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

What is the Cuscuta exaltata ATP synthase subunit C (atpE) and why is it significant for research?

The ATP synthase subunit C, encoded by the atpE gene in the plastid genome of Cuscuta exaltata, is a component of the ATP synthase complex that catalyzes ATP synthesis during photosynthesis. This protein is particularly significant for research because C. exaltata, unlike most other Cuscuta species, retains a more complete set of photosynthesis-related genes in its chloroplast genome. While most Cuscuta species have lost photosynthetic capacity to various extents during their evolution as parasitic plants, C. exaltata maintains 67 genes in its chloroplast genome, including some photosynthesis-related genes that have been lost in other Cuscuta species .

The unique evolutionary status of C. exaltata makes its atpE gene an important model for studying the transition from autotrophy to heterotrophy in parasitic plants. As a parasitic plant that retains more photosynthetic machinery than its relatives, C. exaltata provides insights into the selective pressures that maintain certain plastid genes even as photosynthetic function diminishes. Studying this protein can help researchers understand the minimum requirements for chloroplast function in plants transitioning to a parasitic lifestyle.

How does C. exaltata atpE differ from atpE in non-parasitic plants?

While the search results don't provide specific sequence information about C. exaltata atpE compared to non-parasitic plants, several important differences can be inferred based on the evolutionary context:

First, C. exaltata has retained more chloroplast genes (67 total) than other Cuscuta species, with some species having as few as 31-33 genes . This suggests that C. exaltata represents an intermediate stage in the evolutionary trajectory toward full parasitism, likely reflected in its atpE gene structure and function.

Second, phylogenetic analyses based on conservative single-copy genes show that C. exaltata clusters with other Cuscuta species, suggesting that despite its retention of more photosynthesis-related genes, its genetic makeup still aligns with its taxonomic classification as a parasitic plant . This indicates that even retained genes like atpE have likely undergone parasitism-specific adaptations.

Third, the evolutionary pressures on photosynthesis-related genes in parasitic plants differ from those in autotrophic plants. In parasitic plants, these genes may be under relaxed selective pressure or may be maintained for alternative functions beyond photosynthesis, potentially resulting in sequence and functional divergence in the atpE gene.

What is the role of ATP synthase in parasitic plants like C. exaltata?

Several hypotheses exist regarding the role of ATP synthase in parasitic plants:

• Reduced photosynthetic activity: C. exaltata may maintain limited photosynthetic capacity, requiring functional ATP synthase but with reduced activity compared to fully autotrophic plants.

• Alternative energy metabolism: ATP synthase may function in energy conservation pathways that are not directly linked to photosynthesis, potentially utilizing alternative sources of proton gradients.

• Structural roles: The ATP synthase complex might be retained for its structural contributions to thylakoid membrane integrity even if its energy production role is diminished.

• Developmental regulation: ATP synthase function may be important during specific developmental stages, such as early growth before host attachment is established.

Comparative analysis shows that C. exaltata has retained more photosynthesis-related genes than other Cuscuta species , suggesting it may maintain more photosynthetic functionality than more specialized parasites, potentially including more active ATP synthase complexes.

How do structural modifications in C. exaltata atpE correlate with its functional adaptations in a parasitic lifestyle?

The structural characteristics of ATP synthase subunit C in C. exaltata likely reflect evolutionary adaptations to its parasitic lifestyle. While detailed structural information specific to C. exaltata atpE is not provided in the search results, researchers would approach this question through comparative structural biology methods.

Key structural features that might show adaptations include:

• Transmembrane domains: Potential modifications in hydrophobicity profiles or membrane-spanning regions that could affect proton channeling or c-ring formation.

• Proton-binding sites: Conservation or alteration of the critical glutamate residue involved in proton translocation, which would directly impact ATP synthesis capacity.

• Subunit interaction interfaces: Changes in regions that interact with other ATP synthase components, potentially affecting complex assembly or stability.

• Regulatory sites: Modifications in regions subject to regulation, which might reflect altered regulatory mechanisms in parasitic metabolism.

