Recombinant Buchnera aphidicola subsp. Acyrthosiphon pisum ATP synthase subunit c (atpE)

What is the basic structure and function of Buchnera aphidicola atpE protein?

The ATP synthase subunit c (atpE) from Buchnera aphidicola is a small hydrophobic membrane protein consisting of 79 amino acids with the sequence: MENLNVDMLYIAVAIMVGLASIGAAIGIGILGGKFLEGAARQPDLVPLLRTQFFVVMGLVDAIPMIAVGLGLYMLFAIS . It functions as a critical component of the F0 sector in ATP synthase, participating in proton translocation across the membrane during the process of oxidative phosphorylation. This protein forms the c-ring of the ATP synthase complex, which rotates as protons pass through the membrane, ultimately driving ATP synthesis. In the context of the Buchnera-aphid symbiotic relationship, this protein plays an essential role in energy metabolism that supports the obligate endosymbiont's functions.

How does the genetic composition of atpE in Buchnera aphidicola reflect evolutionary adaptations?

The atpE gene in Buchnera aphidicola exhibits characteristics consistent with genome reduction and AT-bias typical of long-term endosymbionts. Studies have shown marked AT enrichment in Buchnera compared to orthologous genes in E. coli . This AT bias impacts codon usage patterns and potentially amino acid composition while maintaining functional constraints on this essential protein. The conserved nature of atpE across Buchnera strains from different aphid hosts highlights the critical importance of ATP synthesis in maintaining the symbiotic relationship, even as the genome has undergone significant reduction through evolutionary time. This makes atpE an interesting subject for investigating selective pressures in endosymbiont evolution.

What are the optimal storage and handling conditions for recombinant atpE protein?

Recombinant atpE protein is typically supplied as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE . For optimal stability and activity:

• Store the unopened protein at -20°C/-80°C upon receipt.

• Before opening, briefly centrifuge the vial to bring contents to the bottom.

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• For long-term storage, add glycerol to a final concentration of 5-50% (50% is recommended) and store in aliquots at -20°C/-80°C.

• Working aliquots can be stored at 4°C for up to one week.

• Avoid repeated freeze-thaw cycles as they significantly reduce protein stability and activity .

These conditions are essential for maintaining protein integrity, particularly for membrane proteins like atpE that are prone to aggregation when improperly handled.

What expression systems are most effective for producing recombinant Buchnera aphidicola atpE?

The most effective expression system for recombinant Buchnera aphidicola atpE is heterologous expression in E. coli. The recombinant protein is typically expressed with an N-terminal His-tag to facilitate purification . Several methodological considerations improve expression efficiency:

• Vector selection: pET-series vectors under the control of T7 promoter provide high-level expression.

• Host strain optimization: E. coli strains designed for membrane protein expression (such as C41(DE3) or C43(DE3)) typically yield better results than standard BL21(DE3).

• Codon optimization: Since Buchnera has strong AT bias , codon optimization for E. coli expression may significantly improve yields.

• Induction conditions: Lower temperatures (16-25°C) and reduced IPTG concentrations (0.1-0.5 mM) often produce better results for membrane proteins like atpE.

• Growth media supplementation: Addition of glucose (0.5-1%) may help reduce basal expression before induction.

Expression in a full-length form (1-79 amino acids) has been successfully achieved , though expression of fusion proteins with solubility-enhancing tags may improve yields for certain applications.

What purification strategies yield the highest purity and functional integrity of recombinant atpE?

Optimal purification of recombinant His-tagged Buchnera aphidicola atpE involves multiple chromatographic steps while maintaining the protein in a suitable membrane-mimetic environment:

• Cell lysis and membrane preparation:

  • Sonication or French press in buffer containing protease inhibitors

  • Isolation of membrane fraction by ultracentrifugation (100,000 × g, 1 hour)

• Solubilization:

  • Careful selection of detergents (n-dodecyl β-D-maltoside, n-octyl-β-D-glucopyranoside, or digitonin)

  • Solubilization at 4°C with gentle stirring for 1-2 hours

• Immobilized Metal Affinity Chromatography (IMAC):

  • Binding to Ni-NTA resin in buffer containing selected detergent

  • Washing with increasing imidazole concentrations (20-50 mM)

  • Elution with higher imidazole (250-500 mM)

• Size Exclusion Chromatography:

  • Further purification on Superdex 200 column

  • Assessment of oligomeric state and protein homogeneity

This approach typically yields protein with greater than 90% purity as determined by SDS-PAGE , suitable for structural and functional studies.

