Recombinant Marchantia polymorpha ATP synthase subunit 9, mitochondrial (ATP9)

What is ATP synthase subunit 9 and what role does it play in mitochondrial function?

ATP synthase subunit 9 (ATP9) is a crucial component of the F0 portion of the mitochondrial ATP synthase complex. This protein forms an oligomeric ring structure embedded in the inner mitochondrial membrane and functions as a proton channel. The rotation of this c-ring (as it's also known) during proton translocation drives the conformational changes in the F1 portion of ATP synthase that catalyze ATP synthesis.

In Marchantia polymorpha, as in other eukaryotes, ATP9 likely contributes to cellular energy metabolism through oxidative phosphorylation. The functional importance of this protein is reflected in its high degree of evolutionary conservation. Studies in other organisms have shown that ATP9 homologs typically possess between 40-60% identity across diverse species, suggesting similar conservation patterns in Marchantia .

Why is Marchantia polymorpha used as a model organism for studying mitochondrial proteins?

Marchantia polymorpha has emerged as a valuable model organism for studying fundamental biological processes for several compelling reasons. First, as a liverwort, it occupies a basal position in land plant evolution, providing insights into ancestral plant characteristics. Second, Marchantia possesses a relatively simple genome with minimal gene redundancy, making it ideal for functional studies of conserved proteins like ATP9.

The development of efficient transformation protocols for Marchantia, such as the chopped-thallus transformation method, has significantly enhanced its utility for genetic studies . This method allows for scalable generation of transformants from minimal starting material, facilitating large-scale applications including mutant screening. Additionally, Marchantia's haploid gametophyte-dominant life cycle simplifies genetic analysis, as mutations immediately manifest phenotypically without being masked by dominant alleles.

How conserved is ATP synthase subunit 9 across different species compared to Marchantia polymorpha?

ATP synthase subunit 9 is among the most highly conserved mitochondrial proteins across eukaryotic species, reflecting its fundamental role in energy metabolism. Comparative analyses indicate that ATP9 proteins typically show significant sequence conservation, with identity percentages ranging from 40% to 60% between phylogenetically distant organisms .

Interestingly, while ATP9 is encoded by the mitochondrial genome in many species, in some lineages including several plants, the gene has been transferred to the nuclear genome. In the case of Trypanosoma brucei, for example, the ATP9 gene has been identified in the nuclear genome rather than in the kinetoplast DNA (kDNA) . In Marchantia polymorpha, genomic analyses suggest a similar nuclear localization of the ATP9 gene, necessitating mechanisms for post-translational import of the protein into mitochondria.

The conservation table below illustrates typical sequence identity values between ATP9 homologs from different organisms:

What is known about the gene encoding ATP synthase subunit 9 in Marchantia polymorpha?

The gene encoding ATP synthase subunit 9 in Marchantia polymorpha is predicted to be nuclear-encoded based on comparative genomics with other plant species. Like other nuclear-encoded mitochondrial proteins, the ATP9 precursor likely contains an N-terminal mitochondrial targeting sequence for import into the organelle.

Based on patterns observed in other species such as Trypanosoma brucei, the Marchantia ATP9 gene likely exhibits developmental regulation throughout the organism's life cycle . Transcript analysis methods similar to those used for other mitochondrial proteins would be expected to show differential expression patterns between developmental stages, possibly with higher expression in metabolically active tissues.

The predicted structure of the M. polymorpha ATP9 gene would include regions encoding:

• An N-terminal mitochondrial targeting sequence

• The mature protein domain containing transmembrane helices

• Conserved residues critical for proton translocation

What are the effective methods for isolating mitochondria from Marchantia polymorpha tissues?

Isolating intact mitochondria from Marchantia polymorpha tissues requires protocols optimized for plant tissues with suitable modifications for this bryophyte. An effective method involves:

• Tissue preparation: Harvest young thalli (preferably 14-21 days old) to maximize mitochondrial yield. Avoid using older tissues, which contain more secondary metabolites that can interfere with isolation.

