Recombinant Oenothera glazioviana ATP synthase subunit b, chloroplastic (atpF)

What is the function of ATP synthase subunit b in Oenothera chloroplasts?

ATP synthase subunit b (atpF) is a critical component of the chloroplastic ATP synthase complex, which catalyzes ATP synthesis during photosynthesis. In Oenothera species, this protein forms part of the membrane-embedded F0 portion of the ATP synthase, serving as a peripheral stalk that connects the F1 and F0 portions of the complex. The protein plays an essential role in maintaining the structural integrity of the ATP synthase and facilitating the energy conversion process that transforms the proton gradient across the thylakoid membrane into chemical energy in the form of ATP . Research in Oenothera has shown that the atpF gene is particularly important for proper chloroplast function, with mutations potentially affecting plant vigor and photosynthetic efficiency .

How do the five genetically distinct plastomes of Oenothera differ with respect to ATP synthase genes?

The five basic Oenothera plastomes (I-V) show several polymorphisms in ATP synthase-related genes. Comparative analyses of complete nucleotide sequences reveal that these plastomes have evolved distinct genetic characteristics while maintaining core photosynthetic functions. The plastome differences lead to significant physiological variations, particularly regarding chlorophyll a/b ratios, chlorophyll content per leaf area, and chloroplast ATP synthase activity . For instance, plants with plastome I (such as O. elata) demonstrate better adaptation to high light conditions compared to plants with plastome II, which is reflected in their ATP synthase efficiency. These differences are primarily attributed to the plastome rather than the nuclear genetic background, as evidenced by studies comparing AA-I and AA-II plants under varying light conditions .

What research techniques are commonly used to study recombinant atpF in Oenothera?

Several methodological approaches are employed to study recombinant atpF in Oenothera:

• Molecular Cloning and Expression: The atpF gene is amplified using PCR with specific primers, cloned into expression vectors, and expressed in bacterial or plant expression systems.

• Nucleotide Sequencing: Complete sequencing of the chloroplast genome, particularly using next-generation sequencing technologies, has enabled precise identification of variations in the atpF gene across different Oenothera species .

• Northern Blot Analysis: This technique is used to analyze transcript patterns and accumulation in different genetic backgrounds and under varying environmental conditions .

• Western Blot Analysis: Used to quantify protein levels and examine post-translational modifications of the ATP synthase subunit b .

• Run-on Transcription Analysis: This approach helps determine if changes in transcript levels are due to altered transcription rates or changes in transcript stability .

• Chloroplast Isolation and ATP Synthase Activity Assays: These methods allow for direct measurement of ATP synthase function in different plastome backgrounds .

How do frameshift mutations in ATP synthase genes affect protein function in Oenothera, and what recoding mechanisms might be involved?

This recoding phenomenon appears to involve ribosomal frameshifting during translation, which has been confirmed through the analysis of transplastomic tobacco lines where the atpB gene was experimentally modified to contain similar mutations . The mechanism likely involves the ribosome slipping at the oligoA repeat sequence, effectively "correcting" the frameshift and allowing translation to continue in the proper reading frame.

This represents an important adaptive mechanism that may contribute to the genetic flexibility of Oenothera species, potentially allowing them to maintain functional protein synthesis despite genomic mutations. The relevance of similar mechanisms to atpF remains an area for further investigation.

What role does the atpF gene play in plastid-nuclear incompatibility syndromes observed in Oenothera hybrids?

The incompatibility manifests primarily under high light conditions and results in reduced electron transport capacity. Studies show that in incompatible AB-I hybrids, there is light-dependent impairment of transcription for multiple photosynthetic components, which would ultimately affect ATP production as well .

The atpF gene, as part of the essential ATP synthase complex, is indirectly affected by these broader disruptions to chloroplast function. In compatible combinations, ATP synthase activity remains robust even under varying light intensities, while incompatible combinations show compromised energy production, particularly under high light stress .

This finding highlights the complex interactions between nuclear and plastid genomes in maintaining proper chloroplast function and energy production, with atpF functionality being dependent on proper coordination between these genetic compartments.

What methodological approaches can resolve contradictory findings regarding atpF expression and function in different Oenothera species?

When confronted with contradictory findings regarding atpF expression and function across Oenothera species, researchers can employ several methodological approaches:

• Standardized Growth Conditions: Establish strictly controlled growth environments to eliminate environmental variables that might affect atpF expression. This should include standardized light regimes (intensity, duration, quality), temperature, humidity, and nutritional status.

