Recombinant Cuscuta gronovii ATP synthase subunit C, plastid (atpE)

What is Cuscuta gronovii and why is its ATP synthase subunit C significant for research?

Cuscuta gronovii (Common dodder) is a parasitic plant belonging to the genus Cuscuta within the Convolvulaceae family. As a parasitic plant, it obtains nutrients from host plants rather than through photosynthesis, though it retains chlorophyll and photosynthetic capability to varying degrees. The ATP synthase subunit C (atpE) is particularly significant because, despite the parasitic lifestyle of Cuscuta species, they retain functional photosynthetic genes including those encoding ATP synthase components . This retention suggests these genes remain under strong selective pressure even in parasitic contexts, making them excellent models for studying evolutionary adaptations in parasitic plants . The plastid atpE gene encodes a critical component of the chloroplast ATP synthase complex, which is essential for energy conversion during photosynthesis.

How does the atpE gene in Cuscuta gronovii compare to that in photosynthetic relatives?

The atpE gene in Cuscuta gronovii shows strong evolutionary conservation despite the parasitic lifestyle of this plant. Comparative analyses between Cuscuta species and non-parasitic relatives like Ipomoea purpurea (Morning Glory) reveal that photosynthetic genes, including those in the atp gene family, remain under strong selective constraint in Cuscuta . While some relaxation of selective pressure occurred before the evolution of parasitism in this lineage, photosynthesis-related genes continue to be highly conserved in Cuscuta species. This suggests that these genes serve critical functions beyond carbon fixation through the Calvin cycle . The full-length atpE protein in C. gronovii consists of 81 amino acids with the sequence: MNPIISAASVIAAGFAVGLASIGPGIGQGTAAGRAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFI .

What molecular characteristics define the recombinant C. gronovii atpE protein?

The recombinant C. gronovii ATP synthase subunit C protein is characterized by its:

• Complete protein length of 81 amino acids (residues 1-81)

• N-terminal His-tag for purification and detection purposes

• Expression in E. coli expression systems

• Hydrophobic regions typical of membrane-embedded proteins

• Conserved functional domains necessary for ATP synthesis

When produced recombinantly, the protein is typically supplied as a lyophilized powder with >90% purity as determined by SDS-PAGE analysis . For research applications, it can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with recommended addition of 5-50% glycerol for long-term storage .

How has parasitism influenced the evolution of plastid genes like atpE in Cuscuta species?

The evolution of parasitism in Cuscuta has had selective effects on plastid genes, though not as dramatic as might be expected. Research comparing plastid genomes of parasitic and non-parasitic species reveals:

• Selective retention of photosynthetic genes despite parasitism

• Loss of ndh genes (encoding NAD(P)H dehydrogenase) as the primary gene loss associated with the transition to parasitism in Cuscuta

• Differential gene loss between Cuscuta species, with some exhibiting more extensive genome reduction

Interestingly, analysis of selective constraint shows that photosynthetic genes, including atpE and other atp family genes, remain under stronger purifying selection than other plastid genes in Cuscuta species . This indicates functional importance beyond traditional photosynthesis. While there is some relaxation of selection compared to fully autotrophic plants, the pattern differs significantly from fully non-photosynthetic parasites like Epifagus virginiana, which has lost most photosynthetic genes .

Comparative analysis of selection rates between photosynthetic Ipomoea and Nicotiana relative to a common outgroup (Panax) shows significant differences in substitution rates for different gene classes:

This suggests relaxed selection on these genes began before the evolution of parasitism in the Convolvulaceae lineage .

What alternative functions might explain the retention of atpE and other photosynthetic genes in parasitic Cuscuta species?

The strong conservation of photosynthetic genes including atpE in Cuscuta species, despite reduced reliance on photosynthesis for carbohydrate production, suggests these genes serve alternative critical functions:

• Lipid biosynthesis pathway: Recent research indicates RuBisCo may function in lipid biosynthesis independently of its role in the Calvin cycle. This alternative pathway provides 20% more acetyl-CoA for fatty acid biosynthesis while requiring less than 15% of the ATP and NADPH needed for traditional photosynthesis .

• Carbon dioxide recycling: In C. reflexa, photosynthetic apparatus may facilitate recycling of respiratory carbon dioxide, as evidenced by decreased CO2 release in the presence of light .

• Seed development support: Chlorophyll is most concentrated in developing ovules and seeds of Cuscuta species, suggesting photosynthetic machinery may support efficient lipid allocation to seeds .

These functions may explain why the ATP synthase complex, including the atpE subunit, remains essential even as the primary photosynthetic function diminishes. The retention of photosynthetic genes in Cuscuta contrasts with their loss in completely non-photosynthetic parasites like Epifagus, suggesting that Cuscuta species maintain some level of photosynthetic activity that depends on a functional ATP synthase complex .

What are the optimal conditions for reconstitution and storage of recombinant C. gronovii atpE protein?

