Recombinant Zea mays ATP synthase subunit 9, mitochondrial (ATP9)

What is ATP synthase subunit 9 in Zea mays and what is its role in mitochondrial function?

ATP synthase subunit 9 (ATP9) is a critical component of the mitochondrial ATP synthase complex (Complex V) in Zea mays. This 74-amino acid protein is part of the Fo domain, which is embedded in the inner mitochondrial membrane. ATP9 contributes to the c-ring structure that facilitates proton translocation across the membrane. The functional ATP synthase complex utilizes the energy from the proton electrochemical gradient to catalyze the phosphorylation of ADP to ATP, which is the main energy source for intracellular metabolic pathways . The c-ring, which includes ATP9, plays a central role in the rotary mechanism that couples proton flow to ATP synthesis.

What is the structural composition of recombinant Zea mays ATP9 protein?

Recombinant Zea mays ATP9 is a full-length protein comprising 74 amino acids (residues 1-74) with the sequence: MLEGAKLIGAGAATIALAGAAVGIGNVFSSLIHSVARNPSLAKQLFGYAILGFALTEAIALFALMMAFLILFVF . When produced recombinantly, it's typically fused to an N-terminal His tag to facilitate purification. The protein is a component of the Fo domain of ATP synthase complex and is known to have lipid-binding properties, which is reflected in its alternative name "Lipid-binding protein" . It's worth noting that the recombinant version maintains the same amino acid sequence as the native protein but includes the addition of the His tag for research purposes.

What expression systems are suitable for producing recombinant Zea mays ATP9?

E. coli is the predominant expression system for producing recombinant Zea mays ATP9 protein . This bacterial expression system offers several advantages including high yield, cost-effectiveness, and well-established protocols. The recombinant protein is typically expressed with an N-terminal His tag to facilitate purification via affinity chromatography. When working with E. coli expression systems, researchers should optimize codon usage for plant mitochondrial genes and consider the hydrophobic nature of ATP9 which may affect solubility. Expression conditions including temperature, induction timing, and media composition should be optimized to maximize yield while maintaining protein functionality. Following expression, purification typically involves immobilized metal affinity chromatography (IMAC) followed by additional chromatography steps if higher purity is required.

How does the RNA-binding capability of ATP synthase subunits impact mitochondrial function in Zea mays?

Recent studies have revealed that several ATP synthase subunits function as RNA-binding proteins, which may have significant implications for understanding ATP9 function in Zea mays. Though research specifically on ATP9 RNA interactions is limited, studies on related subunits (ATP5A1, ATP5B, ATP5C1) have demonstrated that RNA binding plays a crucial role in mitochondrial protein import . For ATP9 research, investigators should consider implementing techniques such as RNA interactome capture experiments, fluorescent electrophoretic mobility shift assays (EMSA), and RNA immunoprecipitation followed by qPCR (RIP-qPCR) to identify potential RNA-ATP9 interactions.

The functional significance of these interactions may include roles in:

• Facilitating proper mitochondrial localization of ATP synthase components

• Coordinating assembly of the ATP synthase complex

• Regulating translation of mitochondrial-encoded subunits

• Maintaining mitochondrial membrane potential through organized proton flow

Researchers investigating this aspect should design experiments that can distinguish between direct RNA binding and secondary interactions mediated through other complex V subunits.

What approaches should be used for investigating ATP9 assembly into the ATP synthase complex in Zea mays?

Investigating ATP9 assembly into the ATP synthase complex requires a multi-faceted approach focusing on the sequential assembly process and interacting partners. Based on current understanding of ATP synthase assembly, researchers should consider the following methodological approaches:

• BN-PAGE Analysis: Blue Native Polyacrylamide Gel Electrophoresis can be employed to visualize different assembly intermediates. Remember that the ~450 kDa complex containing the c-ring (which includes ATP9) is considered a breakdown product of the complete 550 kDa complex rather than an assembly intermediate under BN-PAGE conditions .