How can researchers effectively reconstitute purified atpE into functional proteoliposomes?

Reconstitution of purified atpE into proteoliposomes for functional studies requires precise methodology:

• Lipid preparation:

  • Mixture of phosphatidylcholine and phosphatidic acid (3:1 ratio)

  • Formation of small unilamellar vesicles by sonication or extrusion

• Protein incorporation:

  • Mixing purified atpE with lipid vesicles at protein:lipid ratio of 1:50-1:100 (w/w)

  • Controlled detergent removal using:

    • Bio-Beads SM-2 adsorbent

    • Dialysis against detergent-free buffer

    • Cyclodextrin-mediated extraction

• Verification of reconstitution:

  • Freeze-fracture electron microscopy

  • Dynamic light scattering for size distribution

  • Sucrose density gradient centrifugation

• Functional assessment:

  • Proton conductance measurements using pH-sensitive fluorescent dyes

  • ATP synthesis assays when combined with F1 components

This reconstitution process is critical for studying the functional properties of atpE in a controlled membrane environment that mimics its native state in Buchnera.

How is atpE research contributing to our understanding of Buchnera-aphid symbiosis?

Research on Buchnera aphidicola atpE contributes to understanding the energetic basis of this obligate endosymbiotic relationship:

• Energy metabolism in symbiosis:
  Studies of ATP synthase components like atpE reveal how energy production in Buchnera supports metabolic integration with its aphid host. As a central component of oxidative phosphorylation, atpE function directly impacts the endosymbiont's ability to produce ATP needed for essential biosynthetic pathways benefiting the host.

• Metabolic integration with host systems:
  The expression and regulation of genes encoding ATP synthase components may respond to changes in host nutritional status . This coordination indicates fine-tuned metabolic integration between host and symbiont.

• Genome reduction consequences:
  Analysis of atpE structure and function provides insights into how essential molecular machines maintain functionality despite genome reduction and AT-bias in endosymbionts , revealing evolutionary constraints on energy metabolism genes.

• Comparative energetics across symbiotic systems:
  Studying atpE across different Buchnera strains from various aphid hosts enables comparative analysis of energy metabolism adaptations in diverse symbiotic relationships.

These studies collectively enhance our understanding of the energetic foundations underlying this ancient and intimate symbiotic relationship.

What methods are used to study transcriptional responses of atpE in relation to host nutritional status?

Several methodological approaches can be employed to investigate transcriptional responses of atpE to changes in host nutritional status:

• Experimental diet manipulations:

  • Rearing aphids on plants with varying nutritional content

  • Artificial diets supplemented with specific amino acids or other nutrients

  • Exposure to solutions of specific compounds (e.g., glutamine) through feeding on cut seedlings

• Transcript quantification techniques:

  • Quantitative RT-PCR with primers designed based on the Buchnera genome sequence

  • Full-genome microarray analysis

  • RNA-Seq for global transcriptomic profiling

• Normalization and reference genes:

  • Use of stable reference genes (e.g., rpsL encoding ribosomal protein) for accurate normalization

  • Multiple reference genes to improve reliability of expression measurements

• Correlation with metabolic parameters:

  • Parallel measurement of metabolite levels

  • Assessment of ATP synthesis rates

  • Correlation with carotenoid biosynthesis, which may share upstream precursors

These approaches allow researchers to assess how energy metabolism in Buchnera responds to changing host conditions, providing insights into the dynamic nature of this symbiotic relationship.

How does atpE function interface with carotenoid biosynthesis in the Buchnera-aphid system?

The relationship between ATP synthase (including atpE) and carotenoid biosynthesis reveals interesting metabolic connections in the Buchnera-aphid system:

• Shared metabolic precursors:
  Both ATP synthesis and carotenoid biosynthesis involve isoprenoid pathways. Specifically, geranylgeranyl diphosphate (GGPP) is a precursor for carotenoid synthesis , while the energy from ATP produced by ATP synthase containing atpE supports GGPP production.

• Differential expression patterns:
  Green morphs of Acyrthosiphon pisum contain higher levels of α-carotene, β-carotene, and γ-carotene, while red morphs additionally contain cis-torulene, trans-torulene, and 3,4-didehydrolycopene . These differences may correlate with different energetic requirements and potentially different regulation of energy metabolism genes including atpE.

