Recombinant Oryza sativa subsp. japonica Peroxisomal membrane protein 11-3 (PEX11-3)

What is the physiological function of PEX11-3 in rice development and metabolism?

PEX11-3 in rice, like other PEX11 family proteins, is primarily involved in peroxisome biogenesis and proliferation. The peroxisome is an essential organelle that participates in various metabolic processes including fatty acid oxidation, reactive oxygen species metabolism, and photorespiration in plants.

Expression profile analysis has revealed that OsPEX11-3 is expressed predominantly in endosperm and germinated seeds, suggesting a specific role during seed development and germination . This pattern differentiates it from other family members; for example, OsPEX11-1 and OsPEX11-4 show higher expression in leaf tissues, while OsPEX11-5 is expressed in all tissues investigated .

Studies in other species indicate that PEX11 proteins function directly in peroxisome division, with their loss potentially affecting multiple peroxisomal metabolic pathways indirectly. For instance, in mammals, PEX11β-deficient mice show deficiencies in ether lipid synthesis and very long chain fatty acid oxidation . By extension, rice PEX11-3 likely plays a critical role in peroxisome biogenesis during seed development, possibly influencing lipid metabolism during germination.

How should experiments be designed to investigate the stress-responsive nature of OsPEX11-3 expression?

To investigate the stress-responsive nature of OsPEX11-3 expression, researchers should consider a comprehensive experimental approach:

• Plant Material Preparation:

  • Use 3-week-old rice seedlings (such as Minghui 63) grown under normal conditions

  • Ensure uniform growth conditions prior to stress application

• Stress Treatments:

  • Apply diverse abiotic stresses:

    • Chemical stresses: ABA (200 μL/L), H₂O₂ (500 μL/L)

    • Environmental stresses: Salt (200 mM NaCl), drought (dehydration until leaves roll)

    • Nutritional stresses: Low nitrogen (1/10 of normal), low phosphorus (1/20 of normal)

• Temporal Analysis:

  • Collect samples at multiple time points (e.g., 0, 1, 3, 6, 12, 24, 48 hours)

  • This allows tracking of the expression dynamics

• Expression Analysis Methods:

  • RT-PCR for preliminary analysis

  • qRT-PCR for precise quantification

  • RNA-seq for genome-wide expression context

  • Include appropriate housekeeping genes as controls

• Tissue-Specific Analysis:

  • Since OsPEX11-3 is predominantly expressed in endosperm and germinated seeds, compare expression in different tissues under stress

Research has shown that OsPEX11-3 is responsive to ABA and H₂O₂ treatments specifically, but not to salt or nitrogen stress, indicating a specialized stress response pattern compared to other family members . This information should guide the experimental design to focus particularly on oxidative and hormone signaling pathways.

What are the optimal conditions for expressing and purifying recombinant Oryza sativa PEX11-3 protein for structural studies?

For optimal expression and purification of recombinant Oryza sativa PEX11-3 protein:

• Expression System Selection:

  • E. coli has been successfully used for expressing full-length PEX11-3 protein

  • Consider BL21(DE3) or other strains optimized for membrane protein expression

  • For complex structural studies, eukaryotic expression systems like insect cells might provide better post-translational modifications

• Construct Design:

  • Include a His-tag for purification (N-terminal tagging has been successful)

  • Consider a cleavable tag if native protein is required

  • Full-length construct (1-242aa) has been successfully expressed

• Expression Conditions:

  • Optimize temperature (typically 16-25°C for membrane proteins)

  • Induce with appropriate IPTG concentration (0.1-0.5 mM)

  • Consider extended expression times (overnight at lower temperatures)

• Purification Protocol:

  • Cell lysis: Use gentle methods to preserve protein structure

  • Membrane protein extraction: Employ detergents (DDM, LDAO, or other mild detergents)

  • Purification steps:

    • Initial Ni-NTA affinity chromatography

    • Size exclusion chromatography to remove aggregates

    • Consider ion exchange chromatography for higher purity

• Storage Considerations:

  • Store in Tris-based buffer with 50% glycerol

  • Aliquot and store at -20°C or -80°C to prevent freeze-thaw cycles

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

• Quality Assessment:

  • SDS-PAGE (aim for >90% purity)

  • Western blot to confirm identity

  • Circular dichroism to verify secondary structure

  • Dynamic light scattering to assess monodispersity

The reconstitution of lyophilized protein should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added for long-term storage .