• Pulse-Chase Experiments: These can track the incorporation of newly synthesized ATP9 into the c-ring and subsequently into the full complex.

• Crosslinking Studies: Chemical crosslinking followed by mass spectrometry can identify binding partners and proximity relationships during assembly.

• Fluorescence Microscopy: Using fluorescently tagged ATP9 to monitor its localization and incorporation into the complex in vivo.

• Mutational Analysis: Strategic mutations in ATP9 can identify regions critical for assembly and function.

The assembly process likely follows the pattern established in yeast and mammalian systems, where the c-ring forms first, followed by binding of the F1 domain, then the stator arm, and finally subunits a and A6L . Researchers should focus on whether plant-specific factors influence this process in Zea mays.

What is the relationship between nuclear and mitochondrial encoded ATP synthase components in Zea mays?

The relationship between nuclear and mitochondrially encoded ATP synthase components involves complex coordination mechanisms that ensure balanced expression and proper complex assembly. In Zea mays, ATP9 is encoded by the mitochondrial genome, while most other ATP synthase subunits are nuclear-encoded . This dual genetic origin necessitates precise coordination between nuclear and mitochondrial gene expression.

Research approaches to investigate this relationship should include:

• Comparative Genomics: Analysis of both the maize mitochondrial genome (which contains approximately 569,630 bp with 58 identified genes) and nuclear genome for ATP synthase components .

• Translational Regulation Studies: In yeast, expression of mitochondrially encoded subunits is translationally regulated by nuclear-encoded F1 components . Similar mechanisms may exist in Zea mays and can be investigated through polysome profiling and translation inhibition experiments.

• Import Pathway Analysis: Nuclear-encoded subunits must be imported into mitochondria, and this process may be coordinated with the expression of mitochondrially encoded subunits. Import assays using isolated mitochondria can elucidate these pathways.

• Retrograde Signaling Investigation: Disruptions in mitochondrial gene expression or ATP synthase assembly likely trigger retrograde signals to the nucleus, altering expression of nuclear-encoded components.

Interestingly, approximately 155,000 bp of maize mitochondrial sequence has been found within the nuclear genome , suggesting potential evolutionary transfer of genetic material and possible mechanisms for coordinating expression.

What are the optimal conditions for storage and reconstitution of recombinant Zea mays ATP9 protein?

The optimal storage and reconstitution conditions for recombinant Zea mays ATP9 are critical for maintaining protein stability and functionality in research applications. Based on established protocols, the following methodology is recommended:

Storage Conditions:

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

• For working samples, store aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they significantly degrade protein quality

• For long-term storage, aliquot the protein and maintain at -80°C

Reconstitution Protocol:

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

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

• Add 5-50% glycerol (final concentration) as a cryoprotectant, with 50% being the recommended standard concentration

• Prepare small working aliquots to minimize freeze-thaw cycles

• Use Tris/PBS-based buffer with 6% Trehalose, pH 8.0 for dilution if necessary, matching the protein's storage buffer

Researchers should validate protein activity after reconstitution using appropriate functional assays specific to ATP synthase activity. For particularly sensitive experiments, optimization of these conditions may be necessary based on specific research requirements.

What analytical methods are most effective for assessing the purity and functionality of recombinant ATP9?

Multiple analytical approaches should be employed to comprehensively assess both the purity and functionality of recombinant Zea mays ATP9:

Purity Assessment:

• SDS-PAGE: Standard method confirming greater than 90% purity, as typically determined for commercial preparations . Use gradient gels (12-15%) for better resolution of this small protein (74 amino acids).

• Western Blotting: Employing anti-His antibodies for tagged protein or specific anti-ATP9 antibodies.

• Mass Spectrometry: For precise molecular weight determination and identification of potential post-translational modifications or truncations.

• Size Exclusion Chromatography: To assess aggregation state and homogeneity.

