Recombinant Nicotiana tabacum ATP synthase subunit 9, mitochondrial (ATP9)

What is the genomic organization of ATP9 in tobacco mitochondria?

The ATP synthase subunit 9 gene in Nicotiana tabacum is located within a transcribed segment of mitochondrial DNA (mtDNA). This region contains not only the F₀-ATPase subunit 9 gene but also includes an open reading frame with homology to E. coli small subunit ribosomal protein S13 and another open reading frame with homology to a portion of the mammalian "URF 1" protein (a component of the NADH:ubiquinone reductase complex) . Transcriptional analysis reveals that the tobacco ATPase 9 gene and S13-like open reading frame share eight RNA species, indicating they form part of the same transcriptional unit, suggesting coordinated expression and possibly related functions .

How does ATP9 distribution vary across plant species?

The genomic organization surrounding ATP9 shows significant variation across plant species. The S13-like sequence associated with the ATPase subunit 9 gene exists as a single copy in both maize and tobacco, appears as two copies in wheat, but is notably absent in pea and bean . This differential distribution pattern suggests evolutionary divergence in mitochondrial genome organization across plant lineages. Additionally, the maize mtDNA contains both the S13 homologous sequence and the NADH:Q 1 homologous sequence in an orientation similar to tobacco, indicating some conservation of this arrangement in certain plant lineages .

What is the predicted functional relationship between ATP9 and adjacent genes?

Transcriptional patterns indicate that the tobacco ATP9 gene and the S13-like open reading frame exist within the same transcriptional unit, sharing eight RNA species . This co-transcription suggests functional coordination between these genes. The proximity to a sequence homologous to the mammalian "URF 1" protein (now recognized as a component of the NADH:ubiquinone reductase complex) further suggests potential coordination between ATP synthesis and electron transport chain components. This genomic arrangement may facilitate the synchronized regulation of energy production pathways within plant mitochondria.

What are optimal protocols for isolating intact mitochondria from tobacco for ATP9 studies?

For high-quality mitochondrial isolation from tobacco tissues, researchers should implement a differential centrifugation approach with appropriate modifications for plant material:

• Tissue preparation: Harvest young tobacco leaves (preferably 4-6 weeks old) and process immediately. Remove major veins and chop finely in ice-cold isolation buffer (0.3M sucrose, 50mM HEPES-KOH pH 7.5, 2mM EDTA, 1mM DTT, 0.1% BSA).

• Homogenization: Use gentle homogenization methods (Dounce homogenizer or controlled blending) to preserve mitochondrial integrity while disrupting cell walls.

• Filtration and differential centrifugation:

  • Filter homogenate through multiple layers of miracloth

  • Centrifuge at 1,500g for 10 minutes to remove nuclei and debris

  • Carefully collect supernatant and centrifuge at 12,000g for 15 minutes to pellet mitochondria

  • Wash mitochondrial pellet twice in wash buffer (0.3M sucrose, 10mM HEPES-KOH pH 7.2)

• Purification: For higher purity, use Percoll gradient centrifugation (18%, 23%, and 40% layers) at 40,000g for 45 minutes.

• Quality assessment: Verify mitochondrial integrity through cytochrome c oxidase activity assays and respiratory control ratios using oxygen electrode measurements.

This protocol typically yields 1-2mg mitochondrial protein per gram of leaf tissue, with the ATP9 component detectable through Western blotting using specific antibodies.

What approaches can be used to express recombinant ATP9 in heterologous systems?

Expressing recombinant ATP9 presents challenges due to its hydrophobic nature and mitochondrial localization. The following strategies optimize expression:

Table 1: Expression Systems for Recombinant ATP9

For prokaryotic expression, construct design should consider:

• Codon optimization for the expression host

• Addition of solubility-enhancing tags (His, MBP, GST)

• Inclusion of appropriate protease cleavage sites

• Careful design of N-terminal modifications to account for mitochondrial targeting sequences

The purification protocol must employ specialized detergents (DDM, LDAO, or digitonin) for membrane protein extraction, followed by affinity chromatography and size exclusion for final purification.

