Recombinant Nicotiana sylvestris ATP synthase subunit c, chloroplastic (atpH)

What is the function of ATP synthase subunit c (atpH) in Nicotiana sylvestris chloroplasts?

ATP synthase subunit c (atpH) forms the critical proton-conducting channel in the CFo portion of the ATP synthase complex. In N. sylvestris, this protein facilitates proton movement from the thylakoid lumen to the stroma, which drives ATP production. The proton motive force (pmf) generated by light-driven electron transport serves as the essential intermediate in photosynthesis, driving ATP synthesis at the thylakoid ATP synthase while simultaneously acting as a central feedback regulatory signal . This process activates photoprotection mechanisms in chloroplasts during excess light exposure via the qE-response and regulates electron transfer through the cytochrome b6f complex .

How is ATP synthase activity regulated in N. sylvestris?

ATP synthase activity in N. sylvestris is primarily regulated through changes in proton conductivity (gH+). The extent of pmf is regulated in part by the activity of the ATP synthase, which affects the proton conductance of the thylakoid membrane . Low proton conductance (gH+) retards proton efflux from the lumen, thereby increasing pmf . Research suggests that ATP synthase activity may be controlled by the availability of inorganic phosphate (Pi) in the stroma, with evidence pointing to Pi availability as a key regulator when photosynthesis is controlled by feedback due to limited capacity to utilize triose-phosphate .

What experimental methods are used to measure ATP synthase activity in N. sylvestris?

Several methodologies are employed to assess ATP synthase function in N. sylvestris:

• Electrochromic shift (ECSt) measurements: This non-invasive optical technique allows for in vivo estimation of steady-state light-driven pmf and proton conductance (gH+) of thylakoid membranes .

• ATP content assays: ATP levels can be quantified using the luciferin/luciferase assay method. The protocol involves extracting leaf tissue in perchloric acid, neutralizing with KOH, and measuring luminescence with a specialized photometer. The initial peak of the luminescence signal is proportional to ATP content .

• Parallel measurements of CO2 assimilation and gross O2 evolution: These measurements, combined with ECSt parameters under varying CO2 and O2 conditions, provide insights into ATP synthase regulation in relation to photosynthetic activity .

What is the relationship between inorganic phosphate (Pi) and ATP synthase regulation?

The relationship between Pi and ATP synthase regulation in N. sylvestris is characterized by several key features:

• The chloroplast ATP synthase has a relatively high KM for Pi (approximately 0.9 mM) .

• The photosynthetic system maintains constancy of stromal inorganic and esterified phosphate in the short term, with no net phosphate flux between cytosol and chloroplast stroma via the phosphate translocator .

• This property connects free Pi concentration in the chloroplast stroma to the rate of Pi recycling from starch and sucrose synthesis, as well as to Pi partitioning between free Pi and esterification in pools of Calvin cycle intermediates .

• When feedback limitation occurs under limited CO2 conditions, there is a decrease in the proton conductivity of the chloroplast ATP synthase (gH+), which retards proton efflux and increases steady-state proton motive force .

What approaches are recommended for heterologous expression of recombinant N. sylvestris atpH?

For efficient heterologous expression of recombinant N. sylvestris atpH:

• Vector selection: Optimize codon usage based on N. sylvestris genome data to improve expression efficiency in the chosen host system. The genomic information from N. sylvestris (with genome size approximately 2,600 Mb) can be utilized to design appropriate expression constructs .

• Expression systems: For chloroplast proteins like atpH, E. coli-based expression systems with specific chloroplast targeting strategies or in vitro chloroplast import systems can be employed. For structural studies, consider membrane protein-specific expression systems.

• Purification strategy: Develop a purification protocol that maintains the native structure of this hydrophobic membrane protein, typically using mild detergents and avoiding harsh conditions that might denature the protein.

• Validation: Confirm protein identity and functionality through mass spectrometry and reconstitution assays measuring proton conductance.

How does ATP synthase proton conductivity differ between wild-type and starchless mutant N. sylvestris?

Research comparing ATP synthase proton conductivity between wild-type and starchless mutant (NS 458, defective in plastid phosphoglucomutase) N. sylvestris reveals significant differences:

• Under feedback limitation conditions, the starchless mutant exhibits a marked reversal of O2 sensitivity compared to wild-type plants .

