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

What is Recombinant Gossypium hirsutum ATP synthase subunit c, chloroplastic (atpH)?

Recombinant Gossypium hirsutum ATP synthase subunit c, chloroplastic (atpH) is a laboratory-synthesized version of the naturally occurring protein found in cotton (Gossypium hirsutum) chloroplasts. This protein is a critical component of the chloroplastic ATP synthase complex, specifically forming part of the CFo subcomplex that facilitates proton transport across the thylakoid membrane. The recombinant form is produced through molecular cloning and protein expression systems to enable detailed biochemical and functional studies outside the native plant environment .

The protein is classified as a subunit of ATP synthase, which plays an essential role in energy conversion within the chloroplast. Unlike naturally extracted proteins, the recombinant form allows for controlled experimental conditions and potentially modified properties to suit specific research needs.

How does the ATP synthase subunit c relate to other ATP synthase subunits in cotton chloroplasts?

The ATP synthase subunit c (atpH) in cotton chloroplasts functions as part of a larger multi-subunit complex that includes various other components. Based on comparative genomic analyses, the chloroplast ATP synthase consists of multiple subunits including atpB, atpE, and atpF, which have shown significant expression differences in various cotton lines . The subunit c forms part of the membrane-embedded CFo domain, which works in conjunction with the catalytic CF1 domain (containing subunits like atpB and atpE).

Research has demonstrated that these subunits are functionally interdependent, with altered expression of one subunit often affecting the others. For example, in Jin A-CMS (cytoplasmic male sterile) cotton lines, the expression levels of atpB, atpE, and atpF were significantly lower compared to the maintainer line at the microspore abortion stage . This interdependence suggests a coordinated regulation mechanism for the entire ATP synthase complex.

What methodologies are commonly used to express recombinant ATP synthase subunits?

The expression of recombinant ATP synthase subunits, including atpH from Gossypium hirsutum, typically employs heterologous expression systems optimized through systematic experimental design. A recommended approach uses Response Surface Methodology (RSM) based on central composite design (CCD) to optimize key expression parameters .

Key methodological steps include:

• Selection of an appropriate expression system (e.g., bacterial systems like E. coli or eukaryotic systems like P. pastoris)

• Optimization of expression conditions through factorial experimental design

• Evaluation of critical parameters at multiple levels (+1, 0, -1) corresponding to high, medium, and low values

• Statistical analysis of expression results to determine optimal conditions

For example, when optimizing recombinant protein expression, researchers typically evaluate parameters such as:

These parameters must be systematically tested with replication (typically three independent experiments per condition) to ensure reproducibility and statistical validity of the optimization process .

How does the expression of atpH correlate with reactive oxygen species (ROS) metabolism in cotton?

The relationship between ATP synthase subunits and ROS metabolism represents a critical area of investigation in cotton research. While the specific role of atpH (ATP synthase subunit c) has not been fully characterized in the provided research, studies on related subunits provide valuable insights. Research has demonstrated that ATP synthase subunit genes atpE and atpF are intimately linked with ROS metabolism in cotton .

Gene silencing experiments for atpE and atpF have resulted in significant accumulation of hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂) in cotton leaves . This suggests that disruption of the ATP synthase complex through alteration of specific subunits can trigger oxidative stress responses. The mechanistic explanation likely involves:

• Disruption of proton gradient maintenance across the thylakoid membrane

• Altered coupling between electron transport and ATP synthesis

• Subsequent electron leakage to oxygen, forming ROS

These findings correlate with observations in Jin A-CMS cotton, where ATP content decreases significantly at the microspore abortion stage, accompanied by excessive ROS accumulation . This indicates that proper functioning of the ATP synthase complex, including all subunits, is essential for ROS homeostasis in cotton chloroplasts.

What genomic variations exist in the atpH gene across different cotton varieties, and how do they impact function?

Comparative genomic analyses between Jin A-CMS and reference Gossypium hirsutum chloroplast genomes have revealed significant variations in ATP synthase subunit genes. While specific data for atpH was not detailed in the provided research, related ATP synthase subunit genes showed notable single-nucleotide polymorphisms (SNPs) resulting in amino acid substitutions .

The following table summarizes observed variations in ATP synthase subunit genes:

These substitutions, though appearing minimal, can significantly impact protein structure and function. The 97.4% similarity in atpF is particularly notable, as this represents a more substantial deviation than observed in atpB and atpE . Such variations likely contribute to functional differences in ATP synthase performance across cotton varieties.

What experimental approaches are most effective for studying the role of ATP synthase subunit c in cotton fertility and stress response?

