Recombinant Agrostis stolonifera ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c in chloroplasts and what is its role in photosynthetic metabolism?

ATP synthase subunit c is a key component of the F₀ channel in the chloroplastic ATP synthase complex. It forms a homomeric ring structure (c-ring) embedded within the thylakoid membrane that plays a direct role in proton translocation across the membrane. This c-ring consists of multiple c-subunits (typically 10-14) and forms the central stalk rotor element together with the F₁ delta and epsilon subunits . During photosynthesis, the rotation of this c-ring is mechanically coupled to ATP synthesis, which provides the necessary energy for photosynthetic metabolism .

The subunit c functions as part of the F-type ATP synthase, which consists of two major structural domains: the F₁ domain containing the extramembraneous catalytic core and the F₀ domain containing the membrane proton channel. These domains are linked by a central stalk and a peripheral stalk . ATP synthesis occurs when protons flow through the F₀ domain along an electrochemical gradient, driving the rotation of the c-ring, which in turn drives the rotation of the γ-stalk in the F₁ region, ultimately catalyzing the ADP + Pi → ATP reaction .

Why is recombinant production of ATP synthase subunit c significant for research applications?

Recombinant production of ATP synthase subunit c is crucial for research for several key reasons:

• Investigation of stoichiometric variation: The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits per oligomeric ring, which differs among organisms . Recombinant production allows researchers to investigate factors affecting this stoichiometric variation.

• Structural studies: Pure, homogeneous samples are essential for detailed structural analysis using techniques like X-ray crystallography or cryo-electron microscopy.

• Functional studies: Recombinant expression enables site-directed mutagenesis to study structure-function relationships.

• Reconstitution experiments: Purified recombinant c₁ subunits can be used in reconstitution experiments to form c-rings in vitro, allowing the study of assembly mechanisms .

• Overcoming limited natural abundance: Natural sources often provide insufficient quantities of the protein for extensive research.

The recombinant approach solves the challenge of studying the highly hydrophobic ATP synthase subunit c by enabling its production in significant quantities with the correct alpha-helical secondary structure .

What molecular characteristics distinguish ATP synthase subunit c from Agrostis stolonifera?

While specific data on Agrostis stolonifera (creeping bentgrass) ATP synthase subunit c is limited in the search results, we can draw comparisons with other plant ATP synthase c subunits, particularly from rice (Oryza sativa) and spinach (Spinacia oleracea), which share similar characteristics as they are all chloroplastic proteins.

The ATP synthase subunit c from chloroplasts typically has the following characteristics:

In creeping bentgrass (Agrostis stolonifera), as with other grasses, the ATP synthase subunit c would be expected to share these general characteristics while potentially exhibiting species-specific variations in amino acid sequence that might influence its assembly, stability, or functional properties .

How does the c-ring stoichiometry affect the bioenergetic efficiency of ATP synthesis?

The c-ring stoichiometry has profound implications for the bioenergetic efficiency of ATP synthesis. The number of c subunits per ring (n) directly determines the coupling ratio between proton translocation and ATP synthesis .

The exact factors that determine c-ring stoichiometry in different organisms remain undefined, though it likely represents evolutionary adaptation to specific environmental and metabolic conditions .

What expression systems are most effective for recombinant production of hydrophobic ATP synthase subunit c?

The production of highly hydrophobic membrane proteins like ATP synthase subunit c presents significant challenges. Based on the search results, the following expression strategies have proven effective:

E. coli-based expression system with fusion proteins:
The most successful approach described in the search results involves expressing the hydrophobic c₁ subunit as a soluble fusion protein with maltose binding protein (MBP) . This method:

• Uses a plasmid with a codon-optimized gene insert for the target organism (e.g., spinach)

• Expresses the c-subunit as an MBP-c₁ fusion protein, enhancing solubility

• Includes a cleavage site for later separation of the target protein

• Employs BL21 derivative E. coli cells (such as SHuffle T7 Express Competent E. coli)

• Uses specialized media optimization for protein expression

The advantages of this approach include:

• Overcoming the insolubility of the hydrophobic membrane protein

• Enabling significant quantities of purified protein

• Maintaining the correct alpha-helical secondary structure of the protein

Alternative expression systems mentioned:

• Pichia pastoris (yeast) expression system with methanol induction

• Specialized E. coli strains designed for membrane protein expression

• Cell-free expression systems for difficult membrane proteins

The selection of expression system should be based on the specific requirements of the research, including necessary yield, downstream applications, and protein characteristics .

