Recombinant Solanum lycopersicum ATP synthase subunit b, chloroplastic (atpF)

How does atpF contribute to photosynthesis and plant stress responses?

The atpF protein is integral to photosynthetic efficiency as part of the cpATPase complex that generates ATP required for carbon fixation. Research indicates that modifications to ATP synthase components can significantly impact plant responses to environmental stressors.

Studies on the β subunit of the ATP synthase complex have shown that overexpression enhances tolerance to low-temperature stress in tomato plantlets. Specifically, transgenic plants with elevated ATP synthase β subunit levels demonstrated:

• Increased antioxidant enzyme activities (SOD levels 17.6-25.4% higher, POD levels 29.5-37.5% higher, and CAT levels 14.5-38.7% higher than wild-type)

• Reduced membrane damage (MDA levels 20.0-22.1% lower than wild-type)

• Enhanced photosynthetic performance under stress conditions (Pn values 42.6-54.9% higher than wild-type)

Though these findings specifically relate to the β subunit, they highlight the importance of the entire ATP synthase complex, including atpF, in maintaining energy production under stress conditions.

What are the recommended protocols for handling recombinant atpF?

For optimal results when working with recombinant Solanum lycopersicum ATP synthase subunit b protein:

The recombinant protein is typically available in 50 μg quantities and may include a tag determined during the production process to facilitate purification and detection .

What experimental approaches can effectively study atpF interactions with other ATP synthase components?

Several complementary techniques can elucidate the interaction network of atpF:

• Yeast Two-Hybrid (Y2H) Assays: This approach has successfully identified interactions between ATP synthase subunits and auxiliary factors. For example, studies have demonstrated specific interactions between YL1 (a plant lineage-specific auxiliary factor) and the AtpB subunit of cpATPase .

• Bimolecular Fluorescence Complementation (BiFC): This technique allows visualization of protein-protein interactions in vivo, providing spatial information about where these interactions occur within the chloroplast .

• Protein Interaction Validation Methods: For comprehensive verification, researchers should consider employing multiple complementary approaches:

A multi-method approach is essential for confirming authentic biological interactions and understanding their functional significance.

How can transcriptome and metabolome analyses be integrated to study ATP synthase function in plant defense?

Integrated -omics approaches provide powerful insights into the connections between energy metabolism and plant immunity:

Recent research has demonstrated that plant defense mechanisms are closely linked to energy production and reactive oxygen species (ROS) signaling. A study on Solanum lycopersicum revealed that SlSYTA modulates ROS responses driving resistance defense by influencing diverse plant defense pathways, including phenylpropanoid metabolism, hormone signaling, and oxidative phosphorylation .

For researching atpF's role in defense responses, a systematic approach would include:

• Experimental Design:

  • Generate transgenic plants with modified atpF expression

  • Challenge with relevant pathogens (e.g., TMV, Phytophthora capsici, Botrytis cinerea, Pseudomonas syringae)

  • Conduct parallel transcriptome and metabolome analyses

  • Integrate datasets to identify correlations

• Key Parameters to Measure:

  • Changes in PTI (Pattern-Triggered Immunity) responses, including ROS burst

  • Stomatal closure dynamics in response to pathogens

  • Callose deposition patterns

  • Expression of defense-related genes

  • Metabolite profiles, particularly focusing on phenylpropanoids and hormones

• Data Integration Strategy:

  • Pathway enrichment analysis focusing on energy metabolism and defense

  • Network analysis to identify regulatory hubs

  • Correlation analysis between transcripts and metabolites

This integrated approach can reveal how ATP synthase components influence energy status and, consequently, defense responses against pathogens .

What methodologies best assess the impact of atpF modifications on photosynthetic efficiency?

A comprehensive approach to evaluating how atpF alterations affect photosynthesis should include multiple complementary methodologies:

• Gas Exchange Parameters:
  Measuring key photosynthetic indicators:

  Parameter	Abbreviation	What it measures	Relevance to ATP synthase function
  Net photosynthetic rate	Pn	CO₂ assimilation	Direct indicator of photosynthetic output
  Stomatal conductance	Gs	Gas exchange capacity	Affects CO₂ availability
  Transpiration rate	Tr	Water loss	Linked to photosynthetic efficiency
  Intercellular CO₂ concentration	Ci	CO₂ available for fixation	Helps identify limiting factors

  Research has shown that under stress conditions, transgenic plants overexpressing ATP synthase β subunit maintained higher Pn, Gs, and Tr values compared to wild-type plants, indicating enhanced photosynthetic capacity .