Functionality Assessment:

• Lipid Binding Assays: Given ATP9's alternative identification as a lipid-binding protein , assess binding to various lipids using:

  • Lipid overlay assays

  • Fluorescence-based binding studies

  • Isothermal titration calorimetry

• Integration into Proteoliposomes: Reconstitution into artificial membrane systems to assess:

  • Proper membrane insertion

  • Oligomerization capability (c-ring formation)

  • Proton conductance measurements

• Complex Formation Analysis:

  • Blue Native PAGE to assess incorporation into ATP synthase complex when combined with other subunits

  • Pull-down assays to verify interactions with other complex V components

• Structural Integrity Validation:

  • Circular dichroism spectroscopy to confirm proper secondary structure

  • Limited proteolysis to verify folding quality

Each method provides complementary information, and researchers should select the combination most appropriate for their specific research applications.

How can researchers effectively study the role of ATP9 in ATP synthase assembly in Zea mays?

Studying ATP9's role in ATP synthase assembly requires strategic experimental approaches that account for its position in the assembly pathway. Based on current understanding of ATP synthase assembly, where the c-ring (containing ATP9) forms early in the process , the following experimental design is recommended:

In vitro Assembly Studies:

• Reconstitution Experiments: Purify individual components of the ATP synthase complex and reconstitute them in a stepwise manner to monitor ATP9 incorporation into the c-ring.

• Site-Directed Mutagenesis: Introduce strategic mutations in ATP9 to identify residues critical for c-ring formation and subsequent assembly steps.

In vivo Assembly Tracking:

• Inducible Expression Systems: Develop maize cell lines with inducible tagged-ATP9 to monitor incorporation into the complex over time.

• Pulse-Chase Experiments: Trace newly synthesized ATP9 as it assembles into the complete complex.

• Assembly Intermediate Analysis: Use gentle solubilization conditions and BN-PAGE to preserve and identify assembly intermediates containing ATP9.

Interaction Network Mapping:

• Crosslinking Mass Spectrometry: Identify proximity relationships between ATP9 and other subunits during assembly.

• Co-immunoprecipitation: Pull down ATP9 and identify interacting partners at different assembly stages.

• Yeast Two-Hybrid or Split-GFP Assays: Verify direct protein-protein interactions with other subunits.

Functional Impact Assessment:

• RNA Interference or CRISPR-Cas9: Reduce ATP9 levels and assess impact on assembly of other components.

• Overexpression Studies: Examine effects of ATP9 overexpression on complex stoichiometry and assembly.

• Respiration Analysis: Measure respiratory capacity and ATP production in cells with altered ATP9 levels.

These approaches should be combined with careful controls and validation experiments to build a comprehensive understanding of ATP9's role in the assembly process.

What experimental approaches can address the potential RNA-binding properties of ATP9 and their functional significance?

While RNA-binding properties have been identified for several ATP synthase subunits , specific research on ATP9's RNA interactions is limited. To investigate this unexplored area, researchers should implement a systematic experimental approach:

RNA-Binding Characterization:

• RNA Interactome Capture: Apply UV crosslinking followed by poly(A) selection to identify if ATP9 associates with mRNAs in vivo.

• Electrophoretic Mobility Shift Assays (EMSA): Test direct binding between recombinant ATP9 and candidate RNAs, including both mitochondrial transcripts and nuclear-encoded RNAs targeted to mitochondria .

• RNA Immunoprecipitation (RIP): Pull down ATP9 and sequence associated RNAs to identify the binding transcriptome.

• Hydrogen-Deuterium Exchange Mass Spectrometry: Map potential RNA-binding domains within the ATP9 protein structure.

Functional Significance Assessment:

• Mutagenesis of Putative RNA-Binding Domains: Create ATP9 variants with altered RNA-binding capacity and assess impacts on:

  • Protein localization

  • Complex V assembly

  • Mitochondrial function

  • Cell viability

• In vitro Translation Assays: Determine if ATP9 affects translation efficiency of specific mitochondrial transcripts.

• Import Assays: Based on findings that RNA promotes mitochondrial import of F1-ATP synthase components , test if ATP9 import is similarly affected by RNA binding.