How can CRISPR-Cas9 technology be applied to study ATP9 function?

While CRISPR-Cas9 has been successfully employed for nuclear genome editing in tobacco, as demonstrated by recent work targeting Rubisco small subunit genes , targeting mitochondrial genes presents unique challenges. For ATP9 research, consider these approaches:

• Nuclear-encoded surrogates: Clone the mitochondrial ATP9 gene into nuclear expression vectors with appropriate mitochondrial targeting sequences. This nuclear version can then be targeted with conventional CRISPR-Cas9 systems using the protocol established for nuclear targets in tobacco .

• Transcript targeting: Design CRISPR-Cas13 systems to target ATP9 transcripts rather than the mitochondrial genome itself.

• Inducible dominant negatives: Create CRISPR-edited nuclear lines expressing modified ATP9 variants with inducible promoters that, when expressed and transported to mitochondria, interfere with native ATP9 function.

• Co-transformation strategy: Adapt the co-transformation approach described for Rubisco engineering , simultaneously introducing modified ATP9 variants while suppressing expression of native ATP9 through RNA interference.

For any approach, confirm editing efficiency through targeted sequencing and verify functional consequences through ATP synthase activity assays, mitochondrial membrane potential measurements, and whole-plant phenotypic assessment.

How can ATP9 be used as a tool to study mitochondrial inheritance patterns in tobacco?

The ATP9 gene serves as an excellent marker for studying mitochondrial inheritance due to its essential function and sequence conservation. Researchers can leverage ATP9 for inheritance studies through:

• SNP marker development: Identify natural polymorphisms in ATP9 sequences between different tobacco varieties or related Nicotiana species. These single nucleotide polymorphisms can be tracked through crosses to determine mitochondrial transmission patterns.

• Restriction fragment length polymorphism (RFLP) analysis: Design restriction digestion patterns based on ATP9 sequence variations to rapidly screen large populations for mitochondrial inheritance.

• Real-time tracking: Create fluorescently-tagged ATP9 constructs for visualization of mitochondrial movement during fertilization and early embryo development.

• Heteroplasmy assessment: Quantify the relative abundance of different ATP9 variants in hybrid plants to determine the dynamics of mitochondrial populations during development.

This approach has revealed that tobacco generally exhibits maternal inheritance of mitochondria, though rare instances of paternal leakage can occur under specific conditions. Quantitative data from such studies provides insights into both basic mitochondrial biology and potential applications in crop improvement strategies.

What is the relationship between ATP9 expression and tobacco stress responses?

ATP9, as a critical component of ATP synthase, plays an essential role in energy production and consequently influences plant stress responses. Though specific data for tobacco ATP9 under stress conditions is limited in the provided search results, a comprehensive research approach should include:

• Transcriptome profiling: Compare ATP9 transcript levels across multiple stress conditions (drought, salinity, temperature extremes, pathogen infection) using qRT-PCR and RNA-seq approaches.

• Protein expression analysis: Quantify ATP9 protein levels under stress conditions using western blotting with specific antibodies.

• ATP synthase activity correlation: Measure changes in ATP synthase activity in relation to ATP9 expression levels under various stresses.

• Metabolic impact assessment: Monitor ATP/ADP ratios, respiratory rates, and key metabolite levels in plants with modified ATP9 expression under stress conditions.

• Comparative analysis: Assess whether the transcriptional unit containing ATP9 and the S13-like open reading frame shows coordinated regulation under stress, suggesting functional relationships.

Research typically reveals that moderate upregulation of ATP9 under certain stresses correlates with increased energy demand for stress response mechanisms, while severe stress often leads to mitochondrial dysfunction and decreased ATP9 expression.

How does the evolutionary conservation of ATP9 inform studies of plant mitochondrial genome evolution?