What purification strategies yield highest purity and recovery of recombinant ATP synthase subunit c?

Purification of recombinant ATP synthase subunit c requires specialized techniques due to its hydrophobicity and membrane association. Based on the search results, the following purification workflow has been successfully employed:

Step-by-step purification protocol:

• Initial fusion protein purification:

  • Affinity chromatography using maltose or amylose resin for MBP-tagged proteins

  • Column washing to remove non-specifically bound proteins

• Fusion protein cleavage:

  • Enzymatic cleavage to separate the c₁ subunit from MBP

  • Optimization of cleavage conditions (time, temperature, enzyme concentration)

• Separation of cleaved proteins:

  • Reversed-phase column chromatography to isolate the hydrophobic c₁ subunit

  • Alternative: ion exchange chromatography using CMC52 column for basic proteins

• Final purification:

  • Dialysis in appropriate buffer (e.g., 10 mM NaPO₄ pH 6.6, 25 mM NaCl, 0.15 mM EDTA)

  • Concentration using centrifugal filter units with appropriate molecular weight cutoffs

• Quality assessment:

  • SDS-PAGE for purity verification

  • Mass spectrometry for identity confirmation

  • Circular dichroism to confirm correct alpha-helical secondary structure

This purification strategy has been reported to yield significant quantities of highly purified c₁ subunit with the correct secondary structure, enabling further investigations into c-ring assembly and structure .

How can researchers verify the correct folding and secondary structure of purified recombinant ATP synthase subunit c?

Verifying the correct folding and secondary structure of purified recombinant ATP synthase subunit c is crucial before using it in functional studies or reconstitution experiments. The following analytical techniques are recommended:

Circular Dichroism (CD) Spectroscopy:

• Most effective method for confirming the alpha-helical secondary structure of ATP synthase subunit c

• Far-UV CD spectra (190-260 nm) can quantify alpha-helical content

• Near-UV CD spectra (250-350 nm) can provide information about tertiary structure

• Comparison with reference spectra of known correctly folded proteins is essential

Fourier Transform Infrared (FTIR) Spectroscopy:

• Complementary technique to CD for secondary structure analysis

• Particularly useful for membrane proteins in lipid environments

• Analysis of amide I band (1600-1700 cm⁻¹) provides information about secondary structure

Thermal Stability Assays:

• Differential scanning calorimetry (DSC) or CD thermal melts

• Monitors unfolding transitions as temperature increases

• Correctly folded proteins typically exhibit cooperative unfolding behavior

Functional Assays:

• Reconstitution into liposomes and measurement of proton translocation

• Assembly into c-rings and verification of correct oligomerization

• Interaction studies with other ATP synthase components

Structural Analysis:

• Limited proteolysis to probe tertiary structure

• NMR for structural characterization (challenging but informative)

• Native gel electrophoresis to assess oligomeric state

These techniques collectively provide comprehensive validation of the structural integrity of recombinant ATP synthase subunit c, ensuring its suitability for downstream applications .

What techniques can be employed for reconstituting functional c-rings from recombinant monomeric subunits?

Reconstitution of functional c-rings from recombinant monomeric subunits is a challenging but crucial step for studying their structure, assembly, and function. Based on the search results and established methodologies in the field, the following techniques can be employed:

Detergent-mediated reconstitution:

• Solubilization of purified c₁ subunits in appropriate detergents (e.g., n-dodecyl-β-D-maltoside, octyl glucoside)

• Incubation under controlled conditions to promote self-assembly

• Gradual removal of detergent using dialysis or adsorption methods

• Verification of c-ring formation using analytical ultracentrifugation or native gel electrophoresis

Lipid-based reconstitution:

• Incorporation of purified c₁ subunits into liposomes or nanodiscs

• Using specific lipid compositions that mimic the native thylakoid membrane

• Thermal cycling or pH shifts to promote c-ring assembly

• Assessment of proton translocation activity to confirm functional reconstitution

Co-assembly with other ATP synthase components:

• Inclusion of subunits a, b, or F₁ components to stabilize c-ring assembly

• Sequential addition approach to build the complex from individual components

• Monitoring assembly intermediates using crosslinking and mass spectrometry

Factors affecting successful reconstitution:

• pH and ionic strength of the reconstitution buffer

• Temperature and incubation time

• Lipid-to-protein ratio

• Presence of specific lipids (e.g., cardiolipin)

• Addition of stabilizing agents

Verification of successful reconstitution should include structural analysis (electron microscopy, atomic force microscopy) and functional assays (proton translocation, ATP synthesis when combined with F₁) .

