Recombinant Arabidopsis thaliana Probable cytochrome c oxidase subunit 5C-3 (At5g61310)

What is the structural composition of the recombinant At5g61310 protein?

The recombinant full-length Arabidopsis thaliana Probable cytochrome c oxidase subunit 5C-3 (At5g61310) is a 64-amino acid protein (residues 1-64) with UniProt ID Q9FLK2. Its amino acid sequence is MAGHKIAHATLKGPSVVKELVIGLTLGLAAGGLWKMHHWNEQRKTRVFYDLLERGEIGVVVTEE . For research applications, the protein is typically expressed in E. coli with an N-terminal His-tag to facilitate purification. The protein is available in lyophilized powder form with greater than 90% purity as determined by SDS-PAGE analysis .

What is the tissue-specific expression pattern of At5g61310?

At5g61310 expression exhibits distinct tissue-specific patterns that can be visualized using the gus reporter gene system. Expression is predominantly localized in:

• Root and shoot meristems

• Actively growing tissues

• Vascular strands

• Floral tissues (specifically anthers, stigma, and receptacle)

• Developing seeds

Researchers investigating tissue-specific expression should note that GUS activity measurements in protein extracts from transformed plants indicate that At5g61310's expression levels exceed those observed with the constitutive CaMV 35S promoter, making it particularly valuable for studies requiring high expression in specific tissues .

What role does the leader intron play in At5g61310 gene expression?

The leader intron in the 5'-non-coding region of At5g61310 plays a critical role in gene expression regulation. Experimental evidence demonstrates that removal of this leader intron results in a significant decrease in expression to levels only slightly higher than those observed with a promoterless gus gene .

Methodologically, this can be investigated through:

• Constructing gene fusions with and without the leader intron

• Transforming plants with these constructs

• Measuring GUS activity quantitatively in protein extracts

• Performing histochemical staining to visualize spatial expression patterns

When the leader intron is removed, expression becomes restricted primarily to pollen, suggesting that regulatory elements capable of directing pollen-specific expression reside upstream of the intron . The intron also appears to enhance translation efficiency of the corresponding mRNA, as evidenced by comparisons of GUS activity values with transcript levels .

What experimental approaches should be employed to investigate At5g61310's role in mitochondrial function?

Investigating At5g61310's role in mitochondrial function requires a multi-faceted experimental approach:

• Protein localization studies: Use fluorescently tagged At5g61310 constructs to confirm mitochondrial localization and association with Complex IV.

• Respiratory measurements: Assess oxygen consumption rates in wild-type plants versus those with altered At5g61310 expression using oxygen electrodes.

• Blue Native PAGE: Analyze intact respiratory complexes to determine if At5g61310 modifications affect assembly or stability of Complex IV.

• Proteomics analysis: Employ comparative proteomics to identify changes in the mitochondrial proteome resulting from altered At5g61310 expression.

• Environmental stress tests: Evaluate the impact of stressors (particularly low temperature) on At5g61310 expression and mitochondrial function.

These approaches should be adapted from experimental designs used in studies of other mitochondrial respiratory chain components, such as those employed in studying complex I and III subunits under various environmental conditions .

How can researchers effectively compare the functional differences between At5g61310 and other COX5c isoforms?

To effectively compare functional differences between At5g61310 (COX5c-3) and other COX5c isoforms, researchers should implement a systematic experimental design following these methodological steps:

• Gene expression profiling: Analyze transcript abundance of all COX5c isoforms across tissues and under various environmental conditions.

• Mutant analysis: Generate single and multiple knockout/knockdown lines for COX5c isoforms using CRISPR/Cas9 or T-DNA insertion lines.

• Complementation studies: Perform cross-complementation experiments by expressing each isoform in knockout backgrounds of other isoforms.

• Protein-protein interaction studies: Use techniques such as yeast two-hybrid or co-immunoprecipitation to identify differential interactions between isoforms and other respiratory complex components.

• Respiratory activity measurements: Compare cytochrome c oxidase activity in plants with altered expression of different isoforms.

When designing these experiments, it's critical to control for extraneous variables and properly formulate testable hypotheses regarding isoform-specific functions . This approach can reveal whether subunit swapping occurs among COX5c isoforms similar to what has been observed in other respiratory complexes .

What protocols should be followed for optimal expression and purification of recombinant At5g61310?

For optimal expression and purification of recombinant At5g61310, researchers should follow this detailed protocol:

• Expression system selection: Use E. coli as the expression host with an N-terminal His-tag fusion for purification purposes .

• Culture conditions: Grow transformed E. coli under standard conditions with appropriate antibiotic selection.

• Induction optimization: Test various IPTG concentrations and induction temperatures to maximize protein yield while maintaining proper folding.