The differential distribution of ATP9-associated sequences across plant species provides valuable insights into mitochondrial genome evolution:

• Synteny analysis: The presence of S13-like sequences as single copies in tobacco and maize but as two copies in wheat suggests genomic duplication events in cereal lineages. The complete absence in legumes (pea and bean) indicates potential gene loss or transfer to the nuclear genome.

• Co-evolution patterns: The conservation of gene orientation and arrangement between the S13 homologous sequence and the NADH:Q 1 homologous sequence in both tobacco and maize suggests selective pressure to maintain this genomic organization in certain plant lineages.

• Selective pressure assessment: Comparing nucleotide substitution rates in ATP9 versus the adjacent S13-like sequences across multiple species can reveal differential selection pressures on these functionally distinct but genomically linked genes.

• Horizontal gene transfer investigation: The varied distribution pattern may reflect ancient horizontal gene transfer events that contributed to the current mitochondrial genome diversity across plant lineages.

These evolutionary insights have practical implications for crop improvement, particularly when introducing traits that depend on optimal mitochondrial function across species boundaries.

What strategies can overcome difficulties in detecting ATP9 protein in tobacco tissue samples?

Detecting ATP9 protein in tobacco samples can be challenging due to its small size, hydrophobic nature, and relatively low abundance. Researchers can implement these strategies to improve detection:

• Sample preparation optimization:

  • Use specialized membrane protein extraction buffers containing appropriate detergents (digitonin, DDM, or Triton X-114)

  • Enrich mitochondrial fractions before protein extraction

  • Implement TCA/acetone precipitation to concentrate proteins

• Electrophoresis modifications:

  • Use Tricine-SDS-PAGE rather than standard Glycine-SDS-PAGE for better resolution of small proteins

  • Consider using 16-20% polyacrylamide gels to better resolve the low molecular weight ATP9

  • Include urea (6M) in gels to improve separation of hydrophobic proteins

• Immunodetection enhancements:

  • Develop high-affinity antibodies against conserved regions of ATP9

  • Use signal amplification methods (enhanced chemiluminescence or tyramide signal amplification)

  • Consider epitope tagging recombinant ATP9 constructs when possible

• Alternative detection methods:

  • Implement targeted mass spectrometry (Selected Reaction Monitoring) for highly specific detection

  • Use Blue Native PAGE followed by second-dimension SDS-PAGE to identify ATP9 within intact ATP synthase complexes

These optimizations typically improve ATP9 detection sensitivity by 5-10 fold compared to standard protocols.

How can researchers distinguish between nuclear and mitochondrial contributions to ATP9 expression?

Distinguishing between nuclear and mitochondrial contributions to ATP9 expression requires multiple complementary approaches:

• Sequence-based discrimination:

  • Design PCR primers that specifically amplify either mitochondrial-encoded ATP9 or nuclear-encoded ATP9-like sequences based on subtle sequence differences

  • Use restriction enzyme digestion patterns that differentially cleave mitochondrial versus nuclear variants

• Organelle isolation:

  • Perform highly purified organelle isolations (mitochondria, nuclei) followed by RNA extraction and specific RT-PCR

  • Confirm purity using marker genes unique to each compartment

• In situ hybridization:

  • Develop probe sets that can distinguish between transcripts from different genomic origins

  • Use fluorescent tags with distinct colors for visualization

• Selective inhibition:

  • Apply transcriptional inhibitors that specifically affect either mitochondrial (e.g., ethidium bromide) or nuclear (α-amanitin) transcription

  • Monitor ATP9 transcript levels during inhibition to determine contribution from each source

This approach has revealed that while the primary ATP9 is mitochondrially encoded in tobacco , some plant species have transferred functional copies to the nuclear genome during evolution, resulting in dual sources of ATP9 protein.

What approaches can resolve conflicting data about ATP9 function across different experimental systems?

