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

What is ATP synthase subunit c in Oryza sativa subsp. japonica and what is its function in chloroplasts?

ATP synthase subunit c, encoded by the atpH gene, is a critical component of the chloroplastic ATP synthase complex in Oryza sativa subsp. japonica (japonica rice). This protein functions as part of the membrane-embedded F0 portion of the ATP synthase, forming an oligomeric ring structure that facilitates proton translocation across the thylakoid membrane. The rotation of this c-ring, driven by the proton gradient established during photosynthesis, enables the catalytic F1 portion to synthesize ATP from ADP and inorganic phosphate.

In chloroplasts, this protein plays an essential role in the final steps of the light-dependent reactions of photosynthesis, converting the energy stored in the proton gradient into chemical energy in the form of ATP. The chloroplastic ATP synthase complex represents a crucial intersection between the light-dependent and light-independent reactions of photosynthesis in rice and other plant species.

What expression systems are most effective for producing recombinant Oryza sativa ATP synthase subunit c?

For researchers seeking to express recombinant ATP synthase subunit c from japonica rice, several expression systems have proven effective, each with distinct advantages depending on experimental objectives:

Bacterial Expression Systems:

• E. coli-based expression systems remain the most common choice for initial studies due to their rapid growth, high protein yield, and cost-effectiveness. BL21(DE3) strains with pET vector systems are particularly useful for expressing the mature protein without the transit peptide sequence.

• For membrane proteins like ATP synthase subunit c, E. coli C41(DE3) and C43(DE3) strains, specifically engineered for membrane protein expression, often yield better results by reducing toxicity issues.

Methodological Approach:

• Clone the atpH coding sequence (without transit peptide) into an expression vector with an appropriate tag (His6, GST, or MBP).

• Transform into the selected E. coli strain.

• Optimize expression conditions: typically induction with 0.1-0.5 mM IPTG at OD600 of 0.6-0.8, followed by incubation at lower temperatures (16-20°C) to enhance proper folding.

• Extract using mild detergents such as DDM (n-Dodecyl β-D-maltoside) or LDAO (Lauryldimethylamine oxide) to maintain protein integrity.

For functional studies requiring proper folding and post-translational modifications, plant-based expression systems like tobacco or rice cell cultures may be preferable, though with lower yields than bacterial systems.

How does the structure of ATP synthase subunit c from japonica rice compare to other plant species?

The chloroplastic ATP synthase subunit c from japonica rice exhibits high sequence conservation with other crop species, reflecting its fundamental role in energy metabolism. Structural analysis reveals:

• A compact protein (approximately 8 kDa) with two transmembrane α-helices connected by a hydrophilic loop

• Highly conserved acidic residue (typically glutamate) in the second transmembrane helix that is essential for proton translocation

• Sequence identity of approximately 95% with other cereal crops and 85-90% with dicotyledonous plants

The high degree of conservation reflects evolutionary constraints on this essential component of the cellular energy production machinery. Minor amino acid variations between species may contribute to differences in thermal stability or environmental adaptation but rarely affect the core functional domains.

What strategies can researchers employ to optimize purification of recombinant ATP synthase subunit c while maintaining structural integrity?

Purification of membrane proteins like ATP synthase subunit c presents unique challenges requiring specialized approaches:

Recommended Purification Protocol:

• Membrane Fraction Isolation: Following cell lysis, use differential centrifugation (40,000-100,000 × g) to isolate membrane fractions.

• Solubilization Optimization: Test a panel of detergents at varying concentrations:

  • DDM (n-Dodecyl β-D-maltoside): 1-2% for initial solubilization

  • LMNG (Lauryl maltose neopentyl glycol): 0.5-1% for enhanced stability

  • SMA (Styrene-maleic acid) copolymers: For native lipid environment preservation

• Affinity Chromatography: Utilize the fusion tag (typically His6) for initial purification on Ni-NTA resin with detergent concentrations maintained above CMC.

• Size Exclusion Chromatography: Further purify using Superdex 75/200 columns to separate monomeric subunit c from oligomeric assemblies.

• Stability Enhancement: Add lipids (POPC or native thylakoid lipids) at 0.1-0.5 mg/ml to stabilize the protein during storage.

Critical Parameter Monitoring:

• pH should be maintained between 7.0-8.0 to prevent aggregation

• Glycerol (10-20%) inclusion can enhance stability

• Samples should be maintained at 4°C throughout purification to prevent degradation

Researchers should validate the structural integrity of purified protein through circular dichroism spectroscopy, which typically shows characteristic α-helical signatures with minima at 208 and 222 nm for properly folded ATP synthase subunit c.