These structural adaptations would be analyzed in the context of C. exaltata's intermediate position in parasitic evolution, as it retains more chloroplast genes (67) than highly specialized Cuscuta species with as few as 31-33 genes . This comparative approach would help identify structural modifications that correlate with the shift from autotrophy to heterotrophy.

What evolutionary pressures might explain the retention of atpE in C. exaltata while other photosynthesis-related genes have been lost?

The retention of atpE in C. exaltata while other photosynthesis-related genes have been lost in many Cuscuta species presents a fascinating evolutionary puzzle. Several hypotheses might explain this retention:

• Functional repurposing: The ATP synthase complex may serve additional functions beyond photosynthetic ATP production, providing selective pressure to maintain atpE even as photosynthesis diminishes.

• Differential rate of gene loss: The loss of chloroplast genes in parasitic plants follows a pattern, with certain gene categories being lost earlier than others. ATP synthase genes might be among the later genes to be lost in the evolutionary trajectory.

• Structural necessity: The ATP synthase complex might be structurally necessary for maintaining chloroplast membrane integrity or other essential functions, even in the absence of photosynthesis.

• Incomplete transition: C. exaltata may represent an intermediate evolutionary stage in the transition to full heterotrophy, retaining partial photosynthetic capacity that requires functional ATP synthase.

The finding that C. exaltata maintains 67 chloroplast genes compared to as few as 31 in other Cuscuta species supports the idea that it represents an earlier stage in the evolutionary reduction of the photosynthetic apparatus . Comparative genomic analysis across multiple Cuscuta species at different stages of parasitic adaptation would be necessary to test these hypotheses.

How can phylogenetic analysis of atpE contribute to understanding the evolutionary history of parasitism in Cuscuta?

Phylogenetic analysis of atpE can provide valuable insights into the evolutionary history of parasitism in Cuscuta through several analytical approaches:

• Divergence timing analysis:

  • Molecular clock approaches can estimate when atpE began to diverge from photosynthetic ancestors

  • Correlation with estimated timing of parasitic adaptation in different Cuscuta lineages

  • Identification of whether atpE evolution accelerated during the transition to parasitism

• Selection signature mapping:

  • Branch-site models to identify episodic selection during parasitic transition

  • Sliding window analysis to identify domains under different selective pressures

  • Comparison with selection patterns in other ATP synthase subunits

• Co-evolutionary analysis:

  • Correlation of atpE evolution with other retained plastid genes

  • Identification of compensatory mutations that maintain protein-protein interactions

  • Detection of co-evolving sites within the protein structure

The search results note that phylogenetic trees constructed based on conservative single-copy gene sequences are more credible than complete genome-based trees . For atpE specifically, its retention in C. exaltata while being potentially lost in more specialized Cuscuta species provides a valuable marker for tracking the progression of plastid genome reduction during parasitic adaptation.

What are the optimal conditions for recombinant expression of C. exaltata atpE protein?

For recombinant expression of C. exaltata ATP synthase subunit C (atpE), researchers should consider the following optimized protocol:

• Expression system selection:

  • For structural studies: E. coli expression systems (BL21(DE3) or C41/C43(DE3) strains specifically designed for membrane proteins)

  • For functional studies: Consider chloroplast-targeted expression in a plant system like Nicotiana benthamiana

• Expression construct design:

  • Codon optimization for the chosen expression system

  • Addition of appropriate tags (His6, Strep-tag II) for purification

  • Consider fusion partners (MBP, SUMO) to enhance solubility

  • Include TEV or PreScission protease sites for tag removal

• Expression conditions:

  • Induction: Low IPTG concentration (0.1-0.5 mM) for E. coli

  • Temperature: Lower temperature (16-20°C) for membrane protein expression

  • Media supplementation: Consider supplementing with lipids to assist membrane protein folding

  • Duration: Extended expression time (16-20 hours) at lower temperatures

• Membrane fraction isolation:

  • Gentle lysis methods (osmotic shock or mild detergents)

  • Differential centrifugation to isolate membrane fractions

  • Solubilization using detergents compatible with ATP synthase structure (DDM, LMNG)

The optimization process would involve testing multiple expression constructs and conditions, followed by validation of proper folding through activity assays. Given the challenges of membrane protein expression, researchers might need to explore advanced approaches such as cell-free expression systems or nanodiscs for stabilizing the protein in a membrane-like environment.

