Recombinant Picea sitchensis Cytochrome P450 716B2 (CYP716B2)

How does CYP716B2 differ structurally and functionally from CYP716B1 in Sitka spruce?

CYP716B2 (Q50EK0) and CYP716B1 (Q50EK1) are closely related Cytochrome P450 enzymes in Sitka spruce with high sequence similarity. Key differences include:

• Sequence variations: While both proteins share high similarity, CYP716B1 is 493 amino acids long compared to CYP716B2's 497 amino acids.

• Functional differences: Though detailed comparative functional analysis is limited in available literature, these proteins likely have overlapping but distinct substrate specificities and catalytic efficiencies.

• Nomenclature: CYP716B2 is alternatively known as Cytochrome P450 CYPA2, while CYP716B1 is alternatively known as Cytochrome P450 CYPA1 .

Their structural variations, even if minor, may contribute to different roles in terpenoid biosynthesis pathways, which are critical for plant defense mechanisms in conifers.

What are the optimal conditions for expressing recombinant CYP716B2 in E. coli systems?

For optimal expression of recombinant CYP716B2 in E. coli systems, researchers should consider the following methodology:

• Expression system selection: The protein has been successfully expressed in E. coli with an N-terminal His-tag fusion .

• Vector design: Incorporate the full-length sequence (1-497 amino acids) with an N-terminal His-tag for purification efficiency.

• Growth conditions:

  • Culture medium: Rich media such as LB or TB with appropriate selection antibiotics

  • Induction: IPTG induction at OD600 of 0.6-0.8

  • Temperature: Lower temperatures (16-20°C) post-induction may improve protein folding

• Purification approach: Immobilized metal affinity chromatography (IMAC) using Ni-NTA columns to capture the His-tagged protein.

• Quality control: Assess purity using SDS-PAGE (>90% purity should be achievable) .

The recombinant protein should be stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 for optimal stability .

What are the critical considerations for maintaining CYP716B2 stability after purification?

Maintaining the stability of purified recombinant CYP716B2 requires careful attention to several factors:

• Storage buffer composition:

  • Use Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • For long-term storage, add glycerol to a final concentration of 5-50% (with 50% recommended for optimal results)

• Storage temperature protocol:

  • Store stock solutions at -20°C to -80°C

  • Aliquot before freezing to avoid repeated freeze-thaw cycles

  • Working solutions can be kept at 4°C for up to one week

• Reconstitution procedure:

  • Briefly centrifuge the vial before opening to collect contents

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to desired final concentration (5-50%)

• Stability considerations:

  • Avoid repeated freeze-thaw cycles as they significantly reduce enzyme activity

  • If working with the protein over multiple days, maintain small working aliquots at 4°C

How is CYP716B2 involved in Sitka spruce defense against white pine weevil?

CYP716B2 appears to be part of the chemical defense system in Sitka spruce (Picea sitchensis) against the white pine weevil (Pissodes strobi). The involvement can be understood through several mechanisms:

• Terpene biosynthesis: Cytochrome P450 enzymes in conifers are involved in terpene biosynthesis, which produces defensive compounds in oleoresin .

• Inducible defense responses: Upon weevil attack, resistant Sitka spruce genotypes show induced formation of traumatic resin ducts, which may involve CYP-mediated reactions .

• Monoterpene production: Research has particularly highlighted the association between resistance and certain monoterpenes. For example, (+)-3-carene is significantly higher in resistant Sitka spruce genotypes and is associated with resistance against the white pine weevil. Biosynthesis of this compound involves terpene synthases and possibly modifying enzymes like cytochrome P450s .

• Tissue-specific expression: CYP716B2 expression may be upregulated in the leader tissue of resistant trees, contributing to the chemical profile that deters weevil feeding and reproduction .

The enzyme likely participates in the conversion or modification of terpenoids that create the specific chemical profile associated with resistance against the white pine weevil in certain Sitka spruce genotypes.

What methodological approaches can be used to assess CYP716B2 activity in terpenoid biosynthesis?

