Recombinant Oryza sativa subsp. japonica Putative expansin-A27 (EXPA27)

What is the structural composition of rice expansin-A27 and how does it compare to other expansins?

Expansins in rice, including EXPA27, typically contain several characteristic structural elements that are highly conserved across the expansin family. Alpha-expansins are characterized by a series of conserved cysteine residues in the N-terminal half of the protein, a histidine-phenylalanine-aspartate (HFD) motif in the central region, and a series of tryptophan residues near the carboxyl terminus . Bioinformatic analyses of rice expansins have revealed that they typically have a molecular weight of approximately 28 kDa with basic isoelectric points (around pI 9.40) and significant levels of alanine and glycine .

The protein structure consists of two domains: an N-terminal domain with some homology to glycoside hydrolase family 45 proteins (though lacking hydrolytic activity) and a C-terminal domain that resembles a grass pollen allergen. These domains work together to facilitate cell wall loosening without enzymatic breakdown of the major structural polysaccharides.

In which tissues and developmental stages is EXPA27 predominantly expressed?

While specific expression data for EXPA27 is limited in the provided literature, research on rice expansins shows tissue-specific and developmental expression patterns. Research on related expansins indicates expression in multiple tissues with varying levels of abundance. For example, OsEXPA7 is highly expressed in the shoot apical meristem, roots, and leaf sheaths, with particularly strong expression in vascular tissues .

Many rice expansins show expression patterns correlated with actively growing tissues. In deepwater rice, various alpha-expansin genes are expressed in internodes and leaves (5 genes), coleoptiles (3 genes), and roots (9 genes), with high transcript levels specifically in the growing regions of these organs . Five alpha-expansin genes were found exclusively in roots . This suggests that different expansins, including potentially EXPA27, may have specialized roles in specific tissues and developmental processes.

What are the known functions of expansins in rice plant growth and development?

Expansins play crucial roles in various aspects of plant growth and development through their ability to facilitate cell wall loosening. They mediate cell expansion during growth, which is particularly important in rapidly elongating tissues. In deepwater rice, expansins are associated with long-term extension of isolated cell walls and contribute to growth regulation .

Specific functions include:

• Cell wall loosening and cell elongation

• Regulation of root development and architecture

• Involvement in shoot elongation and leaf expansion

• Mediation of coleoptile growth during early seedling development

• Contribution to vascular tissue development

Additionally, certain expansins like OsEXPA7 are involved in stress responses, particularly salt stress tolerance, by coordinating sodium transport, reactive oxygen species (ROS) scavenging, and cell-wall loosening .

How does overexpression of EXPA27 affect rice morphology and stress tolerance compared to other expansin family members?

While specific data on EXPA27 overexpression is not provided in the search results, research on related expansins offers valuable insights. Overexpression of OsEXPA7, for example, results in significant morphological changes including increased biomass in both shoots and roots, longer shoot and root lengths, and higher primary root numbers compared to wild-type plants .

Under salt stress conditions, OsEXPA7-overexpressing lines demonstrate remarkable stress tolerance benefits:

• Lower leaf damage

• Decreased electrical conductivity (indicating reduced cell damage)

• Higher chlorophyll content retention

• Improved survival rates under 150 mM salt stress

• Altered ion homeostasis with decreased Na+ and accumulated K+ in leaves and roots

• Enhanced antioxidant activity with lower reactive oxygen species (ROS) accumulation

These findings suggest that strategic overexpression of expansins like EXPA27 might similarly enhance stress tolerance through modification of cell wall properties, alterations in root and shoot architecture, and changes in stress response pathways.

What molecular mechanisms underlie the role of expansins in salt stress tolerance in rice?

The molecular mechanisms through which expansins confer salt stress tolerance involve multiple integrated pathways. Research on OsEXPA7 reveals several key mechanisms that may be relevant to understanding EXPA27 function:

• Ion homeostasis regulation: Expansin overexpression appears to modulate Na+ and K+ distribution, with decreased sodium and increased potassium levels in both leaves and roots, which is critical for salt tolerance .

