Recombinant Oryza sativa ATP synthase subunit c, chloroplastic (atpH)

What is the basic structure and function of ATP synthase subunit c in rice chloroplasts?

ATP synthase subunit c (atpH) is a critical component of the chloroplastic ATP synthase complex in Oryza sativa (rice). This protein forms part of the membrane-embedded F0 portion of the ATP synthase, creating a ring structure that facilitates proton translocation. The F0 component works in conjunction with the F1 catalytic portion to convert the proton motive force generated during photosynthesis into chemical energy in the form of ATP.

The atpH subunit is encoded by the chloroplast genome and contributes to the rotary mechanism of ATP synthesis. When protons flow through the c-ring structure, they drive rotation that ultimately leads to conformational changes in the F1 portion, enabling ATP production. The proper function of this subunit is essential for efficient photosynthetic energy conversion .

What post-translational modifications affect atpH function in chloroplasts?

Post-translational modifications (PTMs) of atpH include phosphorylation, acetylation, and redox-based modifications that fine-tune ATP synthase activity in response to varying environmental conditions. The cysteine residues in atpH can undergo thiol modifications in response to changing redox conditions within the chloroplast, serving as a regulatory mechanism for ATP synthase activity during fluctuating light conditions or oxidative stress.

These modifications affect protein-protein interactions within the ATP synthase complex, potentially altering the efficiency of proton translocation and ATP production. Researchers investigating atpH function should consider these PTMs when designing experiments, as sample preparation methods may affect the modification state of the protein and consequently the observed activity .

What are the optimal methods for heterologous expression of recombinant atpH protein?

The successful heterologous expression of recombinant atpH requires careful consideration of expression systems and conditions. Based on research practices:

• Selection of expression system: Pichia pastoris has proven effective for expressing membrane proteins like OSCA channels from rice, suggesting it may be suitable for atpH as well. This yeast system provides proper protein folding and post-translational modifications closer to those in plants than bacterial systems .

• Fusion construct design: Employing TEV protease-cleavable enhanced GFP fusion constructs facilitates monitoring of expression levels and subsequent purification. This approach has been successful for similar membrane proteins from rice .

• Protein purification strategy: For membrane proteins like atpH:

  • Solubilization with mild detergents (n-dodecyl β-D-maltoside or digitonin)

  • Affinity chromatography using the GFP tag

  • Size-exclusion chromatography for final purification

• Verification of oligomeric state: SEC-MALLS (Size-Exclusion Chromatography coupled to Multi-Angle Laser Light Scattering) analysis should be employed to confirm the proper assembly of the protein, as demonstrated for other multimeric membrane proteins from rice .

The purified protein should undergo structural and functional characterization to verify its native-like properties.

How can researchers effectively measure ATP synthase activity in recombinant systems?

Measuring ATP synthase activity in recombinant systems requires specific methodological approaches:

• Reconstitution into liposomes: Purified atpH and other ATP synthase components should be reconstituted into liposomes with appropriate lipid composition mimicking the chloroplast thylakoid membrane.

• Proton gradient formation: Generate a proton gradient across the liposome membrane using one of the following methods:

  • Acid-base transition

  • Light-driven proton pumping with co-reconstituted bacteriorhodopsin

  • Valinomycin-induced K+ diffusion potential

• ATP synthesis measurement: Monitor ATP production using either:

  • Luciferin-luciferase assay for real-time ATP detection

  • NADP+ reduction coupled to ATP production via hexokinase and glucose-6-phosphate dehydrogenase

• Control experiments: Include controls with specific inhibitors (oligomycin, venturicidin, or DCCD) to confirm ATP synthase-specific activity.

These methods allow for quantitative assessment of ATP synthase function and can be used to compare wild-type and mutant versions of the protein or to evaluate the effects of different environmental conditions on enzyme activity .

What techniques are most effective for studying atpH-protein interactions in the ATP synthase complex?

Several complementary techniques are recommended for studying atpH interactions within the ATP synthase complex:

• Cross-linking coupled with mass spectrometry: This approach can identify direct protein-protein interaction interfaces. Chemical cross-linkers of varying lengths can map the spatial arrangement of subunits.

• Blue native polyacrylamide gel electrophoresis (BN-PAGE): This technique preserves native protein complexes and can be used to analyze the integrity of ATP synthase assemblies containing atpH. Subsequent second-dimension SDS-PAGE can identify individual components.