• Regulatory interactions:
  Silencing GGPPS, which produces the carotenoid precursor GGPP, affects carotenoid levels and the expression of carotenoid biosynthesis genes . While direct links to ATP synthase expression have not been established in the provided references, the shared dependence on metabolic energy suggests potential coordination.

• Experimental approaches to study interactions:

  • RNAi targeting energy metabolism genes followed by assessment of carotenoid production

  • Metabolic flux analysis using isotope-labeled precursors

  • Comparative transcriptomics of energy metabolism and carotenoid biosynthesis genes

This research area represents an important frontier in understanding the integration of energy metabolism with specialized biosynthetic pathways in symbiotic systems.

What structural biology approaches are most effective for studying Buchnera aphidicola atpE?

Several structural biology approaches are particularly valuable for studying Buchnera aphidicola atpE, each offering unique insights:

• X-ray crystallography:

  • Requires purification of highly homogeneous protein

  • May involve lipidic cubic phase crystallization for membrane proteins

  • Provides high-resolution structural information but challenging for membrane proteins

• Cryo-electron microscopy (Cryo-EM):

  • Single-particle analysis of purified ATP synthase complexes containing atpE

  • No crystallization required, proteins visualized in near-native state

  • Increasingly achievable at near-atomic resolution

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Well-suited for small membrane proteins like atpE (79 amino acids)

  • Provides dynamics information not accessible by other methods

  • Can be performed in detergent micelles or lipid bicelles

• Molecular dynamics simulations:

  • In silico analysis of atpE behavior in membrane environments

  • Investigation of proton translocation mechanisms

  • Complements experimental structural data

• Cross-linking mass spectrometry:

  • Maps interaction interfaces between atpE and other ATP synthase subunits

  • Identifies spatial relationships within the assembled complex

The small size of atpE (79 amino acids) makes it amenable to several of these approaches, particularly NMR spectroscopy, which is well-suited for structural analysis of small membrane proteins.

What methods can assess the proton translocation function of recombinant atpE in vitro?

Assessing proton translocation function of recombinant atpE requires specialized techniques:

• Proteoliposome-based fluorescence assays:

  • Reconstitution of purified atpE into liposomes

  • Incorporation of pH-sensitive fluorophores (ACMA, pyranine)

  • Measurement of fluorescence changes in response to imposed pH gradients

  • Quantification of proton flux rates under various conditions

• Patch-clamp electrophysiology:

  • Formation of planar lipid bilayers containing reconstituted atpE

  • Direct measurement of ion currents across the membrane

  • Single-channel recordings to assess conductance properties

  • Determination of ion selectivity and voltage dependence

• Solid-supported membrane electrophysiology:

  • Adsorption of proteoliposomes onto functionalized gold sensors

  • Measurement of transient currents reflecting charge movement

  • High throughput screening of multiple experimental conditions

• ATP synthesis coupling assays:

  • Co-reconstitution of complete ATP synthase complex

  • Application of artificial proton gradient

  • Measurement of ATP production via luciferase assay

  • Assessment of coupling efficiency between proton translocation and ATP synthesis

These complementary approaches provide comprehensive assessment of atpE's functional properties in controlled experimental systems.

How can site-directed mutagenesis be used to identify critical residues in Buchnera aphidicola atpE?

Site-directed mutagenesis offers powerful insights into structure-function relationships in atpE through a systematic approach:

• Identification of target residues:

  • Conserved amino acids identified through sequence alignment

  • Residues predicted to line the proton translocation pathway

  • Amino acids at subunit interfaces within the c-ring

  • Residues potentially involved in interactions with other ATP synthase components

• Mutagenesis strategy:

  • Conservative substitutions (e.g., Asp→Glu) to test specific chemical properties

  • Radical substitutions (e.g., Asp→Ala) to completely remove functional groups

  • Scanning mutagenesis of consecutive residues in key regions

  • Introduction of reporter groups (e.g., Cys for disulfide cross-linking or fluorescent labeling)

• Functional assessment:

  • Expression and purification protocols as established for wild-type protein

  • Comparison of proton translocation activity between mutant and wild-type

  • Structural analysis to detect conformational changes

  • Assembly analysis to assess impact on c-ring formation

• Data integration and modeling:

  • Correlation of sequence, structure, and functional data

  • Computational modeling of mutation effects

  • Development of refined mechanistic models for proton translocation

This approach has been extensively applied to ATP synthase c-subunits from other organisms and can be adapted to identify unique features of Buchnera aphidicola atpE related to its endosymbiotic lifestyle.

