Recombinant Solanum tuberosum ATP synthase subunit 9, mitochondrial (ATP9)

What is the structural composition of ATP synthase subunit 9 in Solanum tuberosum and how does it compare to other plant species?

ATP synthase subunit 9 (ATP9) in Solanum tuberosum is a 74-amino acid mitochondrial protein that forms part of the Fo domain of ATP synthase (Complex V). The complete amino acid sequence is: "MLEGAKLMGAGAATIALAGAAIGIGNVFSSLIHSVARNPSLAKQLFGYAILGFALTEAIA LFALMMAFLILFVF" . This protein is highly conserved across plant species, showing significant homology with ATP9 from organisms including Arabidopsis thaliana, Triticum aestivum, and Nicotiana tabacum .

ATP9 functions as part of the c-ring in the Fo portion of ATP synthase, which is embedded in the inner mitochondrial membrane. This c-ring plays a crucial role in proton translocation across the membrane, thereby contributing to the proton electrochemical gradient that drives ATP synthesis . In contrast to other complex V subunits, ATP9 is encoded by the mitochondrial genome in potato, highlighting its evolutionary significance in energy metabolism.

The mitochondrial location of ATP9 is consistent with its role in oxidative phosphorylation, where it forms part of the rotary motor mechanism that couples proton movement to ATP synthesis. The protein's hydrophobic nature allows it to be properly integrated into the lipid bilayer of the inner mitochondrial membrane, which is essential for maintaining the proton gradient necessary for ATP production .

How is the expression of the atp9 gene regulated in potato mitochondria?

The potato mitochondrial atp9 gene exhibits a relatively simple expression pattern compared to other mitochondrial genes. Transcription initiation occurs at a specific site located 121-128 bp upstream of the atp9 open reading frame . Interestingly, this initiation region does not share homology with proposed consensus sequences for typical plant mitochondrial promoters, suggesting a potentially unique regulatory mechanism for this gene .

Transcript termination occurs 67-71 nucleotides downstream of a putative single-stem loop structure, as determined by nuclease S1 protection analyses . This well-defined transcription unit indicates tight regulation of atp9 expression in potato mitochondria.

The atp9 gene expression likely undergoes developmental regulation, as suggested by transcriptomic studies of potato tuber sprouting, where significant changes in mitochondrial gene expression have been observed during the transition from dormancy to active growth . During sprouting, multiple metabolic pathways are activated, including plant hormone signal transduction and MAPK signaling pathways, which may indirectly influence mitochondrial gene expression .

What is the functional role of ATP9 in the context of the complete ATP synthase complex?

ATP9 (subunit c) is a critical component of the Fo domain of mitochondrial ATP synthase. It assembles into a ring structure (c-ring) within the inner mitochondrial membrane that serves as one of the two rotary motors of the ATP synthase complex . The functional significance of ATP9 lies in its role in proton translocation:

• The c-ring, composed of multiple ATP9 subunits, rotates relative to subunit a when protons pass through the Fo portion via an interface between these subunits.

• This rotation is mechanically coupled to the central stalk (comprising subunits γ, δ, and ε) of the F1 domain.

• The rotation of subunit γ within the F1 α3β3 hexamer provides the conformational energy required for ATP synthesis from ADP and inorganic phosphate .

ATP9's function is essential for maintaining the proton-motive force across the inner mitochondrial membrane, which has two components: a pH differential and an electrical membrane potential (Δψm) . When properly functioning, this system efficiently converts the energy from proton flow into ATP production, which is vital for cellular metabolism in potato tissues.

The integration of ATP9 into the complete ATP synthase complex follows a specific assembly pathway. In yeast models, which have informed our understanding of plant mitochondrial ATP synthase assembly, the c-ring forms early in the assembly process, followed by the addition of the F1 sector, the stator arm, and finally the addition of subunits a and A6L . This orchestrated assembly ensures proper complex formation and function.

How does the mitochondrial genome structure affect ATP9 expression in Solanum tuberosum?