How can site-directed mutagenesis be applied to study structure-function relationships in ATP synthase subunit c?

Site-directed mutagenesis is a powerful approach for investigating structure-function relationships in ATP synthase subunit c. By systematically altering specific amino acid residues, researchers can probe their roles in structure, assembly, and function. Based on the information in the search results and standard practices in the field, here's a comprehensive methodology:

Mutagenesis strategy and workflow:

• Design of mutations:

  • Conservative substitutions (e.g., Glu to Asp) to study subtle functional effects

  • Non-conservative substitutions to dramatically alter properties

  • Alanine scanning to identify essential residues

  • Introduction of reporter groups (e.g., cysteine for labeling)

• Mutagenesis protocol:

  • Using the optimized codon sequence in the expression plasmid

  • PCR-based site-directed mutagenesis

  • Verification of mutations by DNA sequencing

• Expression and purification of mutants:

  • Following the established MBP-fusion protein approach

  • Comparing expression levels and solubility with wild-type

  • Purification using the same protocol as wild-type protein

• Structural characterization of mutants:

  • CD spectroscopy to assess effects on secondary structure

  • Thermal stability analysis to measure effects on protein stability

  • Oligomerization analysis to determine effects on c-ring assembly

• Functional analysis of mutants:

  • Reconstitution into liposomes for proton translocation assays

  • Assembly with other ATP synthase components

  • Measurement of ATP synthesis activity in reconstituted systems

• Data analysis and interpretation:

  • Correlation of structural changes with functional effects

  • Mapping of critical residues onto structural models

  • Integration with computational modeling approaches

This systematic approach allows researchers to develop detailed mechanistic models of ATP synthase function and the specific role of subunit c in energy conversion processes .

What are the unresolved questions regarding c-ring stoichiometry variation across species?

Despite significant advances in ATP synthase research, several fundamental questions regarding c-ring stoichiometry remain unresolved. The search results specifically highlight that "the exact cause of the c(n) variability is not well understood" and that "the cause or purpose of the c-n stoichiometric variation has not yet been defined" .

Key unresolved questions include:

• Evolutionary drivers: What evolutionary pressures lead to different c-ring sizes across species? The search results mention various hypotheses but indicate none have been definitively proven .

• Molecular determinants: What specific molecular features of the c-subunit determine the size of the assembled ring? The search results indicate that further investigations into the relationship between monomeric c₁ and its multimeric ring are needed .

• Functional significance: How does the variation in c-ring size relate to the specific metabolic requirements or environmental adaptations of different organisms? The search results state that "this ratio is inherently related to the metabolism of the organism" but the exact relationship remains unclear.

• Assembly mechanisms: How is the precise stoichiometry of the c-ring maintained during assembly? The search results suggest that reconstitution experiments with recombinant c₁ subunits could help address this question .

• Regulatory influences: Do environmental factors influence c-ring stoichiometry within a single species? This question is not directly addressed in the search results but represents an important area for investigation.

The search results emphasize that "the discovery of additional c-subunit ring stoichiometries in other organisms would also help explain this observation by enabling broader comparisons to be made" , highlighting the need for expanded research across diverse species including Agrostis stolonifera.

What technologies are emerging for high-resolution structural analysis of ATP synthase c-rings?

