Recombinant Ceratophyllum demersum ATP synthase subunit b, chloroplastic (atpF)

What is ATP synthase subunit b, chloroplastic (atpF) in Ceratophyllum demersum?

ATP synthase subunit b, chloroplastic (atpF) is a critical component of the ATP synthase complex in Ceratophyllum demersum (rigid hornwort or coontail), specifically located in the chloroplast. It functions as part of the peripheral stalk in the F-type ATP synthase and is essential for the biogenesis and proper functioning of the entire ATP synthase complex. The full-length protein consists of 184 amino acids and is encoded by the plastid atpF gene. The protein plays a crucial role in energy production during photosynthesis by participating in ATP synthesis .

How is recombinant Ceratophyllum demersum atpF protein typically expressed and purified?

Recombinant Ceratophyllum demersum ATP synthase subunit b, chloroplastic (atpF) protein is typically expressed in Escherichia coli expression systems. For research applications, the protein is often fused with an N-terminal His-tag to facilitate purification using affinity chromatography. The expression involves:

• Cloning the atpF gene into a suitable expression vector

• Transforming E. coli cells with the recombinant vector

• Inducing protein expression under optimized conditions

• Lysing cells and purifying the His-tagged protein using metal affinity chromatography

• Performing quality control through SDS-PAGE analysis to confirm purity (typically >90%)

• Lyophilizing the purified protein for long-term storage

The resulting product is generally supplied as a lyophilized powder that can be reconstituted in an appropriate buffer for experimental use .

What is the significance of Ceratophyllum demersum as a research organism?

Ceratophyllum demersum is a submerged macrophyte found in various aquatic environments, including natural water bodies and artificial anthropogenic reservoirs. It serves as an important research organism for several reasons:

• Bioindicator capabilities: It can be used to monitor environmental pollutants, particularly heavy metals and surfactants in aquatic ecosystems

• Photosynthetic studies: Its well-developed chloroplasts and photosynthetic machinery make it valuable for studying photosynthetic processes

• Stress adaptation: It exhibits measurable physiological responses to environmental stressors, such as changes in pigment composition

• Phytoremediation potential: It has the capacity to absorb and accumulate various pollutants

• Phytochemical profile: It contains numerous bioactive compounds including phenolics and flavonoids with potential medicinal applications

These characteristics make C. demersum a versatile model for research in plant physiology, ecotoxicology, and environmental monitoring .

What methodologies are recommended for analyzing atpF function in Ceratophyllum demersum?

To analyze atpF function in Ceratophyllum demersum, researchers should employ a multi-faceted approach:

• Genetic manipulation techniques:

  • CRISPR-Cas9 gene editing to create knockout or knockdown mutants of atpF

  • Targeted mutagenesis to introduce specific mutations in the atpF gene

  • Comparative analysis with related organisms like Chlamydomonas reinhardtii where atpF mutants have been characterized

• Protein interaction studies:

  • Co-immunoprecipitation to identify protein-protein interactions

  • Blue native PAGE to analyze intact ATP synthase complex assembly

  • Cross-linking studies to map the topology of atpF within the ATP synthase complex

• Functional assays:

  • ATP synthase activity measurements using in vitro reconstitution systems

  • Chloroplast isolation and analysis of ATP synthesis rates

  • Photosynthetic efficiency measurements using chlorophyll fluorescence

• Structural biology approaches:

  • Cryo-electron microscopy of the ATP synthase complex

  • X-ray crystallography of the purified atpF protein

  • Molecular dynamics simulations to understand structural dynamics

These methodologies should be selected based on the specific research question and combined to provide comprehensive insights into atpF function .

How do environmental stressors affect ATP synthase subunit b expression and function in Ceratophyllum demersum?