• Measurements under varying CO2 and O2 conditions (2% vs. 21% O2) show that under limiting CO2:

  • Wild-type plants show inhibition of CO2 assimilation (A) by 21% O2

  • Under CO2 saturated conditions, O2 sensitivity was reversed with slight stimulation of A by 21% O2

  • Under 2% O2, there is a close relationship between A and gross rates of O2 evolution (JO2)

  • Under 21% O2 and limiting CO2, JO2 was much higher than A

• These differences suggest altered regulation of ATP synthase proton conductivity in the starchless mutant, potentially due to disrupted phosphate partitioning affecting the Pi-dependent regulation of ATP synthase .

What experimental approaches can be used to study the effects of site-directed mutations in atpH on proton conductivity?

To investigate structure-function relationships in atpH through site-directed mutagenesis:

• Targeted mutation design:

  • Identify conserved residues in the proton channel region based on sequence alignment with other species

  • Focus on residues likely involved in proton binding and translocation

  • Consider mutations that alter pKa values of key residues

• In vivo assessment methodology:

  • Employ electrochromic shift (ECSt) measurements to quantify changes in proton conductance (gH+)

  • Monitor the effects of mutations on pmf development and decay

  • Compare wild-type and mutant responses to varying light, CO2, and O2 conditions

• Complementation approach:

  • Introduce mutated atpH into ATP synthase-deficient backgrounds

  • Quantify restoration of function using photosynthetic parameters

  • Measure ATP synthase activity through biochemical assays

• Data analysis:

  • Compare kinetic parameters of proton conductance between wild-type and mutant proteins

  • Develop mathematical models relating structural changes to altered function

How can researchers investigate the relationship between atpH and feedback regulation in photosynthesis?

To study the relationship between atpH and photosynthetic feedback regulation:

• Experimental setup: Use a combination of controlled environments with variable CO2 and O2 concentrations. Research shows that limiting CO2 causes a decrease in proton conductivity of the chloroplast ATP synthase (gH+), which increases steady-state proton motive force and activates photoprotective mechanisms .

• Measurement approaches:

  • Simultaneously monitor CO2 assimilation rates, O2 evolution, electrochromic shift parameters, and fluorescence parameters (qE and 1-qL)

  • Assess changes in ATP and RuBP content using extraction and quantification methods described in literature:

    • RuBP content: Measure 14C incorporation into acid-stable product with purified Rubisco

    • ATP content: Use luciferin/luciferase assay method

• Comparative analysis: Compare results between wild-type and mutant plants (e.g., starchless mutant NS 458) to identify key regulatory points. Under similar conditions, these plants show different responses to O2 and CO2 concentrations .

• Data integration: Correlate ATP synthase conductivity changes with photosynthetic parameters to establish causal relationships.

What are the implications of atpH modifications on thylakoid membrane energetics and photoprotection?

Modifications to atpH can significantly impact thylakoid energetics and photoprotection through several mechanisms:

• Effects on non-photochemical quenching: Changes in atpH function directly affect proton conductivity and thus the development of the proton gradient across the thylakoid membrane. This impacts the energy-dependent qE component of non-photochemical quenching, a critical photoprotective mechanism .

• Electron transport regulation: Altered proton conductivity due to atpH modifications affects the down-regulation of electron transfer through the cytochrome b6f complex, which is an important protective mechanism during excess light conditions .

• Energy balance disruption: Changes in ATP synthase efficiency can disrupt the delicate ATP/NADPH ratio required for optimal Calvin cycle operation, potentially leading to photodamage under certain conditions.

• Feedback sensitivity: Modifications to atpH might alter the response to feedback limitation signals, changing how the plant adapts to varying environmental conditions. Research on wild-type and starchless mutant N. sylvestris demonstrates how changes in feedback regulation can lead to different patterns of ATP synthase regulation under various CO2 and O2 conditions .

What growth conditions are optimal for N. sylvestris in experimental settings?

For reproducible results in N. sylvestris research, the following growth conditions are recommended:

Standard growth conditions for N. sylvestris:

• Temperature: 28/22°C (day/night cycle)

• Photoperiod: 14/10 hour day/night cycle

• Humidity: 50% relative humidity

• CO2 concentration: 380 μbar

• Light intensity: Photosynthetic quantum flux density (PFD) of 800 μmol m-2 s-1

• Growth medium: Fertilized potting soil in 8 L pots (one plant per pot)

Verification of plant material:

• For starchless mutants (e.g., NS 458): Confirm phenotype through negative iodine staining for starch

• For wild-type: Verify through expected growth patterns and photosynthetic responses

What analytical techniques are most effective for characterizing recombinant atpH protein?