• Proximity Labeling Approaches: Use BioID or APEX2 fusions with ATP9 to identify proteins in proximity during RNA binding events.

• Live-Cell Imaging: Apply techniques like rnaPLA (RNA proximity ligation assay) to visualize ATP9-RNA interactions in their native cellular context .

This experimental framework would provide the first comprehensive characterization of ATP9's potential role in RNA biology and establish connections to its known functions in ATP synthase assembly and activity.

How does Zea mays ATP9 differ from its homologs in other plant species and model organisms?

Comparative analysis of ATP9 across species reveals important evolutionary patterns and functional conservation. Zea mays ATP9 should be evaluated against homologs from other plants and model organisms across several dimensions:

Sequence Comparison:
The 74-amino acid sequence of Zea mays ATP9 (MLEGAKLIGAGAATIALAGAAVGIGNVFSSLIHSVARNPSLAKQLFGYAILGFALTEAIALFALMMAFLILFVF) shows varying degrees of conservation when compared with homologs. Key comparative features include:

• Sequence Identity with Other Plants: Highest conservation typically occurs with other monocots, particularly in the transmembrane domains that form the c-ring structure.

• Functional Domain Conservation: The hydrophobic regions responsible for membrane insertion and the residues involved in proton translocation show greater conservation than peripheral regions.

• Evolutionary Rate Analysis: ATP9 generally evolves more slowly than nuclear-encoded ATP synthase components due to its critical role in energy production.

Genomic Context Differences:

• In Zea mays, ATP9 is encoded by the mitochondrial genome , but in some species, homologous genes have been transferred to the nuclear genome.

• The organization of genes flanking ATP9 in the mitochondrial genome varies across plant species, reflecting different evolutionary histories of mitochondrial genome rearrangement.

Functional Divergence:

• Proton Conductance Properties: Subtle differences in key residues may affect proton binding and release kinetics.

• Assembly Pathway Variations: While the general assembly process follows similar patterns across species (c-ring formation followed by F1 attachment) , plant-specific factors may influence the efficiency and regulation of this process.

• Protein-Protein Interaction Networks: The specific interactions with assembly factors and other complex V components may differ between species.

This comparative approach not only illuminates evolutionary relationships but also helps identify conserved features essential for function versus species-specific adaptations.

How can knowledge from model systems be applied to understand ATP9 function in Zea mays?

Translating knowledge from model systems to understand ATP9 function in Zea mays requires careful consideration of both conserved mechanisms and species-specific adaptations. The following methodological framework helps researchers leverage model system insights effectively:

Leveraging Yeast Models:
Yeast studies have provided extensive insights into ATP synthase assembly and function that can be applied to Zea mays research:

• The three-module assembly model demonstrated in yeast (c-ring, F1, and Atp6p/Atp8p complex) can guide investigations of ATP9 incorporation into maize ATP synthase.

• Yeast translational regulation mechanisms, where expression of mitochondrially encoded subunits is regulated by F1 components , can inform similar studies in maize.

• Yeast assembly factor homologs should be identified in the maize genome and their interactions with ATP9 characterized.

Mammalian System Applications:

• RNA binding studies of mammalian ATP synthase components suggest similar investigations for maize ATP9, particularly examining if RNA promotes mitochondrial import.

• The detailed structural information available for mammalian ATP synthase can guide structural predictions for maize complex V.

• Respiration assays developed for mammalian cells, such as Seahorse respirometry , can be adapted to assess ATP9 function in maize mitochondria.

Methodological Adaptations Required:

• Plant-specific cellular features (cell walls, plastids, vacuoles) necessitate modifications to protein localization and interaction studies.

• Different codon usage preferences between plants and other model systems require optimization for heterologous expression studies.

• Plant-specific assembly factors and regulatory mechanisms must be identified to fully understand maize ATP9 function.

This translational approach accelerates research by applying established principles while acknowledging the unique aspects of plant systems.

How can multi-omics approaches enhance our understanding of ATP9 function in Zea mays mitochondria?