• Co-immunoprecipitation with tagged atpH: This approach can pull down interaction partners when using antibodies against the tag. Mass spectrometry analysis of the precipitated proteins can identify both known and novel interactors.

• Förster resonance energy transfer (FRET): By tagging atpH and potential interaction partners with appropriate fluorophores, researchers can detect and quantify interactions in vivo.

• Yeast two-hybrid or split-ubiquitin assays: These can be used for initial screening of potential interaction partners, though results should be validated using the methods above.

When interpreting results, researchers should consider that the PPR protein family members, including those like PPR10, may interact with the transcripts of atpH rather than directly with the protein itself, affecting its expression and abundance .

What are effective strategies for overexpressing atpH in rice to enhance photosynthetic efficiency?

Effective strategies for atpH overexpression require careful genetic engineering approaches:

• Promoter selection: Using strong constitutive promoters like rice actin (Act) or tissue-specific promoters such as globulin (Glb) gene promoters has shown success in rice transformation systems .

• Co-expression with transcriptional activators: The rice transcription factor REB has demonstrated significant enhancement of transgene expression. When co-transformed with REB, transgenic rice showed 2.0-2.5 fold higher expression in transient assays and up to 3.7-fold increased expression in stable transformants .

• Signal peptide optimization: Ensuring proper chloroplast targeting by optimizing the transit peptide sequence improves the integration of recombinant atpH into the native ATP synthase complex.

• Selection of appropriate rice variety: The Kitaake variety is recommended for model experiments due to its shorter life cycle and established transformation protocols .

• Homozygosity confirmation: Selecting homozygous transformants (T2 generation) ensures stable inheritance and expression of the transgene, as demonstrated in successful ATP synthase subunit overexpression studies .

Results from related studies on ATP synthase subunit overexpression indicate that this approach can increase the abundance of the entire ATP synthase complex in thylakoid membranes when normalized to chlorophyll content, suggesting similar strategies would be effective for atpH .

How can CRISPR-Cas9 technology be applied to study atpH function in rice?

CRISPR-Cas9 technology offers precise genetic manipulation capabilities for studying atpH function:

• Guide RNA design: Target-specific guide RNAs should be designed to recognize unique sequences within the atpH gene, avoiding off-target effects in other chloroplast genes.

• Delivery methods for chloroplast genome editing:

  • Biolistic transformation of chloroplasts using gold particles coated with CRISPR-Cas9 components

  • Agrobacterium-mediated transformation with chloroplast-targeted Cas9

  • Polyethylene glycol (PEG)-mediated transformation of rice protoplasts

• Homoplasmy confirmation: Because chloroplasts contain multiple genome copies, confirming complete editing (homoplasmy) is essential through techniques such as:

  • Restriction fragment length polymorphism (RFLP) analysis

  • High-resolution melting analysis

  • Deep sequencing of the target region

• Phenotypic analysis framework:

  • Photosynthetic parameter measurements (CO2 assimilation, electron transport rate)

  • Growth analysis under varying light and temperature conditions

  • ATP/ADP ratio quantification

  • Thylakoid membrane organization assessment via electron microscopy

• Complementation studies: To confirm the specificity of observed phenotypes, researchers should perform complementation with wild-type atpH.

This approach allows for precise functional studies of atpH through the creation of specific mutations, deletions, or replacements that would be difficult to achieve through traditional breeding or random mutagenesis .

What are the considerations for designing fusion tags for atpH without disrupting its function?

Designing fusion tags for atpH requires careful consideration of structural and functional constraints:

• Tag position selection:

  • C-terminal tagging is generally preferred as the N-terminus contains critical targeting information for chloroplast localization

  • If N-terminal tagging is necessary, the tag should be inserted after the transit peptide cleavage site

• Tag size and properties:

  • Small tags (e.g., 6×His, FLAG, or Myc) minimize structural interference

  • Fluorescent protein tags should be connected via flexible linkers (3-5 repeats of Gly-Ser) to prevent disruption of assembly

  • Split tags that reconstitute when in proximity can be used to study subunit interactions

• Linker design considerations:

  • Hydrophilic linkers prevent interference with membrane integration

  • Length optimization (15-20 amino acids) provides sufficient flexibility

  • Protease cleavage sites allow tag removal when necessary

• Functional validation assessments:

  • ATP synthase complex assembly analysis via BN-PAGE

  • Proton translocation assays

  • ATP synthesis rate measurements

  • Growth phenotype evaluation

Research has shown that C-terminal Myc-tagging of ATP synthase subunits allows detection without compromising function, as demonstrated in studies with the AtpD subunit in rice . This suggests similar approaches would be suitable for atpH.

