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

What is PEX11-3 and what is its role in plants?

PEX11-3 (Peroxisomal membrane protein 11-3) is one of five members of the PEX11 gene family found in rice (Oryza sativa). It is an integral peroxisomal membrane protein involved in peroxisome biogenesis and proliferation. The primary function of PEX11 proteins across species is to promote peroxisome division, which is essential for maintaining proper cellular metabolism. PEX11-3 specifically contributes to peroxisome membrane dynamics, including membrane expansion, elongation, and division processes that are crucial for peroxisome multiplication .

The importance of PEX11-3 extends beyond simple organelle maintenance. Research has demonstrated that PEX11 proteins act directly in peroxisome division, and their absence can indirectly affect multiple peroxisomal metabolic pathways. Unlike early hypotheses that suggested PEX11 proteins primarily influence fatty acid oxidation, current evidence indicates they have a more fundamental role in peroxisome membrane structure and dynamics .

How is the PEX11-3 gene organized in the rice genome?

Analysis of the genomic structure of the PEX11 gene family in rice has revealed important insights into the organization of PEX11-3. The five members of the rice PEX11 gene family are distributed across chromosomes 3, 4, and 6, with PEX11-3 specifically located on chromosome 3 . Notably, PEX11-2 and PEX11-3 are closely related and appear to have undergone recent duplication events, explaining their genomic proximity and sequence similarity .

Gene structure analysis using the Gene Structure Display Server (GSDS) to compare genomic DNA sequences with cDNA sequences has shown that rice PEX11 genes, including PEX11-3, contain relatively few introns but substantial exonic regions . This genomic architecture may influence the regulation and expression patterns of PEX11-3 under different conditions and developmental stages.

How has PEX11-3 evolved in rice compared to other plant species?

Phylogenetic analysis of PEX11 family members across diverse species has provided insights into the evolutionary history of rice PEX11-3. The evolutionary relationships suggest that rice PEX11 genes have undergone significant diversification not only in their sequences but also in their expression patterns and responses to various environmental conditions .

PEX11-2 and PEX11-3 in rice appear to have arisen from a chromosome 3 insertion following a duplication event, representing a rice-specific evolutionary adaptation . This genetic diversification likely contributed to the specialized functions of these proteins in rice peroxisome biology. While the core function of PEX11 proteins in promoting peroxisome division is conserved across species from yeast to humans, the expansion of the PEX11 gene family in plants like rice suggests adaptation to plant-specific metabolic and developmental needs .

What are the tissue-specific expression patterns of PEX11-3 in rice?

PEX11-3 in rice exhibits a distinct tissue-specific expression pattern that differs from other members of the PEX11 family. According to expression profile analyses, OsPEX11-3 is predominantly expressed in endosperm and germinated seeds, with notable expression also observed in callus tissue, young seedlings, and young panicles at the secondary branch primordium stage .

This expression pattern contrasts with other rice PEX11 family members. For example, OsPEX11-1 and OsPEX11-4 show higher expression in leaf tissues, OsPEX11-2 is detected almost exclusively in germinated seeds, and OsPEX11-5 is expressed more broadly across various tissues . These differential expression patterns suggest specialized roles for each PEX11 family member in rice development and metabolism.

How is PEX11-3 expression affected by environmental stresses?

The expression of rice PEX11 genes, including PEX11-3, shows differential responses to various abiotic stresses, suggesting their involvement in stress adaptation mechanisms. OsPEX11-3 specifically demonstrates responsiveness to abscisic acid (ABA) and hydrogen peroxide (H₂O₂) treatments, indicating its potential role in stress signaling pathways .

What are the best methods for producing recombinant PEX11-3 protein?

For researchers seeking to produce recombinant Oryza sativa PEX11-3 protein, heterologous expression in E. coli has proven effective. The full-length protein (242 amino acids) can be successfully expressed with an N-terminal His tag to facilitate purification . The expression construct should contain the complete coding sequence corresponding to amino acids 1-242 of the native protein.

