Recombinant Triticum aestivum Chymotrypsin inhibitor WCI

What is Triticum aestivum Chymotrypsin Inhibitor (WCI)?

WCI is a novel proteinaceous inhibitor specifically isolated from the endosperm of common wheat (Triticum aestivum). Characterized as a monomeric neutral protein, WCI consists of 119 amino acid residues with a molecular mass of 12,933.40 Da . The inhibitor demonstrates strong, specific inhibitory activity against bovine pancreatic chymotrypsin and chymotryptic-like enzymes from certain insect digestive systems . Structurally, WCI belongs to the cereal trypsin/α-amylase inhibitor superfamily, which includes various defense proteins found in cereal grains that help protect against herbivorous insects and pathogens .

How does WCI compare structurally to other protease inhibitors in cereal crops?

WCI shares significant sequence similarity (45.7-89.1%) with other proteins in the cereal trypsin/α-amylase inhibitor superfamily . While detailed crystallographic data specifically for WCI is not presented in the current literature, related inhibitors like the 0.19 α-amylase inhibitor from wheat have been thoroughly characterized structurally . These related proteins typically feature four major α-helices arranged in an up-and-down pattern, with additional structural elements including a one-turn helix and two short antiparallel β-strands .

The disulfide bonding pattern is highly conserved in this protein family, with the 0.19 α-amylase inhibitor forming five disulfide bonds (C6−C52, C20−C41, C28−C83, C42−C99, and C54−C115) . These covalent linkages are crucial for maintaining the tertiary structure that enables specific protease recognition. Based on sequence similarities, WCI likely shares this characteristic folding pattern and disulfide arrangement, which contributes to its remarkable stability and specific inhibitory function.

What is the molecular heterogeneity observed in purified WCI samples?

An intriguing feature of purified native WCI is its inherent molecular heterogeneity. Automated sequence and mass spectrometry analyses have revealed that approximately 40% of purified WCI samples consist of an isoform called [des-(Thr)WCI] . This variant lacks a threonine residue compared to the full-length protein, suggesting either alternative processing during protein maturation or post-translational modification . This heterogeneity presents both research challenges and opportunities—it complicates purification and standardization efforts but also provides insights into protein processing mechanisms in wheat. Understanding this heterogeneity is crucial for researchers working with native WCI, as it may affect experimental reproducibility and interpretation of structure-function relationships.

What is the substrate specificity profile of WCI?

WCI exhibits a highly selective inhibitory profile that distinguishes it from other protease inhibitors. It strongly inhibits bovine pancreatic chymotrypsin (its namesake target) as well as chymotryptic-like activities isolated from the midgut of agriculturally significant insect pests, including Helicoverpa armigera (cotton bollworm) and Tenebrio molitor (mealworm beetle) . This specific activity against insect digestive enzymes strongly suggests WCI's natural role in plant defense against herbivory.

Notably, WCI demonstrates no inhibitory activity against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase, or human leukocyte elastase . This selective inhibition profile indicates a specialized evolutionary adaptation focused on inhibiting chymotrypsin-like serine proteases. For researchers, this specificity makes WCI a valuable tool for selectively targeting certain proteolytic pathways without broadly disrupting others.

What are the established protocols for isolating native WCI from wheat endosperm?

Successful isolation of native WCI from wheat endosperm requires a multi-step purification process. While specific protocols may vary, effective purification typically involves:

• Initial extraction from wheat endosperm using appropriate buffer systems (often phosphate buffers with pH 7.0-7.5)

• Primary fractionation through ammonium sulfate precipitation to concentrate proteins

• Ion-exchange chromatography (typically anion exchange at neutral pH) to separate proteins based on charge characteristics

• Gel filtration chromatography to separate proteins by molecular size, particularly useful for isolating the ~13 kDa WCI

• Affinity chromatography using immobilized chymotrypsin to selectively capture functionally active WCI

What analytical techniques best demonstrate WCI purity and identity?