How does environmental stress affect atpH expression and ATP synthase assembly in rice?

Environmental stress significantly impacts atpH expression and ATP synthase assembly in rice through multiple mechanisms:

• Drought and osmotic stress effects:

  • Osmotic stress triggers hyperosmolality responses that alter Ca²⁺ signaling pathways affecting chloroplast gene expression

  • OSCA channels, which respond to osmotic stress, may indirectly influence ATP synthase function through altered ion homeostasis

  • Water deficiency typically leads to reduced atpH expression and ATP synthase assembly to conserve resources

• Temperature stress impacts:

  • Heat stress can accelerate translation of chloroplast proteins but may destabilize ATP synthase complexes

  • Cold stress generally decreases expression and slows assembly of ATP synthase components

  • Fluctuations between day and night temperatures affect the stability of atpH mRNA

• Light intensity influence:

  • High light conditions increase demand for ATP synthesis, potentially upregulating atpH

  • Prolonged exposure to excessive light can damage ATP synthase components requiring increased turnover

  • Regulatory mechanisms adjust the stoichiometry of ATP synthase components under varying light conditions

• Salinity stress effects:

  • Salt stress alters thylakoid membrane composition, affecting ATP synthase complex stability

  • Ionic imbalances disrupt proton gradient formation, impairing ATP synthase function

  • Adaptive responses may include post-translational modifications of atpH

These environmental factors should be carefully controlled in experimental designs focused on atpH function to avoid confounding variables affecting experimental outcomes .

What role do PPR proteins play in regulating atpH expression in chloroplasts?

Pentatricopeptide repeat (PPR) proteins play crucial regulatory roles in atpH expression through RNA-binding interactions:

• PPR10 regulatory mechanisms:

  • Binds specifically to the intergenic region between atpI and atpH

  • Stabilizes atpH transcripts by protecting them from 5'→3' exoribonuclease degradation

  • Alters the structure of the 5' end of atpH mRNA to promote translation initiation

  • Creates defined RNA termini through its binding, affecting transcript processing

• BFA2 (a P-class PPR protein) function:

  • Essential for the accumulation of atpH/F transcripts

  • Loss-of-function mutation results in specific reduction of more than 75% of chloroplast ATP synthase

  • Acts as a site-specific binding factor for RNA processing and stabilization

• Regulatory significance in research applications:

  • When designing expression systems for recombinant atpH, consideration of these PPR binding sites is crucial

  • Co-expression of appropriate PPR proteins may enhance recombinant atpH expression

  • Mutations in PPR binding sites can significantly impact atpH abundance independent of promoter strength

This RNA-level regulation by PPR proteins represents a critical layer of post-transcriptional control that researchers must consider when studying atpH expression or designing overexpression strategies .

How does atpH abundance correlate with photosynthetic parameters under varying light conditions?

The relationship between atpH abundance and photosynthetic parameters varies significantly under different light conditions:

Research observations indicate that:

• Under optimal light conditions:

  • Increased ATP synthase abundance through overexpression of components correlates with enhanced photosynthetic capacity

  • Enhanced ATP synthase activity supports increased carbon assimilation

• Under fluctuating light conditions:

  • Plants with higher ATP synthase abundance show improved adaptation to light transitions

  • Faster adjustment of proton motive force prevents photosystem damage

  • More efficient balance between ATP synthesis and NPQ activation

• Under high light stress:

  • Excessive ATP synthase activity may not proportionally increase photosynthesis

  • Regulatory mechanisms adjust actual enzyme activity independent of protein abundance

  • Post-translational modifications become increasingly important regulators

These correlations highlight that optimal photosynthetic performance depends not only on atpH abundance but also on proper stoichiometry with other ATP synthase components and regulatory adjustments under varying environmental conditions .

How can researchers address the stoichiometric challenges when manipulating individual ATP synthase subunits?

