Recombinant Gossypium barbadense ATP synthase subunit c, chloroplastic (atpH)

What is the basic structure and function of ATP synthase subunit c (atpH) in cotton chloroplasts?

ATP synthase in cotton chloroplasts consists of two primary components: CF1 (the catalytic head) and CFo (the membrane-embedded proton channel). The c subunit (atpH) is part of the CFo domain and functions as subunit III. Six subunits of chloroplast ATP synthase, including the c subunit (atpH), are encoded by chloroplast genes located on two gene clusters or operons . In the CFo component, subunit III (atpH) works with subunits I (atpF) and IV (atpI) to form the proton channel that drives ATP synthesis through proton gradient utilization. The c subunit forms a ring structure within the membrane that rotates during proton translocation, driving conformational changes in CF1 that catalyze ATP synthesis .

How does atpH interact with other ATP synthase subunits to facilitate ATP production?

The c subunit (atpH) interacts primarily with other CFo components to form the functional proton channel. Research indicates that interactions between multiple subunits, including the δ subunit and subunits β, γ, ε, I, II, III (atpH), and IV, are responsible for preventing proton leakage . The c-ring rotation, driven by proton movement through the channel, mechanically couples with the central stalk (γ and ε subunits) of CF1, inducing conformational changes in the catalytic sites to synthesize ATP. Specifically, the cross-linking of the I subunit with II-δ inhibits photophosphorylation and ATP hydrolytic activity , demonstrating the intricate functional interconnections between these subunits.

What expression patterns does atpH show during cotton development?

While the search results don't directly address atpH expression patterns, we can infer information from the expression patterns of related ATP synthase subunits. Studies on Jin A-CMS (cytoplasmic male sterile) cotton showed that the expression levels of ATP synthase subunit genes, including atpB, atpE, and atpF, were significantly lower in the sterile Jin A line compared to the maintainer line at the microspore abortion stage . Given that atpH is co-expressed with these genes as part of the same operons in the chloroplast genome, it likely follows similar expression patterns during development, with potential downregulation during microspore abortion in CMS lines.

What are the optimal expression systems for producing recombinant Gossypium barbadense atpH protein?

For recombinant expression of the atpH subunit from Gossypium barbadense, E. coli-based expression systems typically offer the most accessible approach for initial characterization studies. When selecting an expression system, researchers should consider:

• Codon optimization: Chloroplast genes often have codon preferences different from E. coli, necessitating codon optimization of the atpH sequence

• Expression tags: N-terminal or C-terminal affinity tags (such as His6) facilitate purification while minimizing interference with protein function

• Solubility enhancement: Fusion partners like MBP (maltose-binding protein) can improve solubility of the hydrophobic atpH subunit

• Membrane protein expression protocols: As atpH is a membrane protein, specialized protocols using mild detergents for extraction and purification are essential

For functional studies, chloroplast-targeted expression in plant systems may provide a more native environment, though with greater technical complexity.

What techniques are most effective for studying atpH interactions with other ATP synthase subunits?

Multiple complementary approaches are recommended for comprehensive analysis of atpH interactions:

• Co-immunoprecipitation (Co-IP): Using antibodies against atpH or other ATP synthase subunits to pull down protein complexes

• Yeast two-hybrid (Y2H) analysis: Modified membrane Y2H systems can identify direct protein-protein interactions

• Bimolecular fluorescence complementation (BiFC): For visualizing interactions in planta

• Cross-linking studies: Chemical cross-linking coupled with mass spectrometry can identify interaction sites

• Cryo-electron microscopy: For structural determination of the entire ATP synthase complex

Research shows that interactions between multiple ATP synthase subunits, including atpH (subunit III), are critical for preventing proton leakage and maintaining ATP synthesis efficiency . Cross-linking studies have demonstrated that disrupting these interactions can inhibit photophosphorylation and ATP hydrolytic activity .

How can gene silencing approaches be optimized to study atpH function in cotton?

Based on the methods described in the research, virus-induced gene silencing (VIGS) using the tobacco rattle virus (TRV) vector system has proven effective for studying ATP synthase subunits in cotton . When applying this method to atpH:

• Design gene-specific silencing constructs targeting unique regions of atpH to avoid off-target effects

• Use appropriate controls such as TRV2:00 empty vector as demonstrated in the research

• Validate silencing efficiency through qRT-PCR measurement of atpH transcript levels

• Assess phenotypic effects through:

  • ROS measurement assays (see Table 1 for protocols used with other ATP synthase subunits)

  • ATP content determination

  • Chloroplast structure analysis via transmission electron microscopy

  • Photosynthetic efficiency measurements

The research demonstrated that silencing of ATP synthase subunit genes (atpE and atpF) led to significant increases in ROS levels, particularly superoxide (O₂⁻- ) and singlet oxygen (¹O₂) .

