: Nutrient availability (particularly nitrogen) influences CYP735A1 expression

• Post-translational modifications: Potential phosphorylation sites may regulate enzyme activity

• Protein-protein interactions: Association with other ER proteins likely affects enzyme function

• Subcellular localization: The precise ER subdomain localization may be regulated during development

• Metabolic control: Availability of substrate (iP nucleotides) and cofactors regulates the actual conversion rate

How can CYP735A1 antibodies be used to investigate cytokinin biosynthetic enzyme complexes?

CYP735A1 antibodies can reveal potential physical interactions between cytokinin biosynthetic enzymes:

• Co-immunoprecipitation using anti-CYP735A1 antibodies can isolate protein complexes containing CYP735A1

• Immunoprecipitated complexes can be analyzed by mass spectrometry to identify interacting partners

• Bimolecular Fluorescence Complementation (BiFC) can be used with antibody validation to confirm protein-protein interactions in planta

• Proximity-dependent biotin identification (BioID) combined with antibody verification can map the protein interaction network around CYP735A1

• Blue native PAGE followed by immunoblotting can identify native protein complexes containing CYP735A1

• Immunogold electron microscopy can visualize the precise arrangement of enzyme complexes at the ER membrane

How can cytokinin profiles be compared between wild-type and cyp735a1 mutant plants?

Comparing cytokinin profiles requires sensitive analytical techniques:

• Sample preparation:

  • Harvest tissues at consistent developmental stages

  • Flash-freeze in liquid nitrogen immediately

  • Homogenize tissues thoroughly in extraction buffer containing antioxidants

  • Include internal standards for quantification

• Analytical methods:

  • HPLC-MS/MS provides high sensitivity and specificity for cytokinin profiling

  • Ultra Performance Liquid Chromatography (UPLC) coupled with tandem mass spectrometry allows for improved separation

  • Multiple Reaction Monitoring (MRM) increases specificity for target cytokinin species

• Data analysis:

  • Compare ratios of iP-type to tZ-type cytokinins between wild-type and mutants

  • Analyze tissue-specific differences in cytokinin profiles

  • Examine changes in cytokinin conjugates and breakdown products

Note: Values represent typical ranges based on published data for Arabidopsis roots. Actual values vary by tissue type, developmental stage, and growth conditions.

What phenotypic analyses should be performed on cyp735a1 mutant plants?

Comprehensive phenotypic analysis should include:

• Root architecture:

  • Primary root length and growth rate

  • Lateral root number and density

  • Root meristem size and cell division rates

• Shoot morphology:

  • Rosette size and leaf number

  • Leaf expansion and cell size

  • Shoot branching patterns

• Vascular development:

  • Xylem and phloem differentiation

  • Cambial activity

  • Vessel element size and density

• Reproductive development:

  • Flowering time

  • Floral organ development

  • Seed production and viability

• Cellular responses:

  • Cell division rates in meristems

  • Cytokinin response gene expression

  • Changes in cytokinin receptor localization or abundance

• Physiological parameters:

  • Photosynthetic efficiency

  • Nutrient uptake and allocation

  • Drought and stress responses

How can complementation studies verify the function of CYP735A1 in cytokinin biosynthesis?

Complementation studies provide critical validation of gene function:

• Construct design:

  • Native promoter driving CYP735A1 coding sequence

  • Epitope-tagged versions for antibody detection

  • Fluorescent protein fusions for localization studies

• Transformation approaches:

  • Stable transformation of cyp735a1 mutant plants

  • Inducible expression systems to control timing of complementation

  • Tissue-specific promoters to address spatial requirements

• Functional validation:

  • Restoration of tZ-type cytokinin levels in complemented lines

  • Recovery of mutant phenotypes

  • Subcellular localization confirmation using antibodies against CYP735A1 or the epitope tag

• Variant analysis:

  • Structure-function studies with point mutations in catalytic domains

  • Chimeric proteins with CYP735A2 domains to identify functional regions

  • Localization signal mutations to test importance of ER targeting

What are emerging techniques for studying CYP735A1 protein dynamics?

Several cutting-edge approaches are being developed:

• Single-molecule tracking of fluorescently labeled CYP735A1 to study enzyme dynamics within the ER membrane

• CRISPR-mediated endogenous tagging for physiologically relevant expression levels

• Super-resolution microscopy to visualize nanoscale organization of CYP735A1 at the ER

• Cryo-electron microscopy to determine the 3D structure of CYP735A1 and its complexes

• Optogenetic approaches to control CYP735A1 activity with light

• Targeted protein degradation systems to achieve temporal control of CYP735A1 depletion

• Biosensors for real-time monitoring of cytokinin levels and distribution in living tissues

How might CYP735A1 function contribute to environmental adaptation in plants?

CYP735A1's role in cytokinin biosynthesis may have significant implications for plant adaptation:

• Drought response: Changes in the iP/tZ ratio may help plants adapt to water limitation

• Nutrient acquisition: CYP735A1 activity might coordinate root development with nutrient availability

• Temperature adaptation: Cytokinin profiles may shift under temperature stress, involving CYP735A1 regulation

• Pathogen defense: Cytokinin homeostasis plays roles in immune responses, potentially involving CYP735A1

• Seasonal responses: CYP735A1 expression patterns may vary with photoperiod or seasonal cues

• Developmental plasticity: Environmental modulation of CYP735A1 could contribute to phenotypic plasticity