Environmental stressors significantly impact ATP synthase subunit b expression and function in Ceratophyllum demersum through multiple mechanisms:

• Heavy metal stress:

  • Lead ions and other heavy metals can disrupt the pigment composition in C. demersum tissues

  • Under metal stress, chlorophyll a/b ratios change, indicating damage to photosystem II antenna complexes

  • This damage indirectly affects ATP synthase function by reducing the proton gradient necessary for ATP synthesis

• Surfactant exposure:

  • Cationic synthetic surfactants trigger a biphasic response in C. demersum:

    • Initial stress phase: Increased chlorophyll content (1.5-2.2 times higher for chlorophyll a, 1.7-2 times higher for chlorophyll b)

    • Adaptation phase: Membrane stabilization and restoration of ion transport

• Oxidative stress responses:

  • Environmental stressors induce reactive oxygen species (ROS) production

  • During adaptation, mitochondrial and chloroplast activity increases to enhance energy supply

  • ATP synthase gene expression may be upregulated to compensate for energy demands during stress

The following table shows typical pigment content changes in C. demersum tissues under cationic surfactant stress:

*Asterisk indicates statistical significance (p<0.05) compared to control levels .

What role does atpF play in the biogenesis of the chloroplast ATP synthase complex?

The atpF gene product (ATP synthase subunit b) plays a critical role in the biogenesis of the chloroplast ATP synthase complex through several key functions:

• Peripheral stalk formation:

  • AtpF forms part of the peripheral stalk that connects the F₁ and F₀ sectors of ATP synthase

  • It works in conjunction with subunit b' (encoded by the nuclear ATPG gene) to form a stable peripheral stalk

• Complex assembly coordination:

  • Studies in Chlamydomonas reinhardtii have shown that atpF knockout mutants completely prevent ATP synthase function and accumulation

  • This indicates that atpF is essential for the initial stages of complex assembly

• Stability maintenance:

  • The peripheral stalk provides structural stability to the ATP synthase complex during the rotational catalysis of ATP synthesis

  • Without functional atpF, the complex cannot maintain its structural integrity

• Coordinated biogenesis:

  • AtpF participates in the coordinated assembly of plastid-encoded and nuclear-encoded subunits

  • This coordination is essential for the proper stoichiometry of subunits in the functional complex

Research has demonstrated that frame-shift mutations in atpF completely abolish ATP synthase accumulation, highlighting its indispensable role in complex biogenesis and function .

How does the FTSH protease system interact with ATP synthase subunit b in chloroplasts?

The FTSH protease system plays a significant role in regulating ATP synthase subunit b levels and quality control in chloroplasts:

• Proteolytic regulation:

  • FTSH1 is a major thylakoid protease involved in the turnover of photosynthetic proteins

  • Crossing ATP synthase mutants with ftsh1-1 mutants has identified AtpH (ATP synthase subunit c) as an FTSH substrate

  • FTSH significantly contributes to the concerted accumulation of ATP synthase subunits

• Quality control mechanism:

  • FTSH proteases remove damaged or improperly folded ATP synthase subunits

  • This quality control is essential for maintaining functional ATP synthase complexes

  • In the absence of functional FTSH, aberrant ATP synthase subunits may accumulate

• Assembly regulation:

  • FTSH may monitor the stoichiometry of ATP synthase subunits

  • Excess unassembled subunits can be targeted for degradation by FTSH

  • This helps maintain proper assembly of the ATP synthase complex

• Stress response modulation:

  • Under stress conditions, FTSH activity may increase to remove damaged ATP synthase components

  • This allows for the replacement with newly synthesized subunits

  • The process ensures ATP synthase functionality under changing environmental conditions

These interactions highlight the complex regulatory mechanisms that ensure proper ATP synthase biogenesis and maintenance in the chloroplast .

What are the recommended storage and handling protocols for recombinant atpF protein?

