Recombinant Nasturtium officinale ATP synthase subunit b, chloroplastic (atpF)

How does atpF in Nasturtium officinale compare structurally to atpF in other Brassicaceae species?

Comparative analysis reveals high conservation of atpF across the Brassicaceae family. The chloroplast gene encoding ATP synthase subunit b (atpF) in Nasturtium officinale shares significant homology with that of other Brassicaceae members:

Notably, the atpF gene contains introns in Nasturtium officinale as in other related species. These introns play important roles in post-transcriptional regulation and are conserved features in chloroplast genomes across the Brassicaceae family .

What are the optimal methods for expressing recombinant Nasturtium officinale atpF protein?

For successful expression of recombinant Nasturtium officinale atpF, researchers should consider the following methodological approach:

• Vector Selection: pET expression systems with T7 promoter have shown high efficiency for chloroplast proteins. For atpF specifically, include a His-tag or other purification tag at the N-terminus, avoiding the C-terminus where functional domains may be disrupted.

• Expression Host: E. coli BL21(DE3) or Rosetta strains are recommended due to their ability to handle plant codon usage. For atpF, which contains membrane-spanning regions, specialized strains like C41(DE3) or C43(DE3) designed for membrane proteins may provide higher yields.

• Induction Conditions:

  • Temperature: 16-18°C (rather than 37°C)

  • IPTG concentration: 0.1-0.5 mM

  • Duration: Extended induction (overnight)

  • Growth phase: Mid-log phase (OD₆₀₀ = 0.6-0.8)

• Solubilization Strategy: As a membrane protein component, atpF requires proper solubilization using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or CHAPS at concentrations just above their critical micelle concentration.

This approach has been validated for similar chloroplast membrane proteins and can be adapted specifically for Nasturtium officinale atpF .

What techniques are most effective for purifying and characterizing recombinant atpF protein?

For purification and characterization of recombinant Nasturtium officinale atpF, the following methodological workflow is recommended:

• Purification Protocol:

  • Cell lysis: Sonication or pressure-based methods in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and appropriate detergent

  • IMAC purification: Using Ni-NTA resin for His-tagged protein

  • Size exclusion chromatography: For removing aggregates and achieving higher purity

  • Consider on-column refolding if inclusion bodies form

• Characterization Methods:

  • SDS-PAGE and Western blotting: Confirm size (approximately 20 kDa) and identity

  • Circular dichroism: Assess secondary structure content

  • Mass spectrometry: Verify sequence integrity

  • Dynamic light scattering: Evaluate homogeneity

• Functional Verification:

  • ATPase activity assays

  • Reconstitution into liposomes to test proton translocation

  • Binding assays with other ATP synthase subunits

This comprehensive approach ensures both structural integrity and functional activity of the purified recombinant protein .

How does atpF contribute to chloroplast energy metabolism in Nasturtium officinale?

The atpF gene product plays a critical role in chloroplast energy metabolism through its function in the ATP synthase complex:

• Structural Role: ATP synthase subunit b forms part of the peripheral stalk of the F₀ sector, which connects the membrane-embedded proton channel (F₀) to the catalytic portion (F₁) where ATP synthesis occurs.

• Proton Translocation: Studies of chloroplast ATP synthase indicate that subunit b participates in the proton circuit by forming a hydrophilic pathway along which protons move from the thylakoid lumen to the stromal side.

• Integration with Photosynthetic Electron Transport: The function of ATP synthase, including the atpF subunit, is tightly coupled to photosynthetic electron transport, utilizing the proton gradient generated during light reactions.

• Regulatory Function: Evidence suggests that subunit b may have additional regulatory functions, potentially responding to metabolic cues or stress conditions to modulate ATP synthesis rates in chloroplasts.

In the context of Nasturtium officinale as an aquatic plant, the atpF gene shows adaptations that may enhance energy metabolism under varying environmental conditions, particularly in phosphorus-limited environments as indicated by research on phosphorus use efficiency in watercress .

How can researchers study interactions between atpF and other components of the chloroplast ATP synthase complex?

To investigate protein-protein interactions involving atpF in the ATP synthase complex, researchers can employ the following methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Express tagged atpF in plant chloroplasts or heterologous systems

  • Isolate intact complexes using antibodies against the tag

  • Identify interacting partners by mass spectrometry

  • Validate specific interactions with targeted Western blotting

• Yeast Two-Hybrid (Y2H) Assays:

  • Design constructs without transmembrane domains for soluble protein fragments

  • Test binary interactions with other ATP synthase subunits

  • Use split-ubiquitin system for membrane protein variants

• Bimolecular Fluorescence Complementation (BiFC):

  • Transform plant protoplasts with fusion constructs

  • Visualize interactions in vivo through fluorescence microscopy

  • Quantify interaction strength through fluorescence intensity

• Cross-linking Mass Spectrometry:

  • Apply chemical cross-linkers to isolated chloroplasts

  • Identify cross-linked peptides by tandem mass spectrometry

  • Map interaction interfaces at amino acid resolution

These approaches provide complementary data on both the occurrence and spatial arrangement of protein-protein interactions involving atpF within the ATP synthase complex .