Establishing the purity and identity of isolated WCI requires multiple complementary analytical approaches:

• SDS-PAGE analysis provides initial confirmation of molecular weight (~13 kDa) and preliminary purity assessment

• Mass spectrometry is essential for precise molecular weight determination (12,933.40 Da for full-length WCI) and can detect the presence of the [des-(Thr)WCI] isoform

• N-terminal sequencing through Edman degradation confirms protein identity and can detect N-terminal processing or heterogeneity

• Functional assays measuring inhibition of chymotrypsin activity verify that the purified protein retains its biological activity

• Circular dichroism spectroscopy assesses secondary structure content, providing confirmation of proper folding

• Isoelectric focusing can distinguish WCI from other wheat proteins with similar molecular weights

When reporting WCI purification, researchers should document protein yield at each purification stage, final specific activity (inhibitory units per mg protein), and the ratio of full-length to [des-(Thr)WCI] isoforms to ensure experimental reproducibility and facilitate comparative studies between different wheat varieties or recombinant production systems.

How can enzymatic assays be optimized for quantifying WCI inhibitory activity?

Optimizing enzymatic assays for accurate quantification of WCI inhibitory activity requires careful consideration of several parameters:

• Substrate selection is critical—N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide represents an ideal synthetic substrate for measuring chymotrypsin inhibition due to its specificity and chromogenic properties

• Enzyme concentration must be carefully calibrated to ensure linear reaction kinetics within the measurement timeframe

• Pre-incubation conditions between WCI and chymotrypsin significantly impact observed inhibition—typically 5-10 minutes at room temperature allows formation of stable enzyme-inhibitor complexes

• Buffer composition affects both enzyme activity and inhibitor binding; phosphate buffers (pH 7.5) with calcium supplementation provide optimal conditions for chymotrypsin activity

• Temperature control is essential, with 25°C or 37°C being standard assay temperatures that should be precisely maintained

• Data analysis should include determination of IC50 values (inhibitor concentration causing 50% inhibition) and, for more detailed kinetic characterization, Ki values through Lineweaver-Burk or Dixon plots

For comparing WCI variants or analyzing inhibition of different target proteases, researchers should standardize assay conditions and report activity in well-defined units, such as inhibitory units (the amount of inhibitor required to reduce enzyme activity by 50% under standard conditions) rather than simply percent inhibition at arbitrary concentrations.

What expression systems have been successfully employed for recombinant WCI production?

Escherichia coli has been successfully employed as an expression system for recombinant WCI production. The cDNA encoding WCI (wci-cDNA) was isolated from wheat immature caryopses and utilized to generate recombinant protein in E. coli . This bacterial expression system offers advantages including rapid growth, high protein yields, and well-established protocols for genetic manipulation.

While not explicitly described for WCI in the available literature, alternative expression systems that might merit investigation for recombinant WCI production include:

• Yeast systems (Pichia pastoris or Saccharomyces cerevisiae), which may offer improved protein folding

• Insect cell expression systems, which provide eukaryotic post-translational modifications

• Plant-based expression systems, which might be particularly appropriate for a plant-derived protein like WCI

The choice of expression system should be guided by specific research requirements, including needed protein yield, desired post-translational modifications, and downstream applications.

What strategies optimize recovery of active recombinant WCI from inclusion bodies?

Recovering active recombinant WCI from inclusion bodies requires a carefully optimized refolding protocol. Based on experimental evidence with WCI and similar protease inhibitors, the following approach can yield soluble and partially active protein :

• Inclusion body isolation through differential centrifugation following cell lysis

• Solubilization using denaturing agents (typically 6-8M urea or 6M guanidine hydrochloride) with reducing agents (DTT or β-mercaptoethanol) to fully unfold the protein and reduce disulfide bonds

• Protein purification under denaturing conditions using affinity tags or conventional chromatography

• Controlled refolding through:

  • Dilution method: Slowly diluting the denatured protein into refolding buffer containing redox pairs (reduced/oxidized glutathione) to facilitate correct disulfide bond formation

  • Dialysis: Gradually removing denaturants through sequential dialysis against decreasing concentrations

  • On-column refolding: Immobilizing the denatured protein before gradual removal of denaturants

• Optimization of refolding conditions:

  • Protein concentration: Lower concentrations (0.1-0.5 mg/ml) typically reduce aggregation

  • Additives: L-arginine (0.5-1M), glycerol (10-20%), or PEG can enhance folding efficiency

  • Redox environment: Typically 5:1 to 10:1 ratio of reduced:oxidized glutathione

  • Temperature: Lower temperatures (4-16°C) generally reduce aggregation

• Final purification of correctly folded protein through size exclusion chromatography followed by activity assays to confirm functional recovery

The efficacy of refolding should be assessed through both structural (circular dichroism, fluorescence spectroscopy) and functional (inhibitory activity) measurements to ensure the recombinant protein closely resembles native WCI.