The mitochondrial genome of Solanum tuberosum exists as multiple individual molecules rather than a single circular chromosome, which has significant implications for ATP9 expression . This multichromosomal nature leads to several important effects:

• Intra-molecular recombination events can generate multiple isoforms and sub-genomic circles, creating a dynamic and complex genomic environment .

• Studies have shown that different potato clones exhibit variations in their mitochondrial DNA structure, with some assembled into four circular molecules (referred to as group A) .

• These structural differences may affect gene expression patterns, including that of ATP9, across different potato varieties.

The genomic context of ATP9 is further complicated by shared sequences between mitochondrial DNA molecules. For instance, in some potato clones, molecules 1a and 1b share a common sequence of approximately 219 kb, with 77.3 kb and 9.8 kb of unique sequence in each molecule respectively . This genomic arrangement can influence the regulation and expression levels of mitochondrial genes including ATP9.

Research has also identified key differences in mitogenomes through comparative analyses of different potato clones, which may contribute to variability in ATP9 expression and function across different genotypes . This genomic plasticity likely represents an adaptation mechanism that allows for metabolic flexibility in response to environmental conditions.

What are the optimal conditions for expressing and purifying recombinant Solanum tuberosum ATP9 protein?

For successful expression and purification of recombinant Solanum tuberosum ATP9 protein, the following optimized protocol is recommended:

Expression System Selection:
E. coli is the preferred expression system for recombinant ATP9 from Solanum tuberosum . This prokaryotic system offers several advantages:

• High protein yield

• Relatively simple culture conditions

• Compatibility with the hydrophobic nature of ATP9

Vector Design and Tagging Strategy:

• The full-length ATP9 coding sequence (amino acids 1-74) should be cloned into an expression vector with an N-terminal His-tag to facilitate purification .

• The His-tag enables efficient one-step purification using metal affinity chromatography.

Expression Conditions:

• Culture temperature: 30°C (lower temperatures may improve proper folding)

• Induction: 0.5-1.0 mM IPTG when culture reaches OD600 of 0.6-0.8

• Post-induction growth: 4-6 hours for optimal protein accumulation

Purification Protocol:

• Cell lysis should be performed using sonication or pressure-based methods in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10 mM imidazole.

• Following clarification by centrifugation, the lysate should be applied to Ni-NTA resin.

• Washing with increasing imidazole concentrations (20-50 mM) removes non-specifically bound proteins.

• Elution with 250-300 mM imidazole yields purified ATP9 protein.

• Final purification can achieve greater than 90% purity as determined by SDS-PAGE .

Storage Recommendations:

• The purified protein should be stored as a lyophilized powder or in solution with 6% trehalose at pH 8.0 .

• For long-term storage, aliquot and store at -20°C/-80°C.

• Avoid repeated freeze-thaw cycles which can compromise protein integrity .

• For working solutions, reconstitute in deionized sterile water to 0.1-1.0 mg/mL with 5-50% glycerol as a cryoprotectant .

What techniques are most effective for studying ATP9 function in the context of mitochondrial research?

Several advanced techniques have proven particularly valuable for studying ATP9 function in potato mitochondria:

Isolation of Intact Mitochondria:

• Differential centrifugation of potato tissue homogenates using sucrose gradients (0.3-1.0 M) allows isolation of functional mitochondria.

• Mitochondrial integrity can be assessed using cytochrome c oxidase activity assays with and without membrane disruption.

In Vitro Transcription Analysis:
For studying ATP9 gene expression, in vitro capping of primary mitochondrial RNAs followed by hybridization to the 5' flanking sequences of the atp9 gene has been successfully employed to identify transcription initiation regions . This can be complemented with:

• Primer extension analysis to map 5' transcript termini

• Nuclease S1 protection assays to precisely determine both 5' and 3' transcript boundaries

Functional Assessment of ATP Synthase Activity:

• Polarographic measurement of oxygen consumption coupled to ATP synthesis in isolated mitochondria

• Spectrophotometric assays coupling ATP production to NADPH formation through hexokinase and glucose-6-phosphate dehydrogenase

• Chemiluminescent detection of ATP production using luciferase-based assays

Structural Analysis:

• Blue Native PAGE (BN-PAGE) to examine the assembly and oligomerization state of ATP synthase complexes containing ATP9

• Immunoprecipitation using anti-ATP9 antibodies to identify interacting partners

• Cryo-electron microscopy for high-resolution structural analysis of ATP9 within the c-ring

Genetic Manipulation:
RNA interference (RNAi) or CRISPR-Cas9 technologies targeting nuclear genes involved in mitochondrial function can be used to study the impact on ATP9 expression and function, as direct manipulation of the mitochondrial genome remains challenging in plants.