• Mechanical disruption: Grind tissues in isolation buffer (0.3M sucrose, 50mM HEPES-KOH pH 7.5, 1mM EDTA, 0.1% BSA, 1mM DTT, and protease inhibitor cocktail) using a chilled mortar and pestle under cold conditions (4°C).

• Differential centrifugation:

  • Initial centrifugation at 2,500g for 5 minutes to remove cellular debris

  • Collection of supernatant and centrifugation at 10,000g for 15 minutes to pellet mitochondria

  • Washing of mitochondrial pellet in isolation buffer without BSA

• Percoll gradient purification: For higher purity, layer the crude mitochondrial fraction on a discontinuous Percoll gradient (18%, 25%, and 40%) and centrifuge at 40,000g for 45 minutes.

This protocol yields mitochondrial fractions suitable for functional studies, protein analysis, and ATP synthase characterization. Inner mitochondrial membrane vesicles can be isolated from the purified mitochondria for specific studies of membrane-bound proteins like ATP9, similar to techniques applied for T. brucei .

How can recombinant ATP synthase subunit 9 from Marchantia polymorpha be expressed and purified?

Expressing and purifying recombinant ATP synthase subunit 9 from Marchantia polymorpha presents challenges due to its highly hydrophobic nature and membrane integration. A methodological approach involves:

• Expression system selection:

  • E. coli-based expression using specialized strains (C41(DE3) or C43(DE3)) designed for membrane protein expression

  • Use of fusion tags (e.g., MBP or SUMO) to improve solubility and prevent aggregation

• Construct design:

  • Clone the mature ATP9 coding sequence (without mitochondrial targeting sequence) using PCR amplification from M. polymorpha cDNA

  • Insert into expression vectors with N-terminal tags and appropriate protease cleavage sites

  • Include a C-terminal polyhistidine tag for purification

• Expression optimization:

  • Induce at lower temperatures (16-20°C) to prevent inclusion body formation

  • Use lower IPTG concentrations (0.1-0.5 mM)

  • Extend expression time to 16-20 hours

• Solubilization and purification:

  • Solubilize membrane fractions using detergents (DDM, LDAO, or C12E8)

  • Purify using nickel affinity chromatography

  • Perform size exclusion chromatography as a final purification step

• Verification:

  • Confirm identity using mass spectrometry

  • Verify proper folding using circular dichroism spectroscopy

  • Compare with native protein isolated from inner mitochondrial membrane vesicles using co-migration assays, similar to approaches used for T. brucei ATP9

What transformation protocols work best for introducing modified ATP9 genes into Marchantia polymorpha?

The recently developed chopped-thallus transformation method offers significant advantages for introducing modified ATP9 genes into Marchantia polymorpha . This protocol is particularly suitable for large-scale transformations and offers the following methodological benefits:

• Preparation of plant material:

  • Chop entire thalli into small fragments (~1-2 mm²) using a razor blade

  • Regenerate for 4-5 days on ½ Gamborg's B5 medium supplemented with 1% sucrose and 1.3% phytoagar (optimal regeneration period as determined by comparative studies)

• Agrobacterium-mediated transformation:

  • Use Agrobacterium tumefaciens strain EHA101 for highest transformation efficiency

  • Grow bacterial culture overnight and resuspend in ½ Gamborg's B5 medium

  • Co-cultivate plant fragments with Agrobacterium suspension for 48 hours

  • Transfer to selection medium containing appropriate antibiotics and cefotaxime to eliminate Agrobacterium

• Vector construction:

  • Design expression constructs using Marchantia-specific promoters such as the MpEF1α promoter for constitutive expression

  • Use Gateway-compatible destination vectors such as pMpGWB303 for efficient cloning

• Selection and regeneration:

  • Perform selection for 2-3 weeks on appropriate antibiotic medium

  • Transfer transformants to fresh medium for continued growth and analysis

This method generates numerous transformants from minimal starting material, making it ideal for experiments requiring multiple transgenic lines with modified ATP9 variants .

What imaging techniques are most effective for visualizing ATP synthase localization in Marchantia polymorpha mitochondria?