How might transcriptional regulation of atpE differ in Buchnera compared to free-living bacteria?

The transcriptional regulation of atpE in Buchnera aphidicola likely differs substantially from free-living bacteria due to genome reduction and adaptation to the endosymbiotic lifestyle:

• Reduced regulatory networks:
  Buchnera has undergone extensive genome reduction, resulting in loss of many transcriptional regulators present in free-living bacteria . This reduction likely affects regulation of all genes, including those encoding ATP synthase components.

• Experimental approaches to study regulation:

  • Microarray or RNA-Seq analysis under different host nutritional conditions

  • Quantitative RT-PCR targeting atpE transcripts

  • Promoter analysis to identify potential regulatory elements

  • Comparison of expression patterns with co-regulated genes

• Host influence on regulation:
  Expression may respond to signals from the aphid host rather than direct sensing of environmental conditions. Experimental manipulations of host diet can be used to test this hypothesis , for example:

  • Feeding aphids with modified diets (nutrient supplementation or restriction)

  • Measuring Buchnera gene expression changes in response to host signals

• Potential regulatory mechanisms:

  • Post-transcriptional regulation may play a larger role than transcriptional control

  • Polycistronic organization with other ATP synthase genes may enable coordinated expression

  • Subtle changes in mRNA stability might influence effective transcript levels

Understanding these unique regulatory features would provide insights into how essential energy metabolism genes are controlled in this specialized symbiotic context.

What approaches can be used to study the assembly of ATP synthase complexes containing atpE in Buchnera?

Studying ATP synthase assembly in Buchnera presents unique challenges requiring specialized approaches:

• In vitro reconstitution studies:

  • Sequential addition of purified subunits, including recombinant atpE

  • Monitoring of complex formation by native gel electrophoresis

  • Assessment of functional activity at different assembly stages

  • Identification of assembly intermediates by mass spectrometry

• Fluorescent protein tagging and microscopy:

  • Development of genetic tools for fluorescent protein fusion in Buchnera

  • Visualization of ATP synthase assembly in bacteriocytes

  • Live-cell imaging to track assembly dynamics

  • Super-resolution microscopy to resolve individual complexes

• Cross-linking and interaction analysis:

  • Chemical cross-linking of assembled complexes

  • Mass spectrometric identification of cross-linked peptides

  • Mapping of subunit interaction interfaces

  • Comparison with ATP synthase assembly in model organisms

• Proteomic profiling:

  • Quantitative proteomics to measure stoichiometry of subunits

  • Analysis of complex composition under different conditions

  • Identification of potential assembly factors

These approaches would reveal whether Buchnera has evolved unique ATP synthase assembly pathways as part of its adaptation to the endosymbiotic lifestyle, potentially including simplified assembly mechanisms consistent with its reduced genome.

What experimental approaches could differentiate between host and symbiont ATP production in the Buchnera-aphid system?

Differentiating between host and symbiont ATP production requires sophisticated experimental designs:

• Selective inhibition approaches:

  • Application of inhibitors with differential effects on insect vs. bacterial ATP synthases

    • Oligomycin (primarily affects eukaryotic F1F0-ATP synthase)

    • DCCD (affects both but with different potencies)

  • Measurement of total ATP levels before and after inhibitor treatment

  • Calculation of relative contributions to total ATP pool

• Isotope labeling and tracking:

  • Feeding aphids with isotope-labeled glucose or other metabolites

  • Tracing incorporation into ATP molecules

  • Mass spectrometric distinction between differently labeled ATP pools

  • Analysis of labeling kinetics to determine production rates

• Subcellular fractionation:

  • Isolation of bacteriocytes from aphid tissues

  • Separation of Buchnera cells from host cytoplasm

  • Measurement of ATP synthesis capacity in each fraction

  • Assessment of ATP transport between compartments

• Genetic manipulation approaches:

  • RNAi targeting Buchnera ATP synthase components

  • Measurement of effects on total ATP levels

  • Analysis of compensatory responses in host energy metabolism

What are the key technical challenges in expressing and purifying functional recombinant atpE?

