Recombinant Oryza sativa subsp. japonica Endoglucanase 10 (GLU2)

What is Recombinant Oryza sativa subsp. japonica Endoglucanase 10 (GLU2) and what are its basic properties?

Recombinant Oryza sativa subsp. japonica Endoglucanase 10 (GLU2) is a recombinant protein derived from rice, officially designated with UniProt number Q84R49 . It belongs to the family of endoglucanases (EC 3.2.1.4) and is alternatively known as Endo-1,4-beta glucanase 10 or OsGLU2 . The commercially available recombinant form is typically expressed in E. coli expression systems and has a purity of >85% as determined by SDS-PAGE .

The basic properties of this protein include:

How should GLU2 be stored and reconstituted for optimal activity in laboratory settings?

Proper storage and reconstitution are crucial for maintaining GLU2 activity. According to product specifications, the shelf life of the protein varies depending on its form and storage conditions :

• For liquid formulations: 6 months at -20°C/-80°C

• For lyophilized formulations: 12 months at -20°C/-80°C

For reconstitution, the following protocol is recommended :

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is the standard recommended concentration) for long-term storage

• Aliquot and store at -20°C/-80°C for extended storage

Important handling notes :

• Repeated freezing and thawing is not recommended as it may compromise protein activity

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

What is the substrate specificity of GLU2 and how is its enzymatic activity characterized?

GLU2 is an endoglucanase that hydrolyzes β-1,4-glucan linkages in cell wall polysaccharides. While specific substrate preference data for GLU2 itself is limited in the provided sources, research on similar endoglucanases indicates they typically show activity toward:

Enzymatic activity is typically characterized using reducing sugar assays (DNS method), viscosity reduction assays (for CMC), or HPLC analysis of released oligosaccharides . The specific activity is often reported in units/mg protein, where one unit is defined as the amount of enzyme that releases 1 μmol of reducing sugar (glucose equivalent) per minute under standard conditions.

How does temperature and pH affect GLU2 activity and stability?

While the specific temperature and pH optima for GLU2 are not explicitly stated in the provided search results, studies on similar endoglucanases from plants provide insights into their general behavior:

Based on related endoglucanases characterized in research studies, the following parameters can be expected:

It's important to note that specific optimal conditions for GLU2 should be determined experimentally for each research application, as recombinant production and specific buffer components can influence these parameters.

How does GLU2 function in cell wall modification during rice development?

GLU2, as an endoglucanase, plays a critical role in cell wall modification during rice development. Research indicates that endoglucanases within the GLU2 family, along with xyloglucan endotransglucosylases, are involved in regulating or modifying cell wall expansion .

In developing rice grains, GLU2 and related endoglucanases appear to be particularly important in the pericarp tissues. Transcriptome analysis has shown that genes encoding for endoglucanases within the GLU2 family have high relevance scores in association with cell wall-related functions in the pericarp . This suggests that GLU2 is involved in the expansion capability of the pericarp, which may influence endosperm cell expansion and ultimately affect final grain size and weight.

The molecular mechanism involves:

• Hydrolysis of β-1,4-glucan linkages in cell wall polysaccharides, leading to cell wall loosening

• Enabling cell expansion during tissue growth

• Participating in cell wall remodeling during developmental processes

Under stress conditions, such as high post-anthesis temperature, GLU2-related genes are downregulated, which may restrict cell wall assembly in the outer layers of the grain and consequently limit their expansion potential .

Are there differences in GLU2 expression patterns between indica and japonica rice subspecies?

Transcriptome analyses have revealed differences in gene expression patterns between indica and japonica rice subspecies during various developmental processes, including floret opening and closure . While the search results don't specifically address GLU2 expression differences between the subspecies, they do indicate that:

• Considerable differences exist in the expression patterns of cell wall-related genes between indica and japonica rice during developmental transitions

• These differences may contribute to the distinct morphological and physiological characteristics observed between the two subspecies

Research has shown that during floret opening and closure, differentially expressed genes shared between indica and japonica rice are involved in several metabolic pathways, including starch and sucrose metabolism and plant hormone signal transduction . Since GLU2 functions in cell wall modification, which is regulated by hormonal signals and closely related to carbon metabolism, its expression pattern may vary between the subspecies.

To conclusively determine GLU2 expression differences between indica and japonica rice, targeted gene expression analyses through qRT-PCR or RNA-seq would be necessary, focusing specifically on GLU2 expression during various developmental stages and under different environmental conditions.

How is GLU2 involved in rice immune responses during fungal pathogen infection?