How should researchers interpret differential expression patterns of PEX11 family members in rice under various stress conditions?

The differential expression patterns of PEX11 family members under stress conditions require careful interpretation using these methodological approaches:

• Comparative Expression Analysis:

• Functional Interpretation Framework:

  • Gene expression changes should be assessed in the context of peroxisome biology

  • Upregulation may indicate increased need for peroxisome proliferation under specific stresses

  • Tissue-specific patterns suggest specialized roles in different plant organs

• Biological Significance Assessment:

  • OsPEX11-3's response to ABA and H₂O₂ but not other stresses suggests a specific role in hormone and oxidative stress signaling pathways

  • The predominant expression in endosperm and germinated seeds, combined with stress responsiveness, suggests OsPEX11-3 may regulate peroxisome dynamics during seed development under stress conditions

  • Lack of responsiveness in OsPEX11-2 indicates functional specialization within the family

• Evolutionary Context:

  • The diversification of expression patterns among rice PEX11 genes indicates functional specialization during evolution

  • Comparison with PEX11 expression patterns in other plant species can provide insights into conserved and species-specific functions

When interpreting expression data, researchers should consider that changes in PEX11 expression likely directly affect peroxisome division, which then indirectly influences various metabolic processes that occur in peroxisomes . The tissue-specific expression patterns combined with stress-specific responses suggest that the rice PEX11 family has evolved to regulate peroxisome dynamics in different tissues under various environmental conditions.

What is the relationship between PEX11-3 expression, peroxisome proliferation, and metabolic activity in rice cells?

The relationship between PEX11-3 expression, peroxisome proliferation, and metabolic activity is complex and warrants careful analysis:

• Mechanistic Relationship:

  • Evidence suggests that PEX11 proteins directly promote peroxisome division rather than acting through metabolic pathways

  • Studies in mammals have shown that PEX11 overexpression promotes peroxisome division even in the absence of peroxisomal metabolic activity

  • The loss of PEX11 proteins affects multiple, unrelated peroxisomal metabolic activities, suggesting an indirect effect on metabolism

• Expression-Proliferation Connection:

  • Research in Arabidopsis has demonstrated that overexpression of PEX11 genes induces peroxisome proliferation, while reduced expression decreases peroxisome abundance

  • By extension, increased OsPEX11-3 expression in rice, particularly in endosperm and germinated seeds, likely promotes peroxisome division in these tissues

• Metabolic Consequences:

  • Changes in peroxisome number and morphology due to altered PEX11-3 expression would affect:

    • Lipid metabolism during seed germination

    • Reactive oxygen species metabolism, particularly important during stress responses

    • Other peroxisomal metabolic pathways

• Regulatory Circuit Analysis:

  • OsPEX11-3's responsiveness to ABA and H₂O₂ suggests a regulatory circuit where:

    • Stress signals → Increased OsPEX11-3 expression → Peroxisome proliferation → Enhanced metabolic capacity → Improved stress tolerance

  • This circuit may be particularly important during seed germination under stressful conditions

• Tissue-Specific Considerations:

  • The predominant expression of OsPEX11-3 in endosperm and germinated seeds suggests a specialized role in peroxisome-dependent metabolism during these developmental stages

  • Peroxisomes in germinating seeds are involved in converting stored lipids to sucrose through β-oxidation and the glyoxylate cycle

Understanding this relationship requires experimental approaches that can distinguish between direct effects on peroxisome division and indirect effects on metabolism. Techniques such as live cell imaging of peroxisome dynamics combined with metabolomic analysis in PEX11-3 overexpression and knockdown lines would provide valuable insights into these interconnected processes.

How might protein-protein interaction networks involving PEX11-3 regulate peroxisome morphology and division in rice?