What experimental designs are most appropriate for studying the function of recombinant C. exaltata atpE in vitro?

Studying the function of recombinant C. exaltata ATP synthase subunit C in vitro requires specialized experimental designs that address its role in the ATP synthase complex. Appropriate approaches include:

• Reconstitution systems:

  • Liposome reconstitution with defined lipid composition

  • Co-reconstitution with other ATP synthase subunits

  • Proton gradient establishment using pH jumps or light-driven pumps

• Functional assays:

  • ATP synthesis measurements using luciferase-based assays

  • Proton transport assays using pH-sensitive fluorescent dyes

  • ATP hydrolysis measurements with coupled enzyme assays

  • Membrane potential measurements using voltage-sensitive dyes

• Biophysical characterization:

  • Surface plasmon resonance to study interactions with other subunits

  • Isothermal titration calorimetry for binding energetics

  • Fluorescence spectroscopy to monitor conformational changes

• Comparative functional analysis:

  • Side-by-side comparison with atpE from photosynthetic plants

  • Analysis of chimeric proteins with domains from different species

  • Mutagenesis of key residues identified through comparative sequence analysis

Experimental design should include appropriate controls, such as denatured protein samples, inhibitor treatments (oligomycin, DCCD), and comparison with well-characterized ATP synthase components from model organisms. Statistical analysis should account for technical variability in membrane protein assays, with multiple independent protein preparations to ensure reproducibility.

What controls should be included when studying the enzymatic activity of recombinant C. exaltata atpE?

Rigorous experimental controls are essential when studying the enzymatic activity of recombinant C. exaltata ATP synthase subunit C to ensure valid and interpretable results:

Positive controls:

• Well-characterized ATP synthase subunit C from model organisms (E. coli, spinach chloroplast)

• Commercially available F1F0-ATP synthase for activity benchmarking

• Synthetic proton gradient systems with known proton flux rates

Negative controls:

• Heat-denatured C. exaltata atpE protein

• Site-directed mutants with alterations to critical functional residues

• Samples treated with specific inhibitors:

  • DCCD (N,N'-dicyclohexylcarbodiimide) - covalently binds to the critical carboxyl residue in subunit c

  • Oligomycin - binds to the interface between subunit c and a

  • Venturicidin - interacts with the c-ring

System controls:

• Liposomes/nanodiscs without incorporated protein

• No-gradient conditions (equal pH on both sides of membrane)

• ATP hydrolysis controls to account for non-specific activity

Technical controls:

• Multiple independent protein preparations to assess batch-to-batch variability

• Concentration gradients to establish reaction kinetics

• Time-course measurements to ensure linearity of activity assays

For publication-quality data, researchers should report activity measurements from at least three independent protein preparations, with appropriate statistical analysis of variability. Functional differences between C. exaltata atpE and counterparts from photosynthetic plants should be validated using multiple complementary assay methods.

How does C. exaltata atpE compare structurally and functionally with atpE from photosynthetic plants?

Comparing C. exaltata atpE with its counterparts in photosynthetic plants reveals important evolutionary adaptations related to the parasitic lifestyle. While specific structural information for C. exaltata atpE is not provided in the search results, we can outline the approach to this comparative analysis:

Structural comparison:

Functional comparison:

• ATP synthesis rates: Likely reduced in C. exaltata compared to photosynthetic plants, reflecting diminished photosynthetic activity

• Proton translocation efficiency: Potentially modified to accommodate lower energy demands

• Regulatory properties: May show altered response to regulatory factors reflecting integration with host metabolism

• Stability and turnover: Possibly different protein stability reflecting altered selective pressures

Phylogenetic analysis indicates that despite maintaining more genes than other Cuscuta species, C. exaltata still clusters with parasitic plants when conservative single-copy genes are analyzed , suggesting functional adaptations while maintaining core structural features of the ATP synthase complex.