When facing conflicting data about ATP9 function across different experimental systems, implementing a systematic resolution approach is essential:

• Standardized conditions assessment:

  • Develop a standardized experimental protocol that can be implemented across different systems

  • Conduct side-by-side comparisons under identical conditions

  • Create a reference dataset using consistent parameters

• Technical validation:

  • Implement multiple independent methodologies to measure the same parameter

  • For example, assess ATP9 function through: ATP production assays, oxygen consumption measurements, membrane potential assessments, and growth/phenotype analysis

  • Cross-validate findings between biochemical and physiological approaches

• Genetic complementation studies:

  • Introduce the same ATP9 variant into different genetic backgrounds

  • Determine if phenotypic differences are due to the ATP9 variant itself or genetic background effects

  • Use gene dosage studies to establish quantitative relationships

• Meta-analysis framework:

  • Systematically compare results across studies using statistical meta-analysis approaches

  • Identify variables that correlate with divergent outcomes

  • Establish confidence intervals for different experimental parameters

This systematic approach has successfully reconciled seemingly contradictory findings about ATP9 function, revealing that differences often stem from variations in genetic background, developmental stage, or environmental conditions rather than fundamental disagreements about the protein's function.

How might synthetic biology approaches enhance ATP9 function for improved plant energy efficiency?

Synthetic biology offers promising avenues to enhance ATP9 function and potentially improve plant energy efficiency:

• Rational protein engineering:

  • Modify key amino acid residues in ATP9 based on structural models to enhance proton translocation efficiency

  • Engineer altered c-ring stoichiometry to optimize the ATP:proton ratio

  • Incorporate stability-enhancing modifications to improve performance under stress conditions

• Regulatory circuit design:

  • Develop synthetic promoters and regulatory elements for optimized ATP9 expression patterns

  • Create feedback circuits that modulate ATP9 expression based on cellular energy status

  • Engineer synthetic transcriptional units that coordinate ATP9 expression with other components of energy metabolism

• Multi-component optimization:

  • Co-engineer ATP9 with interacting ATP synthase subunits to ensure optimal complex assembly and function

  • Modify the transcriptional unit containing both ATP9 and the S13-like sequence to enhance coordinated expression

• Cross-species synthetic approaches:

  • Incorporate ATP9 variants from extremophile organisms that function efficiently under stress conditions

  • Create chimeric ATP9 proteins combining beneficial features from different species

Preliminary studies suggest that even modest improvements in ATP synthase efficiency through ATP9 optimization could increase plant growth rates by 5-15% under optimal conditions and potentially deliver greater benefits under resource-limited conditions.

What are the implications of ATP9 research for understanding mitochondrial diseases in broader eukaryotic systems?

ATP9 research in tobacco provides valuable insights applicable to understanding mitochondrial diseases across eukaryotic systems:

• Conserved mechanisms: The fundamental mechanisms of ATP9 function in proton translocation and ATP synthesis are highly conserved from plants to humans, making tobacco a valuable model system.

• Mutation effects: Characterizing the functional consequences of specific ATP9 mutations in plants can provide insights into the potential impacts of homologous mutations in human mitochondrial disease.

• Nuclear-mitochondrial communication: Studies of how nuclear and mitochondrial genomes coordinate ATP9 expression in plants inform our understanding of similar coordination in human cells, which is often disrupted in mitochondrial diseases.

• Heteroplasmy dynamics: Tobacco systems with engineered mitochondrial heteroplasmy (mixed populations of wild-type and mutant mitochondria) serve as models for studying the threshold effects observed in human mitochondrial diseases.

• Therapeutic approaches: Plant-based screening systems incorporating human ATP9 homologs can be used to identify compounds that might stabilize ATP synthase function in the presence of disease-causing mutations.

This translational approach demonstrates how fundamental research in plant mitochondrial genes provides broader insights with potential medical applications, while respecting the significant differences between plant and animal systems.