Recombinant Arabidopsis thaliana NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial (At5g47570)

What is the current annotation status of At5g47570 in Arabidopsis thaliana?

At5g47570 has been annotated as an "Unknown protein" in the Arabidopsis genome, with limited functional characterization in earlier studies . While it is classified as part of the NADH dehydrogenase [ubiquinone] 1 beta subcomplex, its precise function within this complex requires further investigation. Unlike well-characterized NADH dehydrogenases such as AtNDB2, which has been extensively studied and shown to be responsible for approximately 90% of external NADH oxidation in isolated mitochondria , At5g47570's specific contribution to mitochondrial electron transport remains to be fully elucidated.

How does At5g47570 differ from other NADH dehydrogenases in Arabidopsis mitochondria?

At5g47570 belongs to the NADH dehydrogenase [ubiquinone] 1 beta subcomplex, which is distinct from the alternative NADH dehydrogenases like the NDB family (including AtNDB2). While AtNDB2 functions as an external NADH dehydrogenase facing the intermembrane space and oxidizing cytosolic NADH , At5g47570 is predicted to be part of the multisubunit Complex I. This fundamental structural and functional difference is important to consider when designing experiments to study At5g47570's specific activities. Unlike AtNDB2, which has been shown to increase in expression under stress conditions and work synergistically with AtAOX1A , the regulation and interacting partners of At5g47570 require further characterization.

What experimental approaches are recommended for initial characterization of At5g47570's subcellular localization?

For confirming the mitochondrial localization of At5g47570, a multifaceted approach is recommended:

• Fluorescent protein fusion studies: Generate N-terminal and C-terminal GFP fusions with At5g47570 and observe their localization in Arabidopsis cell cultures or protoplasts. This approach can help determine whether the protein contains specific targeting signals that might be masked in one fusion orientation.

• Co-localization experiments: Perform co-localization studies with established mitochondrial markers such as MitoTracker dyes or mitochondrial-targeted fluorescent proteins.

• Subcellular fractionation: Isolate mitochondria using established protocols and confirm At5g47570 presence through immunoblotting with specific antibodies.

• In silico prediction validation: Validate bioinformatic predictions of targeting sequences using tools available through databases like ARAMEMNON, which provides consensus prediction of protein localization signals .

How is At5g47570 expression regulated in response to environmental stresses?

While specific information about At5g47570 regulation is limited in the provided literature, research on related mitochondrial electron transport components provides a framework for investigation. Based on studies of AtNDB2, which shows altered expression under stress conditions , the following approaches are recommended:

• Transcriptomic analysis: Monitor At5g47570 transcript levels under various stress conditions (drought, high light, temperature extremes, and pathogen infection) using qRT-PCR.

• Promoter analysis: Characterize the At5g47570 promoter region to identify potential stress-responsive elements.

• Stress treatment experiments: Similar to studies on AtNDB2 and AtAOX1A, which demonstrated increased tolerance to drought and high-light stress when overexpressed , subject At5g47570 mutant and overexpression lines to various stresses to determine its role in stress responses.

• Randomized block design experiments: Implement experimental designs similar to those used in other Arabidopsis studies, with multiple blocks within each experimental replicate to control for environmental variation .

What is known about the tissue-specific expression pattern of At5g47570?

To characterize the tissue-specific expression pattern of At5g47570:

• Promoter-reporter gene fusion studies: Generate transgenic plants expressing reporter genes (GUS, GFP) under the control of the At5g47570 promoter.

• Tissue-specific transcriptome analysis: Analyze publicly available RNA-seq data from different tissues and developmental stages.

• Immunohistochemistry: Develop specific antibodies against At5g47570 and use them for tissue-specific localization studies.

• Single-cell transcriptomics: For more detailed resolution, utilize single-cell RNA-seq data if available to determine cell type-specific expression patterns.

What are the optimal conditions for recombinant expression and purification of At5g47570?