High-resolution structural analysis of ATP synthase c-rings is essential for understanding their assembly, function, and species-specific variations. While the search results don't explicitly detail the latest structural analysis technologies, we can integrate this information with current state-of-the-art approaches in the field:

Cryo-electron microscopy (cryo-EM):

• Enables visualization of ATP synthase complexes in near-native states

• Recent advances allow resolutions approaching 2-3Å for membrane proteins

• Particularly valuable for visualizing intact c-rings within the complete ATP synthase complex

• Can reveal subtle structural differences between species with different c-ring stoichiometries

X-ray crystallography:

• Has historically provided high-resolution structures of isolated c-rings

• Requires large quantities of purified, homogeneous protein

• The recombinant expression system described in the search results could provide sufficient material for crystallization attempts

Solid-state NMR spectroscopy:

• Emerging technique for membrane protein structural analysis

• Can provide atomic-level information about c-subunit packing and interactions

• Particularly useful for studying dynamics and protonation states

Advanced mass spectrometry techniques:

• Native mass spectrometry for determining exact stoichiometry

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for probing dynamics and solvent accessibility

• Crosslinking mass spectrometry to map subunit interfaces

Integrative structural biology approaches:

• Combining multiple structural techniques for comprehensive analysis

• Computational modeling to predict species-specific variations

• Molecular dynamics simulations to understand functional mechanisms

The purification of recombinant c₁ subunits as described in the search results provides the foundation for applying these advanced structural techniques to investigate the specific characteristics of Agrostis stolonifera ATP synthase c-rings compared to other species.

How can genomic and proteomic approaches enhance our understanding of ATP synthase variation across plant species?

Genomic and proteomic approaches offer powerful tools for comprehensive investigation of ATP synthase variation across plant species, including Agrostis stolonifera. Integrating information from the search results with current methodologies in the field:

Genomic approaches:

• Comparative genomic analysis:

  • Sequencing and annotation of atpH genes from diverse plant species

  • Analysis of codon usage patterns that may affect expression (as mentioned in the optimization for E. coli expression)

  • Identification of regulatory elements affecting expression levels

  • Evolutionary analysis to trace the history of atpH gene modifications

• Transcriptomic analysis:

  • RNA-Seq studies to compare atpH expression across tissues and conditions

  • Analysis of alternative splicing or RNA editing events

  • Correlation of expression patterns with environmental or developmental factors

  • Investigation of coordinated expression with other ATP synthase components

Proteomic approaches:

• Large-scale comparative proteomics:

  • Mass spectrometry-based identification of ATP synthase components across species

  • Quantification of stoichiometric relationships between subunits

  • Identification of post-translational modifications

  • Analysis of protein-protein interaction networks

• Structural proteomics:

  • Cross-species comparison of ATP synthase c-subunit structures

  • Analysis of c-ring assembly differences between species

  • Identification of species-specific structural adaptations

Integration of multi-omics data:

By integrating these approaches, researchers can develop comprehensive models of how ATP synthase structure and function vary across species, potentially revealing how plants like Agrostis stolonifera have adapted their energy production mechanisms to specific environmental niches .

What experimental approaches can resolve the relationship between environmental adaptation and ATP synthase efficiency in grasses like Agrostis stolonifera?

Understanding the relationship between environmental adaptation and ATP synthase efficiency in grasses like Agrostis stolonifera requires interdisciplinary approaches combining molecular biology, biochemistry, and ecological physiology. Based on the search results and the broader scientific context:

Field-to-laboratory experimental design:

• Ecological sampling and analysis:

  • Collection of Agrostis stolonifera specimens from diverse environments (different temperatures, light intensities, soil conditions)

  • Measurement of ATP synthase activity and efficiency in these natural samples

  • Correlation with specific environmental parameters

• Controlled environment studies:

  • Growth of plants under defined conditions mimicking various environmental stresses

  • Analysis of ATP synthase structure, composition, and activity

  • Determination of c-ring stoichiometry under different conditions

• Genetic approach:

  • Creation of transgenic plants with modified ATP synthase components

  • Comparative analysis of plants with different c-ring stoichiometries

  • Assessment of growth, photosynthetic efficiency, and stress resistance

• Biochemical characterization:

  • Isolation of ATP synthase from plants grown in different conditions

  • Measurement of ATP synthesis rates, proton translocation efficiency

  • Determination of enzyme kinetics and thermodynamic parameters

• Structural analysis:

  • Investigation of potential structural adaptations in ATP synthase from stress-adapted plants

  • Comparison with ATP synthases from other grass species adapted to different niches

Integration with stress response pathways:

The search results mention that phosphite treatments of Agrostis stolonifera can "improve significantly turfgrass quality and can reduce disease occurrence" . This suggests potential links between nutrient signaling, stress responses, and energy metabolism that could be further explored through:

These integrated approaches would provide insights into how the molecular structure and function of ATP synthase in Agrostis stolonifera has evolved to support its adaptation to specific environmental conditions, with potential implications for improving stress resistance in agriculturally important grasses .

