Recombinant Lolium perenne ATP synthase subunit b, chloroplastic (atpF)

How is the atpF gene organized within the Lolium perenne chloroplast genome?

The atpF gene is located within the chloroplast genome of Lolium perenne, which has been completely sequenced and is 135,282 bp in size with a typical quadripartite structure . The chloroplast genome of L. perenne contains genes for 76 unique proteins, 30 tRNAs, and four rRNAs .

The atpF gene is among the protein-coding genes in the chloroplast genome, specifically designated as LopeCp029 in genome annotation systems . In the Poaceae family, chloroplast genomes typically contain more than 200 mononucleotide repeats of at least 7 bp in length, concentrated mainly in the large single copy region of the genome . These repeats can be useful for genetic diversity studies when designing chloroplast microsatellite markers.

Within the context of chloroplast genetic variation, the atpF gene is located in a region that can show varying levels of conservation across Poaceae species. Studies using chloroplast microsatellite markers have shown that nucleotide composition in these regions varies considerably among subfamilies, with Pooideae (which includes Lolium) biased toward poly A repeats .

What expression systems are commonly used for recombinant Lolium perenne ATP synthase subunit b production?

For the expression of recombinant Lolium perenne ATP synthase subunit b, E. coli expression systems are most commonly employed . The methodology typically involves:

• Vector construction: The atpF gene sequence (coding for amino acids 1-183) is cloned into an appropriate expression vector with an N-terminal His-tag for purification purposes .

• Expression conditions: Transformation into E. coli expression strains, followed by induction of protein expression. While specific conditions are not detailed in the search results, typical protocols would involve:

  • Growth at 37°C until reaching optimal density

  • Induction with IPTG

  • Expression at reduced temperatures (16-25°C) to enhance proper folding

• Purification process: The recombinant protein is typically purified using affinity chromatography targeted at the His-tag, followed by additional purification steps if necessary .

• Final product form: The purified protein is often obtained as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE .

The storage recommendations for the recombinant protein include:

• Storage at -20°C/-80°C upon receipt

• Aliquoting for multiple use to avoid repeated freeze-thaw cycles

• Short-term working aliquots can be stored at 4°C for up to one week

How does phosphorus deficiency affect expression and function of ATP synthase in Lolium perenne?

Phosphorus (P) deficiency has significant effects on ATP synthase expression and function in Lolium perenne, as part of broader metabolic adaptations. Research has shown that after just 24 hours of P deficiency, internal phosphate concentrations are reduced, and significant alterations are detected in both the transcriptome and metabolome of Lolium perenne genotypes .

Methodological approach for studying P deficiency effects:

• Experimental design:

  • Expose seedlings to controlled nutrient solutions with and without P

  • Standard growth conditions: 23°C with 16-h daylight regime (PAR = 360 μmol m⁻² s⁻¹)

  • Comparison of different genotypes (e.g., IRL-OP-2538-P and Cashel-P)

• Transcriptomic analysis:

  • Use of cross-species hybridization with barley microarrays to study gene expression changes

  • Early response mechanisms can be detected after just 24 hours of P deprivation

• Metabolomic profiling:

  • Gas chromatography-mass spectrometry to profile metabolite changes

  • Assessment of metabolic adjustments including changes in key compounds

The data indicate that P deficiency leads to replacement of phospholipids with sulfolipids and utilization of glycolytic bypasses . The table below shows examples of significantly different metabolites in leaves and roots under P-sufficient and P-deficient conditions:

These metabolic changes reflect the adaptation of energy metabolism, including potential adjustments in ATP synthase expression and function, enabling Lolium perenne to maintain energy production under P-limited conditions .

How can chloroplast markers including atpF be used to study genetic diversity in Lolium species?

Chloroplast markers, including those derived from the atpF gene region, provide valuable tools for studying genetic diversity in Lolium species. Research has developed several chloroplast microsatellite markers based on knowledge of variable regions within the Lolium perenne chloroplast genome .

