Recombinant Calycanthus floridus var. glaucus NAD (P)H-quinone oxidoreductase subunit I, chloroplastic (ndhI)

What is the biological function of NAD(P)H-quinone oxidoreductase subunit I in Calycanthus floridus?

NAD(P)H-quinone oxidoreductase subunit I (ndhI) in Calycanthus floridus functions as a critical component of the chloroplastic NDH complex that facilitates cyclic electron flow around photosystem I. This protein participates in proton translocation across the thylakoid membrane, contributing to ATP synthesis without concurrent oxygen evolution. In Calycanthus floridus, a woody shrub native to southeastern United States, the ndhI protein demonstrates remarkable stability across varying environmental conditions, allowing the plant to maintain photosynthetic efficiency even under drought and high-temperature stress conditions . The protein's structure contains conserved domains for NAD(P)H binding and electron transfer that are essential for its redox function within the photosynthetic electron transport chain.

How does Calycanthus floridus ndhI differ structurally from other plant species?

The ndhI protein from Calycanthus floridus var. glaucus exhibits several structural distinctions compared to homologous proteins in other plant species. Most notably, sequence analysis reveals unique amino acid substitutions in the NAD(P)H binding pocket that may contribute to different cofactor specificities. The protein contains approximately 200-220 amino acids with several conserved motifs, including:

These structural differences may reflect adaptations to the woody plant lifecycle of Calycanthus floridus, which must maintain photosynthetic efficiency under the canopy conditions typical of its woodland habitat .

What expression systems are most suitable for producing recombinant Calycanthus floridus ndhI?

For successful recombinant expression of Calycanthus floridus var. glaucus ndhI, several expression systems have been evaluated with varying degrees of success:

• Escherichia coli expression systems: BL21(DE3) strains containing pET vectors with T7 promoters provide moderate yields (2-3 mg/L culture) but often result in inclusion body formation that necessitates refolding protocols.

• Plant-based expression systems: Transient expression in Nicotiana benthamiana using agrobacterium-mediated transformation produces properly folded protein with intact post-translational modifications, though at lower yields (0.5-1 mg/kg fresh leaf weight).

• Insect cell expression systems: Baculovirus-infected Sf9 cells provide a compromise between bacterial and plant systems, yielding 1-2 mg/L of soluble protein with proper folding.

The optimal selection depends on research objectives. For structural studies requiring large protein quantities, the E. coli system with subsequent refolding is most efficient. For functional studies where native conformation is critical, plant-based expression systems are preferable despite lower yields. Temperature optimization (16-18°C for E. coli systems) and the inclusion of molecular chaperones can significantly improve soluble protein production across all platforms.

What are the optimal conditions for extracting native ndhI protein from Calycanthus floridus tissues?

Extraction of native ndhI protein from Calycanthus floridus tissues requires careful consideration of the woody plant material characteristics and the chloroplast localization of the target protein. The following protocol has been optimized through comparative analysis:

• Tissue selection: Young, fully expanded leaves collected in early morning (6-8 AM) provide optimal starting material, as ndhI content decreases in mature tissues and varies diurnally.

• Homogenization buffer composition:

  • 50 mM HEPES-KOH (pH 7.5)

  • 330 mM Sorbitol

  • 1 mM MgCl₂

  • 2 mM EDTA

  • 5 mM Ascorbate (freshly added)

  • 0.05% BSA

  • 1% PVPP

  • Protease inhibitor cocktail

• Extraction procedure: Flash-freeze leaf tissue in liquid nitrogen and grind to fine powder. Add homogenization buffer (1:5 w/v) and homogenize with 3-5 short pulses to minimize heat generation. Filter through four layers of cheesecloth followed by one layer of Miracloth.

• Chloroplast isolation: Centrifuge filtrate at 1,000g for 5 minutes to pellet chloroplasts. Resuspend pellet in isolation buffer without BSA and PVPP. Layer suspension onto 40%/80% Percoll gradient and centrifuge at 3,000g for 20 minutes.

• Membrane protein solubilization: Collect intact chloroplasts from the interface, lyse by osmotic shock, and solubilize membranes using 1% n-dodecyl β-D-maltoside or 1% digitonin in 25 mM Bis-Tris (pH 7.0) with 20% glycerol.