What are the critical parameters for successful codon optimization when expressing plant chloroplast proteins in E. coli?

Codon optimization is crucial for the successful expression of plant chloroplast proteins like ATP synthase subunit c in E. coli. The search results specifically mention codon optimization for the atpH gene from spinach . Based on this information and established principles in the field, the following critical parameters should be considered:

Key parameters for codon optimization:

Optimization protocol based on search results:

This optimization process is particularly important for chloroplast proteins like ATP synthase subunit c, as chloroplast genes often have codon usage patterns significantly different from those preferred by E. coli, which can severely limit heterologous expression .

What strategies effectively overcome the challenges of expressing highly hydrophobic membrane proteins like ATP synthase subunit c?

Expressing highly hydrophobic membrane proteins like ATP synthase subunit c presents significant challenges that require specialized strategies. The search results describe a successful approach for expressing this challenging protein . Based on this information and established principles in the field:

Effective expression strategies:

• Fusion protein approach:

  • The search results highlight expressing ATP synthase subunit c as a "soluble MBP-c₁ fusion protein"

  • Maltose Binding Protein (MBP) acts as a solubility enhancer

  • This approach "enables the soluble expression of an eukaryotic membrane protein in BL21 derivative Escherichia coli cells"

• Specialized expression host selection:

  • Use of specific E. coli strains optimized for membrane or difficult proteins

  • The search results mention "BL21 derivative Escherichia coli cells" and "SHuffle T7 Express Competent E. coli"

  • These strains often have modifications to handle protein folding stress

• Optimization of induction conditions:

  • Careful control of induction temperature (often lower than standard)

  • Reduced inducer concentration to slow expression rate

  • Extended expression time to allow proper folding

• Media and growth condition optimization:

  • Use of enriched media formulations

  • Addition of specific membrane-stabilizing components

  • The search results mention induction protocols with methanol for Pichia pastoris expression

• Post-expression processing:

  • Controlled cleavage of the fusion protein to release the target protein

  • The search results describe cleaving the MBP-c₁ fusion protein and purifying the c₁ subunit

  • Verification that the cleaved protein maintains its native structure

Implementation workflow based on search results:

This comprehensive approach has been demonstrated to yield "significant quantities of highly purified c₁ subunit" with "the correct alpha-helical secondary structure" , making it suitable for further studies of ATP synthase structure and function.

What analytical methods can quantify the stoichiometry of assembled c-rings in experimental samples?

Determining the exact stoichiometry of assembled c-rings is crucial for understanding the relationship between structure and function in ATP synthases from different organisms. While the search results mention that c-ring sizes vary from c₁₀ to c₁₅ among organisms , they don't detail specific analytical methods. Based on this context and established techniques in the field, the following methods can be employed:

Mass spectrometry-based approaches:

• Native mass spectrometry:

  • Allows direct measurement of intact c-ring complexes

  • Can determine exact mass and thus the number of c-subunits

  • Requires specialized instrumentation and careful sample preparation

  • Typical workflow: Extraction in mild detergents → Buffer exchange → Direct infusion into MS

• Crosslinking mass spectrometry:

  • Chemical crosslinking to stabilize the c-ring structure

  • Digestion and MS analysis to identify crosslinked peptides

  • Mapping of subunit interactions to determine arrangement and number

Imaging techniques:

• Atomic Force Microscopy (AFM):

  • Direct visualization of c-ring structure at near-atomic resolution

  • Measurement of ring diameter correlates with subunit number

  • Sample preparation: Reconstituted c-rings in lipid bilayers or native membranes

• Electron Microscopy:

  • Negative stain EM for initial assessment

  • Cryo-EM for high-resolution analysis

  • Image processing to determine symmetry and subunit count

Biochemical and biophysical methods:

• Analytical ultracentrifugation:

  • Determination of molecular weight of assembled c-rings

  • Comparison with known weight of monomeric subunit to calculate stoichiometry

  • Requires purified, detergent-solubilized complexes

• Radiation inactivation:

  • Target size analysis through controlled radiation damage

  • Correlation of functional inactivation with radiation dose to estimate mass

• Quantitative amino acid analysis:

  • Precise determination of protein quantity in purified samples

  • Correlation with functional units to determine stoichiometry

Data analysis and validation:

For Agrostis stolonifera ATP synthase, a combination of these methods would provide the most reliable determination of c-ring stoichiometry, enabling comparison with the known range of c₁₀ to c₁₅ observed in other organisms .