How does atpH contribute to ROS metabolism in cotton chloroplasts?

The ATP synthase c subunit (atpH) likely plays a significant role in ROS metabolism, similar to other ATP synthase subunits. Research has demonstrated that silencing of ATP synthase subunits atpE and atpF led to substantial increases in ROS accumulation in cotton leaves . The following table illustrates ROS content changes in response to ATP synthase subunit silencing:

*p < 0.05; **p < 0.01, Tukey's multiple comparison tests

The mechanism likely involves:

• Disruption of proton gradient maintenance

• Uncoupling of oxidative phosphorylation

• Electron leakage from electron transport chains

• Reduced energy availability for ROS-scavenging systems

As atpH functions in the same complex as these subunits, it likely contributes to ROS regulation through similar mechanisms, maintaining the proton gradient necessary for proper electron transport and preventing ROS formation.

What role does atpH play in cytoplasmic male sterility in cotton?

While the research doesn't directly address atpH's role in cytoplasmic male sterility (CMS), we can infer its potential involvement from studies on related ATP synthase subunits. In Jin A-CMS cotton, ATP synthase genes atpB, atpE, and atpF were significantly downregulated during the microspore abortion stage . This downregulation was associated with:

• Decreased ATP content in anthers

• Disrupted energy metabolism

• Increased ROS accumulation

• Premature programmed cell death in the tapetal layer

Given that atpH is part of the same ATP synthase complex and is co-regulated with these subunits, it likely contributes to CMS through similar mechanisms. The research suggests that altered expression or function of ATP synthase subunits, potentially including atpH, leads to energy deficiency and ROS accumulation, triggering tapetal PCD and pollen abortion .

How do polymorphisms in atpH affect ATP synthase function and plant stress responses?

Comparative analysis of chloroplast genomes revealed single-nucleotide polymorphisms (SNPs) in multiple ATP synthase subunits, including atpB, atpE, and atpF, between Jin A-CMS and reference Gossypium hirsutum . While atpH-specific polymorphisms weren't detailed in the search results, research indicates that:

• Polymorphisms in ATP synthase subunits are associated with maternally inherited enhanced stress recovery

• Such variations can affect:

  • Protein structure and complex assembly

  • Proton conductance efficiency

  • ATP synthesis rates

  • ROS accumulation patterns

The research suggests that polymorphisms in ATP synthase subunits, potentially including atpH, could influence how plants respond to various stresses by affecting energy metabolism and ROS handling capabilities. This has implications for understanding natural variation in stress tolerance among cotton varieties.

How can recombinant atpH be utilized to study the structure-function relationship of ATP synthase in vitro?

Recombinant atpH protein provides a valuable tool for structure-function studies of ATP synthase. Advanced applications include:

• Site-directed mutagenesis to identify critical residues for:

  • Proton binding and translocation

  • Subunit interactions within the c-ring

  • Rotational coupling with other components

• Reconstitution experiments:

  • Incorporation of purified atpH into liposomes to study proton conductance

  • Assembly with other recombinant subunits to reconstruct functional domains

  • Measurement of ATP synthesis activity in controlled conditions

• Structural studies:

  • Crystallography or cryo-EM of atpH alone or in complex with other subunits

  • NMR studies of dynamic regions

  • Molecular dynamics simulations to understand conformational changes during rotation

These approaches can provide insights into how specific amino acid changes, such as those identified in comparative genomic analyses , affect ATP synthase function and plant physiology.

What advanced techniques can be used to visualize atpH localization and dynamics in living plant cells?

Several cutting-edge techniques allow researchers to visualize atpH localization and dynamics:

• Fluorescent protein fusions:

  • Careful design required to maintain protein function

  • C-terminal fusions typically preferred for membrane proteins

  • Use of chloroplast transit peptide to ensure proper targeting

• Super-resolution microscopy:

  • STED (Stimulated Emission Depletion) for imaging beyond the diffraction limit

  • PALM/STORM for single-molecule localization microscopy

  • Allows visualization of c-ring assembly and distribution within thylakoid membranes

• FRAP (Fluorescence Recovery After Photobleaching):

  • Measures protein mobility within membranes

  • Can reveal constraints on atpH movement imposed by complex formation

• FRET (Förster Resonance Energy Transfer):

  • Measures interactions between atpH and other ATP synthase subunits

  • Provides dynamic information about complex assembly and disassembly

These techniques can reveal how atpH distribution and dynamics change in response to developmental cues or stress conditions, potentially connecting to the phenotypes observed in ATP synthase subunit-silenced plants .