• Purification protocol:

  • Perform initial purification using Ni-NTA affinity chromatography

  • Follow with size exclusion chromatography if higher purity is required

  • Aim for >90% purity as assessed by SDS-PAGE

• Storage recommendations:

  • Store as lyophilized powder at -20°C/-80°C

  • After reconstitution in deionized sterile water (0.1-1.0 mg/mL), add glycerol to a final concentration of 5-50% (50% is recommended)

  • Aliquot for long-term storage at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

• Quality control: Verify protein identity by mass spectrometry and assess activity if functional assays are available.

How might environmental stressors affect At5g61310 expression and function?

Based on studies of other mitochondrial respiratory chain components, researchers investigating At5g61310 responses to environmental stressors should consider the following methodological approach:

• Transcriptomic analysis: Monitor At5g61310 transcript levels under various stressors, particularly low temperature (LT), using RT-qPCR or RNA-seq.

• Protein abundance quantification: Use Western blotting or quantitative proteomics to determine if At5g61310 protein levels change in response to stressors.

• Stress treatment regimens:

  • Acute cold stress (e.g., 4°C for 48h)

  • Gradual temperature reduction

  • Varying light conditions during stress

  • Combined stressors (e.g., cold and drought)

• Functional analysis:

  • Measure respiratory rates under stress conditions

  • Assess ROS production

  • Evaluate mitochondrial membrane potential

Current evidence suggests that mitochondrial respiratory complexes undergo compositional changes in response to LT, including potential subunit swapping . For instance, some complex III subunits (UCR1) decrease in abundance under severe cold stress but increase under milder cold conditions . Similar responses might occur with At5g61310, potentially as an adaptive mechanism to maintain respiratory function under stress.

What techniques are most appropriate for studying the regulatory elements in the At5g61310 promoter and leader intron?

To effectively study the regulatory elements in the At5g61310 promoter and leader intron, researchers should employ these methodological approaches:

• Promoter deletion analysis: Create a series of constructs with progressive deletions of the 5' upstream region fused to a reporter gene (e.g., GUS).

• Intron deletion/mutation analysis: Generate constructs with:

  • Complete intron removal

  • Partial intron deletions

  • Site-directed mutations of putative regulatory motifs

• Reporter gene assays: Transform plants with these constructs and assess:

  • Quantitative GUS activity measurements in protein extracts

  • Histochemical staining for spatial expression patterns

• Promoter-intron swapping experiments: Test whether the At5g61310 intron can enhance expression when placed in the context of unrelated promoters, such as the COX5b-1 promoter .

• Transcription factor binding studies:

  • Electrophoretic mobility shift assays (EMSA)

  • Chromatin immunoprecipitation (ChIP)

  • Yeast one-hybrid screens

This experimental approach has revealed that the COX5c-2 intron increases GUS expression levels when fused in the correct orientation with the promoter of the unrelated COX5b-1 gene, suggesting that the intron contains orientation-dependent enhancer elements .

What are the optimal storage and reconstitution conditions for recombinant At5g61310 protein?

For researchers working with recombinant At5g61310 protein, optimal storage and reconstitution protocols are critical for maintaining protein stability and activity:

Storage conditions:

• Store lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, store in aliquots at -20°C/-80°C

• Working aliquots may be stored at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this can compromise protein integrity

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 glycerol to a final concentration of 5-50% (recommended: 50%)

• Prepare multiple small aliquots for long-term storage

The protein is typically supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which helps maintain stability in the lyophilized form .

How can experimental design principles be applied to study At5g61310 function under varying environmental conditions?

When designing experiments to study At5g61310 function under varying environmental conditions, researchers should apply these structured experimental design principles:

• Clearly define variables:

  • Independent variables: Environmental conditions (temperature, light intensity, drought, etc.)

  • Dependent variables: At5g61310 expression levels, protein abundance, respiratory rates

  • Control variables: Growth medium, plant age, time of day for measurements

• Formulate testable hypotheses:

  • Null hypothesis (H0): "Environmental condition X does not affect At5g61310 expression/function"

  • Alternative hypothesis (H1): "Environmental condition X significantly alters At5g61310 expression/function"

• Design treatments with appropriate controls:

  • Include wild-type and mutant/transgenic plants

  • Implement proper randomization

  • Use sufficient biological and technical replicates

  • Include time-course measurements where appropriate

• Control for confounding variables:

  • Standardize plant growth conditions prior to treatments

  • Account for circadian regulation

  • Consider developmental stage effects

• Data analysis considerations:

  • Apply appropriate statistical tests

  • Consider potential interactions between variables

  • Validate findings with alternative approaches

This methodical approach ensures that experiments yield reliable and reproducible results regarding At5g61310's response to environmental stimuli.

How does At5g61310 expression and function compare to other cytochrome c oxidase subunits under stress conditions?