The regulation of peroxisome morphology and division through PEX11-3 protein-protein interactions likely involves several key mechanisms:

• Potential Interaction Partners:

  • Based on studies in other organisms, rice PEX11-3 may interact with:

    • Dynamin-related proteins (DRPs) that mediate membrane fission

    • Fission factor 1 (FIS1) that recruits division machinery

    • Membrane-deforming proteins that facilitate membrane curvature

    • Other PEX proteins involved in peroxisome biogenesis and maintenance

• Stages of PEX11-Mediated Division:

  • Research in human cells has revealed a multi-step process:

    • Initial membrane elongation (observed 4-8 hours after PEX11 expression)

    • Membrane constriction

    • Final fission (resulting in increased peroxisome abundance by 24-48 hours)

  • Each stage likely involves distinct protein-protein interactions

• Investigation Approaches:

  • Yeast two-hybrid screens to identify direct interactors

  • Co-immunoprecipitation followed by mass spectrometry

  • FRET or BiFC to visualize interactions in vivo

  • Split-ubiquitin assays for membrane protein interactions

  • Comparative interactomics between rice PEX11 family members to identify specific versus shared interaction partners

• Functional Validation:

  • Mutagenesis of potential interaction domains

  • Expression of dominant-negative variants

  • Heterologous complementation studies (rice PEX11-3 has potential to complement yeast pex11 null mutant based on Arabidopsis studies)

The sequence diversification within the rice PEX11 family likely contributes to differences in protein-protein interactions, which may explain their specialized functions . Of particular interest would be investigation of whether the stress-responsive nature of OsPEX11-3 (response to ABA and H₂O₂) influences its interaction network under stress conditions, potentially explaining the link between stress responses and peroxisome dynamics.

What are the molecular mechanisms by which PEX11-3 responds to ABA and H₂O₂ but not other stresses, and how does this compare to other PEX11 family members?

The selective responsiveness of PEX11-3 to ABA and H₂O₂ but not other stresses involves sophisticated molecular mechanisms:

• Promoter Architecture Analysis:

  • PEX11-3 promoter likely contains specific cis-regulatory elements:

    • ABA-responsive elements (ABREs)

    • Oxidative stress-responsive elements

    • But lacks elements responsive to salt or nutrient stress

  • Computational analysis of the promoters of all rice PEX11 genes would reveal:

    • Common elements shared by stress-responsive members

    • Unique elements explaining differential responses

• Signaling Pathway Integration:

  • OsPEX11-3 expression appears integrated with:

    • ABA signaling cascade (involving SnRK2 kinases and AREB/ABF transcription factors)

    • Reactive oxygen species (ROS) signaling pathways

  • But not with:

    • Salt stress signaling (SOS pathway)

    • Nutrient deficiency response pathways

• Comparative Response Profiles:

• Molecular Regulation Hypotheses:

  • OsPEX11-3's specific response pattern may result from:

    • Unique transcription factor binding profiles

    • Tissue-specific chromatin accessibility

    • Post-transcriptional regulation by stress-responsive miRNAs

    • mRNA stability differences under various stress conditions

• Functional Implications:

  • The specific responsiveness to ABA and H₂O₂ suggests OsPEX11-3 may be particularly important in:

    • Seed germination under drought conditions (where ABA levels increase)

    • Response to oxidative stress during early development

    • Coordination of peroxisome proliferation with specific stress responses

This selective responsiveness indicates functional specialization among PEX11 family members, with OsPEX11-3 possibly evolved to regulate peroxisome dynamics specifically during hormone and oxidative stress responses in seed tissues .

What are the critical considerations for designing knockout or knockdown experiments to study PEX11-3 function in rice?

When designing knockout or knockdown experiments for rice PEX11-3 functional studies, researchers should consider these critical methodological aspects:

• Strategy Selection:

  • CRISPR/Cas9 gene editing: Most precise for complete knockout

  • RNAi: Effective for knockdown studies with variable suppression levels

  • TILLING: Alternative for obtaining point mutations

  • Artificial microRNA: For tissue-specific knockdown

• Target Design Considerations:

  • Guide RNA design for CRISPR/Cas9:

    • Target early exons to ensure functional disruption

    • Check for potential off-targets within the PEX11 family

    • Design multiple gRNAs to increase targeting efficiency

  • For RNAi constructs:

    • Select regions unique to PEX11-3 to avoid off-target effects on other PEX11 family members

    • Validate specificity using sequence alignment tools

    • Consider trigger length (typically 300-500 bp)

• Control Design:

  • Include wild-type controls

  • Generate complementation lines to confirm phenotype specificity

  • Consider creating knockouts/knockdowns of other PEX11 family members for comparative analysis