• Enhanced antioxidant activity: Under salt stress, expansin-overexpressing lines show lower reactive oxygen species (ROS) accumulation and increased antioxidant activity compared to wild-type plants .

• Transcriptional reprogramming: Differential gene expression analysis reveals that expansin overexpression affects genes involved in:

  • Cation exchange

  • Auxin signaling

  • Cell wall modification

  • Transcriptional regulation

• Sodium transporter upregulation: Notably, salt overly sensitive 1 (SOS1), a sodium transporter, was highly upregulated in expansin-overexpressing lines, suggesting direct involvement in sodium exclusion mechanisms .

• Vascular tissue modification: Structural alterations in root and leaf vasculature observed in expansin-overexpressing lines suggest that changes in transport efficiency may contribute to improved stress tolerance .

What is the relationship between expansin gene expression and phytohormone signaling during stress responses?

Expansin expression and function appear to be integrated with phytohormone signaling networks, particularly in stress response contexts. Research indicates that some expansin genes in rice are responsive to gibberellin (GA) treatment, with five alpha-expansin genes showing induced expression in the internode following GA application . This hormone-responsive expression suggests that expansins may function as downstream effectors in hormone-mediated growth regulation and stress adaptation.

The relationship extends beyond gibberellin, as transcriptional analysis of OsEXPA7-overexpressing lines revealed differential expression of genes involved in auxin signaling . This indicates a potential interaction between expansin function and auxin-mediated processes, which are critical for root development and architecture—key factors in stress adaptation.

Additionally, the induction of some expansin genes by wounding suggests potential crosstalk with jasmonate signaling pathways . These interconnections between expansin expression and various hormone signaling networks provide potential targets for engineering stress-adaptive responses through manipulation of expansin gene expression.

What are the optimal protocols for heterologous expression and purification of recombinant rice EXPA27?

For successful heterologous expression and purification of recombinant rice expansins such as EXPA27, researchers should consider the following methodological approach:

• Expression system selection: Due to the plant origin and potential glycosylation requirements, Pichia pastoris or insect cell expression systems often yield better results than bacterial systems. For bacterial expression, consider specialized E. coli strains optimized for eukaryotic proteins with disulfide bonds (e.g., Origami, SHuffle).

• Construct design considerations:

  • Include the mature protein sequence without the native signal peptide

  • Add appropriate affinity tags (His6 or GST) for purification

  • Consider codon optimization for the expression host

  • Engineer cleavable tags if native protein is required for activity assays

• Expression optimization:

  • Test multiple induction conditions (temperature, inducer concentration, duration)

  • Screen for soluble protein expression rather than inclusion bodies

  • Consider fusion partners that enhance solubility (SUMO, MBP, TRX)

• Purification strategy:

  • Implement a two-step purification approach using affinity chromatography followed by size exclusion

  • Include reducing agents to maintain proper disulfide bond formation

  • Use buffers that mimic plant cell wall pH environment (slightly acidic)

  • Consider detergent addition during purification to maintain stability

• Functional validation:

  • Conduct cell wall extension assays using heat-inactivated cell walls

  • Perform binding assays with cellulose and other cell wall components

  • Verify protein folding using circular dichroism spectroscopy

What experimental design is most effective for analyzing EXPA27 involvement in abiotic stress responses?

An effective experimental design for analyzing EXPA27 involvement in abiotic stress responses should incorporate multiple approaches:

• Genetic manipulation strategies:

  • Generate overexpression lines using constitutive (e.g., CaMV 35S) and tissue-specific promoters

  • Create knockout/knockdown lines using CRISPR-Cas9 or RNAi

  • Develop promoter-reporter fusions (GUS, GFP) to monitor expression patterns under stress

• Stress treatment design:

  • Apply multiple stress intensities (e.g., 100, 150, 200 mM NaCl for salt stress)

  • Implement both short-term (hours to days) and long-term (weeks) stress treatments

  • Consider combinatorial stresses (e.g., salt + drought) to mimic field conditions

  • Include recovery periods to assess resilience

• Comprehensive phenotyping:

  • Monitor growth parameters (shoot/root length, biomass, root architecture)

  • Measure physiological indicators (chlorophyll content, photosynthetic efficiency)