Addressing stoichiometric challenges in ATP synthase subunit manipulation requires sophisticated experimental approaches:

• Coordinated multi-gene expression strategies:

  • Design polycistronic constructs expressing multiple subunits under a single promoter

  • Use self-cleaving 2A peptides to achieve equimolar expression of multiple proteins

  • Employ the CRISPR/Cas9 system for simultaneous editing of multiple ATP synthase genes

  • Consider operon-like expression systems with appropriate spacing between genes

• Feedback-regulated expression systems:

  • Utilize promoters responsive to energy status or redox conditions

  • Design expression cassettes with regulatory elements sensitive to ATP/ADP ratio

  • Incorporate RNA thermosensors or riboswitches that adjust translation in response to metabolite levels

• Analytical techniques for stoichiometry assessment:

  • Blue native PAGE coupled with western blotting for complex composition analysis

  • Targeted mass spectrometry with isotope-labeled reference peptides for absolute quantification

  • Super-resolution microscopy to visualize complex assembly in situ

• Approaches to mitigate unincorporated subunit accumulation:

  • Co-express appropriate chaperones or assembly factors

  • Include conditional degrons for excess unassembled proteins

  • Design constructs with feedback inhibition of translation when free subunits accumulate

Research indicates that overexpression of specific ATP synthase subunits, such as AtpD, can increase the abundance of the entire complex, suggesting that certain subunits may be limiting factors in complex assembly. This understanding can guide strategic manipulation of multiple subunits to maintain proper stoichiometry .

What are the contradictory findings regarding atpH modification and their potential explanations?

Several contradictory findings exist in atpH research with important implications for experimental design:

• Growth phenotype inconsistencies:

  • Some studies report enhanced growth with increased ATP synthase abundance

  • Others show minimal growth improvements despite enhanced photosynthetic parameters

  • Potential explanation: Growth conditions in laboratory settings often don't challenge ATP production sufficiently to manifest phenotypes observed in field conditions

• ATP synthase abundance vs. activity discrepancies:

  • Increased protein abundance doesn't always correlate with proportional increases in activity

  • Potential explanations: Post-translational regulatory mechanisms, assembly constraints, or proton gradient limitations may create activity ceilings independent of protein levels

• Species-specific differences in regulation:

  • Findings from Arabidopsis don't always translate to rice and vice versa

  • Potential explanation: Evolutionary divergence in regulatory mechanisms, different optimization priorities based on ecological niches

• Contradictory effects of PPR proteins:

  • Some PPR protein mutations dramatically reduce atpH abundance while others show minimal effects

  • Potential explanation: Functional redundancy among PPR proteins or context-dependent regulatory roles

Researchers should address these contradictions by:

• Including comprehensive controls across multiple growth conditions

• Measuring both abundance and activity of ATP synthase

• Conducting comparative studies across species when possible

• Considering developmental timing in experimental design

These approaches help resolve apparent contradictions and build a more coherent understanding of atpH function across conditions and species .

What advanced structural biology techniques are revealing new insights about atpH interaction within the ATP synthase complex?

Cutting-edge structural biology techniques are providing unprecedented insights into atpH function:

• Cryo-electron microscopy advances:

  • Near-atomic resolution structures of plant ATP synthase have revealed precise interaction interfaces between subunits

  • Time-resolved cryo-EM is capturing conformational changes during the catalytic cycle

  • Comparative analysis between species highlights conserved and divergent features

  • The technique has been successfully applied to other membrane proteins from rice, suggesting applicability to ATP synthase

• Integrative structural biology approaches:

  • Combination of X-ray crystallography, NMR, and molecular dynamics simulations

  • Cross-linking mass spectrometry (XL-MS) to map interaction networks

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

  • These complement cryo-EM by providing dynamic information not captured in static structures

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to monitor subunit movement in real-time

  • Atomic force microscopy (AFM) for direct visualization of ATP synthase in native membranes

  • Optical and magnetic tweezers to measure forces during rotary catalysis

• In situ structural studies:

  • Cryo-electron tomography of chloroplast membranes capturing ATP synthase in its native environment

  • Correlative light and electron microscopy (CLEM) linking structure to function

  • These approaches reveal how membrane curvature and lipid composition affect ATP synthase organization

These advanced techniques have revealed that the c-ring of ATP synthase (containing atpH) exhibits species-specific adaptations in size and composition that fine-tune the bioenergetic efficiency. This has implications for engineering efforts aimed at optimizing photosynthetic efficiency in crop plants like rice .

What promising approaches might overcome current limitations in atpH research?