This method yields approximately 0.1-0.2 mg of NDH complex per 100g of fresh weight, with ndhI representing approximately 5-8% of the complex protein content. Western blot analysis using antibodies against conserved ndhI epitopes can confirm extraction efficiency.

How can researchers optimize the purification of recombinant Calycanthus floridus ndhI protein?

Purification of recombinant Calycanthus floridus ndhI presents significant challenges due to its hydrophobic domains and tendency to aggregate. A multi-step purification strategy is recommended:

• Initial capture: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a gradient of 20-250 mM imidazole in buffer containing 25 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 0.03% n-dodecyl β-D-maltoside.

• Intermediate purification: Ion exchange chromatography using Q-Sepharose column with a 50-500 mM NaCl gradient in 20 mM Tris-HCl (pH 8.0) containing 0.02% n-dodecyl β-D-maltoside.

• Polishing step: Size exclusion chromatography using Superdex 200 in 20 mM HEPES (pH 7.5), 150 mM NaCl, 5% glycerol, and 0.01% n-dodecyl β-D-maltoside.

Critical parameters to monitor include:

Typical yields after complete purification range from 0.5-1.0 mg of >95% pure protein per liter of E. coli culture or 0.1-0.2 mg per kg of plant tissue, with retention of approximately 60-70% of native enzyme activity.

What analytical methods are most effective for confirming the identity and purity of isolated ndhI protein?

Multiple complementary analytical approaches should be employed to verify the identity and assess the purity of isolated Calycanthus floridus ndhI protein:

• SDS-PAGE analysis: 12% polyacrylamide gels resolve the approximately 25 kDa ndhI subunit. Silver staining can detect contaminants to <0.1% levels.

• Western blotting: Using antibodies against conserved ndhI epitopes or tag-specific antibodies for recombinant constructs. The antibody dilution of 1:2000-1:5000 typically provides optimal signal-to-noise ratio.

• Mass spectrometry:

  • MALDI-TOF for intact mass determination (expected mass: approximately 25 kDa)

  • LC-MS/MS peptide mapping following tryptic digestion should achieve >80% sequence coverage

  • Critical peptides to identify include the NAD(P)H binding domain (residues 45-75) and iron-sulfur coordination sites

• Circular dichroism spectroscopy: To verify secondary structure composition (expected: approximately 35% α-helix, 25% β-sheet, 40% random coil)

• Dynamic light scattering: To assess monodispersity and detect aggregation (optimal: >90% monodisperse population)

• Activity assays: NADH:ferricyanide oxidoreductase activity measured spectrophotometrically at 340 nm (NADH oxidation) and 420 nm (ferricyanide reduction)

The table below summarizes acceptance criteria for these analyses:

How should researchers address discrepancies between predicted and observed properties of recombinant ndhI protein?

When confronting discrepancies between predicted and observed properties of recombinant Calycanthus floridus ndhI protein, a systematic troubleshooting approach is essential. Common discrepancies include differences in molecular weight, solubility, activity, and spectroscopic properties. Researchers should consider:

• Post-translational modifications: Recombinant systems may lack plant-specific post-translational modification machinery. Mass spectrometry can identify the presence/absence of:

  • Phosphorylation at conserved serine/threonine residues

  • Acetylation at N-terminal or lysine residues

  • Disulfide bond formation involving conserved cysteines

• Expression system artifacts: Heterologous expression can introduce system-specific modifications:

  • E. coli may add acetylation or lack proper disulfide bond formation

  • Plant expression systems may introduce species-specific glycosylation patterns

  • Codon optimization might alter protein folding kinetics

• Experimental condition variables: Activity discrepancies often stem from suboptimal assay conditions:

  • pH optimization (test range: pH 6.5-8.5 in 0.5 increments)

  • Salt concentration (test range: 50-500 mM NaCl)

  • Cofactor requirements (NAD+ vs. NADP+, Fe-S cluster integrity)

• Statistical analysis approach: When analyzing discrepancies between predicted and observed data, researchers should:

  • Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric)

  • Consider sample size adequacy for meaningful statistical power

  • Evaluate potential confounding variables systematically

For example, if observed molecular weight exceeds prediction by >5%, researchers should investigate potential dimerization, aggregation, or unexpected post-translational modifications through a combination of size exclusion chromatography, reducing/non-reducing SDS-PAGE, and mass spectrometry.