Investigating the role of ATP synthase subunit c in cotton fertility and stress response requires integrated experimental approaches spanning multiple levels of analysis. Based on successful research on related ATP synthase subunits, the following methodological framework is recommended:

• Transcriptional Analysis

  • Quantitative RT-PCR to measure expression levels across developmental stages, particularly during microsporogenesis

  • RNA-seq for genome-wide transcriptional responses to subunit c manipulation

  • Comparison between sterile and fertile lines at critical developmental points

• Genetic Manipulation

  • VIGS (Virus-Induced Gene Silencing) to downregulate atpH expression

  • CRISPR-Cas9 gene editing for precise manipulation of key residues

  • Complementation studies in mutant backgrounds

• Biochemical Analysis

  • ATP content quantification in anthers and other tissues

  • ROS measurement using specific fluorescent probes for H₂O₂ and ¹O₂

  • Enzymatic activity assays for antioxidant systems

• Structural Analysis

  • Electron microscopy to examine chloroplast ultrastructure

  • Analysis of thylakoid membrane organization

  • Protein interaction studies to identify binding partners

Research on related ATP synthase subunits has demonstrated that these integrated approaches can effectively elucidate the connection between ATP synthase function, ROS metabolism, and fertility. For example, studies have shown that expression levels of ATP synthase subunit genes were significantly lower in sterile cotton lines compared to maintainer lines at critical developmental stages .

How can recombinant atpH be utilized to study ATP synthase assembly in vitro?

Recombinant Gossypium hirsutum ATP synthase subunit c can serve as a powerful tool for investigating ATP synthase assembly processes in controlled in vitro systems. A methodological framework for such studies would include:

• Production of Functional Recombinant Protein

  • Expression optimization using Response Surface Methodology with central composite design

  • Three-level factorial experiments examining temperature, pH, and inducer concentration

  • Purification using affinity chromatography followed by size exclusion chromatography

• Reconstitution Studies

  • Incorporation of purified recombinant atpH into liposomes

  • Sequential addition of other purified ATP synthase subunits

  • Monitoring of complex formation through biophysical techniques

• Structural Analysis of Assembly Intermediates

  • Cryo-electron microscopy of partially assembled complexes

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics

• Functional Assessment

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • ATP synthesis measurements in reconstituted systems

  • Analysis of how mutations affect assembly efficiency and function

This systematic approach allows researchers to dissect the specific contribution of subunit c to the assembly and function of the complete ATP synthase complex, providing insights that may not be accessible through in vivo studies alone.

What are the optimal conditions for expressing recombinant Gossypium hirsutum ATP synthase subunit c?

Optimizing expression conditions for recombinant Gossypium hirsutum ATP synthase subunit c requires systematic evaluation of multiple parameters. Based on established protocols for recombinant protein expression, a Response Surface Methodology (RSM) approach with central composite design (CCD) is highly recommended .

The experimental matrix should test critical parameters at three levels:

Each experimental condition should be tested in triplicate to ensure statistical reliability . The central point (all factors at level 0) should be replicated at least six times to estimate experimental error.

For heterologous expression in Pichia pastoris, which has proven effective for complex membrane proteins, appropriate negative controls include:

• Non-recombinant P. pastoris host cells

• P. pastoris transformed with the native vector without the atpH insert

After expression, optimization of purification conditions represents a secondary but equally important consideration, particularly given the hydrophobic nature of ATP synthase subunit c and its tendency to form oligomeric structures in membrane environments.

What analytical techniques are most appropriate for characterizing the structure and function of recombinant atpH?

Characterization of recombinant Gossypium hirsutum ATP synthase subunit c requires a multi-faceted analytical approach to address its structural properties and functional attributes:

• Structural Characterization

  • Circular Dichroism (CD) spectroscopy to assess secondary structure composition

  • Nuclear Magnetic Resonance (NMR) for atomic-level structural information in solution

  • X-ray crystallography or cryo-electron microscopy for high-resolution structural determination

  • Mass spectrometry for precise molecular weight confirmation and post-translational modifications

• Functional Analysis

  • Reconstitution into liposomes or nanodiscs to measure proton translocation activity

  • Patch-clamp electrophysiology to characterize ion channel properties

  • Binding assays with other ATP synthase subunits to assess complex formation

  • Hydrogen-deuterium exchange to identify dynamic regions and interaction surfaces

• Stability Assessment

  • Differential Scanning Calorimetry (DSC) to determine thermal stability

  • Chemical denaturation studies to assess conformational stability

  • Long-term storage stability under various buffer conditions

• Oligomerization Analysis

  • Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS)

  • Analytical Ultracentrifugation (AUC) to determine oligomeric state in solution

  • Blue Native PAGE to assess native complex formation

These analytical approaches provide complementary information that collectively offers a comprehensive understanding of the recombinant protein's properties, which can then be compared with native protein characteristics or used to investigate the effects of specific mutations.