Research demonstrates that endoglucanases play significant roles in plant-pathogen interactions, particularly during fungal infections of rice. While not directly focused on GLU2, the studies provide important insights into the broader role of endoglucanases in immunity:

• Pathogen-derived endoglucanases: Fungal pathogens like Magnaporthe oryzae secrete endoglucanases (MoCel12A and MoCel12B) during rice infection to target hemicellulose in the rice cell wall . These enzymes release specific oligosaccharides, including trisaccharide 3¹-β-D-Cellobiosyl-glucose and tetrasaccharide 3¹-β-D-Cellotriosyl-glucose .

• Damage-Associated Molecular Patterns (DAMPs): The oligosaccharides released through the action of these fungal endoglucanases function as DAMPs that trigger immune responses in rice . These DAMPs bind to immune receptors like OsCERK1, leading to receptor dimerization and activation of defense responses .

• Host endoglucanases and immune regulation: Plant endoglucanases like GLU2 may be involved in cell wall remodeling during immune responses. The modulation of endoglucanase activity could affect cell wall integrity and the release of signaling molecules during pathogen attack.

Interestingly, transgenic expression of the fungal endoglucanase MoCel12A in rice resulted in:

• Dwarf phenotype

• Spontaneous lesions on leaves

• Constitutive activation of immune responses

• Enhanced disease resistance to M. oryzae

This suggests that endoglucanase activity and the resulting cell wall modifications play important roles in priming and regulating rice immune responses.

What methodologies can be used to study the role of GLU2 in plant immunity?

Several sophisticated methodologies can be employed to investigate GLU2's role in plant immunity:

• Gene expression analysis:

  • qRT-PCR to quantify GLU2 expression changes during pathogen infection

  • RNA-seq to analyze transcriptome-wide changes in GLU2 and related genes

  • In situ hybridization to localize GLU2 expression in infected tissues

• Genetic manipulation:

  • CRISPR/Cas9-mediated knockout of GLU2 to assess its necessity in immune responses

  • Overexpression studies to examine if elevated GLU2 levels enhance resistance

  • Promoter-reporter fusions (e.g., GLU2pro:GUS) to visualize expression patterns during infection

• Biochemical approaches:

  • Enzymatic activity assays to measure GLU2 activity in infected versus healthy tissues

  • Immunolocalization to detect protein abundance and distribution during infection

  • Mass spectrometry to identify cell wall fragments generated by GLU2 during infection

• Interaction studies:

  • Co-immunoprecipitation to identify proteins interacting with GLU2

  • Yeast two-hybrid screening to discover potential immune-related interaction partners

  • In vitro binding assays to test direct interactions with immune receptors

A particularly powerful approach demonstrated in the literature is measuring reactive oxygen species (ROS) bursts in response to cell wall digestion by endoglucanases . For example:

This methodology could be adapted to study GLU2-mediated cell wall modifications and their impact on immune signaling.

What are the optimal conditions for expressing and purifying recombinant GLU2 for research applications?

Based on the search results and established protocols for similar endoglucanases, the following approach is recommended for expression and purification of recombinant GLU2:

Expression Systems:

• E. coli expression: Commonly used for commercial production of GLU2 . Typically utilizes vectors like pET16b with appropriate restriction sites (such as NdeI and BamHI) .

• Pichia pastoris expression: Offers advantages for eukaryotic proteins requiring post-translational modifications .

  • Vector recommendation: pPICZα with XhoI and NotI restriction sites

  • Transformation method: Electroporation after linearization with appropriate restriction enzyme

  • Expression conditions: Mini-jar fermentor with dissolved-oxygen controller

Purification Protocol:

Quality Control:

• SDS-PAGE to assess purity (>85% purity is typical)

• Western blotting for identity confirmation

• Enzyme activity assays using appropriate substrates (e.g., barley β-glucan)

• Deglycosylation analysis with endo-H if expressed in eukaryotic systems

How can GLU2 be used as a tool in studying rice cell wall structure and composition?