How does ATP9 contribute to the oligomerization of ATP synthase and mitochondrial cristae formation in potato cells?

ATP9, as part of the c-ring in the Fo domain, plays a significant role in ATP synthase oligomerization and the subsequent formation of mitochondrial cristae in potato cells. This relationship between structure and function represents an advanced area of mitochondrial biology:

ATP Synthase Oligomerization:
ATP synthase exists not only as individual complexes (monomers) but also forms di- and oligomeric structures in the inner mitochondrial membrane. This oligomerization process confers several advantages:

• Increased stability of the complex, which is continuously subject to dynamic rotor/stator interactions

• Enhanced efficiency of ATP synthesis through cooperative effects between adjacent complexes

• Formation of specialized membrane domains that optimize proton utilization

The presence of ATP9 in the correct stoichiometry and conformation is critical for this oligomerization process. The c-ring, composed of multiple ATP9 subunits, influences the angular association of ATP synthase monomers when they dimerize.

Cristae Formation Mechanism:
The dimerization and oligomerization of ATP synthase directly shapes the inner mitochondrial membrane architecture:

• Due to the angular association of two monomers, dimerization leads to bending of the inner mitochondrial membrane

• This bending creates the characteristic protrusions known as cristae

• Clustering of ATP synthase dimers at the apex of the cristae generates a strong local positive curvature

• This curvature forms a "proton trap" that facilitates ATP synthesis by increasing local proton concentration

Functional Consequences in Potato Cells:
The proper formation of cristae is particularly important in metabolically active potato tissues such as sprouting tubers, where energy demand increases dramatically:

• During dormancy, cristae structure is less developed, corresponding to lower ATP synthase oligomerization

• During sprouting, increased ATP demand triggers structural reorganization of mitochondria, including enhanced cristae development

• This structural adaptation is regulated by the activation of signaling pathways including plant hormone signal transduction and MAPK cascades

The relationship between ATP9, ATP synthase oligomerization, and cristae formation represents a critical aspect of mitochondrial bioenergetics that directly impacts cellular metabolism during key developmental transitions in potato.

What are the unique features of ATP9 assembly into the ATP synthase complex compared to other plant species?

The assembly of ATP9 into the complete ATP synthase complex in Solanum tuberosum exhibits several distinctive features compared to other plant species:

Multichromosomal Mitochondrial Genome Influence:
The unique multichromosomal nature of potato mitochondrial DNA creates special considerations for ATP9 assembly:

• The existence of multiple mitochondrial DNA molecules may affect the coordinated expression of mitochondrial-encoded subunits (including ATP9) and nuclear-encoded subunits

• Intra-molecular recombination events can generate genomic rearrangements that influence the regulation of ATP9 expression

• Different potato genotypes show variations in mitochondrial genome structure, which may lead to differences in ATP synthase assembly efficiency

Assembly Pathway Specificities:
While the general ATP synthase assembly pathway is conserved across species, research suggests potato may have specific adaptations:

Regulatory Factors:
Several factors appear to specifically regulate ATP synthase assembly in potato:

• The expression of nuclear genes for F1 components may exert translational control over the mitochondrial-encoded Fo components, including ATP9, to ensure balanced output

• Environmental stressors such as drought have been shown to affect mitochondrial function in potato , potentially through mechanisms that alter ATP synthase assembly

Developmental Context:
ATP synthase assembly in potato has unique developmental regulation:

• During tuber dormancy, ATP synthase assembly is maintained at basal levels

• The transition to sprouting involves significant upregulation of energy metabolism

• This transition requires coordinated assembly of new ATP synthase complexes, with ATP9 incorporation being a critical step

• Transcriptomic studies have identified differential expression of genes involved in several relevant pathways during this transition, including phenylpropanoid biosynthesis, MAPK signaling, and plant hormone signal transduction

The distinct features of ATP9 assembly in potato reflect adaptations to the unique energy requirements of this storage organ during its developmental cycle of dormancy, sprouting, and growth.