What insights can be gained from comparing atpE sequence conservation across Cuscuta species at different stages of parasitic adaptation?

Comparative analysis of atpE across Cuscuta species at different stages of parasitic adaptation provides a window into the evolutionary trajectory of plastid genes during the transition to heterotrophy. While specific atpE sequence data across multiple Cuscuta species is not provided in the search results, we can outline the expected patterns and their significance:

Expected sequence conservation patterns:

The chloroplast genome analysis of Cuscuta species reveals progressive gene loss associated with increased specialization for parasitism . The patterns of atpE conservation or modification would provide insights into the selective pressures that maintain certain photosynthesis-related genes even as others are lost. This evolutionary window offers a unique opportunity to understand the minimum genetic requirements for maintaining plastid function in the absence of photosynthesis.

The search results indicate that in Cuscuta species, the loss of genes involved in photosynthesis happened gradually, with different species losing photosynthetic capability to various extents . Comparing atpE across this evolutionary gradient would help determine whether its retention in C. exaltata represents an intermediate stage or reflects essential non-photosynthetic functions.

How does the chloroplast genome organization in C. exaltata influence atpE expression and function?

The organization of the chloroplast genome in C. exaltata provides important context for understanding atpE expression and function in this parasitic plant. According to the search results, the chloroplast genome of Ipomoea pes-caprae (a related non-parasitic species in Convolvulaceae) is 161,667 bp with a GC content of 37.56%, containing 141 genes . While specific information about C. exaltata's chloroplast genome organization isn't provided, we know it retains 67 genes, which is fewer than non-parasitic relatives but more than other Cuscuta species .

Several aspects of chloroplast genome organization likely influence atpE expression and function:

• Gene synteny and operons: In most plants, atpE is part of a polycistronic operon that includes other ATP synthase subunit genes. The maintenance or disruption of this operon structure in C. exaltata would directly affect atpE expression.

• Promoter regions: Changes in promoter sequences could alter transcriptional regulation of atpE, potentially adapting expression to parasitic metabolism.

• Intergenic spacers: Modifications in regions between genes might affect mRNA processing and stability, influencing atpE expression levels.

• RNA editing sites: Alterations in RNA editing patterns could modify the final protein sequence, potentially compensating for genomic mutations.

• Repeat sequences: The search results indicate that related plants contain numerous sequence repeats in their chloroplast genomes, with 35 SSRs identified in I. pes-caprae . These repetitive elements might influence genome stability and gene expression in C. exaltata as well.

Understanding these genomic context features is essential for interpreting atpE expression and function in C. exaltata, particularly in comparison with both photosynthetic relatives and more specialized parasitic species.

What are the key challenges in designing primers for PCR amplification of C. exaltata atpE?

Designing effective primers for PCR amplification of C. exaltata atpE presents several challenges that researchers must address:

• Sequence divergence considerations:

  • Parasitic plants like Cuscuta often show accelerated evolution in plastid genes

  • Conventional primers based on photosynthetic plant sequences may fail

  • Optimal approach: Design degenerate primers based on aligned sequences from multiple Cuscuta species and close relatives

• AT-rich regions challenges:

  • Chloroplast genomes typically have high AT content (related Convolvulaceae have approximately 37.56% GC content in the chloroplast genome)

  • AT-rich regions can lead to lower primer stability and specificity

  • Strategies include: increasing primer length (25-30 nucleotides), adding GC clamps, and optimizing annealing temperatures

• Secondary structure interference:

  • Membrane protein genes often contain regions of high hydrophobicity

  • These regions can form stable secondary structures that impede PCR

  • In silico analysis of potential secondary structures before primer design is essential

• Optimization recommendations:

  • Use touchdown PCR protocols to improve specificity

  • Consider adding PCR enhancers (DMSO, betaine) to disrupt secondary structures

  • Validate primers on genomic DNA before attempting cDNA amplification

  • Sequence verify all PCR products before proceeding to cloning

A practical approach would be to design multiple primer pairs targeting different regions of the gene and test them empirically. Additionally, nested PCR approaches may be necessary if initial amplification yields are low, which is common when working with divergent genes from non-model organisms.