• Chlorophyll Fluorescence Analysis:
  This technique serves as an intrinsic indicator of photosynthetic processes and can detect subtle changes in photosystem efficiency resulting from altered ATP synthase function .

• Biochemical Assays:

  • Direct measurement of ATP synthesis rates

  • Quantification of ATP/ADP ratios in chloroplasts

  • Analysis of carbon fixation rates

• Structural Studies:

  • Electron microscopy to assess chloroplast ultrastructure

  • Analysis of thylakoid membrane organization

  • Blue native PAGE to evaluate ATP synthase complex assembly and stability

What are the technical challenges and solutions for producing functional recombinant atpF?

Producing high-quality recombinant atpF presents several technical challenges:

• Membrane Protein Solubility:
  As atpF contains a transmembrane domain, solubility during expression and purification is a significant challenge.

  Solution: Consider expressing only the soluble portion or using specialized systems designed for membrane proteins. Alternatively, employ detergents or amphipols to maintain solubility.

• Expression System Selection:
  The choice of expression system significantly impacts protein yield and functionality.

  Expression System	Advantages	Disadvantages	Suitability for atpF
  E. coli	High yield, cost-effective	Limited post-translational modifications	Moderate; may require optimization
  Yeast	Eukaryotic modifications	Lower yield than bacteria	Good; used successfully for ATP synthase subunits
  Plant-based systems	Native modifications	More complex, lower yield	Excellent for authentic function
  Cell-free systems	Avoids toxicity issues	Expensive, limited scale	Good for initial studies

• Purification Strategy:
  Effective purification while maintaining native structure is essential.

  Solution: Use affinity tags (His-tag is commonly employed for ATP synthase subunits) and optimize purification conditions to minimize protein denaturation .

• Functional Validation:
  Confirming that the purified protein retains native function.

  Solution: Employ interaction studies with partner proteins (e.g., ATP synthase β subunit) and reconstitution experiments to verify functionality .

The recombinant protein can be used for producing specific antibodies against atpF or in research focusing on protein-protein interactions and structural studies .

How might genetic engineering of atpF contribute to developing stress-tolerant crops?

Given the demonstrated role of ATP synthase components in stress responses, particularly the enhanced low-temperature tolerance observed in plants overexpressing the β subunit , strategic modifications of atpF could potentially:

• Enhance Photosynthetic Efficiency Under Stress:
  Optimizing atpF could improve energy production during environmental stress, maintaining higher photosynthetic rates when wild-type plants show significant reductions.

• Strengthen Antioxidant Defense Systems:
  Modified ATP synthase function could support higher activities of antioxidant enzymes (SOD, POD, CAT), reducing oxidative damage during stress conditions.

• Improve Pathogen Resistance:
  Since energy metabolism is linked to defense responses , enhanced ATP production capacity could potentially strengthen pattern-triggered immunity responses.

• Research Priorities:

  • Targeted mutagenesis to identify critical residues for atpF function

  • Field trials of plants with optimized ATP synthase components

  • Analysis of potential trade-offs between enhanced stress tolerance and growth/yield under normal conditions

This research direction has significant implications for developing climate-resilient crop varieties with improved performance under increasingly variable environmental conditions.

What insights can atpF research provide about the evolution of photosynthetic machinery?

The ATP synthase complex represents a fascinating case of evolutionary conservation with specific adaptations in chloroplasts. Comparative studies of atpF across species could reveal:

• Evolutionary Conservation and Divergence:
  Analysis of sequence conservation within the plant kingdom and comparison with bacterial and mitochondrial counterparts.

• Plant-Specific Auxiliary Factors:
  Research has identified plant lineage-specific auxiliary factors like YL1 that are involved in the biogenesis of the cpATPase complex . Further studies on atpF interactions could reveal additional plant-specific adaptations.

• Comparative Functional Studies:
  Examining how atpF function differs across plant species adapted to different environmental niches could provide insights into evolutionary adaptations of photosynthetic machinery.

These evolutionary perspectives could inform both fundamental understanding of photosynthesis evolution and applied approaches to crop improvement.