How can gene silencing approaches be utilized to study atpH function in vivo?

Gene silencing provides a powerful approach for investigating the in vivo function of ATP synthase subunit c in cotton plants. Based on successful studies with related ATP synthase subunits, the following methodological framework is recommended:

• Virus-Induced Gene Silencing (VIGS)

  • Design of specific constructs targeting unique regions of the atpH transcript

  • Transformation of Agrobacterium with VIGS constructs

  • Infiltration of cotton seedlings at optimal developmental stages

  • Confirmation of silencing efficiency through qRT-PCR

• Phenotypic Analysis of Silenced Plants

  • Microscopic examination of chloroplast ultrastructure

  • Analysis of reproductive development, particularly microsporogenesis

  • Measurement of photosynthetic parameters using chlorophyll fluorescence

  • Quantification of ATP content in relevant tissues

• ROS Metabolism Assessment

  • Histochemical staining for H₂O₂ using DAB (3,3'-diaminobenzidine)

  • Fluorescent probe analysis for singlet oxygen (¹O₂) detection

  • Enzymatic assays for antioxidant systems (SOD, CAT, APX)

  • Expression analysis of ROS-responsive genes

• Molecular Complementation

  • Introduction of modified atpH variants to assess functional rescue

  • Analysis of compensatory responses in other ATP synthase subunits

  • Evaluation of downstream metabolic adaptations

This approach has proven effective in related studies, where silencing of ATP synthase genes atpE and atpF resulted in significant ROS accumulation in cotton leaves, demonstrating the critical link between ATP synthase function and ROS metabolism . Similar approaches with atpH would likely reveal its specific contribution to this regulatory network.

How might atpH function be leveraged for improving cotton stress tolerance?

Understanding the role of ATP synthase subunit c in stress responses offers potential avenues for enhancing cotton stress tolerance. Based on research demonstrating the connection between ATP synthase function and reactive oxygen species metabolism, several strategic approaches emerge:

• Targeted Genetic Modifications

  • Identification of naturally occurring atpH variants with enhanced stability under stress conditions

  • Introduction of specific amino acid substitutions to optimize proton translocation efficiency

  • Fine-tuning of expression levels to balance energy production with ROS management

• Screening Methodologies

  • Development of high-throughput screening systems to identify cotton varieties with optimal atpH expression patterns

  • Evaluation of ATP/ROS ratios as predictive markers for stress tolerance

  • Implementation of TILLING (Targeting Induced Local Lesions IN Genomes) approaches to identify beneficial mutations

• Physiological Interventions

  • Application of compounds that stabilize ATP synthase function under stress conditions

  • Modulation of environmental parameters to optimize ATP synthase performance during critical developmental windows

  • Priming treatments to enhance adaptive responses involving ATP synthase regulation

The foundational research supporting these approaches stems from observations that ATP synthase subunits significantly influence ROS metabolism in cotton . In Jin A-CMS cotton, altered expression of ATP synthase genes correlates with increased ROS accumulation and reproductive failure, suggesting that optimal ATP synthase function is essential for stress tolerance and reproductive success .

What are the most promising directions for future research on recombinant Gossypium hirsutum ATP synthase subunit c?

Future research on recombinant Gossypium hirsutum ATP synthase subunit c should focus on several high-priority areas that address current knowledge gaps:

• Structural Biology and Protein Engineering

  • High-resolution structural determination of cotton atpH and comparison with other species

  • Identification of critical residues for function through systematic mutagenesis

  • Engineering of modified variants with enhanced stability or activity

• Systems Biology Integration

  • Comprehensive mapping of atpH interactions within the chloroplast proteome

  • Metabolomic profiling to understand downstream effects of atpH manipulation

  • Integration of transcriptomic, proteomic, and metabolomic data to build predictive models

• Evolutionary and Comparative Studies

  • Comparative analysis of atpH sequences across cotton species and varieties

  • Correlation of sequence variations with physiological traits and environmental adaptations

  • Investigation of coevolution patterns between atpH and other ATP synthase subunits

• Translational Applications

  • Development of diagnostic tools based on atpH sequence or expression analysis

  • Exploration of atpH as a target for enhancing cotton productivity under challenging conditions

  • Utilization of atpH knowledge for comparative studies in other crop species

These research directions build upon the established connections between ATP synthase function, energy metabolism, and reactive oxygen species management in cotton . The significant variations observed in ATP synthase subunit genes between different cotton lines suggest that targeted investigations of atpH could reveal important insights into cotton adaptation and improvement strategies.