What are the main challenges in producing functional recombinant ATP9 protein, and how can they be addressed?

Production of functional recombinant ATP9 protein from Solanum tuberosum presents several significant challenges due to its hydrophobic nature and mitochondrial origin. These challenges and their potential solutions include:

Challenge 1: Protein Solubility
ATP9 is highly hydrophobic, containing multiple transmembrane domains that naturally reside in the lipid environment of the inner mitochondrial membrane.

Solutions:

• Fusion Partners: Utilize solubility-enhancing fusion partners such as MBP (maltose-binding protein) or SUMO (small ubiquitin-like modifier) in addition to the His-tag .

• Detergent Screening: Systematically test different detergents (DDM, CHAPS, OG) to identify optimal solubilization conditions.

• Amphipol Approach: After initial purification, transfer the protein to amphipathic polymers (amphipols) that stabilize membrane proteins in aqueous solutions.

Challenge 2: Proper Folding
Ensuring correct folding of ATP9 outside its native membrane environment is difficult.

Solutions:

• Expression Temperature Optimization: Lower temperature expression (16-20°C) can slow protein synthesis and improve folding.

• Co-expression with Chaperones: Co-express with molecular chaperones like GroEL/GroES to assist proper folding.

• Nanodisc Technology: Reconstitute purified ATP9 into nanodiscs—small patches of lipid bilayer encircled by scaffold proteins—to provide a native-like membrane environment.

Challenge 3: Functional Assessment
Verifying that recombinant ATP9 retains its native function is technically demanding.

Solutions:

• Reconstitution Assays: Develop liposome reconstitution systems where purified ATP9 is incorporated into artificial membrane vesicles.

• Proton Transport Assays: Measure proton transport using pH-sensitive fluorescent dyes in the reconstituted system.

• Co-assembly Testing: Evaluate the ability of recombinant ATP9 to assemble with other ATP synthase components in vitro.

Challenge 4: Yield Optimization
Obtaining sufficient quantities of pure, functional protein for experimental use.

Solutions:

• Codon Optimization: Optimize the ATP9 coding sequence for the expression host (E. coli) .

• Media Formulation: Use auto-induction media to achieve higher cell densities and protein yields.

• Scale-up Strategies: Implement fed-batch fermentation protocols to increase biomass and protein production.

Challenge 5: Storage Stability
Maintaining protein stability during storage.

Solutions:

• Lyophilization: Store as lyophilized powder with appropriate stabilizers like trehalose .

• Glycerol Addition: Add 5-50% glycerol to liquid formulations to prevent freezing damage .

• Aliquoting Strategy: Prepare small single-use aliquots to avoid repeated freeze-thaw cycles .

Implementation of these solutions requires systematic optimization for the specific recombinant construct but can significantly improve the production of functional recombinant ATP9 protein for research applications.

How can researchers effectively study ATP9 function in the context of intact plant systems rather than isolated proteins?

Studying ATP9 function within intact plant systems presents unique challenges but offers more physiologically relevant insights. The following methodological approaches enable effective in vivo analysis:

Genetic Manipulation Strategies:

• Site-directed Mutagenesis of Nuclear-encoded Assembly Factors:

  • Since direct mitochondrial genome editing remains challenging in plants, targeting nuclear genes involved in ATP9 assembly and function provides an indirect approach

  • The CRISPR-Cas9 system can be utilized to modify genes encoding assembly factors like those homologous to ATP12 or TMEM70

  • Analysis of the resulting phenotypes can reveal ATP9's functional significance

• Inducible RNAi Systems:

  • Develop inducible RNA interference constructs targeting nuclear genes that interact with ATP9

  • This approach allows temporal control of the interference, enabling study of acute effects while avoiding lethal phenotypes

Advanced Imaging Techniques:

• In vivo ATP Sensing:

  • Utilize genetically encoded ATP sensors (like ATeam) that can be targeted to different cellular compartments

  • These fluorescent sensors allow real-time visualization of ATP levels in living plant cells

  • Comparing wild-type plants with those having altered ATP9 function reveals its contribution to ATP homeostasis

• Mitochondrial Membrane Potential Measurement:

  • Apply potentiometric dyes like JC-1 or TMRM to intact plant tissues

  • These dyes enable visualization of mitochondrial membrane potential (Δψm), which is directly influenced by ATP9 function

  • Time-lapse imaging during developmental transitions (e.g., dormancy breaking in potato tubers) can reveal dynamic changes in bioenergetics

Physiological and Biochemical Approaches:

• Respiratory Measurements in Tissue Samples:

  • Oxygen consumption analysis using Clark-type electrodes with specific inhibitors of different respiratory chain components

  • This approach allows assessment of coupling efficiency between electron transport and ATP synthesis, which depends on ATP9 function

• Metabolomic Profiling:

  • Comprehensive analysis of metabolites during developmental transitions like potato tuber sprouting

  • Changes in the levels of ATP, ADP, AMP, and other energy-related metabolites can indicate alterations in ATP synthase function

  • Targeted analysis of key metabolites identified in previous studies, such as alkaloids and amino acids whose levels change during sprouting

Systems Biology Integration:

• Multi-omics Correlation Analysis:

  • Combine transcriptomic and metabolomic data to identify correlations between ATP9 expression and metabolic changes

  • This approach has successfully revealed regulatory networks during potato tuber sprouting

  • Principal component analysis and other multivariate statistical methods can identify patterns that might not be apparent in single-omics approaches

• Developmental Time-course Studies:

  • Analyze ATP9 function across developmental stages, particularly during energy-demanding transitions like dormancy breaking in potato tubers

  • This temporal dimension provides insights into the dynamic role of ATP9 in energy metabolism during plant development

These methodologies collectively allow researchers to study ATP9 function within the physiological context of intact plant systems, providing a more complete understanding of its role in plant bioenergetics and development.

What emerging technologies could advance our understanding of ATP9 function in plant mitochondrial bioenergetics?

Several cutting-edge technologies are poised to transform our understanding of ATP9 function in plant mitochondrial bioenergetics:

Cryo-Electron Tomography (Cryo-ET):
This technique allows visualization of macromolecular complexes in their native cellular environment at near-atomic resolution:

• Unlike traditional cryo-EM that works with purified proteins, cryo-ET can examine ATP synthase complexes directly within mitochondrial membranes

• This approach would reveal how ATP9 contributes to c-ring formation and ATP synthase oligomerization in situ

• The resulting 3D reconstructions could demonstrate how ATP9 participates in cristae formation in potato mitochondria, expanding on current theoretical models

Single-Molecule Techniques:
Advances in single-molecule imaging and manipulation provide unprecedented insights into molecular mechanisms:

• Single-molecule FRET (Förster Resonance Energy Transfer) can track conformational changes in ATP synthase during operation

• Optical tweezers can measure the force generated by the ATP synthase rotary motor, quantifying ATP9's contribution to energy conversion

• These approaches would move beyond static structural studies to understand the dynamic aspects of ATP9 function

Mitochondrial Genome Editing:
While technically challenging, emerging methods for plant mitochondrial genome manipulation would revolutionize ATP9 research:

• Mitochondria-targeted TALENs (Transcription Activator-Like Effector Nucleases) could enable precise modifications of the ATP9 gene

• Base editors and prime editors adapted for mitochondrial targeting may allow specific modifications without double-strand breaks

• Direct manipulation of ATP9 would provide definitive insights into structure-function relationships currently inferred from indirect methods

Spatiotemporal Metabolomics:
New approaches enable metabolite analysis with subcellular resolution:

• Mass spectrometry imaging techniques like MALDI-MSI (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging) can map metabolite distributions in tissue sections

• These methods could track ATP distribution within potato tubers during dormancy and sprouting

• Correlation with ATP9 expression and ATP synthase activity would reveal functional relationships between mitochondrial bioenergetics and developmental processes