What are the key considerations for designing site-directed mutagenesis experiments with C. exaltata atpE?

Designing effective site-directed mutagenesis experiments for C. exaltata atpE requires careful planning to target functionally significant residues and interpret the resulting phenotypes:

• Target residue selection strategies:

  • Evolutionary conservation analysis: Identify unusually conserved or divergent residues compared to photosynthetic plants

  • Structural modeling: Focus on residues in functional domains (proton binding, c-ring interface, lipid interaction)

  • Comparative analysis: Target residues that differ between C. exaltata and other Cuscuta species with fewer chloroplast genes

• Mutation design principles:

  • Conservative substitutions: Ala scanning or similar-property substitutions to assess contribution to function

  • Non-conservative substitutions: Charge reversals or polarity changes to disrupt specific interactions

  • Natural variation mimicking: Introducing residues found in other parasitic plants to test evolutionary hypotheses

• Experimental design considerations:

  Mutation Category	Example	Expected Outcome	Control Comparison
  Proton binding site	Glu → Gln substitution	Loss of proton transport	Wild-type activity restoration with reduced pH
  c-ring interface	Gly → Val substitution	Disrupted oligomerization	Size exclusion chromatography profile comparison
  Lipid interaction	Leu → Lys substitution	Altered membrane association	Membrane partitioning assay comparison
  Regulatory site	Ser → Ala substitution	Modified regulation	Phosphorylation assay comparison

• Validation approaches:

  • Expression level verification to ensure altered function isn't due to protein instability

  • Structural validation using circular dichroism or limited proteolysis

  • Multiple functional assays to comprehensively characterize phenotypes

  • Rescue experiments with compensatory mutations to confirm interaction mechanisms

Site-directed mutagenesis experiments should be conducted as part of a larger experimental program that includes comparative studies with atpE from photosynthetic plants and other Cuscuta species. This approach would help distinguish adaptations specific to C. exaltata from general features of ATP synthase function.

What purification strategies are most effective for recombinant C. exaltata atpE?

Purification of recombinant C. exaltata ATP synthase subunit C requires specialized approaches due to its hydrophobic nature as a membrane protein component. An effective purification strategy would include:

• Membrane solubilization:

  • Initial screening of detergents (DDM, LMNG, Digitonin) for optimal solubilization

  • Detergent concentration optimization to maintain protein stability

  • Inclusion of lipids (POPC, cardiolipin) to stabilize the protein

• Affinity chromatography:

  • IMAC (Immobilized Metal Affinity Chromatography) for His-tagged constructs

  • Strep-Tactin chromatography for Strep-tagged constructs

  • Careful optimization of imidazole or desthiobiotin elution gradients

• Secondary purification steps:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Ion exchange chromatography for additional purity if needed

• Protein quality assessment:

  • SDS-PAGE and Western blotting to confirm identity

  • Circular dichroism to verify secondary structure

  • Mass spectrometry for accurate mass determination and post-translational modification analysis

• Functional validation:

  • Reconstitution into liposomes or nanodiscs for functional studies

  • ATPase activity assays to confirm functional folding

For structural studies, additional considerations include detergent exchange procedures optimized for crystallization or cryo-EM sample preparation. The purification protocol would need to be validated for yield, purity, homogeneity, and retention of functional properties before proceeding to detailed characterization studies.

How can structural studies of C. exaltata atpE contribute to understanding energy parasitism in plants?