Multi-omics integration provides a systems-level understanding of ATP9 function that cannot be achieved through any single approach. For comprehensive analysis of ATP9 in Zea mays, researchers should implement the following integrated strategy:

Integrated Multi-omics Framework:

• Genomics + Transcriptomics:

  • Analyze mitochondrial genome variations affecting ATP9 across maize varieties

  • Characterize nuclear genes encoding ATP synthase subunits and assembly factors

  • Quantify ATP9 transcript levels under different physiological conditions

  • Identify potential RNA editing sites in ATP9 transcripts

• Proteomics + Interactomics:

  • Map the complete protein-protein interaction network of ATP9

  • Quantify ATP9 abundance and post-translational modifications

  • Characterize ATP9 assembly intermediates through BN-PAGE coupled with mass spectrometry

  • Identify potential RNA-binding capabilities through interactome capture

• Metabolomics + Fluxomics:

  • Measure ATP/ADP ratios and energy charge in relation to ATP9 function

  • Track metabolic flux changes in response to ATP9 alterations

  • Correlate ATP9 activity with central carbon metabolism pathways

• Structural Biology Integration:

  • Combine cryo-EM structural data with crosslinking mass spectrometry

  • Map ATP9 positioning within the c-ring and complete ATP synthase complex

  • Identify conformational changes during rotary catalysis

Data Integration Methodology:

• Apply machine learning algorithms to identify patterns across omics datasets

• Develop network models that link ATP9 to broader cellular functions

• Use Bayesian approaches to infer causal relationships between changes in ATP9 and downstream effects

This multi-omics strategy provides a comprehensive view of ATP9 function, from its genetic regulation to its impact on cellular metabolism, enabling the identification of both direct mechanisms and systems-level effects.

What computational approaches are most effective for analyzing ATP9 structure-function relationships?

Computational analysis of ATP9 structure-function relationships requires a multi-scale modeling approach that integrates various computational techniques. The following methodological framework is recommended for Zea mays ATP9 research:

Molecular-Level Analysis:

• Homology Modeling and Threading:

  • Generate 3D structural models of Zea mays ATP9 based on solved structures from related organisms

  • Validate models through energy minimization and Ramachandran plot analysis

  • Specific attention should be paid to transmembrane helix packing, as ATP9 forms part of the c-ring

• Molecular Dynamics Simulations:

  • Simulate ATP9 behavior within a lipid bilayer environment

  • Analyze conformational changes during proton binding/release

  • Investigate c-ring rotation dynamics with ATP9 as a component

  • Apply enhanced sampling techniques to capture rare conformational transitions

• Protein-Protein Docking:

  • Model interactions between ATP9 and other ATP synthase subunits

  • Characterize binding interfaces and key interaction residues

  • Evaluate the energetics of these interactions

System-Level Integration:

These computational approaches provide mechanistic insights that are difficult to obtain experimentally, particularly for membrane proteins like ATP9, and generate testable hypotheses for experimental validation.

What are common challenges in working with recombinant ATP9 and how can they be addressed?

Researchers working with recombinant Zea mays ATP9 encounter several technical challenges due to its hydrophobic nature and small size. The following methodological solutions address the most common issues:

Challenge 1: Low Expression Yield

• Problem: ATP9's hydrophobicity often leads to poor expression in standard E. coli systems.

• Solutions:

  • Use specialized E. coli strains designed for membrane protein expression (C41(DE3), C43(DE3), or Lemo21(DE3))

  • Lower induction temperature (16-18°C) and extend expression time

  • Consider fusion tags beyond His, such as SUMO or MBP, to increase solubility

  • Optimize codon usage for E. coli while preserving critical codons

  • Test cell-free expression systems as alternatives

Challenge 2: Protein Aggregation

• Problem: ATP9 tends to aggregate during purification due to hydrophobic interactions.