What molecular techniques are most effective for studying interactions between ATP synthase subunit c and other components of the chloroplastic ATP synthase complex?

To effectively study the interactions between ATP synthase subunit c and other components of the chloroplastic ATP synthase complex, researchers should consider these complementary approaches:

In Vitro Interaction Studies:

• Co-immunoprecipitation (Co-IP): Using antibodies against subunit c or other complex components to pull down interaction partners.

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics between purified components.

• Microscale Thermophoresis (MST): Particularly useful for determining binding affinities in near-native conditions.

In Vivo Interaction Analysis:

• Förster Resonance Energy Transfer (FRET): Tag subunit c and potential interaction partners with appropriate fluorophores to observe real-time interactions in chloroplasts.

• Split-GFP Complementation: Fusion of complementary GFP fragments to subunit c and other complex components to visualize interactions through fluorescence restoration.

• Chemical Cross-linking followed by Mass Spectrometry (XL-MS): To capture transient interactions within the native complex.

Structural Approaches:

• Cryo-electron Microscopy: For visualizing the entire ATP synthase complex with subunit c in its native oligomeric state.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): To identify interaction interfaces through differential solvent accessibility.

When implementing these techniques, researchers should consider using site-directed mutagenesis to systematically alter key residues in subunit c to map interaction surfaces and assess functional consequences of disrupted interactions.

How can researchers effectively analyze gene expression patterns of atpH in response to environmental stresses in japonica rice?

Comprehensive analysis of atpH expression patterns under environmental stress requires a multi-tiered approach:

Transcriptional Analysis Methods:

• Quantitative Real-Time PCR (qRT-PCR):

  • Design primers specific to the atpH transcript region (avoiding homologous sequences)

  • Validate reference genes specific to stress conditions (common choices include Ubiquitin, Actin, or EF1α)

  • Implement the 2^-ΔΔCt method for relative quantification

• RNA-Seq Analysis:

  • For genome-wide expression patterns revealing co-regulated genes

  • Minimum sequencing depth of 20 million reads per sample

  • Use DESeq2 or EdgeR for differential expression analysis with FDR < 0.05

Protein-Level Validation:

• Western Blotting: Using antibodies specific to ATP synthase subunit c

• Multiple Reaction Monitoring (MRM): For targeted proteomics quantification

Experimental Design Considerations:

• Include appropriate time course sampling (early response: 1-6h; acclimation: 24-72h)

• Apply gradual stress treatments when possible to mimic natural conditions

• Include control conditions with proper biological replicates (minimum n=3)

Environmental Stress Conditions Relevant to Rice ATP Synthase Function:

• Temperature stress (heat: 35-42°C; cold: 4-15°C)

• Drought (withholding water or PEG treatment)

• Salinity (100-200 mM NaCl)

• Heavy metal exposure (particularly Cd²⁺ or As)

Integrating transcriptomics with metabolomics data (especially ATP/ADP ratios and photosynthetic parameters) provides a more comprehensive understanding of the physiological significance of atpH expression changes.

How can marker-assisted selection be used to study genes like atpH in rice breeding programs?

Marker-assisted selection represents a powerful approach for incorporating genes of interest, such as atpH, into rice breeding programs. Based on approaches demonstrated in japonica rice research, the following methodology is recommended:

Development of DNA Markers:

• Polymorphism Identification: Begin by identifying SNPs, insertions/deletions, or SSRs in the atpH gene region through comparative sequencing of diverse japonica rice varieties.

• Marker Design: Develop specific markers that can differentiate allelic variants:

  • SNP markers using CAPS (Cleaved Amplified Polymorphic Sequences) or dCAPS approaches

  • Insertion/deletion markers using length polymorphism detection

  • SSR markers based on variable repeat regions

Multiple regression analysis can be used to associate marker data with phenotypic traits, as demonstrated in research on eating quality traits in japonica rice . This approach achieved remarkable prediction accuracy with R² values as high as 0.99 for complex traits.

Validation Process:

• Test markers across diverse germplasm to confirm polymorphism and association with target traits

• Validate marker efficacy in breeding populations through phenotypic evaluation

• Implement in breeding programs through early-generation selection

Table 1: Example of Multiple Regression Analysis for Marker-Trait Association

Note: ** indicates significance at 1% level .

When applying this methodology to genes like atpH, researchers should focus on linking molecular markers to relevant physiological traits such as photosynthetic efficiency, stress tolerance, or growth parameters that might be influenced by ATP synthase function.