  • Assess cell damage markers (electrolyte leakage, ROS accumulation)

  • Analyze ion content (Na+, K+) in different tissues

• Molecular analyses:

  • Perform transcriptomic analysis (RNA-seq) comparing wild-type and modified lines

  • Conduct targeted expression analysis of stress-responsive genes

  • Analyze protein-protein interactions to identify partners

  • Examine changes in cell wall composition and architecture

• Integrative data analysis:

  • Correlate expression data with phenotypic outcomes

  • Compare results across different developmental stages

  • Validate key findings using multiple independent transgenic lines

This multi-faceted approach would allow comprehensive characterization of EXPA27's role in stress responses, similar to studies conducted with OsEXPA7 that revealed its involvement in salt stress tolerance .

How can researchers effectively analyze the cell wall-modifying activities of EXPA27 in vitro and in planta?

Analyzing the cell wall-modifying activities of expansins requires specialized techniques that address both in vitro biochemical activities and in planta effects:

In vitro analysis methods:

• Cell wall extension assays:

  • Prepare cell wall specimens from growing tissues (e.g., rice coleoptiles)

  • Measure extension using a constant-load extensometer

  • Compare extension rates with and without purified EXPA27

  • Test pH-dependency (typically active at acidic pH 4.5-6.0)

• Binding assays:

  • Assess binding to cellulose, hemicellulose, and pectin components

  • Use isothermal titration calorimetry to determine binding parameters

  • Conduct competitive binding assays with other expansins

  • Analyze binding using recombinant protein domains separately

• Polymer mechanics analysis:

  • Measure changes in cell wall creep and stress relaxation

  • Use atomic force microscopy to detect nanoscale changes in wall properties

  • Analyze effects on wall polymer interactions using solid-state NMR

In planta analysis methods:

• Cell wall architecture assessment:

  • Prepare transverse and longitudinal sections of tissues from wild-type and transgenic plants

  • Analyze cell size, shape, and wall thickness using microscopy

  • Perform immunolabeling to detect changes in wall polymer distribution

  • Use electron microscopy to examine ultrastructural changes

• Growth kinematics:

  • Track growth patterns using time-lapse imaging

  • Measure cell expansion rates in specific tissues

  • Correlate expansion with EXPA27 expression using reporter lines

• Wall composition analysis:

  • Fractionate cell wall components (cellulose, hemicellulose, pectin)

  • Determine sugar composition by gas chromatography

  • Assess changes in wall polymer molecular weight distribution

  • Measure alterations in cross-linking between wall components

• Mechanical properties testing:

  • Analyze tissue extensibility using mechanical stretching devices

  • Measure wall elasticity and plasticity

  • Determine breaking strength and other mechanical parameters

  • Compare properties across different tissues and developmental stages

Research on OsEXPA7 has demonstrated that morphological analysis can reveal structural alterations in root and leaf vasculature in overexpressing lines, indicating meaningful changes in cell wall properties that correlate with stress tolerance .

What emerging technologies could advance our understanding of EXPA27 function in rice?

Several cutting-edge technologies hold promise for advancing our understanding of expansin function in rice:

• Single-cell transcriptomics and proteomics:

  • Map expansin expression at unprecedented resolution

  • Identify cell-specific responses to expansin activity

  • Reveal tissue-specific co-expression networks

• CRISPR-based approaches:

  • Implement multiplexed editing to target multiple expansin family members simultaneously

  • Use base editing for precise modification of key amino acids

  • Apply CRISPRa/CRISPRi for temporal control of expansin expression

  • Create conditional alleles to study essentiality

• Advanced imaging techniques:

  • Utilize super-resolution microscopy to visualize expansin localization in cell walls

  • Apply expansion microscopy to reveal nanoscale distribution patterns

  • Implement live-cell imaging with fluorescently tagged expansins

  • Use correlative light and electron microscopy to connect structure and function

• Computational approaches:

  • Implement molecular dynamics simulations of expansin-wall interactions

  • Apply machine learning to predict expansin targets and functions

  • Develop models of cell wall mechanics incorporating expansin activity

• Systems biology integration:

  • Create comprehensive models connecting expansin activity to growth phenotypes

  • Map expansin interactions across the stress response network

  • Integrate multi-omics data to reveal emergent properties

These technologies would help address key questions about the specific functions of EXPA27 and other expansins in rice development and stress responses.