Proper storage and handling of recombinant Ceratophyllum demersum ATP synthase subunit b, chloroplastic (atpF) protein are critical for maintaining its stability and functionality. The following protocols are recommended:

• Long-term storage:

  • Store the lyophilized protein at -20°C to -80°C

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

  • The protein should be stored in Tris/PBS-based buffer containing 6% Trehalose, pH 8.0

• Reconstitution procedure:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

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

  • Add glycerol to a final concentration of 5-50% (recommended: 50%)

  • Aliquot for long-term storage at -20°C to -80°C

• Working conditions:

  • For short-term use, working aliquots can be stored at 4°C for up to one week

  • Repeated freezing and thawing should be avoided as it may cause protein denaturation

  • When conducting experiments, maintain protein samples on ice when not in use

• Quality control measures:

  • Verify protein integrity by SDS-PAGE before experimental use

  • Check for potential aggregation by dynamic light scattering or size exclusion chromatography

  • Functional assays may be performed to ensure the protein maintains its expected activity

Adherence to these protocols will help ensure the reliability and reproducibility of experimental results using the recombinant atpF protein .

What expression systems are optimal for producing functional recombinant atpF protein?

Several expression systems can be used for producing functional recombinant Ceratophyllum demersum ATP synthase subunit b, chloroplastic (atpF) protein, each with specific advantages and limitations:

• Escherichia coli expression system:

  • Advantages: Most commonly used, rapid growth, high yield, cost-effective

  • Optimization strategies:

    • Use BL21(DE3) or Rosetta strains for efficient expression

    • Employ low temperature induction (16-20°C) to enhance proper folding

    • Include molecular chaperones to assist in folding

    • Utilize specialized vectors with T7 or tac promoters

  • Purification approach: N-terminal His-tag facilitated metal affinity chromatography

• Yeast expression systems (Pichia pastoris, Saccharomyces cerevisiae):

  • Advantages: Eukaryotic post-translational modifications, secretion capability

  • Optimization strategies:

    • Use methanol-inducible promoters in P. pastoris

    • Optimize codon usage for yeast expression

    • Include yeast secretion signals for extracellular production

  • Purification approach: Combination of affinity chromatography and ion exchange

• Insect cell/baculovirus expression system:

  • Advantages: Advanced folding machinery, suitable for complex proteins

  • Optimization strategies:

    • Optimize virus-to-cell ratio (MOI)

    • Harvest at optimal time points (48-72 hours post-infection)

    • Use Sf9 or Hi5 cells depending on protein characteristics

  • Purification approach: Multi-step purification including affinity and size exclusion

• Cell-free expression system:

  • Advantages: Rapid production, ability to incorporate unnatural amino acids

  • Optimization strategies:

    • Use wheat germ or E. coli-based extracts

    • Supplement with chaperones and cofactors

    • Optimize reaction conditions (temperature, pH, salt)

  • Purification approach: Direct purification from reaction mixture

Current research indicates that E. coli remains the most reliable system for atpF expression, consistently producing soluble and functional protein with yields suitable for structural and functional studies .

How can researchers effectively analyze the interaction between atpF and other ATP synthase subunits?

To effectively analyze interactions between atpF and other ATP synthase subunits, researchers should employ a multi-technique approach:

• Co-immunoprecipitation (Co-IP) studies:

  • Use antibodies specific to atpF or other subunits to pull down interaction partners

  • Identify co-precipitated proteins by mass spectrometry

  • Verify interactions using reciprocal Co-IP experiments

  • Include appropriate controls to rule out non-specific binding

• Yeast two-hybrid (Y2H) and split-ubiquitin systems:

  • Test direct binary interactions between atpF and other ATP synthase subunits

  • Map interaction domains through truncation or deletion constructs

  • Validate positive interactions through multiple reporter systems

  • Quantify interaction strength through beta-galactosidase assays

• Blue native polyacrylamide gel electrophoresis (BN-PAGE):

  • Analyze intact ATP synthase complexes under native conditions

  • Compare complex formation in wild-type and atpF mutant samples

  • Combine with second-dimension SDS-PAGE for subunit composition analysis

  • Use antibody detection to confirm the presence of specific subunits

• Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC):

  • Create fluorescently tagged versions of atpF and interacting partners

  • Analyze their proximity and interaction in vivo

  • Perform live-cell imaging to observe dynamic interactions

  • Quantify interaction strength through fluorescence intensity measurements

• Cross-linking coupled with mass spectrometry (XL-MS):

  • Use chemical cross-linkers to capture transient or weak interactions

  • Identify cross-linked peptides by mass spectrometry

  • Generate distance constraints for structural modeling

  • Map the topology of the ATP synthase complex

By combining these complementary approaches, researchers can build a comprehensive understanding of how atpF interacts with other subunits to form a functional ATP synthase complex in Ceratophyllum demersum chloroplasts .