When investigating how At5g61310 expression and function compare to other cytochrome c oxidase subunits under stress conditions, researchers should implement comparative experimental approaches:

• Simultaneous expression analysis:

  • Perform RT-qPCR or RNA-seq to quantify transcript levels of multiple COX subunits

  • Compare expression patterns across tissues and stress conditions

  • Analyze correlation between expression of different subunits

• Protein abundance comparisons:

  • Use quantitative proteomics to measure changes in multiple COX subunits simultaneously

  • Apply western blotting with subunit-specific antibodies

  • Assess stoichiometric relationships between subunits

• Functional analysis:

  • Compare phenotypes of mutants affected in different COX subunits

  • Evaluate respiratory parameters in these mutants under stress

  • Assess mitochondrial morphology and dynamics

Available data on other respiratory chain components suggest that different subunits can show distinct responses to stress. For example, while some complex III subunits (UCR1) decrease in abundance under severe cold stress, others like QCR7−1 and CYC1−1 increase, while their isoforms QCR7−2 and CYC1−2 decrease . This suggests the possibility of subunit swapping as an adaptive response to stress, a phenomenon that might also apply to cytochrome c oxidase subunits including At5g61310.

What methodologies are most effective for comparing the tissue-specific roles of different COX5c isoforms?

To effectively compare the tissue-specific roles of different COX5c isoforms, researchers should employ these methodological approaches:

• Promoter-reporter gene fusions:

  • Create promoter-GUS constructs for each COX5c isoform

  • Transform plants and perform histochemical staining

  • Compare spatial expression patterns across tissues and developmental stages

• Isoform-specific antibodies:

  • Develop antibodies specific to each COX5c isoform

  • Perform immunolocalization studies

  • Use western blotting to compare protein levels across tissues

• Isoform-specific knockout/knockdown lines:

  • Generate CRISPR/Cas9 or RNAi lines for each isoform

  • Compare phenotypes, particularly in tissues where expression overlaps

  • Assess respiratory parameters in affected tissues

• Complementation studies with tissue-specific promoters:

  • Express each isoform under the control of tissue-specific promoters in knockout backgrounds

  • Determine if isoforms can functionally substitute for each other in specific tissues

• Cell-type specific transcriptomics:

  • Use fluorescence-activated cell sorting or laser capture microdissection

  • Compare isoform expression in specific cell types

  • Identify co-expressed genes that might indicate functional specialization

This comparative approach has revealed that regulatory elements in the 5'-non-coding regions, particularly leader introns, are essential for the tissue-specific expression patterns of COX5c genes .

What are promising approaches for investigating potential post-translational modifications of At5g61310?

Investigating potential post-translational modifications (PTMs) of At5g61310 requires sophisticated analytical techniques:

• Mass spectrometry-based approaches:

  • Perform LC-MS/MS analysis of purified At5g61310 protein

  • Use multiple protease digestions to achieve comprehensive sequence coverage

  • Apply PTM-specific enrichment strategies (e.g., phosphopeptide enrichment)

  • Quantify modification stoichiometry under different conditions

• Site-directed mutagenesis:

  • Mutate potential PTM sites identified by mass spectrometry

  • Express mutant proteins in planta

  • Assess functional consequences of preventing specific modifications

• In vitro modification assays:

  • Test susceptibility of purified At5g61310 to various modifying enzymes

  • Determine how modifications affect protein-protein interactions or activity

• Modification-specific antibodies:

  • Develop antibodies that recognize specific PTMs on At5g61310

  • Use these for western blotting and immunoprecipitation experiments

  • Assess changes in modification status under different conditions

Given that respiratory complex subunits often undergo PTMs in response to environmental stresses , investigating how such modifications might regulate At5g61310 function represents an important area for future research.

How might advanced gene editing approaches be applied to study At5g61310 function?

Advanced gene editing approaches offer powerful tools for studying At5g61310 function:

• CRISPR/Cas9-mediated genome editing:

  • Generate complete knockout lines by introducing frameshift mutations

  • Create specific amino acid substitutions to study structure-function relationships

  • Develop conditional knockout systems (e.g., with inducible promoters)

  • Implement multiplexed editing to target multiple COX5c isoforms simultaneously

• Base editing approaches:

  • Introduce specific point mutations without double-strand breaks

  • Target conserved residues predicted to be functionally important

  • Create subtle mutations that may not completely abolish function

• Epitope tagging at endogenous loci:

  • Add fluorescent protein or affinity tags to the endogenous At5g61310 gene

  • Maintain native expression patterns and regulatory elements

  • Enable visualization of subcellular localization and protein complex formation

• Promoter editing:

  • Modify regulatory elements in the promoter or leader intron

  • Assess effects on tissue-specific expression patterns

  • Create plants with altered expression levels but maintaining tissue specificity

These advanced gene editing approaches, combined with proper experimental design principles , will enable more precise dissection of At5g61310 function than traditional methods like T-DNA insertions or constitutive overexpression.