How do native and recombinant WCI differ in structural and functional properties?

Comparative analysis reveals several important differences between native wheat-derived WCI and its recombinant counterpart produced in E. coli:

• Activity profile: Recombinant WCI recovered from inclusion bodies through refolding procedures is described as "partially active" , suggesting incomplete recovery of the native inhibitory capacity. This reduced activity likely results from suboptimal folding or disulfide bond formation during the in vitro refolding process.

• Structural heterogeneity: Native WCI exhibits inherent molecular heterogeneity due to the presence of the [des-(Thr)WCI] isoform, which accounts for approximately 40% of purified samples . In contrast, recombinant WCI expressed from a single cDNA sequence would produce a homogeneous protein population (barring unexpected processing in E. coli), potentially simplifying some analyses but not accurately representing the natural state of the inhibitor.

• Post-translational modifications: While WCI is not extensively modified, any wheat-specific modifications would be absent in the E. coli-produced protein. The missing modifications might contribute to the observed activity differences.

• Folding efficiency: The requirement for in vitro refolding of recombinant WCI likely results in a portion of misfolded or partially folded protein in the final preparation. Native WCI, having folded within the wheat endosperm's specialized environment, likely represents a more homogeneously folded population.

These differences highlight important considerations for researchers. While recombinant production offers advantages in terms of yield and genetic manipulation potential, native WCI may be preferable for certain applications requiring full activity or natural heterogeneity. Ideally, both sources should be characterized in parallel to establish equivalence or document differences for specific experimental contexts.

What kinetic parameters best characterize WCI's inhibitory mechanism?

Comprehensive characterization of WCI's inhibitory mechanism requires determination of several key kinetic parameters:

• Inhibition constant (Ki): This fundamental parameter quantifies the strength of binding between WCI and its target proteases. Lower Ki values indicate stronger binding. While specific values for WCI are not provided in the available literature, related inhibitors in the same family typically exhibit Ki values in the nanomolar range for their target proteases .

• Inhibition type: Enzyme kinetic analysis using Lineweaver-Burk or Dixon plots can determine whether WCI acts as a competitive, noncompetitive, or uncompetitive inhibitor. Based on the mechanism of related protease inhibitors, WCI likely functions as a competitive inhibitor, directly blocking substrate access to the protease active site .

• Association rate constant (kon): Measuring the rate at which WCI forms complexes with target proteases provides insights into the kinetics of inhibition. Fast association rates (typically 10⁵-10⁷ M⁻¹s⁻¹ for protease inhibitors) suggest efficient capture of target enzymes.

• Dissociation rate constant (koff): This parameter indicates the stability of the enzyme-inhibitor complex. Slow dissociation rates result in more effective and sustained inhibition.

• Stoichiometry of inhibition: Determining whether WCI binds proteases in a 1:1 ratio or if higher-order complexes form can reveal important mechanistic details.

For comprehensive kinetic characterization, researchers should employ multiple complementary methods, including spectrophotometric assays with chromogenic substrates, stopped-flow techniques for rapid kinetics, and direct binding measurements using techniques like isothermal titration calorimetry or surface plasmon resonance.

How can molecular modeling approaches elucidate WCI's structure-function relationships?

Molecular modeling provides valuable insights into WCI's structure-function relationships when experimental structural data is limited . A comprehensive modeling approach should include:

• Homology modeling: Generating a three-dimensional model of WCI based on related proteins with solved structures, such as the 0.19 α-amylase inhibitor from wheat (with only 2.06 Å resolution) . This approach leverages the significant sequence similarity (45.7-89.1%) between WCI and other members of the cereal trypsin/α-amylase inhibitor superfamily .

• Reactive site identification: Computational analysis of the model can predict the likely reactive site residues that interact directly with the protease active site. For chymotrypsin inhibitors, this typically involves residues complementary to chymotrypsin's preference for large hydrophobic side chains at the P1 position.

• Molecular docking: Simulating the interaction between WCI and target proteases (bovine chymotrypsin and insect proteases) can reveal binding modes, contact residues, and the structural basis for WCI's specificity.

• Molecular dynamics simulations: Analyzing the dynamic behavior of free and protease-bound WCI can provide insights into conformational changes upon binding and identify flexible regions critical for function.