• Functional Redundancy Consideration:

  • Given that rice has five PEX11 family members, consider:

    • Creating multiple knockout combinations

    • Analyzing expression compensation by other family members

    • Targeting domains unique to PEX11-3

• Phenotypic Analysis Focus:

  • Based on PEX11-3's expression pattern, focus analysis on:

    • Seed development and germination processes

    • Response to ABA and oxidative stress

    • Peroxisome abundance and morphology in endosperm tissue

    • Lipid metabolism during germination

• Technical Challenges and Solutions:

  • Challenge: Lethality - if complete knockout is lethal

    • Solution: Use inducible or tissue-specific promoters

  • Challenge: Subtle phenotypes due to redundancy

    • Solution: Apply stress conditions (ABA, H₂O₂) to reveal conditional phenotypes

  • Challenge: Distinguishing direct from indirect effects

    • Solution: Combine with time-course studies and early molecular markers

• Validation Approaches:

  • Confirm mutation/knockdown by sequencing and expression analysis

  • Analyze peroxisome numbers using fluorescent markers

  • Examine peroxisomal metabolic activities

  • Measure stress response parameters

Given that OsPEX11-3 is expressed predominantly in endosperm and germinated seeds and responds to ABA and H₂O₂ , studies should particularly focus on peroxisome dynamics during seed development and germination under normal and stress conditions.

What are the most effective approaches for visualizing and quantifying changes in peroxisome dynamics in response to altered PEX11-3 expression in rice cells?

For effective visualization and quantification of peroxisome dynamics in response to altered PEX11-3 expression:

• Fluorescent Protein-Based Visualization Systems:

  • Generate transgenic rice expressing peroxisome-targeted fluorescent markers:

    • YFP-PTS1 (Yellow Fluorescent Protein with peroxisomal targeting signal type 1)

    • CFP-PEX11-3 (Cyan Fluorescent Protein fused to PEX11-3) for co-localization studies

    • Photoactivatable fluorescent proteins for tracking individual peroxisomes

  • Establish stable transgenic lines with these markers in:

    • Wild-type background

    • PEX11-3 overexpression lines

    • PEX11-3 knockout/knockdown lines

• Advanced Microscopy Techniques:

  • Confocal microscopy for high-resolution 3D imaging

  • Super-resolution microscopy for detailed morphological analysis

  • Spinning disk confocal for rapid live-cell imaging

  • TIRF microscopy for membrane dynamics

  • FRAP (Fluorescence Recovery After Photobleaching) to study protein mobility

• Quantitative Parameters to Measure:

  • Number of peroxisomes per cell

  • Size distribution of peroxisomes

  • Morphological categories (spherical, elongated, constricted)

  • Peroxisome movement dynamics (velocity, directional changes)

  • Fission and fusion events per unit time

  • Protein turnover rates using pulse-chase approaches

• Temporal Analysis Approaches:

  • Time-lapse imaging to capture the complete process of:

    • Initial peroxisome elongation (typically observed 4-8 hours after increased PEX11 expression)

    • Subsequent division events

    • Final increase in peroxisome abundance (24-48 hours after expression)

• Image Analysis and Quantification Tools:

  • Open-source software (ImageJ/Fiji with appropriate plugins)

  • Machine learning approaches for automated detection and classification

  • Custom analysis pipelines for high-throughput analysis

  • 3D reconstruction and volumetric analysis

• Correlative Approaches:

  • Combine fluorescence imaging with:

    • Electron microscopy for ultrastructural details

    • Biochemical assays for metabolic activities

    • Expression analysis to correlate with molecular changes

• Experimental Designs for Rice:

  • For seedlings: Root tips and coleoptiles are amenable to live imaging

  • For mature tissues: Develop thin-section live imaging approaches

  • For endosperm (where PEX11-3 is predominantly expressed): Establish protocols for isolated endosperm cell imaging

The visualization approach should be designed to capture the three kinetically distinct steps observed in peroxisome proliferation: initial peroxisome targeting of PEX11-3, peroxisome elongation, and increased peroxisome abundance , with particular attention to how these processes might be affected by ABA and H₂O₂ treatments that have been shown to induce OsPEX11-3 expression .