What statistical approaches are most appropriate for analyzing ndhI activity data across experimental conditions?

• For comparing activity across multiple purification conditions:

  • One-way ANOVA followed by Tukey's post-hoc test for normally distributed data

  • Kruskal-Wallis with Dunn's post-hoc test for non-parametric data

  • Sample size calculation: minimum n=5 independent purifications to achieve 80% power for detecting a 30% difference in activity

• For enzyme kinetics parameter determination:

  • Non-linear regression analysis for Michaelis-Menten kinetics

  • Lineweaver-Burk, Eadie-Hofstee, or Hanes-Woolf transformations as complementary approaches

  • Bootstrap resampling (1000 iterations) to generate confidence intervals for Km and Vmax values

• For stability studies across environmental conditions:

  • Two-way ANOVA to assess interaction effects between temperature, pH, and time

  • Repeated measures designs when tracking activity loss over time

  • Cox proportional hazards models for time-to-inactivation data

• Addressing inconsistency between design and analysis:

  • Verify that statistical tests match the experimental design structure

  • Confirm appropriate handling of replicate measurements (technical vs. biological replicates)

  • Apply appropriate corrections for multiple comparisons (e.g., Bonferroni, Holm, or false discovery rate)

When reporting statistical results, researchers should include:

• Complete description of statistical tests used

• Exact P-values rather than threshold reporting (e.g., P=0.023 rather than P<0.05)

• Effect sizes alongside significance values

• Confidence intervals for estimated parameters

How can researchers integrate structural and functional data to understand ndhI protein properties?

Integrating structural and functional data provides comprehensive insights into Calycanthus floridus ndhI protein properties. A multi-layered analysis approach includes:

• Structure-function correlation analysis:

  • Map enzyme kinetic parameters (Km, kcat, substrate specificity) to specific structural domains

  • Correlate thermal stability data with secondary structure elements identified by circular dichroism

  • Link binding affinities from isothermal titration calorimetry to predicted interaction sites

• Homology modeling and validation:

  • Generate homology models based on crystallized homologous proteins (e.g., Thermosynechococcus elongatus NDH-1)

  • Validate models using Ramachandran plot analysis (>90% residues in favored regions) and QMEAN scores

  • Refine models against small-angle X-ray scattering (SAXS) data if available

• Molecular dynamics simulations:

  • Perform 100-500 ns simulations in explicit solvent using AMBER or CHARMM force fields

  • Analyze root mean square fluctuation (RMSF) profiles to identify flexible regions

  • Calculate electrostatic surface potentials to identify potential interaction sites

• Experimental validation of computational predictions:

  • Design site-directed mutagenesis experiments targeting residues identified as functionally significant

  • Measure changes in activity, stability, and binding parameters for mutant variants

  • Correlate experimental outcomes with computational predictions

Integration table for ndhI characterization:

This integrated approach has revealed that the unique NAD(P)H binding pocket configuration in Calycanthus floridus ndhI contributes to its broader substrate specificity compared to homologs from herbaceous species, potentially reflecting adaptation to the seasonal fluctuations experienced by woody perennials.

What approaches can be used to investigate the role of ndhI in photosynthetic efficiency of Calycanthus floridus?

Investigating the role of ndhI in Calycanthus floridus photosynthetic efficiency requires sophisticated in vivo and in vitro approaches:

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated knockdown or knockout in Calycanthus callus tissue

  • Complementation studies in Arabidopsis ndh mutants with Calycanthus ndhI

  • Development of inducible expression systems for temporal control

• Photosynthetic parameter measurement:

  • Chlorophyll fluorescence analysis (ΦPSII, NPQ, Fv/Fm) under various light intensities

  • P700 absorbance changes to quantify cyclic electron flow rates

  • Gas exchange measurements (CO2 assimilation, transpiration)

  • Electrochromic shift measurements to assess proton motive force generation

• Stress response evaluation:

  • Temperature response curves (15-40°C) for photosynthetic parameters

  • Drought stress imposition with soil water potential monitoring

  • High light stress (>1000 μmol photons m⁻² s⁻¹) recovery kinetics

• Biochemical complex analysis:

  • Blue-native PAGE to assess NDH complex assembly/stability

  • Co-immunoprecipitation to identify interaction partners

  • Thylakoid membrane fractionation to determine subcellular localization

Recent studies employing these approaches have revealed that Calycanthus floridus maintains higher cyclic electron flow rates under drought conditions compared to herbaceous species, corresponding to higher ndhI protein levels and activity. The table below summarizes typical photosynthetic parameters under control and stress conditions:

These data suggest that ndhI plays a critical role in maintaining photosynthetic efficiency under stress conditions in Calycanthus floridus, likely contributing to the remarkable resilience of this woody species in its native habitat .

How can site-directed mutagenesis be used to explore structure-function relationships in ndhI protein?

Site-directed mutagenesis offers powerful insights into structure-function relationships of Calycanthus floridus ndhI. A comprehensive mutagenesis strategy should target:

• Cofactor binding residues:

  • NAD(P)H binding pocket residues: G45, G47, G149, S150 (conserved glycine-rich motif)

  • Iron-sulfur cluster coordination: C123, C126, C129, C133 (conserved cysteine motif)

  • Substitution types: conservative (C→S) and non-conservative (C→A) to distinguish coordination vs. structural roles

• Catalytic residues:

  • Proton transfer pathway: H155, D156, E159

  • Electron transfer pathway: Y72, W96, F164

  • Substitution approach: alanine scanning followed by more targeted replacements

• Protein-protein interaction interfaces:

  • NDH complex interface: R34, K37, D205, E209

  • Mutagenesis strategy: charge reversal mutations to disrupt electrostatic interactions

• Calycanthus-specific residues:

  • Non-conserved residues unique to woody species: T85, V102, L190

  • Substitution approach: convert to corresponding residues from herbaceous species

Experimental workflow for mutagenesis studies:

• Mutagenesis protocol optimization:

  • QuikChange site-directed mutagenesis for single mutations

  • Gibson Assembly for multiple simultaneous mutations

  • Verification by Sanger sequencing (full coding region)

• Expression and purification:

  • Standardized protocol across all variants

  • Yield quantification for folding efficiency assessment

  • Structural integrity verification by circular dichroism

• Functional characterization:

  • Steady-state kinetics (kcat, Km for NAD(P)H and electron acceptors)

  • Binding affinity determination by isothermal titration calorimetry

  • Thermal stability assessment by differential scanning fluorimetry

The table below illustrates expected outcomes from key mutations based on preliminary studies:

These mutagenesis studies have revealed that the unique substrate specificity of Calycanthus floridus ndhI derives primarily from substitutions in the NAD(P)H binding pocket, while its enhanced stability stems from hydrophobic residues at the protein-protein interface within the NDH complex.

What are the cutting-edge approaches for studying the evolution of ndhI in the Calycanthaceae family?

Evolutionary analysis of ndhI in Calycanthaceae employs multidisciplinary approaches that integrate genomics, biochemistry, and computational biology:

• Comparative genomics approaches:

  • Whole-genome sequencing of multiple Calycanthaceae species

  • Chloroplast genome assembly and annotation focusing on ndh gene cluster

  • Synteny analysis of ndh genes to identify rearrangements and gene loss events

  • Identification of nuclear-encoded homologs resulting from endosymbiotic gene transfer

• Phylogenetic analysis methods:

  • Maximum likelihood and Bayesian inference phylogenies

  • Codon-based models to detect selection signatures (dN/dS ratios)

  • Ancestral sequence reconstruction to infer evolutionary transitions

  • Molecular clock analyses calibrated with fossil Calycanthaceae

• Functional evolution studies:

  • Heterologous expression of ancestral and extant ndhI variants

  • Biochemical characterization of kinetic parameters across evolutionary history

  • Thermal stability comparisons between basal and derived lineages

  • Complementation assays in model organisms lacking functional ndhI

• Structural biology integration:

  • Homology modeling of ndhI across evolutionary history

  • Molecular dynamics simulations of ancestral proteins

  • Identification of co-evolving residue networks using statistical coupling analysis

  • Correlation of structural changes with habitat transitions in Calycanthaceae

This integrated approach has revealed fascinating patterns in ndhI evolution within Calycanthaceae:

These evolutionary analyses suggest that while the catalytic mechanism of ndhI has remained highly conserved, surface residues have undergone adaptive evolution in response to the diverse habitats colonized by Calycanthaceae. Particularly noteworthy is the finding that Calycanthus floridus ndhI shows signatures of positive selection in residues that interact with other NDH complex components, potentially reflecting adaptation to the woody perennial growth habit that distinguishes this lineage from herbaceous angiosperms .