How does the stoichiometry of atpH within the c-ring differ between Gossypium species, and what are the functional implications?

The c-ring stoichiometry (number of c subunits forming the ring) can vary between species and has significant functional implications. For Gossypium species:

• Determination methods:

  • Atomic force microscopy of isolated c-rings

  • Mass determination via native mass spectrometry

  • Structural studies via cryo-electron microscopy

• Functional implications of differing stoichiometries:

  • Affects the H⁺/ATP ratio (bioenergetic efficiency)

  • Influences ATP synthesis rate and ATP yield per proton

  • May reflect adaptations to different environmental conditions

• Evolutionary considerations:

  • Comparison between Gossypium species can reveal selective pressures

  • May correlate with differences in photosynthetic efficiency or stress tolerance

While the search results don't provide specific data on c-ring stoichiometry in Gossypium barbadense, this represents an important area for comparative research that could explain functional differences in ATP synthase between cotton species and varieties with different cytoplasmic backgrounds.

What are the major technical challenges in purifying functional recombinant atpH protein?

Purification of functional recombinant atpH presents several technical challenges:

• Hydrophobicity: As a membrane protein, atpH is highly hydrophobic and prone to aggregation

  • Solution: Use specialized detergents (e.g., DDM, LMNG) for extraction and purification

  • Consider using amphipols or nanodiscs for maintaining stability in solution

• Proper folding: Ensuring correct folding in heterologous expression systems

  • Solution: Lower expression temperatures (16-20°C) to slow protein synthesis

  • Co-expression with chaperones can improve folding efficiency

• Functional assessment: Verifying that purified protein retains native activity

  • Solution: Develop reconstitution assays with proton gradient measurements

  • Use complementation assays in bacterial ATP synthase mutants

• Yield limitations: Typically low expression levels for membrane proteins

  • Solution: Scale-up cultures and optimize induction conditions

  • Consider cell-free expression systems for toxic proteins

These challenges require methodical optimization but are surmountable with contemporary protein biochemistry approaches.

How can researchers effectively study interactions between nuclear and chloroplast-encoded components of ATP synthase?

Studying interactions between nuclear and chloroplast-encoded ATP synthase components requires specialized approaches:

• Bi-genomic complementation systems:

  • Chloroplast transformation in combination with nuclear transformation

  • Analysis of genetic suppression or synthetic lethality

• Split-reporter systems adapted for chloroplast-nuclear protein interactions:

  • Modified BiFC systems with appropriate targeting sequences

  • Dual-genome expression controls

• Co-evolution analysis:

  • Computational approaches to identify co-evolving residues between nuclear and chloroplast-encoded subunits

  • Validation through targeted mutagenesis

• Proteomic approaches:

  • Affinity purification of intact ATP synthase complexes

  • Cross-linking mass spectrometry to identify interaction interfaces

The interaction between nuclear and chloroplast genomes is particularly relevant to understanding cytoplasmic male sterility, as research indicates that CMS results from genetic interactions between nuclear and cytoplasmic genes .

What are the most promising directions for engineering atpH to improve plant stress tolerance or productivity?

Based on the research findings linking ATP synthase function to ROS metabolism and stress responses , several promising engineering approaches emerge:

• Strategic mutations:

  • Modify proton-binding residues to optimize H⁺/ATP ratio

  • Engineer interfaces between subunits to enhance complex stability under stress

  • Target amino acid differences identified in comparative genomics of cotton varieties

• Expression modulation:

  • Fine-tune atpH expression levels to balance energy production and ROS generation

  • Develop stress-responsive promoters for contextual regulation

• Heterologous expression:

  • Introduce atpH variants from stress-tolerant species

  • Create chimeric proteins incorporating beneficial features from multiple species

• Directed evolution approaches:

  • Develop screening systems for atpH variants with enhanced function

  • Focus on improving thermotolerance or salt tolerance of ATP synthase

These approaches could potentially address the energy deficiency and ROS accumulation observed in ATP synthase-compromised plants , improving stress tolerance, photosynthetic efficiency, and ultimately crop productivity.