• Solutions:

  • Include appropriate detergents (DDM, LDAO, or Fos-choline) in all buffers

  • Apply gradient purification with increasing detergent concentrations

  • Consider purification under denaturing conditions followed by controlled refolding

  • Use size exclusion chromatography as a final purification step to remove aggregates

  • Maintain glycerol (5-10%) in purification buffers to enhance stability

Challenge 3: Functional Assessment Difficulties

• Problem: Verifying ATP9 functionality is challenging outside the complete ATP synthase complex.

• Solutions:

  • Develop reconstitution assays with other ATP synthase components

  • Use lipid binding assays as proxy measures for proper folding

  • Apply circular dichroism to confirm secondary structure

  • Develop mini-complex assembly assays focusing on c-ring formation

  • Utilize proton flux assays in proteoliposomes containing reconstituted ATP9

Challenge 4: Stability Issues

• Problem: Purified ATP9 may rapidly lose activity during storage.

• Solutions:

  • Store as lyophilized powder for long-term stability

  • Add 6% trehalose to storage buffers as a stabilizing agent

  • Avoid repeated freeze-thaw cycles

  • Store small working aliquots at 4°C for up to one week

  • For long-term storage, maintain at -80°C with 50% glycerol

These methodological solutions derive from established protocols and can be adapted to specific research requirements, ensuring successful work with this challenging but important protein.

How can researchers troubleshoot inconsistencies in ATP synthase assembly experiments involving ATP9?

Troubleshooting inconsistencies in ATP synthase assembly experiments involving ATP9 requires systematic analysis of multiple variables that can affect experimental outcomes. The following methodological framework addresses common sources of variability and provides solutions:

Experimental Design Factors:

• Sample Preparation Variability

  • Problem: Inconsistent solubilization of mitochondrial membranes leads to variable ATP9 recovery.

  • Solution: Standardize detergent type, concentration, and solubilization time; consider digitonin for gentler extraction that preserves supercomplexes.

• Assembly Intermediate Stability

  • Problem: The c-ring containing ATP9 can dissociate during BN-PAGE, creating artifacts.

  • Solution: Remember that the ~450 kDa complex containing the c-ring is considered a breakdown product rather than an assembly intermediate under standard BN-PAGE conditions ; use milder conditions and shorter run times.

• Detection Method Sensitivity

  • Problem: Small size of ATP9 (74 amino acids) makes detection challenging.

  • Solution: Employ epitope tagging strategies compatible with assembly; consider using antibodies against the c-ring rather than individual ATP9 subunits.

Biological Sources of Variation:

• Developmental Stage Effects

  • Problem: ATP synthase assembly pathways may vary with plant developmental stage.

  • Solution: Strictly control for developmental stage; conduct time-course experiments to map stage-specific assembly patterns.

• Environmental Condition Impacts

  • Problem: Growth conditions affect mitochondrial biogenesis and ATP synthase assembly.

  • Solution: Standardize growth conditions (light, temperature, nutrients); consider controlled stress experiments to understand condition-dependent assembly.

• Genetic Background Influence

  • Problem: Different maize varieties may exhibit subtly different assembly patterns.

  • Solution: Use isogenic lines; compare results across multiple genotypes to identify conserved versus variable assembly steps.

Analytical Troubleshooting Approach:

• Control Experiment Matrix

  • Develop a systematic control matrix varying one parameter at a time

  • Include positive controls with known assembly patterns

  • Use biochemical complementation to verify specific assembly step requirements

• Multi-method Validation

  • Confirm BN-PAGE results with orthogonal methods such as sucrose gradient fractionation

  • Apply both Western blotting and mass spectrometry for detection

  • Validate in vitro findings with in vivo approaches

This systematic troubleshooting framework allows researchers to identify and address sources of inconsistency in ATP9 assembly experiments, leading to more reproducible and reliable results.

What are emerging research areas for ATP9 that could lead to breakthroughs in understanding plant mitochondrial function?