Several innovative approaches show promise for overcoming current research limitations:

• Synthetic biology platforms:

  • Designer ATP synthase with optimized c-subunit composition

  • Orthogonal expression systems segregated from native regulation

  • Minimal chloroplast genome approaches to eliminate regulatory complexity

  • These may allow precise manipulation of ATP synthase without confounding variables

• Single-cell omics for heterogeneity assessment:

  • Single-cell proteomics to quantify cell-to-cell variation in ATP synthase composition

  • Spatial transcriptomics to map expression patterns across different cell types in rice leaves

  • These techniques can reveal how cellular specialization affects ATP synthase optimization

• Artificial intelligence for design optimization:

  • Machine learning algorithms to predict optimal subunit combinations

  • Structure-based design of improved atpH variants with enhanced performance

  • Systems biology modeling to predict complex emergent properties from subunit modifications

• Optogenetic approaches:

  • Light-controlled expression or degradation of atpH

  • Photoswitchable ATP synthase inhibitors for precise temporal control

  • These tools enable dynamic studies of ATP synthase function with unprecedented temporal resolution

• Organelle isolation and manipulation techniques:

  • Improved methods for chloroplast isolation maintaining functional integrity

  • Microfluidic platforms for single-organelle analysis

  • These approaches reduce system complexity and enable more controlled experimentation

Implementation of these approaches could significantly advance our understanding of atpH function and guide more effective strategies for enhancing photosynthetic efficiency in rice and other crop plants .

How might atpH research contribute to climate-resilient rice varieties?

AtpH research has significant potential to contribute to climate-resilient rice varieties through several pathways:

• Enhanced photosynthetic efficiency under heat stress:

  • Optimized atpH variants with improved thermostability

  • Modified regulatory elements to maintain ATP synthase function during temperature fluctuations

  • These modifications could maintain productivity during increasingly frequent heat waves

• Improved water-use efficiency under drought conditions:

  • ATP synthase modifications that maintain function under lower stomatal conductance

  • Integration with OSCA channel research to coordinate osmotic stress responses

  • Potential to maintain energy production even with reduced water availability

• Salinity tolerance mechanisms:

  • Variants that maintain proton gradient integrity despite ionic imbalances

  • Coordination with ion transport systems to mitigate salt stress effects

  • These adaptations would support rice cultivation in increasingly saline soils

• Fluctuating light adaptation:

  • Optimized regulatory responses to rapidly changing light conditions

  • Enhanced photoprotection through coordinated ATP synthase and NPQ responses

  • Particularly valuable for maintaining productivity under unpredictable weather patterns

• CO₂ response optimization:

  • Adjusting ATP/NADPH production ratio to match demands of carbon fixation under elevated CO₂

  • Supporting increased productivity potential under future atmospheric conditions

Research already demonstrates that modified ATP synthase abundance affects photosynthetic parameters , suggesting that targeted atpH modifications could be a valuable component of multi-trait breeding strategies for developing climate-resilient rice varieties essential for food security in changing environments.

What interdisciplinary approaches might accelerate breakthroughs in ATP synthase engineering for rice improvement?

Accelerating breakthroughs in ATP synthase engineering requires strategic interdisciplinary collaboration:

• Integration of computational and experimental biology:

  • Molecular dynamics simulations to predict effects of mutations

  • Quantum mechanical calculations of proton transfer energetics

  • Machine learning prediction of protein-protein interactions

  • These computational approaches can guide targeted experimental design and reduce trial-and-error

• Combining synthetic biology with traditional breeding:

  • Precision engineering of ATP synthase followed by traditional crossing

  • CRISPR-based promotion of recombination at beneficial haplotypes

  • These approaches blend cutting-edge technology with established breeding methods

• Merging biophysics with crop physiology:

  • Linking molecular-level energetics to whole-plant performance

  • Field-deployable sensors for real-time ATP/ADP ratio monitoring

  • Translation of laboratory findings to relevant agricultural conditions

• Ecological perspectives in molecular design:

  • Consideration of plant-microbe interactions affecting photosynthesis

  • Environmental adaptation principles guiding genetic engineering

  • These perspectives ensure modifications perform well in complex ecosystems

• Integration with systems biology:

  • Metabolic flux analysis to understand system-wide impacts

  • Multi-omics integration (genomics, proteomics, metabolomics)

  • Identification of unexpected compensatory mechanisms

• Collaboration between basic research and agricultural extension:

  • Rapid translation of foundational discoveries to field applications

  • Farmer feedback informing research priorities

  • These connections ensure research addresses practical challenges

Successful interdisciplinary approaches have already demonstrated potential, as seen in studies where manipulation of ATP synthase components positively affected photosynthetic parameters , suggesting that continued integration across disciplines will accelerate practical applications of atpH research.