Visualizing ATP synthase localization in Marchantia polymorpha mitochondria requires specialized imaging techniques that provide sufficient resolution while preserving structural integrity. The most effective approaches include:

• Confocal microscopy with fluorescent protein fusions:

  • Generate ATP9-GFP fusion constructs under native promoter control

  • Co-localize with established mitochondrial markers (such as MitoTracker dyes)

  • Use proMpANT:GFP as a control for transformation and expression efficiency in different tissues

  • Perform live-cell imaging to monitor dynamic changes in mitochondrial morphology and ATP synthase distribution

• Super-resolution microscopy:

  • Employ STED (Stimulated Emission Depletion) or STORM (Stochastic Optical Reconstruction Microscopy) for nanoscale resolution

  • These techniques can resolve individual ATP synthase complexes within mitochondrial cristae

• Immunogold electron microscopy:

  • Use specific antibodies against ATP9 or epitope tags

  • Visualize with gold particle-conjugated secondary antibodies

  • Provides ultrastructural context for ATP synthase localization

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence microscopy with electron microscopy for comprehensive analysis

  • Particularly useful for studying ATP synthase distribution in different mitochondrial subcompartments

For studying ATP9 expression patterns in different tissues and developmental stages, a transcriptional fusion approach similar to that used for proMpANT:GFP would be valuable, allowing visualization of expression patterns in specific tissues such as meristematic regions .

How does the expression of ATP synthase subunit 9 change during different developmental stages of Marchantia polymorpha?

The expression of ATP synthase subunit 9 in Marchantia polymorpha likely exhibits developmental regulation, similar to patterns observed for other mitochondrial proteins in diverse organisms. Based on studies of ATP9 in Trypanosoma brucei, we can anticipate significant variations in expression levels across different developmental stages .

Methodological approach for studying developmental regulation:

• Transcript analysis:

  • Perform quantitative RT-PCR to measure ATP9 mRNA levels at different developmental stages

  • Compare expression between gemmae, young thalli, mature thalli, and reproductive structures

  • Expected pattern: higher expression in metabolically active tissues and developmental stages

• Protein level analysis:

  • Use western blotting with specific antibodies to quantify ATP9 protein levels

  • Compare with other ATP synthase subunits to identify coordinated regulation

  • Expected result: 5-10 fold higher expression in young, rapidly growing tissues compared to mature tissues

• Reporter gene assays:

  • Generate promoter-reporter constructs (proATP9:GFP) similar to the proMpANT:GFP approach

  • Analyze expression patterns in different tissues and developmental stages

  • Compare with patterns of other energy metabolism genes

In T. brucei, ATP9 transcript levels were shown to be 10-14 fold higher in procyclic forms compared to bloodstream forms . By analogy, we might expect significant upregulation of ATP9 expression during periods of active growth and development in Marchantia, particularly in meristematic regions where energy demands are high.

What post-translational modifications have been identified in Marchantia polymorpha ATP synthase subunit 9?

The post-translational modifications (PTMs) of ATP synthase subunit 9 in Marchantia polymorpha remain largely unexplored, but based on studies in other organisms, several PTMs are likely present and functionally significant. A comprehensive methodological approach to identifying these modifications would include:

• Mass spectrometry-based PTM mapping:

  • Isolate ATP9 from purified mitochondria using immunoprecipitation

  • Perform tryptic digestion followed by LC-MS/MS analysis

  • Use specialized software (e.g., MaxQuant with PTM search) to identify modifications

• Expected modifications:

  • N-terminal processing to remove mitochondrial targeting sequence

  • Acetylation of lysine residues (regulatory function)

  • Phosphorylation of serine/threonine residues (activity modulation)

  • Possibly methylation or other modifications

• Functional analysis of PTMs:

  • Generate site-directed mutants at modified residues

  • Express modified variants in Marchantia using the chopped-thallus transformation method

  • Assess impact on ATP synthase assembly, activity, and mitochondrial function

The predicted PTM profile for M. polymorpha ATP9 based on conservation with other species:

How does the function of ATP synthase subunit 9 in Marchantia polymorpha compare to that in higher plants?