For optimal experimental outcomes when working with recombinant C. gronovii ATP synthase subunit C protein, researchers should follow these research-validated protocols:

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to ensure all material is at the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is standard) to prevent freeze-thaw damage

• Mix gently until completely dissolved

Storage Recommendations:

• For short-term use: Store working aliquots at 4°C for up to one week

• For long-term storage: Store at -20°C/-80°C in single-use aliquots

• Avoid repeated freeze-thaw cycles, which can compromise protein integrity

• Store in Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 for stability

These conditions are optimized to maintain the structural and functional integrity of the protein for research applications.

What experimental approaches are most effective for studying atpE function in parasitic plant biology?

To study the function of atpE in parasitic plant biology, several complementary experimental approaches have proven effective:

Comparative Genomic Analysis:

• Sequence comparisons of atpE between parasitic and non-parasitic relatives

• Analysis of selective constraints using dN/dS ratios

• Phylogenetic analysis to track gene evolution across parasitic lineages

Functional Studies:

• Heterologous expression systems to produce recombinant protein for biochemical characterization

• In vitro ATPase activity assays under varying conditions

• Reconstitution experiments in liposomes to study membrane integration and function

Structural Biology:

• X-ray crystallography or cryo-EM studies of the ATP synthase complex

• Molecular dynamics simulations to understand protein-membrane interactions

• Structure-function relationship studies using site-directed mutagenesis

Physiological and In Vivo Studies:

• Localization studies using tagged proteins or immunohistochemistry

• Metabolic studies comparing ATP production in different tissues

• Developmental expression analysis throughout the parasite life cycle

When designing experiments, researchers should consider the high hydrophobicity of the atpE protein and its membrane-embedded nature, which can present challenges for solubility and handling in aqueous solutions. The His-tagged recombinant version facilitates purification and detection in many experimental contexts .

How can researchers differentiate between photosynthetic and non-photosynthetic functions of atpE in Cuscuta species?

Differentiating between photosynthetic and non-photosynthetic functions of atpE in Cuscuta requires sophisticated experimental approaches:

Metabolic Labeling Studies:

• Use of 13C-labeled carbon dioxide to track carbon flow in light/dark conditions

• Comparative analysis between different tissues with varying chlorophyll content

• Pulse-chase experiments to determine ATP utilization pathways

Inhibitor Studies:

• Selective inhibition of photosynthetic electron transport (using DCMU)

• ATP synthase inhibitors (oligomycin) to block ATP production

• Comparative metabolomic analysis with and without inhibitors

Tissue-Specific Analysis:

• Isolation of plastids from green versus non-green tissues

• Analysis of atpE expression correlation with photosynthetic versus lipid synthesis genes

• Comparison of ATP synthase activity between reproductive tissues (high chlorophyll content) and vegetative tissues

Genetic Approaches:

• RNA interference (RNAi) or CRISPR-based knockdown/knockout of atpE

• Overexpression studies with detailed phenotypic characterization

• Complementation experiments in plants with compromised ATP synthase function

Research on Cuscuta species has shown that chlorophyll is most concentrated in developing ovules, seeds, and stressed seedlings, suggesting tissue-specific functions . Comparing these tissues metabolically could reveal distinct roles for ATP synthase in energy production versus biosynthetic pathways.

What insights can recombinant C. gronovii atpE provide about the transition from autotrophy to heterotrophy in parasitic plants?

Recombinant C. gronovii atpE offers a unique window into evolutionary processes during the transition from autotrophy to heterotrophy:

• Evolutionary Intermediates: Cuscuta species represent evolutionary intermediates that retain photosynthetic machinery despite parasitism, unlike completely non-photosynthetic parasites like Epifagus. Studies of atpE can reveal how key photosynthetic components are maintained during this transition .

• Selective Pressures: Analysis of sequence conservation in atpE compared to other plastid genes helps identify differential selection pressures. While ndh genes were lost early in Cuscuta evolution, atp genes remain under strong selection, indicating functional importance .

• Functional Repurposing: The retention of photosynthetic genes despite reduced photosynthesis suggests functional repurposing. Experimental studies with recombinant atpE can test hypotheses about alternative functions, such as supporting lipid biosynthesis .

• Taxonomic Comparisons: By comparing atpE characteristics across different Cuscuta species with varying photosynthetic capacities (from subgenera Monogyna, Cuscuta, and Grammica), researchers can track progressive changes during heterotrophic adaptation.

Experiments with the recombinant protein might include comparative binding studies with components of different metabolic pathways, activity assays under conditions mimicking those in parasitic plant tissues, and structural analyses to identify adaptations specific to the parasitic lifestyle.

How might C. gronovii atpE be used in synthetic biology applications for studying energy metabolism?

The unique characteristics of C. gronovii atpE make it a valuable component for synthetic biology applications focused on energy metabolism:

• Minimal ATP Synthase Models: As parasitic plants potentially maintain ATP synthase for specialized functions with minimal energy input, C. gronovii atpE could inform design of stripped-down, energy-efficient ATP synthase complexes for synthetic systems.

• Alternative Energy Coupling: Understanding how ATP synthase functions in plants with reduced photosynthetic capacity could inspire novel ways to couple different metabolic pathways to ATP production in engineered organisms.