Synthetic Biology Approaches:
Reconstitution of minimal ATP synthase systems would enable precise mechanistic studies:

• Bottom-up assembly of simplified ATP synthase containing ATP9 and minimal essential components

• Incorporation into synthetic membrane systems with controlled composition

• These reductionist approaches would isolate ATP9's specific contribution to proton translocation and energy conversion

Computational Prediction and Modeling:
Advanced computational methods offer powerful predictive capabilities:

• Molecular dynamics simulations can model ATP9 behavior within the membrane environment

• Machine learning approaches integrating multi-omics data could predict ATP9 functional networks

• These in silico methods complement experimental approaches by generating testable hypotheses about ATP9 function

The integration of these emerging technologies promises to provide a comprehensive understanding of ATP9's role in plant mitochondrial bioenergetics, potentially leading to applications in crop improvement for enhanced energy efficiency.

How might research on potato ATP9 contribute to agricultural improvements in crop stress resistance and post-harvest storage?

Research on potato ATP9 has significant potential to contribute to agricultural improvements, particularly in developing crops with enhanced stress resistance and improved post-harvest characteristics:

Energy Efficiency and Stress Tolerance:
Understanding the role of ATP9 in mitochondrial energy production provides avenues for crop improvement:

• Heat Stress Resistance:

  • ATP synthase efficiency is critical during heat stress when respiratory demands increase

  • Plants with optimized ATP9 expression may maintain energy homeostasis under elevated temperatures

  • This could translate to potato varieties with improved tolerance to heat waves, an increasingly important trait under climate change scenarios

• Drought Tolerance Enhancement:

  • Studies have shown that mitochondrial function is significantly affected during drought stress in potato

  • ATP9's role in maintaining ATP synthase efficiency during water limitation could be leveraged to develop varieties with improved drought tolerance

  • Targeted approaches could optimize the coordination between ATP9 and other subunits to maintain energy production under water-limited conditions

Post-Harvest Quality and Storage Longevity:

• Tuber Dormancy Regulation:

  • ATP9 function is linked to energy metabolism changes during potato tuber dormancy and sprouting

  • Transcriptomic and metabolomic analyses have revealed that sprouting involves significant changes in energy-related pathways

  • Modulating ATP9 expression or activity could potentially extend dormancy periods by influencing the energy status that triggers sprouting

• Sprout Inhibition Strategies:

  • Current research on sprout inhibitors like methyl jasmonate and 1,4-dimethylnaphthalene shows they impact phytohormonal and stress protective pathways

  • Understanding how these compounds affect mitochondrial function and ATP9 specifically could lead to more targeted approaches to sprout control

  • This knowledge could result in storage protocols that maintain tuber quality while minimizing chemical treatments

Metabolic Engineering Applications:

• Enhanced Nutritional Quality:

  • Energy metabolism influences the synthesis and accumulation of various metabolites in potato tubers

  • Research shows that during sprouting, significant changes occur in alkaloids, amino acids, and flavonoids

  • Targeted manipulation of ATP9 could potentially redirect energy flux to favor the accumulation of beneficial compounds while minimizing undesirable ones

• Reduced Glycoalkaloid Accumulation:

  • Sprouting potatoes produce increased levels of potentially harmful glycoalkaloids

  • Understanding the energetic basis of these metabolic shifts could lead to varieties that maintain low glycoalkaloid levels even under conditions that would normally promote sprouting

  • This would enhance food safety while extending usable storage life

Implementation Pathways:

• Marker-Assisted Selection:

  • Identification of nuclear genes that interact with ATP9 or influence ATP synthase assembly could provide selection markers for breeding programs

  • This indirect approach circumvents the challenges of directly manipulating the mitochondrial genome

• Modulation of Assembly Factors:

  • Targeting nuclear-encoded assembly factors that influence ATP synthase assembly and stability

  • This approach could achieve desired changes in ATP9 function without direct mitochondrial genome modification

• Post-Harvest Treatment Optimization:

  • Development of storage protocols that maintain optimal ATP synthase function

  • Physical or chemical treatments that specifically target energy-related pathways involved in breaking dormancy