To assess CYP716B2 activity in terpenoid biosynthesis, researchers can employ several methodological approaches:

• In vitro enzyme assays:

  • Reconstitute purified CYP716B2 with NADPH-cytochrome P450 reductase

  • Provide potential terpenoid substrates (monoterpenes, sesquiterpenes, diterpenes)

  • Measure substrate consumption and product formation via GC-MS or LC-MS

  • Determine enzyme kinetics parameters (Km, Vmax, kcat)

• Genetic manipulation approaches:

  • Perform heterologous expression in yeast or tobacco systems

  • Create knockdown/knockout lines using RNAi or CRISPR-Cas9 in model plant systems

  • Conduct complementation studies with the recombinant protein

• Metabolomic analysis:

  • Compare terpenoid profiles between tissues with differential CYP716B2 expression

  • Use gas chromatography (GC) analysis to quantify monoterpenes like (+)-α-pinene, (−)-limonene, (+)-sabinene, and (+)-3-carene

  • Track changes in terpenoid profiles after insect attack or mechanical damage

• Transcriptome-proteome correlation:

  • Conduct transcript profiling to identify expression patterns

  • Perform targeted selected reaction monitoring to confirm expression at the proteome level

  • Correlate expression levels with metabolite abundance using multivariate statistics

This multi-faceted approach can provide comprehensive insights into the enzymatic function of CYP716B2 in terpenoid biosynthesis pathways relevant to plant defense.

How can researchers design experiments to determine substrate specificity of CYP716B2?

Designing experiments to determine substrate specificity of CYP716B2 requires a systematic approach combining biochemical, analytical, and computational methods:

• Substrate screening methodology:

  • Prepare a library of potential substrates including various terpenes (monoterpenes, sesquiterpenes, diterpenes)

  • Conduct high-throughput enzyme assays with purified recombinant CYP716B2

  • Monitor conversion rates using LC-MS, GC-MS, or coupled spectrophotometric assays

  • Create a substrate preference profile based on relative activity levels

• Structure-activity relationship analysis:

  • Test structurally related compounds to identify essential molecular features for recognition

  • Perform competitive inhibition assays to determine binding affinities

  • Use site-directed mutagenesis of key residues in the enzyme's active site to confirm interactions

• Computational prediction approaches:

  • Perform homology modeling based on structurally characterized plant P450s

  • Conduct molecular docking simulations with potential substrates

  • Use molecular dynamics to simulate enzyme-substrate interactions

• In vivo validation experiments:

  • Express CYP716B2 in heterologous systems like yeast or tobacco

  • Supply various substrates and analyze metabolite profiles

  • Compare with native Sitka spruce tissues expressing CYP716B2

  • Use metabolic profiling to identify endogenous substrates in planta

This comprehensive approach will establish not only which compounds serve as substrates but also the relative efficiency of the enzyme toward different substrate classes.

What are the best approaches for studying the transcriptional regulation of CYP716B2 in response to pest attack?

To study transcriptional regulation of CYP716B2 in response to pest attack, researchers should implement the following methodological approaches:

• Time-course expression analysis:

  • Subject Sitka spruce seedlings to controlled white pine weevil exposure or mechanical damage

  • Collect tissue samples at defined intervals (e.g., 2, 5, 10, 15, and 22 days post-treatment)

  • Extract RNA and perform RT-qPCR targeting CYP716B2

  • Include reference genes for normalization and resistant/susceptible genotypes for comparison

• Promoter analysis and regulation:

  • Isolate the CYP716B2 promoter region (approximately 2kb upstream of transcription start site)

  • Identify putative regulatory elements using bioinformatic tools

  • Create promoter-reporter gene constructs (e.g., GUS or luciferase)

  • Test promoter activity in transiently transformed tissues upon pest exposure or treatment with defense-related hormones (jasmonic acid, ethylene)

• Chromatin immunoprecipitation (ChIP) analysis:

  • Identify transcription factors potentially involved in pest-induced expression

  • Perform ChIP followed by qPCR or sequencing to identify protein-DNA interactions

  • Validate interactions with electrophoretic mobility shift assays (EMSA)

• Comparative analysis between resistant and susceptible genotypes:

  • Compare CYP716B2 copy number, sequence variation, and expression patterns

  • Analyze differences in promoter sequences and epigenetic modifications

  • Correlate differences with variation in transcript and protein abundance

This multi-faceted approach will provide insights into how CYP716B2 transcription is regulated in response to pest attack and how this regulation differs between resistant and susceptible Sitka spruce genotypes.

How does CYP716B2 expression correlate with monoterpene profiles in resistant vs. susceptible Sitka spruce genotypes?