What methods are effective for genetic transformation of the atpF gene in Nasturtium officinale?

Recent advances have enabled efficient genetic transformation systems for Nasturtium officinale that can be applied to the atpF gene. The following methodological approach has demonstrated success:

• Agrobacterium-mediated Transformation:

  • Select young stems as explant material

  • Use Agrobacterium tumefaciens strain GV3101 or EHA105

  • Employ a binary vector system with appropriate selection markers

  • Culture explants in MS medium containing 4 mg/L 6-BA + 1.5 mg/L TDZ + 1.5 mg/L 2,4-D

  • Confirm transformation by PCR and observe GFP fluorescence (when using a GFP reporter)

• Chloroplast-specific Transformation Strategy:

  • Design constructs with homologous recombination regions flanking the atpF gene

  • Include a selectable marker (e.g., aadA gene conferring spectinomycin resistance)

  • Use biolistic delivery methods for chloroplast transformation

  • Select transformants on medium containing appropriate antibiotics

  • Verify homoplasmy through multiple rounds of selection

• Regeneration Protocol:

  • Induce callus formation under specific hormone concentrations

  • Transfer to shoot induction medium with 3 mg/L 6-BA + 3 mg/L TDZ

  • Root in MS medium (pH 5.7)

  • Acclimate plantlets before transfer to soil

This transformation system achieves efficiency rates of 65-72% for nuclear transformation and can be adapted for chloroplast-specific modification of the atpF gene .

How can CRISPR-Cas9 be utilized for targeted editing of the atpF gene in Nasturtium officinale?

For CRISPR-Cas9 editing of the chloroplast-encoded atpF gene in Nasturtium officinale, researchers should implement the following specialized approach:

• Chloroplast-specific CRISPR System Design:

  • Develop chloroplast-specific promoters (e.g., PpsbA) to drive Cas9 expression

  • Design sgRNAs targeting conserved regions of atpF while avoiding off-targets

  • Include chloroplast-specific markers for selection

• Delivery Methods:

  • Biolistic transformation using gold particles coated with CRISPR components

  • Alternative: chloroplast-targeted peptides fused to Cas9 for protein-based delivery

• Target Site Selection:

  • Focus on non-intronic regions for higher editing efficiency

  • Avoid regions critical for core function if studying subtle modifications

  • Consider accessibility of the target region within the nucleoid structure

• Screening Protocol:

  • PCR amplification of target region followed by restriction digestion

  • Sanger sequencing to confirm precise edits

  • Next-generation sequencing for detecting low-frequency editing events

  • Observation of phenotypic changes associated with ATP synthesis

• Homoplasmy Achievement:

  • Multiple rounds of selection on spectinomycin-containing media

  • Single-cell propagation to enrich for edited plastome copies

  • PCR verification of homoplasmic state

This approach represents the cutting edge of chloroplast genome editing and can be applied for both functional studies and potential improvement of energy metabolism in Nasturtium officinale .

How does environmental stress affect atpF expression and function in Nasturtium officinale?

Research on environmental stress responses in Nasturtium officinale has revealed significant impacts on atpF expression and function:

• Phosphorus Limitation Response:

  • Under phosphorus deficiency (P-), plants maintain shoot yield through enhanced root biomass development

  • Transcriptomic analysis reveals differential expression of genes involved in ATP metabolism

  • The atpF gene shows altered expression patterns as part of the plant's phosphorus use efficiency (PUE) mechanisms

  • Two distinct breeding lines (line 60 and line 102) demonstrate different strategies for maintaining ATP synthesis under P limitation

• Temperature Stress Effects:

  • Cold stress (below 15°C) induces changes in ATP synthase activity

  • Heat stress (above 30°C) affects assembly of the ATP synthase complex

  • Post-translational modifications of atpF increase under temperature extremes

• Oxidative Stress Implications:

  • High light conditions trigger protective mechanisms involving ATP synthase

  • Antioxidant capacity increases under stress conditions that affect ATP synthesis

  • Glucosinolate concentration remains stable despite altered energy metabolism

• Methodological Approaches for Stress Studies:

  • RNA-seq analysis to quantify transcriptional changes

  • Proteomic analysis to detect post-translational modifications

  • Biochemical assays to measure ATP synthase activity under varying conditions

  • Comparative analysis of different watercress genotypes to identify stress-tolerant variants

This research provides valuable insights into the adaptive mechanisms of energy metabolism in watercress under environmental stress conditions .

