CRTISO Antibody

What is CRTISO and why is it important in plant research?

CRTISO is a redox-type enzyme structurally related to the bacterial-type phytoene desaturase CRTI. It plays a crucial role in the carotenoid biosynthesis pathway by isomerizing adjacent cis-double bonds at C-7 and C-9 pairwise into the trans-configuration, converting cis-lycopenes to all-trans-lycopene. This isomerization is a prerequisite for lycopene cyclization in plants. Unlike in bacteria where a single enzyme (CrtI) catalyzes the desaturation steps from phytoene to lycopene, plants require multiple enzymes including CRTISO for this process .

CRTISO is particularly important because mutations in this gene result in the accumulation of prolycopene and other cis-isomers of upstream precursors, which can dramatically alter plant phenotypes, especially petal and fruit coloration. The enzyme has been cloned from various plants including Arabidopsis (ccr2 mutant) and tomato (tangerine mutant) .

How do CRTISO mutations affect plant phenotypes?

CRTISO mutations produce distinctive phenotypic effects across different plant species:

• In Arabidopsis, the ccr2 mutant exhibits partial inhibition of lutein synthesis in light-grown tissue and accumulation of poly-cis-carotene precursors in dark-grown tissue .

• Etioplasts of dark-grown crtiso mutants accumulate acyclic poly-cis-carotenoids instead of cyclic all-trans-xanthophylls and notably lack prolamellar bodies (PLBs), demonstrating that carotenoid biosynthesis is required for PLB formation .

• In tomato, tangerine mutants with defective CRTISO accumulate prolycopene, resulting in orange-colored fruits instead of red .

• In calendula (Calendula officinalis), variations in CRTISO activity determine petal color, with orange petals associated with accumulation of 5-cis-carotenoids due to reduced CRTISO activity .

The table below summarizes the phenotypic effects observed in different plants with CRTISO mutations:

What are the primary applications of CRTISO antibodies in research?

CRTISO antibodies serve several critical functions in carotenoid biosynthesis research:

• Detection and quantification of CRTISO protein expression in different tissues and under various conditions

• Immunolocalization studies to determine the subcellular distribution of CRTISO

• Isolation of protein complexes through immunoprecipitation to identify interaction partners

• Validation of gene expression studies at the protein level

• Confirmation of successful genetic modifications such as knockout or knockdown experiments

• Investigation of post-translational modifications that may regulate CRTISO activity

• Distinction between different CRTISO homologs when studying their specific functions

For researchers investigating carotenoid biosynthesis pathways, CRTISO antibodies provide direct evidence of protein expression and localization that complements molecular techniques focused on gene expression.

What methods are most effective for CRTISO protein detection?

Several methods can be employed for detecting CRTISO protein in plant tissues, each with specific advantages:

• Western blotting: The most common method for detecting and semi-quantifying CRTISO protein levels in tissue extracts. This technique requires careful protein extraction to maintain enzyme integrity and specific antibodies against CRTISO.

• Immunohistochemistry/Immunofluorescence: These techniques allow visualization of CRTISO localization within tissue sections or cells, providing spatial information about protein distribution.

• Enzyme-linked immunosorbent assay (ELISA): Useful for quantitative measurement of CRTISO protein in tissue extracts when calibrated with purified standards.

• Immunoprecipitation: For isolating CRTISO complexes from plant tissues to study protein-protein interactions.

For optimal detection, researchers should consider using extraction buffers similar to those described for isolating CRTISO from calendula petals, which utilize cetyltrimethylammonium bromide method for initial RNA isolation .

How can researchers generate specific antibodies against CRTISO?

Generating specific antibodies against CRTISO requires careful consideration of several factors:

• Antigen selection: Researchers can use either full-length recombinant CRTISO protein or unique peptide sequences specific to CRTISO. For distinguishing between homologs (like the four CoCRTISO homologs in calendula), peptides from non-conserved regions should be selected.

• Expression system: For recombinant CRTISO production, bacterial expression systems using vectors like pMAL-c2x (as used for CoCRTISO1 mutants) can be employed . E. coli expression systems have been successfully used for CRTISO enzymatic analysis .

• Purification strategy: Fusion tags such as maltose-binding protein can facilitate purification of recombinant CRTISO for immunization.

• Validation methods: Specificity of generated antibodies should be verified using:

  • Western blots with recombinant CRTISO and plant extracts

  • Comparison between wild-type and crtiso mutant tissues

  • Peptide competition assays

  • Cross-reactivity tests with related carotenoid pathway enzymes

When generating antibodies against specific CRTISO homologs, researchers should design antigenic peptides that avoid homologous regions, similar to the approach used for designing qPCR primers for expression analysis of the four CoCRTISO homologs .

How should researchers design experiments to study CRTISO function using antibodies?