• In silico mutagenesis: Computational prediction of how specific amino acid substitutions might affect WCI's structure and inhibitory properties can guide experimental design for structure-function studies.

• Electrostatic surface mapping: Visualizing the charge distribution on WCI's surface can help explain its selectivity for certain proteases over others.

These modeling approaches are particularly valuable for guiding experimental work, including site-directed mutagenesis to confirm predicted functional residues and protein engineering efforts to enhance WCI's inhibitory properties or modify its specificity.

What techniques can assess WCI stability under different environmental conditions?

Comprehensive stability assessment of WCI under various environmental conditions requires multiple complementary techniques:

• Temperature stability:

  • Differential scanning calorimetry (DSC) precisely determines the melting temperature (Tm) and thermodynamic parameters of unfolding

  • Circular dichroism (CD) spectroscopy monitors changes in secondary structure content during thermal denaturation

  • Activity assays at various temperatures measure retention of functional inhibitory capacity

• pH stability:

  • Activity measurements across a pH range (typically pH 2-10) quantify the functional pH profile

  • Spectroscopic methods (CD, fluorescence) detect structural changes that might precede activity loss

  • Isoelectric focusing identifies any pH-dependent changes in charge properties

• Chemical stability:

  • Resistance to denaturants (urea, guanidine hydrochloride) through activity retention and spectroscopic assessment

  • Response to oxidizing agents that might affect cysteine residues and disulfide bonds

  • Stability in the presence of organic solvents or detergents relevant to experimental protocols

• Proteolytic resistance:

  • Incubation with various proteases followed by SDS-PAGE analysis to identify susceptible regions

  • Mass spectrometry to characterize proteolytic fragments and map vulnerable sites

  • Time-course studies to determine the kinetics of proteolytic degradation

• Long-term storage stability:

  • Activity retention during extended storage at different temperatures (4°C, -20°C, -80°C)

  • Effects of freeze-thaw cycles on structural integrity and function

  • Formulation optimization with stabilizing excipients

Similar protease inhibitors have demonstrated remarkable stability across broad pH ranges (pH 4-8) and temperatures (20-80°C) , suggesting that WCI might exhibit comparable robustness. This stability is likely conferred by its compact structure and disulfide bonds, making it particularly suitable for applications requiring resistance to harsh environmental conditions.

How effective is WCI against digestive enzymes from agricultural pest insects?

WCI demonstrates significant inhibitory activity against digestive enzymes from agricultural pest insects, particularly those relying on chymotrypsin-like proteases for protein digestion . In vitro studies have confirmed WCI's effectiveness against chymotryptic-like activities isolated from the midgut of two economically important pests:

• Helicoverpa armigera (cotton bollworm): This major lepidopteran pest causes extensive damage to cotton, tomato, chickpea, and other crops worldwide. WCI effectively inhibits its digestive enzymes in vitro, suggesting potential for disrupting its digestive physiology .

• Tenebrio molitor (mealworm beetle): A coleopteran pest of stored grains and flour, T. molitor's digestive enzymes are also susceptible to inhibition by WCI .

While specific inhibition percentages for WCI are not provided in the available literature, related protease inhibitors have demonstrated remarkable efficacy against insect digestive enzymes. For example, PmTKI (Piptadenia moniliformis trypsin inhibitor) shows inhibitory activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%) .

The ability of WCI to target digestive enzymes from taxonomically diverse pests (spanning both Lepidoptera and Coleoptera) suggests broad potential for agricultural applications. Future research should expand testing to additional pest species and conduct in vivo feeding trials to confirm that enzyme inhibition translates to reduced insect growth, development, and survival.

What methodologies best evaluate WCI's potential for transgenic crop protection?