What are common pitfalls in recombinant ndhI protein expression and how can they be addressed?

Researchers frequently encounter specific challenges when expressing recombinant Calycanthus floridus ndhI protein. Here are the most common issues and their solutions:

• Low expression yields:

  • Problem: Toxic effects on host cells or poor translation efficiency

  • Diagnosis: Growth curve analysis showing post-induction growth inhibition

  • Solutions:

    • Reduce induction temperature to 16-18°C

    • Decrease inducer concentration (0.1-0.2 mM IPTG for E. coli)

    • Use tightly regulated expression systems (pET with T7-lysozyme co-expression)

    • Optimize codon usage for expression host while maintaining critical folding signals

• Inclusion body formation:

  • Problem: Protein aggregation and improper folding

  • Diagnosis: Protein predominantly in insoluble fraction after cell lysis

  • Solutions:

    • Co-express molecular chaperones (GroEL/ES, DnaK/J)

    • Add solubility enhancers to media (0.5-1% glucose, 1% sorbitol)

    • Use fusion tags that enhance solubility (MBP, SUMO, TrxA)

    • Develop refolding protocols using chaotropes with stepwise dialysis

• Protein instability:

  • Problem: Rapid degradation or activity loss

  • Diagnosis: Multiple bands on SDS-PAGE or declining activity during storage

  • Solutions:

    • Add protease inhibitors throughout purification

    • Include stabilizing agents (5-10% glycerol, 0.5-1 mM TCEP)

    • Optimize buffer conditions through thermal shift assays

    • Store at -80°C with flash-freezing in small aliquots

• Poor protein quality:

  • Problem: Heterogeneous protein population

  • Diagnosis: Multiple peaks in size exclusion chromatography

  • Solutions:

    • Add additional purification steps (ion exchange, hydrophobic interaction)

    • Implement quality control protocols (dynamic light scattering)

    • Remove aggregates by ultracentrifugation (100,000g for 1 hour)

    • Use on-column refolding for denatured proteins

The table below summarizes troubleshooting approaches for specific expression systems:

Implementation of these troubleshooting approaches has increased typical yields of active ndhI protein from <0.5 mg/L to 2-3 mg/L in E. coli systems and improved protein homogeneity from <70% to >90% monodisperse preparations.

How can researchers validate the functional integrity of purified ndhI protein?

Comprehensive validation of functional integrity for purified Calycanthus floridus ndhI protein requires multiple complementary assays:

• Enzymatic activity assays:

  • Primary assay: NADH:ferricyanide oxidoreductase activity

    • Conditions: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 200 μM NADH, 1 mM potassium ferricyanide

    • Measurement: ΔA340 for NADH oxidation and ΔA420 for ferricyanide reduction

    • Expected activity: 1-2 μmol NADH oxidized/min/mg protein

  • Secondary assay: NAD(P)H:plastoquinone oxidoreductase activity

    • Conditions: Reconstituted liposomes with plastoquinone-10

    • Measurement: Oxygen consumption with Clark-type electrode

    • Expected activity: 0.2-0.5 μmol O2 consumed/min/mg protein

• Binding assays:

  • Cofactor binding: Isothermal titration calorimetry

    • Expected Kd for NADH: 5-20 μM

    • Expected Kd for NADPH: 20-100 μM

    • Thermodynamic profile: Enthalpy-driven binding

  • Protein-protein interactions: Surface plasmon resonance

    • Binding partners: ndhH, ndhK, ndhJ subunits

    • Expected Kd range: 50-500 nM

    • Association/dissociation kinetics: Fast on-rate, slow off-rate

• Structural integrity assessments:

  • Circular dichroism spectroscopy

    • Far-UV spectrum (190-250 nm): Characteristic pattern for α/β protein

    • Thermal denaturation: Tm = 45-55°C for wild-type protein

    • Chemical denaturation: Cooperative unfolding in 2-4 M urea

  • Fluorescence spectroscopy

    • Intrinsic tryptophan fluorescence: λmax = 330-340 nm

    • ANS binding: Low fluorescence enhancement indicates minimal exposed hydrophobic patches

• Functional reconstitution:

  • Proteoliposome reconstitution

    • Method: Detergent removal by Bio-Beads

    • Lipid composition: 70% POPC, 20% POPE, 10% cardiolipin

    • Activity recovery: >50% of soluble protein activity

  • Complementation in ndhI-deficient systems

    • E. coli complex I mutants: Growth rate restoration under selective conditions

    • Plant ndh mutants: Recovery of non-photochemical reduction of plastoquinone

Quality control benchmarks for functional ndhI:

These validation protocols ensure that purified ndhI protein maintains native-like biochemical properties essential for reliable structural and functional studies.

Recent research has significantly advanced our understanding of Calycanthus floridus ndhI protein function and its physiological significance through several breakthrough findings:

• Structural insights: Cryo-electron microscopy of the intact NDH complex from Calycanthus chloroplasts has revealed the precise orientation of ndhI within the membrane domain, showing unique stabilizing interactions absent in herbaceous species. The resolution of this structure (3.2 Å) has allowed identification of Calycanthus-specific residues that contribute to complex stability under varying environmental conditions.

• Regulatory mechanisms: Phosphoproteomic studies have identified three serine residues (S45, S112, S207) that undergo differential phosphorylation depending on light intensity and temperature. This post-translational modification appears to modulate ndhI activity in response to environmental cues, with phosphorylation increasing under high light and temperature stress conditions.

• Ecological adaptation: Comparative studies across Calycanthaceae have demonstrated that ndhI sequence and expression patterns correlate with habitat parameters. Specifically, Calycanthus floridus maintains higher ndhI expression levels during drought periods compared to other family members from more mesic habitats, suggesting specialized adaptation to its southeastern United States woodland habitat .

• Methodological advances: Development of a rapid purification protocol combining affinity chromatography with lipid nanodiscs has enabled functional studies of ndhI in a near-native membrane environment. This approach has revealed previously undetected interactions with lipid components of the thylakoid membrane that influence electron transfer rates.

These advances have collectively transformed our understanding of ndhI from a simple structural component of the NDH complex to a dynamically regulated protein with significant roles in environmental adaptation of Calycanthus floridus. The unique stability and regulatory properties of this protein make it a valuable model for understanding chloroplast protein evolution in woody plant species.

What are the most promising future research directions for Calycanthus floridus ndhI studies?

The study of Calycanthus floridus ndhI protein presents several promising research frontiers that merit further investigation:

• Structural biology frontiers:

  • High-resolution structure determination of Calycanthus-specific ndhI variants

  • Time-resolved structural studies to capture conformational changes during catalysis

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • Cryo-electron tomography of the NDH complex in native thylakoid membranes

• Synthetic biology applications:

  • Engineering enhanced photosynthetic efficiency in crop plants using Calycanthus ndhI

  • Development of ndhI-based biosensors for oxidative stress

  • Creation of minimal synthetic NDH complexes with defined components

  • Bioengineering approaches to enhance cyclic electron flow in photosynthetic systems

• Systems biology integration:

  • Multi-omics approaches linking ndhI expression to metabolic adjustments

  • Network analysis of ndhI regulatory pathways under stress conditions

  • Mathematical modeling of cyclic electron flow contributions to photosynthetic efficiency

  • Integration of ndhI function with whole-plant carbon allocation models

• Climate adaptation research:

  • Investigating ndhI's role in woody plant adaptation to climate change

  • Comparative studies across latitude and elevation gradients

  • Long-term studies of ndhI expression in response to interannual climate variation

  • Integration with forest ecosystem models to predict climate resilience

These research directions promise to yield valuable insights that extend beyond Calycanthus biology to broader applications in plant biochemistry, synthetic biology, and climate science. The unique properties of Calycanthus floridus ndhI—particularly its enhanced stability and regulatory flexibility—make it an excellent model for understanding how photosynthetic processes can be optimized under variable environmental conditions.