What advanced analytical techniques can be used to characterize post-translational modifications of ATP synthase subunit c?

Post-translational modifications (PTMs) of ATP synthase subunit c can significantly influence protein function, stability, and interactions. Researchers should employ these advanced analytical techniques for comprehensive PTM characterization:

Mass Spectrometry-Based Approaches:

• Bottom-up Proteomics:

  • Enzymatic digestion (typically trypsin) followed by LC-MS/MS analysis

  • Precursor ion scanning for specific PTM-related mass shifts

  • Parallel reaction monitoring (PRM) for targeted analysis of modified peptides

• Top-down Proteomics:

  • Analysis of intact protein to maintain PTM combinations and stoichiometry

  • High-resolution instruments (Orbitrap or FTICR) are essential

  • Electron transfer dissociation (ETD) fragmentation preserves labile modifications

PTM Enrichment Strategies:

• Phosphorylation: TiO₂ or IMAC (Fe³⁺/Ga³⁺) enrichment

• Glycosylation: Lectin affinity chromatography or hydrazide chemistry

• Acetylation: Anti-acetyllysine antibody immunoprecipitation

Data Analysis Considerations:

• Use multiple search algorithms (MASCOT, SEQUEST, MaxQuant) with appropriate PTM settings

• Apply false discovery rate control (typically <1% at peptide level)

• Validate unexpected modifications through synthetic peptide standards

Complementary Methods:

• 2D gel electrophoresis with specific PTM staining (Pro-Q Diamond for phosphorylation)

• Site-directed mutagenesis of modified residues to assess functional significance

• Antibodies against specific modifications for immunodetection

ATP synthase subunit c may undergo several PTMs that affect its function, including phosphorylation, acetylation, and oxidative modifications. Mapping these modifications under different physiological conditions provides insights into regulatory mechanisms governing ATP synthase activity in chloroplasts.

How can researchers design effective CRISPR/Cas9 experiments to study the function of atpH gene in japonica rice?

Designing effective CRISPR/Cas9 experiments for studying the atpH gene requires careful consideration of the unique challenges associated with chloroplast-targeted proteins and essential metabolic functions:

Strategic Considerations:

• Gene Essentiality Management: Complete knockout of atpH may be lethal, so consider:

  • Inducible CRISPR systems (using dexamethasone-inducible promoters)

  • Tissue-specific promoters (mesophyll-specific or developmentally regulated)

  • Creating knockdown rather than knockout (CRISPRi approach with dCas9)

  • Heteroplasmy management strategies for chloroplast genome editing

• Guide RNA (gRNA) Design:

  • Target regions with minimal off-target potential in the rice genome

  • Design 2-3 gRNAs per target region to increase editing efficiency

  • Avoid regions with secondary structure that might impede Cas9 access

  • Use rice-optimized U6 or U3 promoters for gRNA expression

Implementation Protocol:

• Vector Construction:

  • Clone rice codon-optimized Cas9 and gRNAs into a binary vector

  • Include appropriate selectable markers (hygromycin or kanamycin resistance)

• Delivery Methods:

  • Agrobacterium-mediated transformation of rice callus

  • Protoplast transformation for transient expression and validation

  • Biolistic delivery for direct chloroplast transformation

• Screening and Validation:

  • Initial PCR screening followed by Sanger sequencing

  • T7 Endonuclease I assay for mutation detection

  • Deep sequencing for comprehensive mutation profiling

Phenotypic Analysis Approach:

• Monitor photosynthetic parameters (Fv/Fm, ETR, NPQ) using chlorophyll fluorescence

• Assess ATP/ADP ratios in leaf tissue using bioluminescence assays

• Measure growth parameters under different light intensities

• Evaluate stress responses, particularly to high light and temperature fluctuations

Table 2: Potential gRNA Target Sites for atpH in Japonica Rice

Note: Efficiency and off-target scores are theoretical predictions based on typical CRISPR design algorithms.

For studying subtle functional changes, researchers should consider creating specific point mutations rather than complete gene disruption, as this may reveal structure-function relationships while maintaining cell viability.

What are the most common challenges in recombinant expression of ATP synthase subunit c and how can they be addressed?