When expressing recombinant PEX11-3, several methodological considerations are important:

• Expression system selection: E. coli systems like BL21(DE3) are commonly used for initial expression trials, though eukaryotic systems may be preferable for certain applications requiring post-translational modifications.

• Optimal induction conditions: As a membrane protein, expression may benefit from lower induction temperatures (16-25°C) and reduced IPTG concentrations to prevent inclusion body formation.

• Solubilization strategies: Since PEX11-3 is a membrane protein, appropriate detergents should be used during extraction and purification steps.

• Purification protocol: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective for His-tagged constructs, followed by size exclusion chromatography for higher purity.

What are the recommended storage conditions for recombinant PEX11-3?

After purification, recombinant PEX11-3 protein requires specific storage conditions to maintain stability and activity. The lyophilized protein powder should be stored at -20°C or -80°C upon receipt . For working solutions, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended .

To enhance stability during storage, the addition of glycerol to a final concentration of 5-50% is advisable, with 50% being a standard recommendation for long-term storage at -20°C or -80°C . Once reconstituted, working aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can compromise protein integrity .

How does PEX11-3 contribute to peroxisome membrane dynamics?

PEX11 proteins, including PEX11-3, play crucial roles in peroxisome membrane dynamics through several coordinated mechanisms. Although the specific activities of rice PEX11-3 have not been as extensively characterized as mammalian PEX11β, studies across multiple species suggest conserved functional mechanisms .

PEX11 proteins promote peroxisome division through a multistep process:

• Membrane expansion: PEX11 proteins promote the elongation of pre-existing peroxisomal membranes, which is a prerequisite for division .

• Membrane remodeling: The N-terminal amphipathic α-helix of PEX11 proteins inserts into the outer leaflet of the peroxisomal membrane, causing membrane asymmetry and driving membrane bending and curvature .

• Recruitment of division machinery: PEX11 proteins interact with membrane adaptors like FIS1 and MFF, which recruit dynamin-related protein 1 (DRP1) to mediate membrane scission .

• Stimulation of GTPase activity: PEX11 proteins can stimulate the GTPase activity of DRP1, promoting its function in membrane division .

The self-interaction and oligomerization of PEX11 proteins are also important for their membrane remodeling activities. PEX11 forms homo-dimers and higher-order oligomers, which may act as scaffold proteins to mediate membrane bending and expansion through interactions with membrane lipids .

How does PEX11-3 function relate to peroxisomal metabolism?

Contrary to earlier hypotheses that suggested PEX11 proteins primarily influence peroxisomal metabolism, particularly fatty acid oxidation, current evidence indicates that PEX11 proteins act directly in peroxisome division, with their loss having indirect effects on multiple metabolic pathways .

Studies in mammalian systems have shown that cells lacking PEX11β display reduced peroxisome abundance even in the absence of peroxisomal metabolic substrates, supporting the view that PEX11 proteins have a primary role in peroxisome division rather than metabolism . Furthermore, PEX11β-deficient mice exhibit partial deficiencies in multiple distinct peroxisomal metabolic pathways, including ether lipid synthesis and very long chain fatty acid oxidation .

In rice, the differential expression of PEX11 family members, including PEX11-3, under various stress conditions suggests that these proteins may modulate peroxisome abundance and function in response to environmental challenges . This adaptive regulation of peroxisome dynamics could influence critical metabolic processes housed in plant peroxisomes, such as β-oxidation of fatty acids, photorespiration, and reactive oxygen species metabolism.

How can functional studies of PEX11-3 be designed to elucidate its specific role in rice?

To investigate the specific functions of PEX11-3 in rice, researchers can employ several complementary approaches:

• Gene knockout/knockdown studies: CRISPR-Cas9 genome editing or RNAi approaches can be used to generate PEX11-3-deficient rice plants. Phenotypic analysis of these plants under normal and stress conditions can reveal the physiological impact of PEX11-3 loss.

• Fluorescent protein tagging: Fusion of PEX11-3 with fluorescent proteins (e.g., GFP) can enable real-time visualization of its subcellular localization and dynamics using confocal microscopy. This approach can reveal how PEX11-3 behaves during peroxisome elongation and division events.