Research comparing resistant and susceptible Sitka spruce genotypes reveals important correlations between CYP716B2 expression and monoterpene profiles:

• Expression patterns and monoterpene correlation:

  • In resistant Sitka spruce genotypes (e.g., H898), CYP716B2 may be differentially expressed compared to susceptible genotypes (e.g., Q903)

  • This differential expression correlates with distinct monoterpene profiles, particularly the presence of defense-associated compounds like (+)-3-carene

• Temporal dynamics of expression and monoterpene production:

  • Upon weevil attack, monoterpene profiles remain relatively stable over a 22-day period, though some compounds show dynamic changes

  • (+)-3-carene, a compound associated with resistance, decreases significantly at days 15 and 22 in resistant genotypes exposed to weevil feeding, possibly due to volatilization from feeding sites exceeding the rate of biosynthesis

• Quantitative differences in terpene composition:

  Compound	Resistant Genotype	Susceptible Genotype	Association with Resistance
  (+)-3-carene	High levels	Trace or undetectable	Strong positive correlation
  (+)-α-pinene	Present	Present	No clear correlation
  (−)-limonene	Present	Present	No clear correlation
  (+)-sabinene	Present	Present	No clear correlation

• Functional implications:

  • The differential expression of CYP716B2 and related enzymes may contribute to the distinct chemical profiles that deter weevil feeding, affect ovary development in female weevils, and prevent successful reproduction on resistant trees

This correlation between enzyme expression and monoterpene profiles provides insights into the molecular basis of resistance against the white pine weevil in Sitka spruce.

What methodological approaches can be used to compare CYP716B2 function across different conifer species?

To compare CYP716B2 function across different conifer species, researchers should employ a comprehensive comparative analysis approach:

• Phylogenetic analysis and sequence comparison:

  • Identify CYP716B2 orthologs from multiple conifer species using bioinformatic tools

  • Construct phylogenetic trees to visualize evolutionary relationships

  • Calculate sequence identity/similarity percentages

  • Identify conserved domains and variable regions that may confer species-specific functions

• Heterologous expression and functional characterization:

  • Clone CYP716B2 genes from different conifer species

  • Express recombinant proteins in a common system (E. coli, yeast, or tobacco)

  • Compare enzymatic activities, substrate preferences, and kinetic parameters

  • Identify species-specific functional differences

• Metabolomic profiling:

  • Analyze terpenoid profiles from different conifer species with known CYP716B2 variants

  • Correlate metabolite abundance with CYP716B2 sequence variations

  • Perform correlation network analysis to identify species-specific metabolic pathways

• Transcriptomic analysis:

  • Compare expression patterns of CYP716B2 orthologs across species

  • Identify co-expressed genes that may form species-specific functional modules

  • Analyze expression in response to common stressors (pests, pathogens, abiotic stress)

• Cross-species complementation:

  • Express CYP716B2 from one species in another species or model plant

  • Assess changes in metabolite profiles and resistance phenotypes

  • Determine functional conserved and divergent aspects of the enzyme

This multi-faceted approach will provide insights into how CYP716B2 function has evolved across conifer species and its role in species-specific defense mechanisms.

How has CYP716B2 evolved in relation to pest pressure in different Sitka spruce populations?

The evolution of CYP716B2 in relation to pest pressure in Sitka spruce populations reveals important insights into plant-insect coevolution:

• Genetic variation patterns:

  • Certain Sitka spruce populations (e.g., from Haney and Squamish areas) show fewer than average weevil attacks despite being in high-weevil-hazard areas, suggesting localized evolution of resistance

  • Resistant genotypes like H898 represent extreme resistance in an otherwise highly susceptible species

  • This suggests that strong selection pressure from the white pine weevil has driven the evolution of resistance traits in specific populations

• Molecular evolution mechanisms:

  • CYP716B2 and related genes show evidence of evolution through several mechanisms:

    • Gene duplication events

    • Copy number variation

    • Differential gene expression

    • Sequence diversification

  • For example, in the related 3-carene biosynthesis pathway, resistant trees have a specific gene (PsTPS-3car2) that is absent in susceptible trees, demonstrating the importance of copy number variation in resistance

• Selection pressures:

  • Long-term interactions with herbivorous insects have likely exerted selective pressure on defense-related genes

  • The complex terpenoid defense system, including CYP716B2, has evolved in response to sustained herbivory pressure over the extremely long lifespan of conifer trees

Understanding these evolutionary patterns can provide insights into the molecular basis of adaptation and guide breeding programs for developing weevil-resistant Sitka spruce varieties.

What methods should be used to investigate the role of CYP716B2 in climate adaptation mechanisms in Sitka spruce?