How can researchers differentiate between the effects of mutations in atpF versus ATPG genes?

Differentiating between the effects of mutations in atpF (encoding ATP synthase subunit b) versus ATPG (encoding ATP synthase subunit b') requires a systematic comparative analysis approach:

• Phenotypic characterization:

  • Light sensitivity: Both atpF and ATPG mutants typically show high light sensitivity, but the severity may differ

  • Growth rates: Compare growth curves under various light intensities and carbon sources

  • Photosynthetic parameters: Measure photosynthetic efficiency using PAM fluorometry

• Biochemical analysis:

  • ATP synthase accumulation:

    • atpF knockout mutants: Complete absence of ATP synthase complex

    • ATPG knockdown mutants: Small accumulation of functional ATP synthase

    • ATPG knockout mutants: No ATP synthase accumulation

  • Complex assembly: Analyze using BN-PAGE followed by immunoblotting with antibodies against various ATP synthase subunits

• Proteomic approach:

  • Mass spectrometry analysis:

    • In atpF mutants: Look for absence of b subunit and potential changes in other subunits

    • In ATPG mutants: Look for absence of b' subunit and potential changes in other subunits

  • Quantitative proteomics: Identify differential accumulation of other ATP synthase subunits

• Genetic complementation tests:

  • Cross atpF and ATPG mutants to determine if they belong to the same or different complementation groups

  • Perform targeted rescue experiments with wild-type copies of each gene

• Transcriptomic analysis:

  • Compare gene expression profiles of atpF versus ATPG mutants

  • Identify differentially regulated genes that may indicate specific cellular responses

Research has demonstrated that while both atpF and ATPG knockout mutations prevent ATP synthase function and accumulation, ATPG knockdown mutants (such as those with transposon insertions in the 3'UTR) may retain some ATP synthase activity, allowing for differentiation between the two gene defects .

What analytical techniques are most effective for assessing ATP synthase activity in Ceratophyllum demersum samples?

To effectively assess ATP synthase activity in Ceratophyllum demersum samples, researchers should employ multiple complementary techniques:

• Oxygen evolution/consumption measurements:

  • Light-dependent oxygen evolution: Measures photosynthetic electron transport chain activity

  • Dark respiration: Measures mitochondrial oxygen consumption

  • Methodology:

    • Use Clark-type oxygen electrodes

    • Measure in the presence/absence of ATP synthase inhibitors (oligomycin, DCCD)

    • Calculate activity rates based on chlorophyll content

• ATP production assays:

  • Luciferin-luciferase bioluminescence assay:

    • Highly sensitive method for quantifying ATP

    • Can be performed on isolated chloroplasts or thylakoid membranes

  • Coupled enzyme assays:

    • Utilize ATP-dependent enzymes (hexokinase/glucose-6-phosphate dehydrogenase)

    • Monitor NADPH production spectrophotometrically

• Proton gradient measurements:

  • Fluorescent probes: Use pH-sensitive fluorescent dyes (ACMA, 9-aminoacridine)

  • Light-induced pH changes: Monitor ΔpH formation across thylakoid membranes

  • Correlation with ATP synthesis: Link proton gradient formation to ATP production rates

• Electrochemical techniques:

  • Patch-clamp analysis: Directly measure ion currents through ATP synthase

  • Artificial membrane systems: Reconstitute purified ATP synthase in liposomes

• Functional proteomics approach:

  • Activity-based protein profiling: Use activity-based probes to assess functional ATP synthase

  • Native gel electrophoresis: Combine with in-gel activity assays to visualize active complexes

Each technique provides complementary information, and combining multiple approaches yields the most comprehensive assessment of ATP synthase activity in Ceratophyllum demersum. For comparing wild-type and mutant samples, normalizing activities to total chlorophyll content or protein concentration is essential for accurate interpretation .