• Protein-protein interaction studies: Techniques such as yeast two-hybrid, co-immunoprecipitation, or proximity labeling approaches can identify interaction partners of PEX11-3, providing insights into its molecular network.

• Structure-function analysis: Site-directed mutagenesis of key domains, particularly the N-terminal amphipathic helix, can help determine which regions are critical for PEX11-3 function in peroxisome division.

• Comparative studies with other PEX11 family members: Expression of different rice PEX11 proteins in PEX11-3 knockout backgrounds can assess functional redundancy or specialization among family members.

What are the implications of PEX11-3 research for crop improvement strategies?

Understanding the functions of PEX11-3 and other PEX11 family members in rice has potential implications for crop improvement strategies, particularly in enhancing stress resilience:

• Stress tolerance enhancement: Given that PEX11-3 is responsive to ABA and H₂O₂ treatments , modulating its expression might enhance resilience to specific stresses. Peroxisomes are crucial for detoxifying reactive oxygen species generated during environmental stress, and optimized peroxisome dynamics could improve stress tolerance.

• Metabolic engineering: Peroxisomes house critical metabolic pathways relevant to plant development and stress responses. Manipulating PEX11-3 expression could potentially enhance beneficial metabolic activities like β-oxidation of fatty acids during seed germination or jasmonic acid biosynthesis during defense responses.

• Developmental improvements: The high expression of PEX11-3 in endosperm and germinated seeds suggests its importance in seed development and germination. Engineering optimal expression patterns could potentially improve seed quality or germination efficiency.

• Comparative genomics applications: Analysis of PEX11-3 variants across rice varieties with different stress tolerance profiles could identify natural variants with enhanced functions, which could be introduced into elite cultivars through breeding or genetic engineering approaches.

How does PEX11-3 function integrate with broader cellular processes in rice?

PEX11-3 function likely integrates with multiple cellular processes in rice beyond peroxisome division:

• Hormone signaling networks: The responsiveness of PEX11-3 to ABA suggests its involvement in hormone signaling cascades, potentially linking peroxisome dynamics to stress response pathways.

• Oxidative stress response: PEX11-3's response to H₂O₂ treatment indicates its potential role in oxidative stress management, which could involve coordination with antioxidant systems and stress signaling pathways.

• Developmental regulation: The predominant expression of PEX11-3 in endosperm and germinated seeds suggests a role in seed development and germination processes, potentially linking peroxisome function to nutrient mobilization during these critical developmental phases.

• Membrane contact sites: Research in other systems has shown that peroxisome membrane proteins, including PEX11 proteins, can participate in membrane contact sites with other organelles like the endoplasmic reticulum . These contacts facilitate lipid transfer and coordinate interorganellar communication.

• Metabolic adaptation: Through its effects on peroxisome abundance and distribution, PEX11-3 may indirectly influence multiple metabolic pathways housed in peroxisomes, allowing metabolic adaptation to changing environmental conditions.

What are the key unanswered questions about PEX11-3 function in rice?

Despite advances in understanding PEX11 proteins, several important questions about rice PEX11-3 remain unanswered:

• Functional specificity: What specific aspects of peroxisome biology does PEX11-3 regulate that distinguish it from other rice PEX11 family members? Does its predominant expression in endosperm and germinated seeds reflect specialized functions in these tissues?

• Regulatory mechanisms: What transcription factors and regulatory elements control PEX11-3 expression under normal and stress conditions? How is its activity modulated at the post-translational level?

• Protein interactions: What specific proteins interact with PEX11-3 in rice peroxisomes? Are these interactions similar to or distinct from those observed with other PEX11 proteins?

• Evolutionary adaptation: How has PEX11-3 evolved specifically in rice compared to other plant species, and does this reflect adaptation to specific environmental challenges or metabolic requirements?

• Physiological significance: What are the phenotypic consequences of PEX11-3 deficiency or overexpression in rice under various environmental conditions? How do these effects translate to agronomically relevant traits?

Addressing these questions will require integrated approaches combining molecular biology, cell biology, biochemistry, and whole-plant physiology studies.