Investigating the role of CYP716B2 in climate adaptation mechanisms requires an integrative approach combining molecular, physiological, and ecological methods:

• Geographic analysis of genetic variation:

  • Sample CYP716B2 sequences from Sitka spruce populations across diverse climatic gradients

  • Correlate sequence polymorphisms with climate variables

  • Identify signatures of selection using population genetic approaches

  • Analyze haplotype distributions in relation to climate zones

• Climate chamber experiments:

  • Expose resistant and susceptible genotypes to controlled climate conditions

  • Monitor CYP716B2 expression and enzymatic activity under various temperature and moisture regimes

  • Analyze changes in terpenoid profiles in response to climate variables

  • Test interactions between climate factors and pest resistance

• Seasonal expression profiling:

  • Monitor CYP716B2 expression throughout annual cycles

  • Correlate expression patterns with seasonal changes in climate

  • Analyze potential roles in both pest resistance and freezing acclimation

  • Integrate with analysis of other climate-responsive genes

• Systems biology approach:

  • Apply tools such as Automated Layout Pipeline for Inferred NEtworks (ALPINE)

  • Visualize co-expression networks of CYP716B2 with climate adaptation genes

  • Identify functional modules and signaling pathways involving CYP716B2

  • Correlate network structure with adaptive phenotypes

This methodology can reveal how CYP716B2 functions not only in pest resistance but potentially also in broader climate adaptation mechanisms, particularly considering evidence of its involvement in processes like freezing acclimation in Sitka spruce.

What are the common challenges in expressing active CYP716B2 and how can they be addressed?

Expressing active CYP716B2 presents several technical challenges that require careful troubleshooting:

• Poor expression levels:

  • Challenge: CYP450 proteins often express poorly in bacterial systems

  • Solution: Optimize codon usage for E. coli, use specialized strains (e.g., Rosetta, Arctic Express), or try alternative expression systems like yeasts or insect cells

  • Validation: Confirm expression levels via western blot using anti-His tag antibodies

• Protein misfolding and inclusion body formation:

  • Challenge: Membrane-associated CYPs frequently misfold in heterologous systems

  • Solution: Lower induction temperature (16-18°C), reduce inducer concentration, co-express molecular chaperones, or use solubilization tags (SUMO, thioredoxin, GST)

  • Validation: Perform solubility analysis comparing total vs. soluble fractions

• Loss of heme incorporation:

  • Challenge: Proper heme incorporation is essential for activity

  • Solution: Supplement growth medium with δ-aminolevulinic acid (ALA), add hemin during induction, ensure sufficient iron availability

  • Validation: Measure CO-difference spectra to confirm proper heme incorporation

• Instability during purification:

  • Challenge: CYP enzymes often lose activity during purification

  • Solution: Include glycerol (20-50%) and reducing agents in all buffers, minimize time at room temperature, use disposable columns to reduce processing time

  • Validation: Perform activity assays at each purification step to track retention of function

• Storage stability issues:

  • Challenge: Rapid activity loss during storage

  • Solution: Store in Tris/PBS-based buffer with 6% trehalose at pH 8.0 and 50% glycerol, prepare small aliquots to avoid freeze-thaw cycles

  • Validation: Test activity after various storage conditions and durations

Implementing these strategies can significantly improve the yield and activity of recombinant CYP716B2.

How can researchers address the challenge of identifying specific substrates for CYP716B2?

Identifying specific substrates for CYP716B2 presents significant challenges that can be addressed through a systematic multi-technique approach:

• Untargeted metabolomic screening:

  • Challenge: The natural substrate(s) of CYP716B2 remain unidentified

  • Approach: Perform comparative metabolomics between tissues/genotypes with different CYP716B2 expression levels

  • Method: Use LC-MS/MS or GC-MS with untargeted analysis to identify compounds that correlate with CYP716B2 expression

  • Validation: Confirm metabolite identities using authentic standards

• Activity-guided fractionation:

  • Challenge: Complex plant extracts contain numerous potential substrates

  • Approach: Create biochemical activity assays using recombinant CYP716B2

  • Method: Fractionate plant extracts and test each fraction for substrate activity, progressively narrowing down to individual compounds

  • Validation: Confirm activity with purified compounds and determine kinetic parameters

• Substrate candidate prediction:

  • Challenge: The substrate scope is potentially very broad

  • Approach: Use structural modeling and docking simulations to predict likely substrates

  • Method: Create a homology model of CYP716B2 based on crystallized plant P450s, then perform in silico docking with terpenoid libraries

  • Validation: Test top-ranking candidates in vitro with the recombinant enzyme

• Comparative genomic approaches:

  • Challenge: Limited functional annotation of plant P450s

  • Approach: Identify functionally characterized homologs in other species

  • Method: Perform phylogenetic analysis of CYP716 family and identify the most closely related enzymes with known substrates

  • Validation: Test the identified substrate classes with recombinant CYP716B2

• Pathway reconstruction:

  • Challenge: CYP716B2 likely functions within a complex biosynthetic pathway

  • Approach: Reconstruct the relevant pathways in heterologous systems

  • Method: Co-express CYP716B2 with potential pathway partners in yeast or tobacco

  • Validation: Analyze the resulting metabolite profiles to identify pathway intermediates and products

This comprehensive approach maximizes the chances of identifying the physiological substrates of CYP716B2 and placing the enzyme within its correct metabolic context.

What are the most promising applications of CYP716B2 in conifer improvement programs?

The potential applications of CYP716B2 in conifer improvement programs are significant and multifaceted:

• Pest resistance enhancement:

  • Approach: Introducing or upregulating CYP716B2 in susceptible Sitka spruce genotypes to enhance resistance against white pine weevil

  • Potential impact: Could allow reintroduction of commercially valuable Sitka spruce to its native range in coastal British Columbia, where it is currently limited by weevil susceptibility

  • Implementation strategy: Use marker-assisted selection to identify natural variants with enhanced CYP716B2 expression or function

• Forest restoration and climate adaptability:

  • Approach: Integrating CYP716B2 markers into selection programs for more resilient forest trees

  • Potential impact: Development of trees with enhanced defense capabilities against multiple stressors (pests, pathogens, climate extremes)

  • Implementation strategy: Screen diverse populations to identify beneficial CYP716B2 variants that correlate with multiple stress tolerance traits

• Metabolic engineering of defense compounds:

  • Approach: Engineer enhanced production of resistance-associated terpenes like (+)-3-carene

  • Potential impact: Create trees with optimized defense compound profiles that deter multiple pests

  • Implementation strategy: Modulate CYP716B2 expression in coordination with other terpene biosynthesis genes

• Biomarker development:

  • Approach: Use CYP716B2 variants as biomarkers for pest resistance in breeding programs

  • Potential impact: Accelerate selection of resistant genotypes without requiring lengthy field trials

  • Implementation strategy: Develop high-throughput genotyping methods to screen for favorable CYP716B2 alleles

The integration of CYP716B2 knowledge into tree improvement programs could significantly enhance forest resilience and productivity in the face of increasing biotic and abiotic stresses.

What experimental approaches should be prioritized to better understand the role of CYP716B2 in the broader terpenoid biosynthesis network?

To elucidate the role of CYP716B2 within the broader terpenoid biosynthesis network, several experimental approaches should be prioritized:

• Multi-omics integration:

  • Approach: Combine transcriptomics, proteomics, and metabolomics data to place CYP716B2 in its pathway context

  • Methodology: Apply network analysis tools like ALPINE (Automated Layout Pipeline for Inferred NEtworks) to visualize and analyze integrated datasets

  • Expected outcome: Identification of co-regulated genes, proteins, and metabolites that function together with CYP716B2

• CRISPR-based genome editing:

  • Approach: Create precise knockouts or modifications of CYP716B2 in conifer tissue culture systems

  • Methodology: Develop optimized delivery methods for CRISPR-Cas9 components into conifer tissues

  • Expected outcome: Definitive assessment of CYP716B2's role in terpenoid production and pest resistance

• Protein-protein interaction studies:

  • Approach: Identify proteins that physically interact with CYP716B2

  • Methodology: Apply techniques such as yeast two-hybrid, co-immunoprecipitation, or proximity labeling

  • Expected outcome: Discovery of metabolic complexes or regulatory proteins that influence CYP716B2 function

• Subcellular localization studies:

  • Approach: Determine the precise subcellular location of CYP716B2

  • Methodology: Create fluorescent protein fusions and use confocal microscopy to visualize localization

  • Expected outcome: Insights into the spatial organization of terpenoid biosynthesis and transport

• Comparative analysis across resistant genotypes:

  • Approach: Study multiple independently-evolved resistant Sitka spruce genotypes

  • Methodology: Compare CYP716B2 sequence, expression, and associated metabolite profiles

  • Expected outcome: Identification of convergent evolutionary solutions to pest pressure and common mechanisms of resistance

These prioritized approaches would significantly advance our understanding of how CYP716B2 functions within the complex terpenoid biosynthesis network of conifers and could reveal new strategies for enhancing tree resistance to pests and environmental stressors.