When designing experiments to study CRTISO function using antibodies, researchers should consider:

• Proper controls: Include both positive controls (recombinant CRTISO protein) and negative controls (tissue from crtiso knockout mutants) in immunodetection experiments.

• Tissue selection: Different tissues may express varying levels of CRTISO. For example, in calendula, CRTISO expression is petal color-specific , while in Arabidopsis, expression patterns differ between leaves and flowers. Target tissues with known CRTISO expression.

• Developmental timing: CRTISO expression may vary during development, so collect samples at appropriate developmental stages.

• Extraction conditions: Optimize protein extraction methods to maintain CRTISO integrity and activity. Methods similar to those used for extracting carotenoids from calendula petals (using hexane and aqueous 90% methanol) may need to be adapted for protein extraction .

• Co-localization studies: Consider co-immunodetection with other carotenoid pathway enzymes to understand spatial relationships.

• Functional assays: Combine immunodetection with enzyme activity assays, such as the HPLC detection of carotenoid isomerization, to correlate protein levels with activity.

How can CRTISO antibodies be used to distinguish between different CRTISO homologs?

Distinguishing between different CRTISO homologs (such as CoCRTISO1-4 in calendula) requires specialized approaches:

• Homolog-specific antibodies: Generate antibodies against unique epitopes that differ between homologs. This approach requires careful sequence analysis to identify divergent regions, similar to how researchers designed specific primers for RT-PCR to avoid homologous regions when studying expression patterns of the four CoCRTISO homologs in calendula .

• Two-dimensional western blotting: Separate homologs based on both molecular weight and isoelectric point differences before immunodetection.

• Immunoprecipitation followed by mass spectrometry: Isolate CRTISO proteins using a general anti-CRTISO antibody, then identify specific homologs through peptide mass fingerprinting.

• Tissue-specific expression analysis: Some homologs may be preferentially expressed in specific tissues. For example, in calendula, certain CRTISO homologs are expressed in a petal color-specific manner . Target tissues known to express specific homologs.

• Mutant complementation studies: Use antibodies to confirm expression of specific homologs in complementation experiments with crtiso mutants.

What techniques can be combined with CRTISO antibodies for studying protein-protein interactions?

Several techniques can be combined with CRTISO antibodies to investigate protein-protein interactions:

• Co-immunoprecipitation (Co-IP): Use CRTISO antibodies to pull down protein complexes from plant extracts, followed by western blotting or mass spectrometry to identify interacting partners.

• Proximity ligation assay (PLA): This technique allows visualization of protein interactions in situ by generating fluorescent signals when two proteins of interest are in close proximity.

• Bimolecular fluorescence complementation (BiFC): While not directly using antibodies, this technique can validate interactions identified through antibody-based methods.

• Yeast two-hybrid screening followed by Co-IP validation: Potential interactions identified through Y2H can be confirmed using CRTISO antibodies.

• Chromatin immunoprecipitation (ChIP): If studying transcription factors that regulate CRTISO expression, this technique can identify DNA-protein interactions.

• Cross-linking immunoprecipitation: For capturing transient or weak interactions between CRTISO and other proteins.

These approaches can help elucidate the interactome of CRTISO and better understand its regulation and function in the carotenoid biosynthesis pathway.

How can CRTISO antibodies be used in conjunction with CRISPR/Cas9 gene editing experiments?

CRTISO antibodies can be valuable tools in CRISPR/Cas9 gene editing studies:

• Validation of knockout efficiency: Confirm the absence of CRTISO protein in gene-edited lines, complementing molecular analysis of DNA mutations.

• Protein level analysis in partial knockouts: Quantify reduced CRTISO expression in heterozygous mutants or when using promoter modifications.

• Analysis of truncated proteins: Detect and characterize truncated CRTISO proteins resulting from frameshift mutations.

• Confirmation of complementation: Verify protein expression when reintroducing wild-type or modified CRTISO genes into mutant backgrounds.

• Introgression monitoring: Track the presence of CRTISO protein when crossing edited lines into different genetic backgrounds.

• Mosaic analysis: In experiments inducing somatic recombination at the CRTISO locus (as described in tomato ), antibodies could confirm CRTISO expression in tissues showing gain-of-function phenotypes.

• Functional domain studies: When creating targeted mutations in specific CRTISO domains, antibodies can confirm protein expression while functional assays assess activity changes.

What are common challenges in CRTISO antibody experiments and how can they be addressed?