How can researchers optimize reconstitution conditions to study ATP synthase function in artificial membrane systems?

Reconstitution of ATP synthase components, particularly the c-ring, into artificial membrane systems is essential for functional studies. While the search results don't provide specific reconstitution protocols, they mention the goal of using recombinant c₁ for "reconstitution of the multimeric ring (c₁₄)" . Based on this context and established methodologies:

Optimization parameters for reconstitution:

• Lipid composition selection:

  • Mimic native thylakoid membrane composition

  • Test various phospholipid mixtures (e.g., DOPC, DOPE, DOPG)

  • Include thylakoid-specific lipids like monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)

  • Optimize lipid:protein ratio (typically 10:1 to 100:1 w/w)

• Membrane platform selection:

  • Liposomes: Spherical lipid bilayers enclosing aqueous compartments

  • Nanodiscs: Disc-shaped lipid bilayers stabilized by scaffold proteins

  • Planar lipid bilayers: Flat membranes suitable for electrical measurements

  • Giant unilamellar vesicles (GUVs): Large vesicles amenable to microscopy

• Reconstitution method optimization:

  • Detergent-mediated reconstitution with controlled detergent removal

  • Direct incorporation during liposome formation

  • Fusion of protein-containing vesicles

  • Optimization of pH, temperature, and ionic conditions

• Functional gradient establishment:

  • Creation of pH gradients across the membrane

  • Establishment of electrical potential differences

  • ATP/ADP concentration gradients for reverse mode studies

Workflow for reconstitution optimization:

Functional assay methods:

• Proton pumping assays:

  • pH-sensitive fluorescent dyes (ACMA, pyranine)

  • Continuous monitoring of proton movement across the membrane

  • Correlation with ATP hydrolysis activity

• ATP synthesis measurements:

  • Luciferase-based ATP detection

  • Radiolabeled phosphate incorporation

  • Enzyme-coupled continuous assays

• Rotational analysis:

  • Single-molecule fluorescence techniques

  • Attachment of probes to specific subunits

  • Direct visualization of rotor movement

These approaches would enable researchers to study the functional properties of reconstituted ATP synthase from Agrostis stolonifera and investigate how its specific c-ring structure influences its bioenergetic properties compared to other species .

How does the structure and function of ATP synthase subunit c differ between photosynthetic and non-photosynthetic organisms?

ATP synthase subunit c exhibits important structural and functional differences between photosynthetic organisms like Agrostis stolonifera and non-photosynthetic organisms. Based on the search results and contextual knowledge of the field:

Structural comparisons:

• c-ring stoichiometry:

  • Photosynthetic organisms (including plants like Agrostis stolonifera) typically have larger c-rings (c₁₄-c₁₅)

  • Non-photosynthetic bacteria often have smaller c-rings (c₁₀-c₁₁)

  • Mitochondrial ATP synthases generally have intermediate sizes (c₈-c₁₀)

• Primary sequence differences:

  • Chloroplastic c-subunits (like those in Agrostis stolonifera) have distinct sequences reflecting their endosymbiotic origin

  • The search results mention that chloroplastic ATP synthase subunit c in spinach has 81 amino acids

  • Non-photosynthetic bacterial homologs often have slightly different lengths

  • Mitochondrial c-subunits may have organism-specific adaptations

• Post-translational modifications:

  • Differing patterns of modification between organisms

  • Some bacteria have specific modifications of the proton-carrying residues

Functional implications:

• Bioenergetic efficiency:

  • The H⁺/ATP ratio varies according to c-ring size: 4.7 for c₁₄ rings versus 3.3 for c₁₀ rings

  • Photosynthetic organisms with larger rings require more protons per ATP synthesized

  • This lower efficiency may be advantageous in photosynthetic conditions where light energy is abundant

• Operating conditions:

  • Chloroplast ATP synthases operate with the proton motive force generated by photosynthesis

  • Bacterial ATP synthases often work with different respiratory chains

  • Mitochondrial ATP synthases function within the constraints of cellular respiration

• Regulatory mechanisms:

  • Different regulatory mechanisms have evolved in different organisms

  • Some photosynthetic organisms have specific regulatory proteins or modifications

Evolutionary perspective:

These differences reflect the diverse evolutionary histories and energy management strategies across different domains of life, with photosynthetic organisms like Agrostis stolonifera having adapted their ATP synthases to the specific requirements of photosynthetic energy conversion .