Evaluating WCI's potential for transgenic crop protection requires a systematic, multi-stage approach:

• In vitro enzyme inhibition assays:

  • Testing WCI against gut extracts from target pest species to establish inhibitory efficacy

  • Determining IC50 values against specific insect proteases

  • Comparing WCI potency with other protease inhibitors being considered for crop protection

• Artificial diet bioassays:

  • Incorporating purified WCI into artificial insect diets at various concentrations

  • Measuring effects on key parameters including:

    • Larval weight gain and growth rate

    • Mortality at different developmental stages

    • Development time and pupal weights

    • Adult emergence and reproductive capacity

  • Calculating LC50 (lethal concentration) and EC50 (effective concentration) values

• Plant expression system optimization:

  • Testing different promoters for appropriate tissue-specific expression

  • Evaluating signal peptides for efficient secretion or subcellular targeting

  • Optimizing codon usage for improved expression in the target crop

• Transgenic plant characterization:

  • Quantifying WCI expression levels in different plant tissues

  • Confirming proper folding and activity of plant-produced WCI

  • Assessing any unintended effects on plant growth or development

• Insect challenge experiments:

  • Whole-plant bioassays with target pests under controlled conditions

  • Field trials to evaluate protection under natural infestation

  • Multi-generation studies to assess potential resistance development

• Safety assessment:

  • Allergenicity evaluation through sequence comparison with known allergens

  • Digestibility studies in simulated mammalian digestive conditions

  • Toxicity testing in appropriate non-target organisms

Similar protease inhibitors like SKTI (soybean Kunitz trypsin inhibitor) have shown promising results in such evaluations, with diets containing 500 μM SKTI reducing larval weight by up to 64% and causing increased mortality and developmental deformities in cotton boll weevil . These approaches would provide comprehensive data on WCI's potential effectiveness and safety for transgenic crop protection.

How might insect populations develop resistance to WCI-based crop protection?

Understanding potential resistance mechanisms to WCI-based crop protection is crucial for developing sustainable pest management strategies. Several pathways by which insect populations might develop resistance include:

• Digestive enzyme diversification: Insects may evolve to produce alternative digestive proteases that are not efficiently inhibited by WCI. This adaptation has been observed in response to other protease inhibitors, with insects shifting from chymotrypsin-like to trypsin-like enzymes or inducing novel protease isoforms .

• Increased protease expression: Insects might compensate for inhibited enzymes by overexpressing digestive proteases, effectively overwhelming the inhibitory capacity of plant-produced WCI. This quantitative response can enable insects to maintain sufficient digestive function despite partial inhibition.

• Protease structural modifications: Mutations in the insect protease genes might produce enzymes with altered binding sites that maintain catalytic activity but exhibit reduced affinity for WCI, directly reducing inhibitor effectiveness.

• Enhanced gut proteolytic degradation: Insects could evolve to produce proteases specifically capable of degrading WCI before it can inhibit digestive enzymes, essentially neutralizing the plant's defense mechanism.

• Behavioral adaptations: Insects might develop feeding behaviors that minimize exposure to plant tissues with high WCI concentrations or adjust their feeding physiology to compensate for reduced digestive efficiency.

To mitigate these resistance risks, researchers should consider:

• Pyramiding multiple inhibitors with different specificities in transgenic plants

• Combining protease inhibitors with other insecticidal proteins (e.g., Bt toxins) with different modes of action

• Expression of WCI alongside synergistic factors that might enhance its inhibitory effectiveness

• Implementing appropriate refuge strategies to maintain susceptible insect populations

• Monitoring for early signs of resistance development through regular sampling and enzyme profiling of target pest populations

These proactive approaches can help ensure the long-term effectiveness of WCI-based crop protection strategies.

How can protein engineering enhance WCI specificity toward target pest proteases?

Enhancing WCI's specificity toward target pest proteases through protein engineering requires sophisticated approaches that leverage both structural knowledge and evolutionary principles:

• Reactive site modification: The primary approach involves targeted mutations in WCI's reactive site loop that directly interacts with proteases. Based on molecular modeling results , researchers can identify specific residues for substitution to better complement the active site geometry of target insect proteases while reducing affinity for non-target proteases.

• Directed evolution strategies:

  • Phage display libraries of WCI variants can be screened against immobilized target proteases

  • Yeast surface display allows for quantitative screening using fluorescence-activated cell sorting

  • Error-prone PCR generates random mutations throughout the WCI sequence for screening unexpected beneficial changes

• Chimeric inhibitor design: Creating fusion proteins that combine the reactive site region of WCI with structural scaffolds from other inhibitors that might offer enhanced stability or binding kinetics.

• Computational design approaches:

  • In silico modeling of WCI-protease complexes to predict mutations that enhance complementarity

  • Molecular dynamics simulations to identify dynamically important residues not obvious from static structures

  • Machine learning algorithms trained on protease-inhibitor interaction data to suggest non-intuitive modifications

• Secondary binding site engineering: Beyond the reactive site, introducing modifications in secondary contact regions that enhance specificity through species-specific interactions with surface loops of target proteases.