Researchers may encounter several challenges when working with CRTISO antibodies:

• Cross-reactivity with related enzymes: CRTISO shares structural similarity with other carotenoid pathway enzymes, particularly bacterial-type phytoene desaturase CRTI . To address this:

  • Use peptide-specific antibodies targeting unique regions of CRTISO

  • Include appropriate controls (crtiso mutants)

  • Perform peptide competition assays to confirm specificity

• Low protein abundance: CRTISO may be expressed at low levels in some tissues. To overcome this:

  • Optimize protein extraction methods

  • Use concentration techniques like immunoprecipitation before detection

  • Consider amplification methods for immunohistochemistry

• Post-translational modifications: These may affect epitope recognition. Strategies include:

  • Generate antibodies against multiple regions of CRTISO

  • Use phosphorylation-specific antibodies if phosphorylation is suspected

  • Compare native and denatured protein detection

• Protein degradation during extraction: CRTISO may be sensitive to proteolysis. Solutions include:

  • Include protease inhibitors in extraction buffers

  • Perform extractions at low temperatures

  • Optimize extraction protocols specific to plant tissue type

• Background signal in plant tissues: High background can obscure specific signals. Approaches include:

  • Optimize blocking conditions

  • Increase washing stringency

  • Use monoclonal antibodies when possible

  • Pre-absorb antibodies with plant extracts from crtiso mutants

How should researchers interpret contradictory results between CRTISO expression and carotenoid profiles?

When faced with contradictory results between CRTISO protein detection and carotenoid profiles, researchers should consider:

• Post-translational regulation: CRTISO may be present but inactive due to post-translational modifications or protein-protein interactions.

• Enzymatic redundancy: Alternative isomerization pathways may exist. For example, light-mediated photoisomerization can partially substitute for CRTISO activity in some tissues .

• Substrate availability: CRTISO may be present but lacking appropriate substrates due to upstream enzyme deficiencies.

• Protein localization issues: CRTISO may not co-localize with its substrates due to altered subcellular targeting.

• Antibody specificity problems: The antibody may be detecting related proteins rather than CRTISO itself.

• Different CRTISO homologs: Different homologs may have varying activities against different substrates. For example, in calendula, there are four CRTISO homologs with potentially different substrate specificities .

• Experimental conditions: Extraction methods may affect enzyme stability or activity. HPLC analysis conditions can influence carotenoid detection (e.g., method used for calendula: solvent A, methanol/methyl tert-butyl ether/H2O = 95:1:4; solvent B, methanol/methyl tert-butyl ether/H2O = 25:71:4) .

Table 3: Comparison of CRTISO Activity and Expression Across Different Plant Genotypes

*Data significantly different from wild type (P < 0.05)

How can CRTISO antibodies contribute to understanding carotenoid pathway regulation?

CRTISO antibodies can provide valuable insights into carotenoid pathway regulation through:

• Protein stability studies: Tracking CRTISO protein levels under different environmental conditions or developmental stages to understand post-transcriptional regulation.

• Identification of regulatory protein interactions: Using co-immunoprecipitation with CRTISO antibodies to identify proteins that may regulate CRTISO activity.

• Post-translational modification analysis: Developing modification-specific antibodies (phospho-specific, etc.) to understand how CRTISO activity is regulated.

• Tissue-specific expression patterns: Mapping CRTISO protein distribution across different tissues and developmental stages to correlate with carotenoid accumulation patterns.

• Response to environmental stimuli: Monitoring changes in CRTISO protein levels in response to light, temperature, or other environmental factors.

• Protein turnover studies: Using antibodies in pulse-chase experiments to determine CRTISO protein half-life under different conditions.

• Chromatin regulation studies: Investigating how modifications in chromatin around the CRTISO locus (as observed in ccr1 alleles ) affect protein expression.

What novel approaches could enhance CRTISO antibody specificity and utility?

Several innovative approaches could improve CRTISO antibody specificity and utility:

• Nanobody development: Creating single-domain antibody fragments derived from camelid antibodies, which may offer improved access to conformational epitopes.

• CRISPR-engineered cell lines expressing tagged CRTISO: Generating plant lines with epitope-tagged endogenous CRTISO to enable detection with highly specific commercial tag antibodies.

• Proximity labeling techniques: Combining CRTISO antibodies with proximity labeling methods (BioID, APEX) to identify proteins in the vicinity of CRTISO in vivo.

• Single-molecule detection methods: Adapting super-resolution microscopy techniques for visualizing individual CRTISO molecules and their interactions.

• Antibody engineering approaches: Using techniques like CDR walking to optimize antibody affinity and specificity. This approach has successfully improved antibody affinity in other systems, such as the development of high-affinity anti-HIV gp120 Fab with a 420-fold increase in affinity (Kd=1.5x10-11 M) .

• Computational antibody design: Utilizing in silico approaches like OptCDR, OptMAVEn, or RosettaAntibodyDesign to create optimized antibodies based on CRTISO structure .

• Conformation-specific antibodies: Developing antibodies that specifically recognize active or inactive conformations of CRTISO to study its regulation.

• Cross-linking mass spectrometry: Combining antibody-based purification with cross-linking mass spectrometry to map CRTISO interaction interfaces with high resolution.