Methodological approach for chloroplast marker development and application:

• Marker development:

  • Screen chloroplast genomes of Poaceae taxa for mononucleotide microsatellite repeat regions

  • Design primers for amplification from targeted loci

  • Validate markers on diverse germplasm collections

• Sample collection and diversity assessment:

  • Evaluate markers on multiple accessions (e.g., 16 Irish and 15 European L. perenne ecotypes, 9 L. perenne cultivars, other Lolium taxa)

  • Extract DNA using standard protocols

  • Perform PCR amplification and product analysis through sequencing, polyacrylamide gel electrophoresis, or agarose gel electrophoresis

• Data analysis:

  • Apply locus-by-locus AMOVA (Analysis of Molecular Variance) to test genetic structure

  • Calculate fixation indices (FST) to assess population differentiation

  • Evaluate within-population and among-population variance components

Research has shown that certain chloroplast markers can effectively distinguish between Lolium species. For example, the TeaCpSSR28 marker can distinguish between all tested Lolium species and Lolium multiflorum due to an elongation of an A8 mononucleotide repeat in L. multiflorum . The marker TeaCpSSR31 has demonstrated high levels of microsatellite length variation and single nucleotide polymorphisms, while TeaCpSSR27 can reveal variation within some L. perenne accessions due to a 44-bp indel that can be detected by simple agarose gel electrophoresis .

The table below summarizes the performance of different chloroplast markers for genetic diversity studies:

These markers serve as valuable tools for plant breeding companies, seed testing agencies, and researchers for monitoring genetic diversity within breeding pools, tracing maternal inheritance, and distinguishing closely related species .

What functional assays can be used to study the activity of recombinant ATP synthase subunit b?

To assess the activity and functional properties of recombinant Lolium perenne ATP synthase subunit b, several methodological approaches can be employed:

• Reconstitution assays:

  • Reconstitute the recombinant subunit b with other ATP synthase components in liposomes

  • Measure ATP synthesis rates using luciferase-based luminescence assays

  • Assess proton translocation using pH-sensitive fluorescent dyes

• Binding and interaction studies:

  • Analyze interactions with other ATP synthase subunits using surface plasmon resonance

  • Employ co-immunoprecipitation to identify binding partners

  • Use isothermal titration calorimetry to determine binding constants

• Structural studies:

  • Circular dichroism spectroscopy to assess secondary structure elements

  • Nuclear magnetic resonance (NMR) for structural determination of the recombinant protein

  • X-ray crystallography if crystals can be obtained

• Functional complementation:

  • Express the recombinant protein in E. coli strains with defective ATP synthase

  • Assess restoration of ATP synthesis capacity

  • Measure growth rates under conditions requiring ATP synthase function

When working with the recombinant protein, recommended handling procedures include:

• Brief centrifugation prior to opening the vial

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) for long-term storage

• Aliquoting to avoid repeated freeze-thaw cycles

How do environmental stressors affect ATP synthase expression and function in Lolium perenne?

Environmental stressors have significant impacts on ATP synthase expression and function in Lolium perenne as part of broader metabolic adaptations. Research has shown that Lolium perenne undergoes substantial metabolic adjustments when exposed to various stressors including xenobiotics, heavy metals, and nutrient deficiencies .

Methodological approaches to study stress responses:

• Experimental design for stress studies:

  • Exposure to subtoxic levels of diverse stressors:

    • Herbicide glyphosate (inhibits aromatic amino acid production)

    • AMPA (aminomethylphosphonic acid, glyphosate degradation product)

    • Triazole fungicide tebuconazole

    • PAH molecule fluoranthene

    • Heavy metal copper

  • Different exposure modalities to reflect realistic environmental conditions

  • Controlled growth conditions with precise monitoring

• Multi-omics analysis:

  • Transcriptomics to measure changes in gene expression

  • Metabolomics to profile metabolite adjustments

  • Physiological analysis to assess plant performance

• Physiological measurements:

  • Chlorophyll fluorescence to assess photosynthetic efficiency

  • Oxygen evolution measurements

  • Growth parameters

Research indicates that long-term adjustment to and survival of perennial ryegrass at subtoxic levels of diverse xenobiotic and heavy-metal stresses are associated with major flexibility and complex regulations of central carbon and nitrogen metabolisms . These adaptations likely involve changes in ATP synthase expression and function to maintain energy homeostasis under stress conditions.

In the case of phosphorus deficiency specifically, plants show rapid responses (within 24 hours) at the transcriptome and metabolome levels, including changes in phospholipid composition and activation of glycolytic bypasses , which would significantly impact energy production pathways involving ATP synthase.

What are the challenges and solutions in expressing and purifying functional recombinant Lolium perenne ATP synthase subunit b?