Structural studies of C. exaltata ATP synthase subunit C can provide unique insights into the molecular basis of energy parasitism in plants through several research avenues:

• Structural adaptation mechanisms:

  • Crystallographic or cryo-EM structures would reveal whether C. exaltata atpE retains canonical ATP synthase architecture or has evolved structural modifications

  • Molecular dynamics simulations could identify altered dynamics relevant to reduced photosynthetic function

  • Comparison with structures from photosynthetic plants would highlight parasitism-specific adaptations

• Protein-protein interaction landscape:

  • Structural characterization of interactions with other ATP synthase subunits

  • Identification of modified interfaces that may reflect altered complex assembly or regulation

  • Potential discovery of novel protein interactions unique to parasitic plants

• Functional insights from structure:

  • Correlation of structural features with altered catalytic properties

  • Identification of modified proton translocation pathways

  • Structural basis for potential repurposed functions in parasitic metabolism

The unique position of C. exaltata as a parasitic plant that retains more chloroplast genes (67) than other Cuscuta species makes its ATP synthase components particularly valuable for understanding the structural transitions that accompany the evolution of parasitism. Structural studies could reveal whether C. exaltata represents an intermediate stage with partially modified ATP synthase architecture, providing insights into the progressive adaptation of energy metabolism during the transition to heterotrophy.

How might understanding C. exaltata atpE inform strategies for controlling parasitic plants?

Knowledge of C. exaltata atpE and its role in parasitic plant metabolism could potentially inform novel strategies for controlling parasitic plants, which represent significant agricultural challenges worldwide. Understanding the molecular adaptations in ATP synthase could lead to targeted approaches:

• Target identification:

  • If atpE in parasitic plants has unique structural or functional features compared to host plants, these differences could be exploited for selective inhibition

  • Comparative analysis of binding sites could inform the design of parasite-specific inhibitors

• Vulnerability assessment:

  • Understanding the role of ATP synthase in parasitic plant energy metabolism might reveal metabolic vulnerabilities

  • If C. exaltata and other parasitic plants depend on ATP synthase function during critical life stages (e.g., germination or initial host attachment), this could present an intervention point

• Biocontrol approaches:

  • Knowledge of parasitic plant biology can inform biological control strategies, as outlined in search result

  • Understanding ATP synthase function in parasitic plants could help assess their vulnerabilities to various environmental conditions, potentially informing timing of control measures

The search results indicate that biological control is environmentally friendly, reduces pesticide use, reduces contamination, reduces health risks, is relatively cheap, and is self-sustaining . Molecular insights into parasitic plant metabolism, including the function of atpE, could enhance the effectiveness of biological control by identifying specific vulnerabilities in parasitic plant physiology.

What future research directions could build upon our understanding of C. exaltata atpE?

Research on C. exaltata ATP synthase subunit C opens several promising future directions that could significantly advance our understanding of parasitic plant biology and chloroplast evolution:

• Comparative genomics expansion:

  • Sequencing additional Cuscuta species to create a finer-grained evolutionary series

  • Multi-gene analysis to identify co-evolutionary patterns among ATP synthase subunits

  • Pan-parasitic plant analysis to identify convergent adaptations in ATP synthase components

• Functional diversification investigation:

  • Exploration of potential alternative functions for ATP synthase in parasitic plants

  • Investigation of interactions with host plant metabolism

  • Analysis of tissue-specific expression patterns across the parasitic plant body

• Synthetic biology approaches:

  • Development of minimal ATP synthase systems incorporating C. exaltata components

  • Engineering chimeric ATP synthases to identify functionally important adaptations

  • Creation of model systems to study the transition from autotrophy to heterotrophy

• Ecological studies:

  • Investigation of how ATP synthase function correlates with host specificity and parasitic capacity

  • Analysis of environmental factors that influence gene expression in parasitic plants

  • Exploration of how ATP synthase function relates to success in different ecological niches

The search results indicate that C. exaltata represents a valuable model for studying the evolutionary transition to parasitism, as it retains more chloroplast genes than other Cuscuta species while still clustering phylogenetically with parasitic plants . Future research that integrates structural, functional, and evolutionary approaches would provide comprehensive insights into the molecular mechanisms underlying this fascinating biological transition.