How should researchers interpret changes in photosynthetic pigment composition in relation to ATP synthase function?

Changes in photosynthetic pigment composition provide valuable insights into ATP synthase function in Ceratophyllum demersum. Researchers should interpret these changes using the following framework:

• Chlorophyll a/b ratio as a stress indicator:

  • Normal ratio: Typically 2.5-3.0 in healthy C. demersum

  • Elevated ratio (>5.0): Indicates stress and potential damage to photosystem II antenna complexes

  • Relationship to ATP synthase: Damaged photosystems reduce electron transport, limiting proton gradient formation and ATP synthesis

• Carotenoid content and photoprotection:

  • Increased carotenoids: Often a photoprotective response to stress

  • Xanthophyll cycle: Activation indicates excessive light energy that cannot be utilized for ATP synthesis

  • Interpretation: High carotenoid/chlorophyll ratio may indicate impaired ATP synthase function causing backup of electron transport

• Total chlorophyll content and energy capture:

  • Decreased total chlorophyll: Reduces light-harvesting capacity

  • Correlation with ATP production: Directly impacts the energy available for ATP synthesis

  • Adaptation responses: Plants may increase chlorophyll temporarily during stress adaptation to enhance energy capture

• Pigment composition changes under pollutant stress:

  • Under cationic surfactant stress, C. demersum exhibits:

    • Initial increase in chlorophyll a (1.5-2.2 times higher than control)

    • Increased chlorophyll b (1.7-2 times higher)

    • Elevated carotenoids (2.4 times higher)

  • These changes reflect the plant's attempt to maintain energy production despite stress

• Interpretation framework for ATP synthase function:

Researchers should consider these pigment changes as part of an integrated response to maintain cellular energy homeostasis when ATP synthase function is compromised or when the plant faces environmental stressors that indirectly affect ATP synthesis .

What are the future research directions for studying Ceratophyllum demersum atpF in the context of environmental adaptation?

Future research on Ceratophyllum demersum atpF should focus on several promising directions that integrate molecular function with ecological adaptation:

These research directions will not only advance our fundamental understanding of ATP synthase function but also contribute to practical applications in environmental monitoring, bioremediation, and adaptation to changing aquatic ecosystems .

How can researchers utilize Ceratophyllum demersum atpF studies to advance broader knowledge of chloroplast energy metabolism?

Research on Ceratophyllum demersum atpF can significantly advance our understanding of chloroplast energy metabolism through several key approaches:

• Comparative studies across photosynthetic organisms:

  • Use C. demersum as a model to identify conserved and divergent features of ATP synthase

  • Compare atpF function in submerged aquatic plants versus terrestrial plants

  • Investigate evolutionary adaptations of chloroplast energy systems in aquatic environments

• Integration of photosynthetic and respiratory energy systems:

  • Study the coordination between chloroplast ATP synthase and mitochondrial ATP production

  • Investigate energy partitioning under different environmental conditions

  • Develop models of whole-cell energy homeostasis in photosynthetic cells

• Application to synthetic biology and biotechnology:

  • Engineer optimized ATP synthase systems based on C. demersum adaptations

  • Develop bioenergy applications utilizing robust aquatic plant ATP production systems

  • Create synthetic chloroplasts with enhanced energy production capabilities

• Advanced structural biology:

  • Determine high-resolution structures of C. demersum ATP synthase complex

  • Compare structural adaptations across plants from different habitats

  • Use structural insights to explain functional differences in energy metabolism

• Educational and training opportunities:

  • Develop C. demersum as an accessible model system for studying chloroplast bioenergetics

  • Create teaching modules using this abundant aquatic plant

  • Train next-generation scientists in integrating molecular, physiological, and ecological approaches