GLU2 can serve as a valuable enzymatic tool for studying rice cell wall structure and composition through several sophisticated applications:

• Selective degradation for structural analysis:

  • GLU2 can selectively hydrolyze β-1,4-glucan linkages in cell wall polysaccharides

  • Sequential enzymatic digestion with GLU2 followed by other cell wall-degrading enzymes helps map structural relationships between cell wall components

  • Released fragments can be analyzed by mass spectrometry to determine structural features

• Probe for accessibility of cell wall components:

  • Differential digestion patterns between cell types or developmental stages reveal changes in cell wall architecture

  • Comparing GLU2 activity on cell walls from different rice varieties helps identify structural variations

• Generation of oligosaccharides as analytical standards:

  • GLU2-generated oligosaccharides can serve as standards for cell wall analysis

  • These standards enable quantification of similar structures in cell wall hydrolysates

• Synergistic studies with other enzymes:
  Research on similar endoglucanases demonstrates synergistic effects with cellobiohydrolases . For GLU2, experimental design might include:

  Enzyme Combination	Ratio	Substrate	Expected Outcome
  GLU2 alone	100:0	PASC	Limited release of soluble oligosaccharides
  GLU2:Cellobiohydrolase	75:25	PASC	Increased hydrolysis compared to individual enzymes
  GLU2:Cellobiohydrolase	50:50	PASC	Optimal synergistic effect
  GLU2:Cellobiohydrolase	25:75	PASC	Moderate synergistic effect
  Cellobiohydrolase alone	0:100	PASC	Limited release of cellobiose

• Cell wall imaging studies:

  • Fluorescently labeled GLU2 can be used to visualize accessible β-1,4-glucan structures in situ

  • Comparing binding patterns before and after various treatments helps map cell wall modifications

These approaches collectively provide powerful tools for detailed characterization of rice cell wall architecture, which is essential for understanding developmental processes and responses to environmental stresses.

What are the emerging applications of GLU2 in biotechnology beyond basic research?

While the search results focus primarily on basic research aspects of GLU2, several emerging biotechnological applications can be extrapolated based on the properties and functions of endoglucanases:

• Biofuel production enhancement:

  • GLU2 could be utilized in enzymatic cocktails for rice straw saccharification

  • Research on cellobiose utilization pathways suggests GLU2 could be integrated with engineered yeast fermentation systems

  • Potential synergistic effects with other enzymes could improve cellulosic ethanol production efficiency

• Crop improvement strategies:

  • Modulating GLU2 expression could potentially influence grain size and weight through its effects on cell wall expansion

  • Engineering GLU2 variants with enhanced activity under stress conditions might improve crop resilience

  • Utilizing GLU2 promoter elements in genetic engineering applications for tissue-specific expression

• Biomaterial development:

  • GLU2-generated oligosaccharides could serve as building blocks for novel biomaterials

  • Controlled partial digestion of cellulosic materials could create modified polysaccharides with unique properties

  • Cell wall fragments with immune-stimulating properties could be developed as agricultural biologicals

• Analytical tool development:

  • GLU2 could be incorporated into biosensors for detecting specific cell wall structures

  • Enzyme-linked immunosorbent assays (ELISAs) utilizing GLU2 could provide sensitive methods for analyzing cell wall composition

How might climate change affect GLU2 expression and function in rice, and what are the implications for research?

Climate change, particularly rising temperatures, may significantly impact GLU2 expression and function in rice, with important implications for future research:

• Effects of high temperature on GLU2 expression:
  Research has shown that high post-anthesis temperature affects the expression of genes involved in cell wall modification, including endoglucanases in the GLU2 family . Specifically:

  • Downregulation of cell wall-related genes in the pericarp under high temperature conditions

  • Reduced expansion capability of the pericarp, potentially limiting grain size and weight

  • Altered cuticle development, which may affect water retention and stress tolerance

• Research implications and future directions:

  Climate Factor	Potential Effect on GLU2	Research Implications
  High temperature	Altered expression patterns	Need for temperature-tolerant variants
  Drought stress	Changes in cell wall composition	Study of GLU2 role in water stress adaptation
  CO₂ concentration	Modified carbon allocation patterns	Investigation of GLU2 regulation under elevated CO₂
  Combined stresses	Complex regulatory changes	Multi-stress experimental designs needed

• Methodological considerations for future research:

  • Development of field-relevant stress treatments that mimic predicted climate scenarios

  • Integration of -omics approaches to understand GLU2 regulation in changing environments

  • Creation of rice varieties with modified GLU2 expression to test adaptation potential

  • Establishment of high-throughput phenotyping systems to assess cell wall modifications under stress

• Breeding implications:

  • Identification of natural GLU2 variants with enhanced stability under temperature stress

  • Exploration of GLU2 expression differences between indica and japonica subspecies for adaptability traits

  • Potential use of GLU2 as a molecular marker for selecting climate-resilient rice varieties

Understanding how GLU2 responds to climate factors will be crucial for developing strategies to maintain rice productivity in changing environments.