• Membrane Protein Engineering: The sequence characteristics of C. gronovii atpE, evolved under unique selective pressures, may provide insights for engineering membrane proteins with enhanced stability or functional efficiency.

• Sensing and Regulatory Systems: ATP synthase components from parasitic plants could be adapted as biosensors or regulatory elements in synthetic pathways, especially those that need to function under energy-limited conditions.

Experimental approaches might include:

• Creating chimeric ATP synthase complexes with components from parasitic and non-parasitic organisms

• Engineering minimal synthetic membranes with reconstituted C. gronovii ATP synthase

• Developing reporter systems based on ATP synthase activity for metabolic engineering applications

These applications build on the natural "experiment" of parasitic plant evolution, where ATP synthase has been maintained under alternative selective pressures compared to photosynthetic plants.

What are the primary technical challenges in studying recombinant C. gronovii atpE protein?

Researchers face several technical challenges when working with recombinant C. gronovii atpE:

• Membrane Protein Challenges: As a highly hydrophobic membrane protein, atpE presents difficulties in:

  • Achieving proper folding during recombinant expression

  • Maintaining structural integrity during purification

  • Preventing aggregation in aqueous solutions

• Functional Reconstitution: To study native activity:

  • The protein must be properly integrated into lipid membranes

  • Assembly with other ATP synthase components may be necessary

  • Maintaining the proton gradient required for function presents technical hurdles

• Parasitic Plant Material Limitations:

  • Obtaining sufficient endogenous comparison material is difficult

  • Cuscuta species are challenging to cultivate without host plants

  • Different tissues show variable expression patterns

• Experimental Design Complications:

  • Distinguishing between photosynthetic and non-photosynthetic functions requires sophisticated approaches

  • Interpreting results in the context of parasitic lifestyle adds complexity

  • Potential plant-specific post-translational modifications may not be reproduced in E. coli expression systems

To address these challenges, researchers typically employ specialized techniques such as:

• Detergent screening for optimal solubilization

• Liposome reconstitution for functional studies

• Advanced imaging techniques to study membrane integration

• Host-parasite co-culture systems for comparative analyses

What emerging technologies could advance understanding of plastid ATP synthase function in parasitic plants?

Several cutting-edge technologies hold promise for advancing our understanding of plastid ATP synthase function in parasitic plants:

• Cryo-Electron Microscopy (Cryo-EM):

  • Allows visualization of ATP synthase structure in near-native conditions

  • Can reveal parasite-specific structural adaptations

  • Enables study of the complete ATP synthase complex architecture

• Single-Molecule Techniques:

  • Fluorescence resonance energy transfer (FRET) can track conformational changes

  • Optical tweezers can measure mechanical forces during ATP synthesis

  • Single-molecule tracking can reveal dynamics in membrane environments

• Advanced Metabolomics:

  • Stable isotope labeling to track metabolic fluxes

  • Mass spectrometry imaging to map metabolites in different tissues

  • Integration with transcriptomics for comprehensive pathway analysis

• CRISPR-Based Technologies:

  • Precise genome editing in Cuscuta species

  • Optogenetic control of ATP synthase components

  • Base editing for targeted mutagenesis of specific residues

• Computational Approaches:

  • Molecular dynamics simulations of membrane-protein interactions

  • Machine learning analysis of evolutionary patterns

  • Systems biology models of energy metabolism in parasitic contexts

These technologies, applied in combination, could reveal how ATP synthase function has been modified and maintained throughout the evolution of parasitism in plants, potentially uncovering novel biological principles relevant to both basic science and biotechnological applications.

How might comparative studies between different Cuscuta species inform our understanding of atpE evolution?

Comparative studies across Cuscuta species at different stages of parasitic adaptation offer powerful insights into atpE evolution:

• Subgeneric Comparisons: Cuscuta comprises multiple subgenera with varying degrees of photosynthetic capacity:

  • Subgenus Monogyna (e.g., C. exaltata) - retains visible chlorophyll throughout stems and inflorescences

  • Subgenus Cuscuta (e.g., C. europaea) - intermediate photosynthetic capacity

  • Subgenus Grammica (e.g., C. obtusiflora, C. gronovii) - chlorophyll mainly in reproductive structures

• Correlation with Genome Reduction: Research has shown differential gene loss between species:

  • C. exaltata has lost ndh genes but retained most photosynthetic genes

  • C. obtusiflora shows more extensive gene loss, including genes for plastid-encoded RNA polymerase

  • Both species retain all photosynthetic genes with only minor exceptions

• Tissue-Specific Expression Patterns: Comparing atpE expression across:

  • Green versus non-green tissues

  • Reproductive versus vegetative structures

  • Developmental stages from seedling to mature plant

• Selection Analysis Approaches:

  • Calculating dN/dS ratios across different branches of Cuscuta phylogeny

  • Codon-based tests for positive selection at specific sites

  • Analysis of coevolution between interacting protein components

Such comparative approaches could reveal whether atpE adaptation follows predictable patterns during heterotrophic transition or shows lineage-specific innovations. This evolutionary perspective provides context for interpreting experimental results with recombinant proteins and informs hypotheses about functional shifts in ATP synthase during parasitic adaptation.