Expression and purification of functional recombinant Buchnera aphidicola atpE presents several technical challenges:

• Membrane protein expression barriers:

  • Potential toxicity to host cells when overexpressed

  • Proper membrane insertion and folding requirements

  • Formation of inclusion bodies rather than functional protein

• Solubilization and stability issues:

  • Finding optimal detergents for extraction without denaturation

  • Maintaining stability during purification procedures

  • Preventing aggregation during concentration steps

• Yield limitations:

  • Typically lower expression levels compared to soluble proteins

  • Losses during multiple purification steps

  • Challenges in scaling up production

• Functional verification complexities:

  • Difficulty assessing activity of isolated atpE subunit

  • Need for reconstitution into appropriate membranes

  • Complex assay systems required for functional testing

• Solutions and innovations:

  • Use of specialized expression strains (C41/C43)

  • Fusion partners to enhance solubility and folding

  • Screening multiple detergents and buffer conditions

  • Application of amphipols or nanodiscs for improved stability

  • Development of high-throughput screening for optimal conditions

When properly addressed, these challenges can be overcome to produce high-purity recombinant atpE with >90% purity suitable for structural and functional studies.

How can researchers effectively study protein-protein interactions involving atpE in the ATP synthase complex?

Several complementary techniques enable effective study of protein-protein interactions involving atpE:

• Chemical cross-linking coupled with mass spectrometry (XL-MS):

  • Application of bifunctional cross-linkers to stabilize transient interactions

  • Enzymatic digestion of cross-linked complexes

  • Mass spectrometric identification of cross-linked peptides

  • Computational modeling of interaction interfaces

  • Advantage: captures interactions in native or near-native environments

• Co-immunoprecipitation with tagged constructs:

  • Expression of His-tagged atpE or other tagged ATP synthase components

  • Precipitation with tag-specific antibodies

  • Identification of co-precipitated proteins by Western blotting or mass spectrometry

  • Advantage: relatively straightforward technique with good specificity

• Surface plasmon resonance (SPR) and biolayer interferometry (BLI):

  • Immobilization of purified atpE on sensor chips

  • Measurement of real-time binding kinetics with other subunits

  • Determination of affinity constants and binding dynamics

  • Advantage: provides quantitative binding parameters

• Förster resonance energy transfer (FRET):

  • Labeling of atpE and potential interaction partners with fluorophore pairs

  • Detection of energy transfer indicating close proximity

  • Live-cell applications possible with fluorescent protein fusions

  • Advantage: can detect interactions in living cells or reconstituted systems

These approaches collectively provide comprehensive characterization of the interaction network involving atpE within the ATP synthase complex.

What innovative approaches can overcome the challenges of studying uncultivable endosymbionts like Buchnera?

Innovative approaches to study uncultivable endosymbionts like Buchnera include:

• Advanced isolation techniques:

  • Development of methods to purify intact bacteriocytes from aphid tissues

  • Flow cytometry-based sorting of Buchnera cells

  • Microfluidic systems for manipulation of individual symbiont cells

  • Single-cell genomics and transcriptomics approaches

• Genetic manipulation strategies:

  • RNA interference (RNAi) through host-mediated delivery

  • Development of transformation protocols using specialized vectors

  • CRISPR-Cas delivery systems adapted for endosymbionts

  • Conditional expression systems responsive to external stimuli

• Ex vivo culture approximations:

  • Development of cell-free systems containing Buchnera components

  • Short-term maintenance of isolated bacteriocytes

  • Creation of artificial membrane systems mimicking host environment

  • Co-culture with insect cell lines to provide essential factors

• Heterologous expression systems:

  • Reconstruction of Buchnera metabolic pathways in E. coli

  • Expression of complete Buchnera protein complexes in culturable hosts

  • Creation of minimal cells incorporating essential Buchnera features

  • Synthetic biology approaches to recreate symbiotic functions

These innovative approaches are gradually overcoming the historical barriers to studying uncultivable endosymbionts, enabling deeper investigation of their biology and symbiotic relationships.

How might systems biology approaches integrate atpE function into comprehensive models of the Buchnera-aphid symbiosis?