For comprehensive characterization of recombinant atpH:

• Structural analysis:

  • Circular dichroism (CD) spectroscopy: To assess secondary structure content

  • NMR spectroscopy or X-ray crystallography: For detailed structural information

  • Cryo-electron microscopy: Particularly useful for membrane protein complexes

• Functional assessment:

  • Reconstitution into liposomes: To measure proton translocation activity

  • ATPase activity assays: To quantify ATP hydrolysis rates

  • Proton conductance measurements: Using techniques such as electrochromic shift (ECSt) measurements

• Interaction studies:

  • Cross-linking experiments: To identify binding partners

  • Co-immunoprecipitation: To confirm protein-protein interactions

  • Surface plasmon resonance: To measure binding kinetics with other ATP synthase subunits

How can genomic data improve recombinant atpH research?

The genomic data available for N. sylvestris provides several advantages for recombinant atpH research:

• Sequence optimization: The complete genome of N. sylvestris represents 82.9% of its expected size with N50 sizes of about 80 kb . This information allows for codon optimization strategies when designing recombinant expression constructs.

• Transcriptome analysis: Approximately 44,000-53,000 transcripts are expressed in roots, leaves, or flowers of N. sylvestris . This data can help identify optimal tissues for gene expression studies and protein purification.

• Comparative genomics: Comparing atpH sequences across Nicotiana species (such as between N. sylvestris and N. tomentosiformis) can highlight conserved regions critical for function versus variable regions that might confer species-specific properties .

• Regulatory element identification: Genomic data helps identify promoters and regulatory elements controlling atpH expression, which can be useful in designing expression constructs or understanding native regulation.

What are the major challenges in expressing and purifying functional recombinant atpH?

Researchers commonly encounter several challenges when working with recombinant atpH:

• Expression obstacles:

  • Membrane protein toxicity to expression hosts

  • Protein misfolding and aggregation

  • Low expression yields

• Purification difficulties:

  • Maintaining protein stability during extraction from membranes

  • Selecting appropriate detergents that preserve native structure

  • Achieving sufficient purity without compromising activity

• Functional assessment complications:

  • Reconstituting purified protein into appropriate membrane environments

  • Distinguishing specific atpH activity from background ATPase activity

  • Correlating in vitro measurements with in vivo function

Recommended solutions:

• Use specialized expression hosts designed for membrane proteins

• Optimize detergent selection through systematic screening

• Consider fusion protein approaches to enhance solubility

• Validate protein functionality through multiple complementary assays

How can researchers address data inconsistencies between in vitro and in vivo atpH studies?

When facing discrepancies between in vitro and in vivo results:

What emerging technologies will advance recombinant atpH research?

The future of recombinant atpH research will likely be shaped by several cutting-edge technologies:

• CRISPR-Cas9 genome editing: Precise modification of the native atpH gene in N. sylvestris to study structure-function relationships in the native genomic context.

• Single-molecule techniques: Direct visualization of atpH function within the ATP synthase complex using high-resolution microscopy techniques.

• Computational approaches: Advanced molecular dynamics simulations to model proton conductance through the atpH channel and predict the effects of mutations.

• Synthetic biology: Design of novel atpH variants with altered properties for enhanced photosynthetic efficiency or adapted function under specific environmental conditions.

• Multi-omics integration: Combining genomics, transcriptomics, proteomics, and metabolomics data to understand the system-level consequences of atpH modifications on photosynthetic metabolism and plant fitness.

How might recombinant atpH research contribute to improving photosynthetic efficiency?

Research on recombinant atpH has significant potential to enhance photosynthetic efficiency through:

• Optimized proton conductance: Engineering atpH to fine-tune proton conductivity (gH+) could optimize the balance between ATP synthesis and photoprotection mechanisms.

• Improved feedback regulation: Modifying atpH response to regulatory signals might reduce limitations imposed by feedback inhibition, particularly under changing environmental conditions where studies show complex interactions between CO2 availability, O2 concentration, and ATP synthase function .

• Enhanced stress tolerance: Engineered atpH variants could potentially maintain better photosynthetic performance under abiotic stress conditions by regulating non-photochemical quenching and electron transport more effectively.

• Energy balance optimization: Modifications to atpH could help maintain optimal ATP/NADPH ratios under fluctuating environmental conditions, potentially reducing photodamage and improving carbon fixation efficiency.

These advances could ultimately contribute to creating crop plants with improved photosynthetic performance and yield under challenging environmental conditions.