Recombinant expression of ATP synthase subunit c poses several challenges due to its hydrophobic nature and role as a membrane protein. Here are the most common issues and their methodological solutions:

Challenge 1: Protein Toxicity to Expression Hosts

• Solution: Use tightly controlled induction systems (e.g., pBAD or T7lac promoters) with minimal leaky expression

• Methodology: Begin with low inducer concentrations (0.01-0.1 mM IPTG) and short induction periods (3-4 hours)

• Alternative Approach: Use specialized E. coli strains like C41(DE3) and C43(DE3) specifically developed for toxic membrane proteins

Challenge 2: Inclusion Body Formation

• Solution: Reduce expression temperature to 16-20°C after induction

• Methodology: Add fusion partners that enhance solubility (MBP, SUMO, or Trx tags)

• Recovery Protocol: If inclusion bodies form, implement a specialized refolding protocol using a urea gradient (8M to 0M) in the presence of lipids or detergents

Challenge 3: Low Yield

• Solution: Optimize codon usage for the expression host

• Methodology: Increase scale while maintaining optimal cell density (OD600 = 0.6-0.8 at induction)

• Enhancement Strategy: Supplement growth media with specific components (0.2% glucose pre-induction, 1% ethanol post-induction)

Challenge 4: Improper Folding

• Solution: Co-express with appropriate chaperones (GroEL/ES, DnaK/J)

• Methodology: Include lipids or detergent mixtures in the growth media

• Verification Technique: Use circular dichroism to confirm secondary structure integrity

Table 3: Optimization Matrix for Recombinant ATP Synthase Subunit c Expression

A systematic approach testing multiple conditions is recommended, with small-scale expression tests (5-10 mL cultures) before scaling up to larger volumes.

How can researchers effectively analyze the impact of point mutations on ATP synthase subunit c function?

Analyzing the impact of point mutations on ATP synthase subunit c requires a systematic approach combining in vitro and in vivo methodologies:

In Vitro Functional Assessment:

• ATP Hydrolysis/Synthesis Assays:

  • Reconstitute purified mutant proteins into liposomes

  • Measure ATP synthesis using luciferin/luciferase assays

  • Assess ATP hydrolysis through phosphate release detection

  • Calculate enzymatic parameters (Km, Vmax, kcat) for comparative analysis

• Proton Translocation Assays:

  • Use pH-sensitive fluorescent dyes (ACMA or pyranine)

  • Measure proton pumping efficiency across proteoliposome membranes

  • Determine H⁺/ATP ratios for mutant versus wild-type proteins

In Vivo Functional Studies:

• Complementation Assays:

  • Express mutant variants in atpH-deficient backgrounds

  • Measure growth rates under photosynthetic conditions

  • Assess ATP levels in transformed lines

• Physiological Measurements:

  • Chlorophyll fluorescence analysis (Fv/Fm, NPQ, ETR)

  • Photosynthetic oxygen evolution measurements

  • Growth analysis under varying light conditions

Structural Impact Assessment:

• Molecular Dynamics Simulations:

  • Model the effect of mutations on protein stability and interactions

  • Calculate free energy differences between wild-type and mutant structures

  • Analyze proton access pathways through the c-ring

• Experimental Structure Determination:

  • Cryo-EM analysis of assembled ATP synthase complexes

  • HDX-MS to probe conformational changes induced by mutations

Table 4: Critical Residues in ATP Synthase Subunit c and Their Functional Significance

Note: Exact residue positions would depend on the specific sequence of japonica rice ATP synthase subunit c.

When analyzing mutations, researchers should prioritize residues that are highly conserved across species, as these are most likely to serve critical functional roles in the ATP synthase complex.

What statistical approaches are most appropriate for analyzing data from ATP synthase functional studies?

For Comparative Studies (Wild-type vs. Mutant/Treatment):

• Parametric Tests:

  • Student's t-test for two-group comparisons (with normality verification)

  • ANOVA with post-hoc tests (Tukey's HSD or Bonferroni) for multiple group comparisons

  • Paired t-tests for before/after comparisons on the same samples

• Non-parametric Alternatives:

  • Mann-Whitney U test (for non-normal two-group comparisons)

  • Kruskal-Wallis with Dunn's post-hoc test (for multiple non-normal groups)

  • Wilcoxon signed-rank test (for paired non-normal data)

For Correlation and Regression Analysis:

• Simple/Multiple Linear Regression:

  • For predicting continuous outcomes based on experimental variables

  • Example application: Predicting ATP synthesis rates based on multiple factors

  • Model validation through residual analysis and cross-validation

• Specialized Regression Models:

  • Non-linear regression for enzyme kinetics data (Michaelis-Menten, Hill equation)

  • Mixed-effects models for repeated measures designs

As demonstrated in the research on japonica rice eating quality, multiple regression analysis can effectively relate marker genotypes to phenotypic traits with high predictive power (R² values up to 0.99) . This approach can be adapted for analyzing relationships between ATP synthase genetic variants and functional outcomes.