For successful recombinant expression and purification of At5g47570:

• Expression system selection:

  • For structural studies: E. coli-based expression systems with solubility-enhancing tags (MBP, SUMO)

  • For functional studies requiring post-translational modifications: Insect cell or plant-based expression systems

• Purification strategy:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tag

  • Secondary purification: Size exclusion chromatography

  • Verification: SDS-PAGE and Western blotting

• Buffer optimization:

  Buffer Component	Recommended Range	Purpose
  pH	7.0-8.0	Maintain protein stability
  NaCl	150-300 mM	Reduce non-specific interactions
  Glycerol	5-10%	Enhance protein stability
  Reducing agent	1-5 mM DTT or TCEP	Prevent oxidation of cysteine residues

• Quality control: Assess protein homogeneity using dynamic light scattering and thermal shift assays to optimize buffer conditions.

What methods are most effective for measuring the enzymatic activity of At5g47570?

Based on studies of related NADH dehydrogenases , the following approaches are recommended:

• Spectrophotometric assays: Monitor NADH oxidation at 340 nm in isolated mitochondria or with purified protein. This approach can be used to determine:

  • Kinetic parameters (Km, Vmax)

  • Substrate specificity (NADH vs. NADPH)

  • Effects of inhibitors

• Oxygen consumption measurements: Using oxygen electrodes to measure the contribution of At5g47570 to mitochondrial respiration.

• In vivo NADH/NAD+ ratio measurements: Implement fluorescent protein sensors (such as iNAP and SoNar) that have been successfully used to monitor NADH/NAD+ ratios in Arabidopsis .

• Electron paramagnetic resonance (EPR) spectroscopy: For detailed analysis of electron transfer reactions and redox centers.

How can I generate and validate knockout or knockdown lines of At5g47570?

For generating and validating At5g47570 mutant lines:

• T-DNA insertion line screening:

  • Obtain T-DNA insertion lines from stock centers

  • Confirm homozygosity through PCR genotyping

  • Verify loss of expression through RT-PCR and Western blotting

• CRISPR/Cas9-based genome editing:

  • Design specific sgRNAs targeting At5g47570

  • Screen transformants for mutations using sequencing

  • Confirm protein loss using immunoblotting

• RNAi or artificial microRNA approaches:

  • For partial knockdown if complete knockout is lethal

  • Confirm knockdown efficiency using qRT-PCR and Western blotting

• Complementation testing:

  • Transform knockout lines with wild-type At5g47570 to confirm phenotype rescue

  • Include both native promoter and constitutive promoter versions

• Phenotypic validation:

  • Assess growth parameters under normal and stress conditions

  • Measure mitochondrial respiration rates

  • Quantify NADH/NAD+ ratios using fluorescent sensors

How does At5g47570 integrate into the mitochondrial electron transport chain?

To determine the integration of At5g47570 into the mitochondrial electron transport chain:

• Blue native PAGE and proteomics: Isolate mitochondrial complexes through blue native PAGE and identify proteins that co-migrate with At5g47570 using mass spectrometry.

• Respiratory complex activity assays: Compare complex I activity in wild-type and At5g47570 mutant plants using standard biochemical assays.

• Electron flow analysis: Utilize specific inhibitors of different respiratory complexes to determine where At5g47570 functions in the electron transport chain.

• Redox state measurements: Monitor changes in NAD(P)H/NAD(P)+ ratios in wild-type versus At5g47570 mutant plants using fluorescent sensors .

• Oxygen consumption patterns: Analyze respiratory control ratios and the effects of specific inhibitors on isolated mitochondria from wild-type and mutant plants.

What proteins interact with At5g47570 and how can these interactions be characterized?

To identify and characterize protein-protein interactions:

• Co-immunoprecipitation: Use antibodies against At5g47570 or epitope-tagged versions to pull down interacting proteins, followed by mass spectrometry identification.