Comparing ATP synthase subunit 9 function between Marchantia polymorpha and higher plants provides insights into the evolution of energy metabolism in land plants. A systematic methodological approach would involve:

• Comparative sequence and structural analysis:

  • Align ATP9 sequences from Marchantia, mosses, ferns, gymnosperms, and angiosperms

  • Identify conserved functional domains and species-specific variations

  • Construct phylogenetic trees to trace evolutionary relationships

  • Expected finding: Core functional residues involved in proton transport would show highest conservation

• Functional complementation studies:

  • Express Marchantia ATP9 in Arabidopsis ATP9 mutants

  • Express Arabidopsis ATP9 in Marchantia ATP9 knockdown lines generated using CRISPR-Cas9

  • Assess restoration of ATP synthase function and mitochondrial activity

  • Expected outcome: Partial functional complementation due to conserved mechanistic roles

• Biochemical characterization:

  • Compare proton translocation efficiency

  • Measure ATP synthesis rates in isolated mitochondria

  • Analyze subunit stoichiometry in ATP synthase complexes

  • Expected result: Similar fundamental mechanisms with potential differences in regulatory features

The evolutionary position of Marchantia as a basal land plant makes it particularly valuable for understanding the ancestral features of plant ATP synthase. While the core function of ATP9 in proton translocation and ATP synthesis is likely conserved, regulatory mechanisms may show significant divergence between Marchantia and higher plants, reflecting adaptation to different ecological niches and energetic demands.

What is the impact of environmental stress on ATP synthase subunit 9 expression and function in Marchantia polymorpha?

Environmental stresses significantly affect mitochondrial function and energy metabolism in plants, with potential specific impacts on ATP synthase subunit 9 expression and function in Marchantia polymorpha. A comprehensive methodological approach to studying these effects would include:

• Transcriptional regulation under stress conditions:

  • Expose Marchantia plants to various stresses (drought, salt, temperature extremes, oxidative stress)

  • Measure ATP9 transcript levels using qRT-PCR at different time points post-stress

  • Compare with other mitochondrial and nuclear stress-responsive genes

  • Expected pattern: Initial downregulation during acute stress followed by upregulation during recovery phases

• Protein level and post-translational modifications:

  • Analyze ATP9 protein abundance using western blotting under stress conditions

  • Identify stress-specific PTMs using mass spectrometry

  • Expected findings: Altered phosphorylation patterns in response to specific stresses

• Functional impacts:

  • Measure ATP synthase activity in mitochondria isolated from stressed plants

  • Assess mitochondrial membrane potential and ROS production

  • Expected results: Modulation of ATP synthase activity to balance energy production and oxidative stress

• Comparative stress responses:

  • Compare ATP9 stress responses in Marchantia versus higher plants

  • Identify conserved and divergent regulatory mechanisms

  • Expected outcome: More direct and rapid responses in Marchantia due to simpler regulatory networks

This research would provide valuable insights into the evolution of stress response mechanisms in plant mitochondria and could potentially identify novel regulatory features specific to basal land plants like Marchantia polymorpha.

What are the current technical challenges in studying ATP synthase assembly in Marchantia polymorpha?

Studying ATP synthase assembly in Marchantia polymorpha presents several technical challenges that require innovative approaches:

• Visualization of assembly intermediates:

  • Challenge: Capturing transient assembly intermediates in vivo

  • Solution approach: Develop split-fluorescent protein tags for different ATP synthase subunits to visualize assembly in real-time

  • Expected outcome: Identification of assembly pathways and kinetics

• Purification of intact ATP synthase complexes:

  • Challenge: Maintaining structural integrity during isolation

  • Solution approach: Optimize detergent conditions and employ gentle extraction methods such as native electrophoresis

  • Critical factors: Temperature control, use of stabilizing agents, and rapid processing

• Functional reconstitution:

  • Challenge: Reconstituting purified ATP synthase into liposomes for functional studies

  • Solution approach: Develop Marchantia-specific protocols based on methods used for other plant systems

  • Expected application: In vitro assessment of ATP synthesis rates and proton pumping efficiency

• Distinguishing between nuclear and mitochondrially-encoded components:

  • Challenge: Determining the origin and regulation of all ATP synthase components

  • Solution approach: Combine genomic, transcriptomic, and proteomic approaches

  • Relevant precedent: Studies in T. brucei demonstrated nuclear encoding of ATP9, contradicting expectations of mitochondrial encoding

Future technical advancements, particularly in cryo-electron microscopy and advanced fluorescence techniques, will facilitate more detailed structural and functional studies of ATP synthase assembly in Marchantia polymorpha.