• Stability optimization: Engineering enhanced thermostability or resistance to proteolytic degradation in the insect gut environment while maintaining inhibitory activity.

For all engineering approaches, iterative rounds of design, production, and functional testing are essential. Success should be measured not only by enhanced inhibition of target proteases but also by maintained structural stability and reduced activity against non-target proteases, particularly those in non-pest organisms.

What insights might comparative genomics provide about WCI evolution and diversity?

Comparative genomics approaches offer powerful insights into WCI evolution and diversity that can inform both fundamental understanding and applied research:

• Evolutionary trajectory mapping: Analyzing WCI homologs across diverse grass species (Poaceae) can reveal the evolutionary history of this inhibitor family. This might identify when specific functional adaptations arose and correlate them with the emergence of particular insect pests, suggesting co-evolutionary relationships.

• Functional diversification patterns: Some cereal species contain multiple WCI-like genes with potentially diverse inhibitory profiles. Genomic analysis can reveal whether these arose through gene duplication events followed by neofunctionalization, subfunctionalization, or other evolutionary processes.

• Selection pressure analysis: Calculating the ratio of non-synonymous to synonymous substitutions (dN/dS) across WCI codons can identify regions under positive selection, which often correspond to functionally important sites adapting to changing target proteases.

• Structural conservation mapping: Mapping sequence conservation onto the predicted three-dimensional structure of WCI can distinguish between structurally critical residues (highly conserved) and specificity-determining residues (variable between homologs).

• Cultivar-specific variation: Analyzing WCI gene sequences across diverse wheat cultivars might reveal natural variants with enhanced efficacy against specific pests, potentially identifying superior alleles for breeding programs.

• Horizontal gene transfer assessment: Determining whether WCI genes show evidence of horizontal transfer between plant lineages, which could indicate particularly adaptive inhibitor functions.

• Regulatory element analysis: Examining promoter regions of WCI genes across species can reveal how expression patterns have evolved, potentially correlating with specific ecological pressures.

These comparative genomics approaches would significantly enhance our understanding of WCI's role in plant defense and potentially identify natural WCI variants with superior properties for agricultural applications.

What physiological mechanisms explain WCI's role in wheat's natural defense system?

Understanding the physiological mechanisms underlying WCI's role in wheat's natural defense system requires investigation of multiple interconnected processes:

• Tissue-specific expression patterns: WCI is detected in wheat endosperm , suggesting a primary role in protecting developing seeds. Comprehensive expression analysis across tissues and developmental stages would reveal whether WCI also functions in protecting vegetative tissues or is specifically deployed for reproductive success.

• Induction dynamics: While constitutively expressed in seeds, WCI expression might be induced or enhanced in response to specific stimuli, including:

  • Herbivore feeding or oviposition

  • Mechanical wounding

  • Exposure to insect oral secretions

  • Pathogen infection

  • Abiotic stresses

• Signaling pathway integration: WCI expression likely integrates with broader defense signaling networks involving:

  • Jasmonic acid signaling typical of anti-herbivore responses

  • Salicylic acid pathways activated during certain pathogen infections

  • Ethylene-mediated stress responses

  • MAPK cascade signaling transducing external threats to gene expression changes

• Synergistic interactions: WCI may function synergistically with other defense proteins in wheat, including:

  • Additional protease inhibitors with complementary specificities

  • Lectins that bind to insect gut structures

  • Amylase inhibitors disrupting carbohydrate digestion

  • Oxidative enzymes that reduce nutrient bioavailability

• Evolutionary trade-offs: Resources allocated to WCI production likely represent trade-offs with growth or yield potential, suggesting that wheat has evolved optimal expression levels balancing defense with productivity.

• Systemic responses: Investigating whether localized herbivory induces WCI expression in distal tissues would reveal if it contributes to systemic acquired resistance.

• Environmental modulation: Understanding how environmental factors (drought, temperature, nutrient availability) affect WCI expression would clarify its role in context-dependent defense strategies.

These investigations would provide a comprehensive picture of how wheat utilizes WCI as part of its integrated defense strategy against herbivores and potentially other threats, informing both evolutionary ecology and applied crop protection research.