• Yeast two-hybrid screening: Use At5g47570 as bait to screen an Arabidopsis cDNA library for interacting proteins.

• Bimolecular fluorescence complementation (BiFC): Verify specific interactions in planta by fusing candidate interacting proteins with complementary fragments of a fluorescent protein.

• Proximity-dependent biotin identification (BioID): Fuse At5g47570 with a biotin ligase to identify proteins in close proximity in vivo.

• Cross-linking mass spectrometry: Use chemical cross-linkers to capture transient interactions followed by mass spectrometry analysis.

How does At5g47570 contribute to plant responses to environmental stresses?

Building on insights from studies of related proteins like AtNDB2 , investigate At5g47570's role in stress responses through:

• Stress tolerance phenotyping: Subject At5g47570 knockout and overexpression lines to multiple stresses (drought, high light, temperature, pathogens) using standardized protocols.

• Stress-induced redox changes: Monitor NAD(P)H/NAD(P)+ ratio changes during stress using fluorescent sensors .

• ROS production and scavenging: Measure reactive oxygen species levels and antioxidant enzyme activities in wild-type versus mutant plants under stress conditions.

• Metabolomic analysis: Identify metabolic shifts that occur in response to stress in wild-type versus At5g47570 mutant plants.

• Transcriptomic response: Compare stress-induced transcriptional changes between wild-type and mutant plants to identify affected pathways.

What is the role of At5g47570 in the crosstalk between mitochondria and chloroplasts?

The interaction between mitochondria and chloroplasts is critical for plant energy metabolism. To study At5g47570's role in this crosstalk:

• Photosynthetic parameters: Measure photosynthetic efficiency (Fv/Fm, electron transport rate) in At5g47570 mutants versus wild-type plants.

• Photorespiratory flux: Analyze the impact of altered At5g47570 expression on photorespiratory metabolism, particularly through the malate-OAA shuttle that connects chloroplast and mitochondrial redox states .

• Dual organelle imaging: Implement simultaneous monitoring of mitochondrial and chloroplastic redox states using organelle-targeted fluorescent sensors .

• Metabolite exchange analysis: Track the movement of key metabolites between organelles using isotope labeling and metabolomics.

• Light response experiments: Compare responses to changing light conditions between wild-type and At5g47570 mutant plants, focusing on parameters like:

  • NADPH levels in chloroplasts

  • NADH/NAD+ ratios in mitochondria

  • ATP production dynamics

How does post-translational modification affect At5g47570 function and stability?

To investigate post-translational modifications (PTMs) of At5g47570:

• Identification of modifications:

  • Phosphoproteomics to identify phosphorylation sites

  • Redox proteomics to detect oxidative modifications

  • Ubiquitin/SUMO proteomics for ubiquitination/SUMOylation

• Functional impact analysis:

  • Generate site-directed mutants of modified residues

  • Compare activity, stability, and interactions of wild-type versus mutant proteins

  • Monitor changes in PTM status under different environmental conditions

• Structural impact modeling:

  • Use molecular dynamics simulations to predict how PTMs affect protein structure

  • Validate predictions through biophysical methods (CD spectroscopy, thermal shift assays)

How has At5g47570 evolved across plant species and what does this reveal about its function?

For evolutionary analysis of At5g47570:

• Phylogenetic analysis:

  • Identify orthologs across diverse plant species

  • Construct phylogenetic trees to trace evolutionary history

  • Identify conserved domains and residues

• Selective pressure analysis:

  • Calculate dN/dS ratios to identify residues under positive or purifying selection

  • Correlate conserved regions with functional domains

• Comparative functional studies:

  • Express orthologs from different species in Arabidopsis At5g47570 knockout backgrounds

  • Test for functional complementation

  • Identify species-specific functional differences

• Correlation with ecological adaptations:

  • Compare sequence variations with the ecological niches of source species

  • Identify potential adaptations to specific environmental conditions