• Comparative Transcriptomics: Implement RNA-Seq analyses across multiple Oenothera species and under varying conditions to generate comprehensive transcription profiles of atpF and related genes. This can reveal species-specific expression patterns and regulatory mechanisms.

• Protein Quantification and Activity Assays: Employ a combination of western blot analysis, mass spectrometry, and ATP synthase activity assays to directly measure both protein abundance and functional activity.

• Plastome Swapping Experiments: Create cybrid plants containing the nuclear genome of one species and the plastome of another to directly test the effects of different plastome versions of atpF in standardized nuclear backgrounds .

• Site-Directed Mutagenesis: Generate specific mutations in the atpF gene to test the functional significance of observed polymorphisms between species.

• Chloroplast Isolation and In Vitro Reconstitution: Isolate chloroplasts and ATP synthase complexes from different species to test functional properties in controlled biochemical environments.

By combining these approaches, researchers can develop a more complete understanding of atpF function across the Oenothera genus and resolve apparent contradictions in previous findings.

How should researchers design experiments to study the light-dependent regulation of atpF in Oenothera species?

Designing experiments to study light-dependent regulation of atpF in Oenothera requires a multifaceted approach:

Experimental Design Framework:

This comprehensive experimental design allows for detailed characterization of how light regulates atpF expression and function across different Oenothera genetic backgrounds.

What are the optimal conditions for expressing and purifying recombinant Oenothera glazioviana ATP synthase subunit b for structural studies?

The expression and purification of recombinant Oenothera glazioviana ATP synthase subunit b requires careful optimization due to the membrane-associated nature of this protein and its chloroplastic origin.

Recommended Protocol for Optimal Expression and Purification:

• Expression System Selection:

  • E. coli BL21(DE3) strains optimized for membrane protein expression (C41/C43)

  • Alternative: Cell-free expression systems to avoid toxicity issues

  • For native conformation studies: Chlamydomonas or tobacco chloroplast transformation systems

• Vector Design:

  • Codon optimization for the expression system

  • Addition of N-terminal His6 or Strep-II tag for purification

  • Inclusion of a cleavable signal sequence to enhance membrane integration

  • Consider fusion with solubility-enhancing partners (MBP, SUMO) for initial expression trials

• Expression Conditions:

  Parameter	Bacterial Expression	Chloroplast Expression
  Temperature	16-18°C	22-25°C
  Induction	0.1-0.5 mM IPTG	N/A
  Duration	16-20 hours	3-4 weeks
  Media	2xYT with 5% glycerol	N/A
  Supplements	1% glucose	N/A

• Membrane Protein Solubilization:

  • Initial screening of detergents: DDM (n-dodecyl β-D-maltoside), LDAO, OG

  • Detergent concentration: 1-2% for solubilization, 0.05-0.1% for purification

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, 1 mM DTT

• Purification Strategy:

  • IMAC (Immobilized Metal Affinity Chromatography) as initial capture step

  • Ion exchange chromatography for intermediate purification

  • Size exclusion chromatography for final polishing and buffer exchange

  • Consider nanodisc reconstitution for structural studies

• Quality Assessment:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Mass spectrometry to verify protein integrity

  • Circular dichroism to assess secondary structure content

  • Thermal shift assays to evaluate stability in different buffer conditions

This optimized protocol provides a foundation for obtaining high-quality recombinant Oenothera glazioviana ATP synthase subunit b suitable for structural and functional studies.

How can researchers distinguish between direct effects on atpF expression and secondary effects due to broader photosynthetic dysfunction?

Distinguishing direct effects on atpF expression from secondary effects caused by broader photosynthetic dysfunction requires a systematic approach integrating multiple lines of evidence:

• Temporal Analysis:

  • Monitor atpF expression changes over a detailed time course following experimental treatments

  • Compare timing of atpF changes with other photosynthetic parameters

  • Early responses (minutes to hours) are more likely to represent direct effects, while delayed responses (days) often indicate secondary effects

• Comparative Expression Analysis:

  • Examine expression patterns of nuclear-encoded ATP synthase subunits in parallel

  • Analyze co-regulated genes in the chloroplast genome

  • Compare with expression changes in functionally unrelated chloroplast genes

  • Identify genes showing similar or divergent expression patterns across treatments

• Targeted Genetic Approaches:

  • Utilize specific promoter modifications to directly affect atpF expression

  • Employ inducible expression systems to control expression timing

  • Use site-directed mutagenesis to alter regulatory elements while maintaining coding sequences

• Physiological Uncoupling:

  Technique	Application	Outcome Measurement
  DCMU treatment	Blocks electron flow from PSII	ATP synthesis continues via cyclic electron flow
  Antimycin A	Inhibits cyclic electron flow	Isolates linear electron transport effects
  Uncouplers (FCCP)	Dissipates proton gradient	Separates ATP synthesis from electron transport
  Inhibitors (oligomycin)	Blocks ATP synthase	Distinguishes ATP synthesis from assembly effects

• Isolated Chloroplast Studies:

  • Perform in vitro transcription/translation assays with isolated chloroplasts

  • Supply exogenous ATP to bypass photosynthetic generation

  • Analyze atpF expression under controlled redox and energetic states

• Integration with Metabolic Data:

  • Correlate atpF expression changes with ATP/ADP ratios

  • Monitor other energy-related metabolites (NADPH/NADP+)

  • Assess retrograde signaling metabolites that may mediate nuclear-chloroplast communication

By implementing this multifaceted approach, researchers can effectively differentiate between direct regulatory effects on atpF and secondary consequences of altered photosynthetic function in Oenothera species .

What bioinformatic approaches are most effective for analyzing atpF sequence variation across Oenothera species and its evolutionary implications?

Analyzing atpF sequence variation across Oenothera species requires sophisticated bioinformatic approaches to extract meaningful evolutionary insights:

• Comparative Sequence Analysis Pipeline:

  • Whole plastome sequencing and assembly from multiple Oenothera species and populations

  • Multiple sequence alignment using MAFFT or MUSCLE with chloroplast-specific gap penalties

  • Extraction of atpF gene sequences and flanking regulatory regions

  • Identification of conserved domains, variable regions, and potential regulatory elements

• Polymorphism Characterization:

  Analysis Type	Tools	Output Metrics
  SNP/Indel Detection	GATK, FreeBayes	Position, type, frequency
  Haplotype Analysis	DnaSP, Arlequin	Haplotype diversity, linkage disequilibrium
  Selection Analysis	PAML, HyPhy	dN/dS ratios, selection coefficients
  Recombination Detection	RDP4, LDhat	Recombination breakpoints, rates
  RNA Editing Site Prediction	PREPACT, CURE-Chloroplast	Editing efficiency, conservation

• Phylogenetic Approaches:

  • Maximum likelihood tree construction using RAxML or IQ-TREE

  • Bayesian inference using MrBayes or BEAST2

  • Reconciliation with species trees to identify potential horizontal transfer events

  • Molecular clock analyses to date divergence events

• Structural Bioinformatics:

  • Homology modeling of ATP synthase subunit b using SWISS-MODEL or I-TASSER

  • Prediction of transmembrane domains using TMHMM or Phobius

  • Assessment of structural impacts of sequence variations using molecular dynamics simulations

  • Identification of coevolving residues using statistical coupling analysis

• Association Analysis:

  • Correlation of sequence variants with phenotypic traits

  • Identification of plastome-specific sequence signatures

  • Mapping of incompatibility-related polymorphisms

  • Integration with transcriptomic data to connect sequence variation with expression differences

• Visualization and Interpretation:

  • Circos plots for whole-plastome comparison

  • Interactive phylogenetic trees with mapped traits using iTOL

  • Protein structure visualization with PyMOL or Chimera

  • Custom R scripts for integrating and visualizing multiple data types

What are the primary challenges in producing functional recombinant Oenothera atpF protein, and how can they be overcome?

Producing functional recombinant Oenothera atpF protein presents several technical challenges due to its membrane-associated nature and origin from the chloroplast genome. Here are the key challenges and strategic solutions:

• Challenge: Membrane Protein Solubility

  Solutions:

  • Develop fusion constructs with highly soluble partners (MBP, SUMO, Trx)

  • Screen multiple detergents systematically (DDM, LDAO, Brij-35, C12E8)

  • Employ amphipols or nanodiscs for maintaining native conformation

  • Consider cell-free expression systems that allow direct incorporation into liposomes

• Challenge: Proper Folding in Heterologous Systems

  Solutions:

  • Decrease expression temperature to 16-18°C to slow translation rate

  • Co-express with chloroplast-specific chaperones

  • Utilize specialized E. coli strains (SHuffle, Origami) with oxidizing cytoplasmic environment

  • Include natural binding partners in co-expression systems

  • Explore in vitro refolding protocols with gradual detergent exchange

• Challenge: Post-translational Modifications

  Solutions:

  • Characterize native modifications using mass spectrometry

  • Select expression systems capable of performing required modifications

  • Employ enzymatic treatments for in vitro modification

  • Design constructs that accommodate lack of modifications when appropriate

• Challenge: Codon Usage Bias

  Solutions:

  • Optimize codons for expression host while maintaining key regulatory features

  • Utilize strains with expanded tRNA repertoires

  • Consider synthetic gene synthesis with optimized sequences

  • Test multiple optimization algorithms and select empirically superior performers

• Challenge: Functional Verification

  Solutions:

  Verification Method	Application	Expected Outcome
  ATP Synthase Reconstitution	Mix with other purified subunits	ATP synthesis activity
  Proteoliposome Incorporation	Membrane insertion assay	Proton pumping activity
  Circular Dichroism	Secondary structure analysis	Proper folding confirmation
  Binding Assays	Interaction with partner proteins	Complex formation
  Limited Proteolysis	Structural integrity assessment	Defined digestion pattern

• Challenge: Low Expression Yield

  Solutions:

  • Scale up using fermentation systems with tight control of growth parameters

  • Optimize media composition with supplemental amino acids and carbon sources

  • Implement two-phase expression systems with initial biomass accumulation

  • Explore alternative expression hosts (Pichia pastoris, insect cells)

  • Consider chloroplast transformation systems for native-like expression

By systematically addressing these challenges, researchers can significantly improve the production of functional recombinant Oenothera atpF protein for structural and functional studies .

How can researchers effectively study interactions between nuclear-encoded assembly factors and chloroplast-encoded ATP synthase subunit b in Oenothera?

Investigating interactions between nuclear-encoded assembly factors and chloroplast-encoded ATP synthase subunit b requires specialized approaches that bridge the gap between the two genetic compartments:

• In vivo Interaction Approaches:

  • Split-GFP/BiFC Systems: Adapt for chloroplast-nuclear protein interactions by targeting fusion proteins to appropriate compartments

  • In vivo Crosslinking: Employ formaldehyde or photo-crosslinking to capture transient interactions

  • Co-immunoprecipitation: Use antibodies against atpF or tagged assembly factors to pull down intact complexes

  • Proximity Labeling: Employ APEX2 or BioID systems fused to atpF to identify proximal proteins

• Genetic Approaches:

  • Nuclear Mutant Screening: Identify nuclear mutants affecting ATP synthase assembly

  • Complementation Studies: Transform nuclear assembly factor genes into mutant backgrounds

  • Plastome Engineering: Create tagged versions of atpF in transplastomic plants

  • Interspecific Hybrids: Study different combinations of nuclear and plastid genomes

• Biochemical Approaches:

  Technique	Application	Expected Outcome
  Blue Native PAGE	Separate intact complexes	Assembly intermediates identification
  2D-PAGE	Resolve complex components	Subunit composition analysis
  Size Exclusion Chromatography	Purify complexes by size	Assembly state determination
  Sucrose Gradient Centrifugation	Separate by density	Complex integrity assessment
  Mass Spectrometry	Identify interacting proteins	Complete interactome mapping

• Real-time Assembly Monitoring:

  • Pulse-chase experiments with radiolabeled amino acids

  • Time-course analysis of complex formation

  • Inducible expression systems for assembly factors

  • In organello protein import assays with isolated chloroplasts

• Structural Biology Integration:

  • Cryo-electron microscopy of isolated ATP synthase complexes

  • Crosslinking mass spectrometry (XL-MS) to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify binding regions

  • Fluorescence resonance energy transfer (FRET) to measure proximity relationships

• Systems Biology Approaches:

  • Correlative transcriptomics of nuclear and chloroplast genes

  • Proteomics profiling under varying assembly conditions

  • Network analysis to identify coordinated expression patterns

  • Integration of genetic, biochemical, and structural data in comprehensive models

This multifaceted strategy enables researchers to thoroughly characterize the complex interactions between nuclear-encoded assembly factors and chloroplast-encoded ATP synthase subunit b in Oenothera, providing insights into the coordinated biogenesis of this essential energy-transducing complex .