What insights can comparative analysis of c-ring structures provide about evolutionary adaptation in plants?

Comparative analysis of c-ring structures across plant species offers valuable insights into evolutionary adaptation mechanisms. While the search results don't provide direct comparative data for Agrostis stolonifera, they mention that "the ratio of protons translocated to ATP synthesized varies according to the number of c-subunits (n) per oligomeric ring (c(n)) in the enzyme, which is organism dependent" and that this ratio "is inherently related to the metabolism of the organism" .

Evolutionary insights from c-ring structural variation:

• Metabolic adaptation:

  • Different c-ring sizes reflect adaptation to specific energetic requirements

  • The search results indicate that the c-ring stoichiometry affects the H⁺/ATP ratio, ranging from 3.3 to 5.0 among organisms

  • Plants adapted to different light environments may have evolved different c-ring structures to optimize energy conversion under their specific conditions

• Environmental specialization:

  • The search results suggest that Agrostis stolonifera (creeping bentgrass) has specific adaptations for turfgrass environments

  • C-ring structure may be optimized for the specific bioenergetic challenges of these habitats

  • Comparative analysis could reveal correlations between habitat and c-ring structure

• Evolutionary constraints and trade-offs:

  • The search results indicate that the exact cause of c-ring variability remains unclear

  • Comparative analysis could reveal whether c-ring size correlates with phylogenetic relationships or represents convergent evolution

  • Analysis could identify whether certain c-ring structures represent fitness trade-offs between energy efficiency and other physiological requirements

Methodological approach for comparative analysis:

Evolutionary hypotheses to test:

• Do closely related plant species have similar c-ring stoichiometries, suggesting phylogenetic conservation?

• Do plants from similar environments show convergent evolution in c-ring structure?

• Is c-ring stoichiometry correlated with photosynthetic efficiency or stress tolerance?

• Does the ATP synthase c-subunit show different patterns of selection compared to other chloroplast genes?

This comparative approach could reveal how plants like Agrostis stolonifera have fine-tuned their energy conversion machinery through evolutionary time to match their specific ecological niches .

How does the efficiency of ATP production in Agrostis stolonifera compare with other agriculturally important grasses?

While the search results don't provide direct comparative data on ATP production efficiency in Agrostis stolonifera versus other grasses, we can develop a research framework based on the information provided about ATP synthase function and the specific characteristics of Agrostis stolonifera.

Comparative framework for ATP production efficiency:

• Molecular determinants of efficiency:

  • The search results indicate that the H⁺/ATP ratio varies according to c-ring size

  • If the c-ring size in Agrostis stolonifera differs from other grasses, this would directly affect ATP production efficiency

  • The exact molecular details of ATP synthase in Agrostis stolonifera would need to be determined experimentally

• Physiological measurements:

  • Direct comparison of ATP production rates under standardized conditions

  • Measurement of photosynthetic parameters (photosynthetic efficiency, electron transport rate)

  • Assessment of ATP/ADP ratios in different cellular compartments

• Environmental response patterns:

  • The search results indicate that Agrostis stolonifera shows specific responses to treatments like phosphite

  • Comparative analysis of how ATP production responds to environmental stressors across grass species

  • Measurement of ATP synthase activity under various light, temperature, and nutrient conditions

Potential comparative analysis table:

Research implications:

• Understanding differences in ATP production efficiency could explain the specific environmental adaptations of Agrostis stolonifera as a turfgrass.

• The search results indicate that phosphite treatments improve turfgrass quality and reduce disease occurrence in Agrostis stolonifera , which could be related to effects on energy metabolism.

• Comparative studies could identify unique adaptations in ATP synthase structure or regulation that could be targets for improving stress tolerance in agriculturally important grasses.

This comparative approach would provide valuable insights into how variations in the molecular machinery of ATP production contribute to the specific ecological adaptations and agricultural performance of different grass species .