Several cutting-edge research directions for Zea mays ATP9 hold promise for transformative insights into plant mitochondrial function and energy metabolism:

ATP9 in Mitochondrial RNA Biology

• Building on discoveries that ATP synthase subunits can bind RNA , investigate whether ATP9 has similar capabilities

• Explore if ATP9-RNA interactions regulate assembly, localization, or activity of ATP synthase

• Develop methodologies to identify the complete RNA interactome of ATP9 in plant mitochondria

ATP9 in Supramolecular Organization

• Investigate ATP9's role in ATP synthase dimerization and cristae formation

• Explore potential interactions between ATP synthase and respiratory chain supercomplexes

• Apply advanced cryo-electron tomography to visualize the spatial arrangement of ATP synthase in plant mitochondrial membranes

Post-translational Modifications of ATP9

• Map the complete PTM landscape of ATP9 under different conditions

• Determine how PTMs regulate ATP9 function, particularly in response to metabolic status

• Develop targeted methods to identify low-abundance but functionally significant modifications

ATP9 in Plant Stress Responses

• Characterize ATP9 expression, assembly, and function under various abiotic stresses

• Investigate if ATP9 variants contribute to stress tolerance in different maize varieties

• Develop ATP9 modifications that could enhance energy efficiency under stress conditions

ATP9 in Mitochondrial-Nuclear Communication

• Explore how disruptions in ATP9 trigger retrograde signaling to the nucleus

• Investigate coordination between nuclear-encoded assembly factors and mitochondrial ATP9

• Determine if nuclear-encoded ATP9 homologs (resulting from mitochondrial DNA transfer to the nucleus ) have acquired new functions

ATP9 in Hybrid Vigor Mechanisms

• Examine mitochondrial-nuclear interactions involving ATP9 in hybrid maize lines

• Investigate potential roles of ATP9 variants in cytoplasmic male sterility and fertility restoration

• Develop molecular markers based on ATP9 sequence or expression patterns for hybrid breeding programs

These emerging research areas combine fundamental biological questions with potential applications in crop improvement and stress tolerance.

What technological advances would enable better characterization of ATP9 structure and function in plant mitochondria?

Technological innovations across multiple disciplines would significantly advance our understanding of ATP9 structure and function in plant mitochondria. The following methodological advances represent high-priority developments:

Advanced Imaging Technologies:

• Cryo-Electron Tomography Adaptations for Plant Samples

  • Development of sample preparation methods that overcome plant-specific challenges

  • In situ visualization of ATP9 within intact mitochondrial membranes

  • Correlation with functional states to capture dynamics of ATP synthase rotation

• Super-Resolution Microscopy for Mitochondrial Proteins

  • Techniques adapted for small proteins like ATP9 with spatial resolution below 10 nm

  • Multiplexed imaging to simultaneously visualize ATP9 with other ATP synthase components

  • Live-cell imaging capabilities to track assembly dynamics in real-time

Structural Biology Innovations:

• Nuclear Magnetic Resonance Methods for Membrane Proteins

  • Development of improved techniques for small, hydrophobic proteins like ATP9

  • Methodologies to study dynamics and conformational changes during proton transport

  • Integration with computational methods for complete structural models

• Mass Spectrometry Advances

  • Ion mobility MS techniques to maintain native-like structure of ATP9-containing complexes

  • Enhanced detection of post-translational modifications on small membrane proteins

  • Development of quantitative proteomic approaches with single-residue resolution

Functional Analysis Technologies:

• Single-Molecule Biophysical Techniques

  • Methods to measure proton transport through individual ATP9 channels

  • Single-molecule FRET to detect conformational changes during function

  • Force microscopy to measure rotational torque in reconstructed ATP synthase systems

• Genome Engineering Tools for Plant Mitochondria

  • Development of mitochondrial genome editing capabilities for plants

  • Site-specific mutagenesis of ATP9 in its native context

  • Inducible expression systems for mitochondrially encoded genes

• Microfluidic Systems for Mitochondrial Assessment

  • Platforms for high-throughput analysis of ATP9 variants

  • Real-time monitoring of ATP synthesis in isolated plant mitochondria

  • Integration with imaging for structure-function correlations