What are the implications of atpF research for improving phosphorus use efficiency in watercress cultivation?

Recent genomic investigations have identified atpF as a potential target for enhancing phosphorus use efficiency (PUE) in Nasturtium officinale cultivation:

• Genomic Insights:

  • Watercress plants grown without additional phosphorus (P-) show distinctive gene expression patterns

  • RNA-seq analysis has identified a suite of genes, including those involved in ATP metabolism, as potential targets for enhanced PUE

  • Two contrasting breeding strategies have emerged:

    • Line 60: Highly plastic root systems with increased root growth under P-

    • Line 102: Maintaining high yield regardless of P supply, but less plastic

• Physiological Adaptations:

  • Under P- conditions, watercress sustains shoot yield through enhanced root biomass

  • Plants develop shorter stems and smaller leaves

  • Antioxidant capacity and concentration of sugars and starch in shoot tissue are enhanced

  • Glucosinolate concentration remains unaffected by P limitation

• Research Applications:

  • Targeted modification of atpF could enhance energy efficiency under P limitation

  • Breeding programs can utilize natural variation in atpF expression for selection

  • Biotechnological approaches may modulate atpF to improve sustainable cultivation

• Recommended Methodological Approach:

  • Candidate gene identification through comparative transcriptomics

  • Validation using CRISPR-based gene editing

  • Field trials in phosphorus-limited conditions

  • Metabolomic analysis to assess downstream effects

This research directly contributes to sustainable agricultural practices by reducing the need for phosphate-based fertilizers that can contribute to eutrophication of aquatic habitats downstream of watercress farms .

How has the atpF gene evolved within the Brassicaceae family?

Evolutionary analysis of the atpF gene across the Brassicaceae family reveals important patterns:

• Sequence Conservation and Divergence:

  • The core functional domains of atpF show high conservation across Brassicaceae

  • Intron regions display higher variability and provide phylogenetic signals

  • The gene structure (containing introns) is preserved across the family

• Comparative Genomic Analysis:

  • In chloroplast genomes of Brassicaceae, atpF is consistently located in the Large Single Copy (LSC) region

  • The atpF gene in Nasturtium officinale shares significant structural similarities with that of other Brassicaceae species

  • Phylogenetic analysis places Nasturtium officinale atpF in close relationship with other aquatic and semi-aquatic Brassicaceae species

• Selection Pressure Analysis:

  • The Ka/Ks ratio for atpF suggests purifying selection across the Brassicaceae family

  • Transmembrane domains show stronger conservation than stromal-facing regions

  • Species adapting to specific environments (like the aquatic habitat of watercress) show subtle adaptive changes in atpF

• Methodological Approach for Evolutionary Studies:

  • Whole chloroplast genome sequencing using next-generation sequencing

  • Multiple sequence alignment of atpF genes across Brassicaceae

  • Calculation of nucleotide diversity (Pi) to identify variable regions

  • Estimation of Ka/Ks ratios to detect selection pressures

This evolutionary perspective provides context for understanding the significance of atpF in the adaptation of Nasturtium officinale to its aquatic environment.

What can comparative analysis of atpF across plant species reveal about chloroplast evolution?

Comparative analysis of ATP synthase subunit b (atpF) across diverse plant species provides valuable insights into chloroplast evolution:

• Structural Conservation Across Plant Kingdom:

  • Core functional domains of atpF show remarkable conservation from algae to flowering plants

  • Presence and positioning of introns in atpF vary between major plant lineages

  • Transmembrane domains show highest conservation, reflecting evolutionary constraints on membrane proteins

• Adaptation Signatures in Specialized Plants:

  • Aquatic plants like Nasturtium officinale show adaptations in atpF related to underwater photosynthesis

  • Plants from high-stress environments exhibit modifications in ATP synthase components

  • C4 plants show distinctive patterns in energy metabolism genes including atpF

• Evolutionary Rate Analysis:

  • atpF evolves at a slower rate than many other chloroplast genes

  • Nonsynonymous (Ka) and synonymous (Ks) substitution rates reveal selection patterns

  • Domains interacting with nuclear-encoded ATP synthase subunits show co-evolutionary patterns

• Methodological Framework for Comparative Studies:

  • Whole chloroplast genome sequencing and assembly

  • Identification of shared genes across species after alignment

  • Calculation of nucleotide diversity and Ka/Ks ratios

  • Protein structure prediction and comparison across diverse species