Data Visualization Recommendations:

• Box plots or violin plots for group comparisons

• Scatter plots with regression lines for correlation analysis

• Heatmaps for visualizing patterns across multiple variables

• Forest plots for meta-analysis of replicated experiments

Statistical Power Considerations:

• Conduct a priori power analysis to determine appropriate sample sizes

• For typical biochemical assays, aim for n≥3 biological replicates

• For physiological measurements with higher variability, consider n≥5

• Report effect sizes (Cohen's d, η²) alongside p-values

Table 5: Recommended Minimum Sample Sizes for Different Experimental Designs

Researchers should employ appropriate statistical software (R, GraphPad Prism, or SPSS) and report complete statistical parameters including test statistics, degrees of freedom, p-values, and confidence intervals.

What emerging technologies hold the most promise for advancing our understanding of ATP synthase subunit c function and regulation?

Several cutting-edge technologies are positioned to significantly advance our understanding of ATP synthase subunit c function and regulation in the coming years:

Structural Biology Advances:

• Time-resolved Cryo-EM: Capturing intermediate states during the rotational catalysis cycle of ATP synthase with millisecond temporal resolution.

• Integrative Structural Biology: Combining multiple techniques (X-ray crystallography, NMR, SAXS) to build comprehensive models of the entire ATP synthase complex.

• In-cell NMR: Examining conformational dynamics of ATP synthase components within intact chloroplasts.

Single-Molecule Techniques:

• High-Speed Atomic Force Microscopy: Visualizing conformational changes of individual ATP synthase complexes during catalysis.

• Optical Tweezers and Magnetic Tweezers: Measuring torque generation and mechanochemical coupling in reconstituted ATP synthase systems.

• Single-molecule FRET: Tracking distance changes between labeled subunits during rotational catalysis.

Genome Engineering Approaches:

• Base Editing and Prime Editing: Making precise nucleotide changes in the atpH gene without double-strand breaks.

• Chloroplast-specific CRISPR Systems: Directly editing the chloroplast genome to modify atpH in situ.

• Synthetic Biology Redesign: Engineering optimized versions of ATP synthase subunit c with enhanced performance characteristics.

Advanced Imaging:

• Super-resolution Microscopy: Visualizing ATP synthase distribution and clustering in thylakoid membranes below the diffraction limit.

• Correlative Light and Electron Microscopy (CLEM): Connecting functional states with structural arrangements in native membranes.

• Label-free Imaging: Using techniques like Coherent Raman Scattering to observe ATP synthase without potentially disruptive labels.

These technologies promise to resolve longstanding questions about how this small but critical protein contributes to the remarkable efficiency of biological energy conversion in chloroplasts.

How might comparative genomics approaches enhance our understanding of atpH evolution and function across different rice varieties?

Comparative genomics approaches offer powerful frameworks for understanding the evolutionary history and functional diversity of the atpH gene across rice varieties:

Methodological Approaches:

• Pan-genome Analysis:

  • Construct a rice pan-genome incorporating multiple high-quality japonica and indica assemblies

  • Identify core, dispensable, and variety-specific elements related to atpH

  • Analyze structural variations in the gene region (CNVs, presence/absence variants)

• Phylogenomic Analysis:

  • Reconstruct the evolutionary history of atpH across Oryza species and close relatives

  • Apply selection pressure analysis (dN/dS ratios) to identify adaptively evolving sites

  • Use ancestral sequence reconstruction to trace the evolutionary trajectory of key functional residues

• Population Genomics:

  • Analyze atpH sequence variation across diverse rice populations (3,000 Rice Genomes Project data)

  • Identify SNPs, haplotypes, and linkage blocks associated with environmental adaptations

  • Conduct genome-wide association studies linking atpH variants to phenotypic traits

Implementation Strategy:

• Extract and align atpH sequences from multiple rice varieties and wild relatives

• Calculate sequence diversity parameters (π, θ, Tajima's D) to identify signatures of selection

• Correlate sequence variants with environmental parameters from collection sites

• Validate functional consequences through targeted experiments

The PCR marker-based evaluation approach demonstrated for eating quality traits in japonica rice provides a model for relating genetic markers to functional outcomes that could be adapted to study ATP synthase function across varieties.

Table 6: Example of atpH Sequence Diversity Parameters Across Rice Varieties

Note: Values in this table are hypothetical and would be determined through actual sequence analysis.

Comparative genomics approaches can reveal how natural selection has optimized ATP synthase function across different environments and provide insights into the molecular basis of adaptation to diverse ecological niches.