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

Basic Research Questions

• What is the role of ATP synthase subunit b in Oenothera parviflora chloroplasts?

  ATP synthase subunit b (atpF) is a critical component of the chloroplastic ATP synthase complex in Oenothera parviflora. This protein plays an essential role in energy transduction during photosynthesis by facilitating proton movement across the thylakoid membrane, which drives ATP synthesis. In Oenothera species, the atpF gene is particularly significant as it is part of a photosynthesis operon in the chloroplast genome that has been implicated in speciation mechanisms .

• How does the structure of the atpF gene differ between Oenothera species?

  The atpF gene structure varies between Oenothera species, with differences in both coding and promoter regions. These variations contribute to plastome-genome incompatibility observed in certain hybrid combinations. For example, specific deletions in the promoter regions of operons containing atpF have been found to affect transcription rates in a light-dependent manner. Sequence analysis has revealed that while the coding region is relatively conserved, the upstream regulatory elements can differ significantly, especially in the 5' untranslated region and promoter elements located 7bp upstream of the -35 box .

• What is plastome-genome incompatibility (PGI) in Oenothera, and how does it relate to ATP synthase?

  Plastome-genome incompatibility (PGI) in Oenothera refers to dysfunctional interactions between the plastid genome (plastome) and the nuclear genome in certain hybrid combinations. The AB-I incompatibility (where nuclear genome AB is combined with plastome type I) results in a yellow-green (lutescent) leaf phenotype due to disturbed photosynthetic capacity. Research has shown that misregulation of the psbB operon, which influences photosynthetic complex formation, plays a major role in this incompatibility. While atpF is not directly part of this operon, ATP synthase activity is significantly affected in incompatible hybrids, with reduced ATP synthase levels observed under high light conditions .

• What are the five basic plastome types in Oenothera, and how do they relate to ATP synthase genes?

  The five basic plastome types in Oenothera (designated I through V) represent genetically distinct chloroplast genomes. These plastomes differ in their nucleotide sequences, including regions encoding ATP synthase components. The complete nucleotide sequences of these plastomes have been determined and compared, revealing specific polymorphisms that contribute to plastome-genome incompatibility. In particular, plastome I shows distinctive features in photosynthesis-related gene regions that affect ATP synthase function when combined with certain nuclear backgrounds .

Technical Methodologies

• What are the most effective protocols for isolating recombinant ATP synthase subunit b from Oenothera parviflora chloroplasts?

  Effective isolation of recombinant ATP synthase subunit b from Oenothera parviflora chloroplasts involves a multi-step process:

  1. Chloroplast Isolation: Harvest 8-10 week old leaves and isolate intact chloroplasts using differential centrifugation in sorbitol-containing buffer.

  2. Thylakoid Membrane Preparation: Lyse chloroplasts in hypotonic buffer and centrifuge to obtain thylakoid membranes.

  3. Protein Extraction: Solubilize membrane proteins using detergents such as n-dodecyl-β-D-maltoside or digitonin.

  4. Purification: Apply the solubilized sample to ion exchange chromatography followed by size exclusion chromatography.

  5. Immunoprecipitation: For specific isolation of ATP synthase subunit b, use antibodies against the subunit for immunoprecipitation.

  6. Western Blot Verification: Confirm the identity and purity of the isolated protein using Western blot with specific antibodies.

  For in vitro transcription and hybridization analysis, isolate approximately 4.9 x 10⁷ chloroplasts, centrifuge at 5,000 g for 1 min, and resuspend in transcription buffer containing 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 0.2 mM CTP, GTP, and ATP, 0.01 mM UTP, and 10 mM 2-mercaptoethanol .

• How can one create plastome-genome incompatible Oenothera hybrids for studying ATP synthase function?

  Creating plastome-genome incompatible Oenothera hybrids for ATP synthase function studies requires a systematic breeding approach:

  1. Select Parent Lines: Choose parents with known plastome types and nuclear genomes (e.g., AB associated with plastome I as seed parent and homozygote CC-II as pollen donor).

  2. Emasculation: Remove immature anthers from the seed parent the day before flower maturation.

  3. Controlled Pollination: Pollinate the emasculated flower with desired pollen, repeating the next day to ensure successful fertilization.

  4. Seed Collection: Harvest seeds approximately 6 weeks after pollination and dry at room temperature.

  5. Verification: Determine the plastome type of resulting plants by pooling material from three successive bracts and using molecular markers.

  6. Screening: Select plants with the desired plastome-genome combination (e.g., AB-I) for experimental studies.

  For crossing experiments involving incompatible combinations, it may be necessary to nurture plants with additional compatible plastomes (e.g., AC-I/IV, CC-I/VI, and CC-II/IV) to ensure viability .

• What molecular marker systems are most effective for genotyping Oenothera ATP synthase genes?

  The most effective molecular marker systems for genotyping Oenothera ATP synthase genes include:

  1. CAPS (Cleaved Amplified Polymorphic Sequences): Particularly useful for distinguishing plastome types. Design primers flanking restriction site polymorphisms in ATP synthase genes.

  2. SSLP (Simple Sequence Length Polymorphisms): Effective for identifying variations in repetitive regions within or near ATP synthase genes.

  3. AFLP (Amplified Fragment Length Polymorphisms): Useful for genome-wide analyses, including regions containing ATP synthase genes.

  4. PCR-Based Markers: Using conserved primer pairs such as rbcLfor (5′-TGTGGCATATGCCTGCTCTG-3′) and psaI_IVP11rev (5′-GGAGAAATCCATTCTTGTCGTC-3′) for plastome typing.

  5. RNA Editing Site Analysis: For distinguishing potential RNA editing differences that affect ATP synthase function.

  These marker systems allow researchers to track the inheritance of specific plastomes and nuclear genomes during crossing experiments, facilitating the study of ATP synthase gene expression and function in different genetic backgrounds .

Comparative Analysis Questions