Expressing and purifying functional recombinant Lolium perenne ATP synthase subunit b presents several challenges, each requiring specific methodological solutions:

• Challenge: Membrane protein solubility

  • Solution:

    • Use of fusion tags (His-tag has been successfully employed)

    • Expression in cell-free systems to bypass membrane integration issues

    • Optimization of detergents for extraction and purification

• Challenge: Proper folding

  • Solution:

    • Expression at lower temperatures (15-25°C)

    • Co-expression with molecular chaperones

    • Use of specialized E. coli strains designed for membrane protein expression

• Challenge: Maintaining stability during purification

  • Solution:

    • Inclusion of appropriate stabilizing agents in all buffers

    • Working at 4°C throughout the purification process

    • Using Tris/PBS-based buffer with 6% Trehalose, pH 8.0 for storage

• Challenge: Long-term storage

  • Solution:

    • Lyophilization to produce a stable powder form

    • Storage at -20°C/-80°C upon receipt

    • Aliquoting to avoid repeated freeze-thaw cycles

    • Addition of 5-50% glycerol for long-term storage at -20°C/-80°C

• Challenge: Functional assessment

  • Solution:

    • Reconstitution into liposomes or nanodiscs for functional studies

    • Coupling with other ATP synthase subunits to assess interactions

    • Development of specific activity assays

When reconstituting the protein:

• Briefly centrifuge prior to opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol (typically 50% final concentration) for stability

How can RNA editing sites in the atpF transcript be identified and what is their functional significance?

RNA editing is a post-transcriptional process that can alter the nucleotide sequence of RNA molecules, potentially affecting protein structure and function. In Lolium perenne chloroplast transcripts, including atpF, RNA editing sites can be identified and characterized using several methodological approaches:

• Experimental approach to identify RNA editing sites:

  • Extract total RNA from plant tissue

  • Synthesize cDNA using reverse transcription

  • Amplify the atpF transcript region using RT-PCR

  • Compare the cDNA sequence with the corresponding genomic DNA sequence

  • Identify C-to-U or other editing events

• Comprehensive analysis methodology:

  • Use high-throughput sequencing of both genomic DNA and cDNA

  • Employ computational tools to identify discrepancies between DNA and RNA sequences

  • Validate editing sites using targeted Sanger sequencing

Research on the Lolium perenne chloroplast genome has identified 31 mRNA editing sites across 33 genes, with five of these editing sites being unique to Lolium . While the search results don't specifically mention editing sites in the atpF transcript, this methodological approach can be applied to study potential editing in this gene.

The functional significance of RNA editing in chloroplast transcripts can be substantial:

• Editing may restore conserved amino acids critical for protein function

• It can create start or stop codons, affecting protein length

• Editing may influence RNA secondary structure and stability

• Changes in codon usage might affect translation efficiency

To assess the functional impact of identified editing sites:

• Perform comparative analysis across species to identify conserved editing events

• Use site-directed mutagenesis to mimic or prevent editing at specific sites

• Express edited and unedited versions of the protein and compare their functional properties

• Apply structural modeling to predict the impact of amino acid changes resulting from editing

What roles does ATP synthase subunit b play in herbicide resistance mechanisms in Lolium species?

While ATP synthase subunit b itself is not directly implicated in herbicide resistance mechanisms in Lolium species, understanding its context in chloroplast function is important for research on herbicide resistance, particularly for herbicides targeting energy production pathways.

Methodological approaches to study potential connections:

• Comparative transcriptomics:

  • Compare atpF expression levels between herbicide-resistant and susceptible populations

  • RNA extraction followed by quantitative RT-PCR or RNA-Seq

  • Analysis of expression patterns under herbicide stress

• Functional genomics:

  • CRISPR-based approaches (if available for chloroplast genes) to modify atpF expression

  • Assess changes in herbicide sensitivity

  • Measure ATP production capacity under herbicide stress

• Metabolic analysis:

  • Measure ATP levels in resistant vs. susceptible plants under herbicide stress

  • Analyze energy charge and adenylate kinase equilibrium

  • Assess respiratory and photosynthetic capacities

Lolium species have evolved multiple- and cross-resistance to at least 14 herbicide mechanisms of action in more than 21 countries, with reports of multiple herbicide resistance to at least seven mechanisms of action in a single population . The primary resistance mechanisms include:

• Target-site resistance (TSR) - mutations in herbicide target proteins

• Non-target-site resistance (NTSR) - including:

  • Enhanced herbicide metabolism

  • Reduced herbicide absorption and translocation

  • Protection-based resistance

Within these mechanisms, ATP synthase and energy metabolism play critical contextual roles, particularly in NTSR mechanisms:

• ATP-binding cassette (ABC) transporters, which require ATP, may actively transport and compartmentalize herbicide conjugates and metabolites

• Cytochrome P450s, which can be involved in herbicide metabolism, require energy for their function

• Metabolic adjustments to maintain energy homeostasis under herbicide stress may involve changes in ATP synthase expression and function