How can CRISPR-Cas9 gene editing be optimized for targeting ATP synthase subunit 9 in Marchantia polymorpha?

Optimizing CRISPR-Cas9 gene editing for ATP synthase subunit 9 in Marchantia polymorpha requires specific considerations due to the essential nature of this gene and the unique aspects of Marchantia transformation:

• Guide RNA design and validation:

  • Select target sites unique to ATP9 to avoid off-target effects

  • Design multiple gRNAs targeting different exons or the exon-intron boundary similar to the approach used for MpANT

  • Test gRNA efficiency using in vitro cleavage assays before plant transformation

  • Recommended approach: Target the second intron-third exon boundary region, following the successful strategy used for MpANT

• Transformation and delivery optimization:

  • Utilize the chopped-thallus transformation method for high-efficiency transformation

  • Optimize Agrobacterium strain selection (EHA101 shows high efficiency)

  • Implement the optimal regeneration period of 4-5 days before co-cultivation

  • Culture on ½ Gamborg's B5 medium supplemented with 1% sucrose and 1.3% phytoagar

• Strategies for studying essential genes:

  • Develop inducible CRISPR systems to control editing timing

  • Create partial knockdown alleles rather than complete knockouts

  • Design complementation constructs to be co-transformed with CRISPR constructs

  • Expected challenge: Complete loss of ATP9 function may be lethal, requiring careful experimental design

• Screening and validation methods:

  • Implement high-throughput genotyping using PCR and sequencing

  • Develop phenotypic screens for mitochondrial function (e.g., using mitochondrial dyes)

  • Perform functional validation through biochemical assays of ATP synthesis

  • Verify editing efficiency through next-generation sequencing

The optimization of CRISPR-Cas9 protocols specifically for Marchantia polymorpha, combined with the chopped-thallus transformation method, provides powerful tools for investigating ATP9 function through targeted genetic modifications.

What are promising approaches for studying the interaction between nuclear and mitochondrial genomes in regulating ATP synthase in Marchantia polymorpha?

The intergenomic communication between nuclear and mitochondrial genomes in regulating ATP synthase components presents a fascinating research area in Marchantia polymorpha. Promising methodological approaches include:

• Retrograde signaling analysis:

  • Create specific mitochondrial perturbations (e.g., electron transport inhibitors, uncouplers)

  • Monitor nuclear gene expression changes for ATP synthase components

  • Identify retrograde signaling components specific to energy metabolism

  • Expected findings: Novel mitochondria-to-nucleus signaling pathways potentially distinct from those in higher plants

• Anterograde signaling identification:

  • Characterize nuclear transcription factors regulating mitochondrial gene expression

  • Identify nuclear-encoded proteins involved in mitochondrial gene expression and RNA processing

  • Expected outcome: Identification of master regulators coordinating ATP synthase component expression

• Evolutionary comparative analysis:

  • Compare nuclear-mitochondrial coordination between Marchantia and higher plants

  • Analyze gene transfer events from mitochondria to nucleus during land plant evolution

  • Expected insight: Evolutionary trajectory of intergenomic communication systems

• Integrative multi-omics approaches:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Implement network analysis to identify coordinated regulation patterns

  • Apply systems biology modeling to predict regulatory interactions

  • Expected output: Comprehensive model of nuclear-mitochondrial coordination in regulating bioenergetics

This research area is particularly relevant given the evidence from other organisms, such as T. brucei, showing nuclear encoding of traditionally mitochondrial genes like ATP9 . Understanding the mechanisms underlying this genomic reorganization and its functional consequences would provide valuable evolutionary insights.