Systems biology approaches offer powerful frameworks for integrating atpE function into holistic models of the Buchnera-aphid symbiosis:

• Genome-scale metabolic modeling:

  • Construction of stoichiometric models of Buchnera metabolism

  • Integration of ATP synthase activity (including atpE function) with metabolic pathways

  • Flux balance analysis to predict energy allocation across different functions

  • Multi-scale models incorporating both host and symbiont metabolism

• Network analysis approaches:

  • Co-expression network analysis incorporating atpE and other ATP synthase genes

  • Integration with carotenoid biosynthesis and amino acid production pathways

  • Identification of regulatory hubs coordinating energy production with biosynthetic activities

  • Comparison of network structures across different aphid species and their Buchnera symbionts

• Multi-omics data integration:

  • Correlation of atpE expression data with proteomics, metabolomics, and host transcriptomics

  • Development of statistical frameworks to identify causative relationships

  • Machine learning approaches to predict system responses to perturbations

  • Visualization tools for complex multi-layered data

• In silico prediction of energetic requirements:

  • Computational prediction of ATP needs for various Buchnera functions

  • Modeling of energy allocation under different host nutritional states

  • Simulation of evolutionary trajectories under different energetic constraints

These approaches will provide unprecedented insights into how ATP production via atpE-containing complexes is integrated into the broader symbiotic system.

What evolutionary questions about ATP synthase could be answered through comparative studies of Buchnera from different aphid species?

Comparative studies of ATP synthase components including atpE from Buchnera in different aphid species could address several fascinating evolutionary questions:

• Co-evolutionary dynamics:

  • Correlation between ATP synthase sequence divergence and host phylogeny

  • Identification of co-evolving residues between interacting subunits

  • Detection of parallel evolutionary changes in host mitochondrial ATP synthase

• Selective pressures on energy metabolism:

  • Analysis of dN/dS ratios to detect selection signatures on atpE and other components

  • Comparison of evolutionary rates between different ATP synthase subunits

  • Identification of subunit interfaces under stronger purifying selection

• Functional consequences of sequence divergence:

  • Structure-function analysis of atpE variants from different Buchnera strains

  • Experimental testing of ATP synthesis efficiency across variants

  • Correlation of sequence differences with host ecological niches

• Molecular clock applications:

  • Calibration of evolutionary rates using fossil-dated aphid divergences

  • Estimation of timing for key adaptive changes in ATP synthase

  • Comparison with other endosymbiont systems to identify convergent evolution

These comparative approaches would provide unique insights into the evolution of this essential molecular machine in the context of endosymbiosis.

How could understanding Buchnera atpE contribute to synthetic biology applications?

Understanding Buchnera atpE could inform several innovative synthetic biology applications:

• Minimalist ATP synthase design:

  • Using insights from Buchnera's streamlined ATP synthase to design simplified energy-producing modules

  • Creation of minimal ATP synthase complexes with reduced subunit composition

  • Engineering of more efficient c-rings based on Buchnera adaptations

• Synthetic endosymbiont development:

  • Design of artificial bacterial endosymbionts with optimized energy production

  • Engineering of ATP synthase variants that function efficiently in host cytoplasmic environments

  • Creation of controllable energy production modules for synthetic symbiotic systems

• Biomimetic nanotechnology:

  • Development of nanoscale rotary motors inspired by the F1F0-ATP synthase

  • Creation of artificial molecular machines with improved efficiency based on evolutionary insights

  • Design of proton-gradient powered devices for various applications

• Agricultural applications:

  • Engineering of beneficial microorganisms with enhanced energy efficiency

  • Development of targeted approaches to modify insect-microbe symbioses

  • Creation of synthetic pathways linking energy production to valuable metabolite synthesis

These applications represent the potential translation of fundamental research on Buchnera atpE into technologies addressing challenges in medicine, agriculture, and materials science.

Table 1: Comparative Analysis of Carotenoid Content in Red and Green Morphs of Acyrthosiphon pisum

*Appears to be tentatively identified carotenoids based on the asterisk notation in the source .

Table 2: Effects of GGPPS Silencing on Carotenoid Biosynthesis Genes in Acyrthosiphon pisum

Data derived from gene expression analysis following RNAi targeting of GGPPS in both red and green morphs of Acyrthosiphon pisum .

Table 3: Recombinant Buchnera aphidicola atpE Protein Specifications

Specifications for commercially available recombinant Buchnera aphidicola subsp. Acyrthosiphon pisum ATP synthase subunit c (atpE) protein with His-tag .