How might comparative studies across rice varieties enhance our understanding of expansin evolution and functional diversity?

Comparative studies across rice varieties offer significant potential for understanding expansin evolution and functional diversity:

• Evolutionary analysis benefits:

  • Identify conserved versus rapidly evolving expansin family members

  • Detect signatures of selection during domestication

  • Trace expansin family expansion and subfunctionalization

  • Recognize lineage-specific adaptations in stress-responsive expansins

• Methodological approach:

  • Sequence expansin gene families across diverse rice varieties

  • Compare expression patterns in matching tissues and developmental stages

  • Analyze promoter regions to identify cis-regulatory evolution

  • Examine protein sequence variations in functional domains

  • Test activity of orthologous expansins from different varieties

• Functional implications:

  • Connect sequence variations to differences in stress tolerance

  • Identify superior alleles for breeding applications

  • Understand the genetic basis for growth differences between varieties

  • Map expansin contribution to adaptive traits in different environments

Research has already demonstrated variety-specific differences in expansin function, as seen in the study of boron toxicity tolerance where different rice cultivars (IR36, Nekken 1, Wataribune, Nipponbare) showed varying responses due to allelic differences in related pathways . Similarly, expression analysis revealed that expansin gene expression levels differed between sensitive cultivars (Wataribune, IR36, Kasalath) and tolerant cultivars (Koshihikari, Nipponbare) .

What are the potential applications of EXPA27 modification for improving rice resilience to combined abiotic stresses?

Modification of expansins like EXPA27 presents multiple avenues for improving rice resilience to combined abiotic stresses:

• Potential applications:

  • Develop varieties with enhanced tolerance to multiple concurrent stresses

  • Improve recovery capacity after stress events

  • Engineer modified root architecture for drought and salinity tolerance

  • Enhance seedling establishment under adverse conditions

  • Optimize biomass production in marginal environments

• Strategic approaches:

  • Target tissue-specific expression using stress-inducible or tissue-specific promoters

  • Stack multiple beneficial expansin modifications

  • Combine expansin engineering with complementary stress tolerance mechanisms

  • Fine-tune expression levels to balance growth and stress adaptation

• Specific stress combinations addressable:

  • Drought + heat stress

  • Salinity + flooding

  • Nutrient deficiency + drought

  • Cold + high light intensity

Research on OsEXPA7 has already demonstrated that overexpression can enhance tolerance to salt stress through multiple mechanisms, including improved ion homeostasis, enhanced antioxidant activity, and structural modifications . These findings suggest that strategic modification of EXPA27 and other expansins could similarly contribute to improved stress resilience, particularly when engineered to address multiple concurrent stresses that are increasingly common with climate change.

What are the key considerations for designing gene editing experiments targeting EXPA27?

Successful gene editing of EXPA27 requires careful experimental design addressing several key considerations:

• Target site selection criteria:

  • Prioritize coding regions with essential functions (e.g., the conserved HFD motif)

  • Avoid regions with high similarity to other expansin family members

  • Consider targeting regulatory regions for expression modulation

  • Verify target uniqueness using whole-genome alignments

  • Check for potential off-target sites with similar sequences

• Guide RNA design parameters:

  • Optimize GC content (40-60%) for stable guideRNA-DNA interactions

  • Evaluate on-target efficiency scores using prediction algorithms

  • Avoid seed region polymorphisms between rice varieties

  • Check secondary structure formation potential

  • Design multiple guides targeting the same region

• Delivery method selection:

  • Callus-based Agrobacterium-mediated transformation

  • Protoplast transfection for initial validation

  • Biolistic delivery for recalcitrant varieties

  • Consider ribonucleoprotein (RNP) delivery to minimize off-targets

• Validation strategy:

  • Implement targeted sequencing of the edited region

  • Perform whole-genome sequencing to detect off-target mutations

  • Conduct phenotypic analysis of multiple independent lines

  • Verify altered expression or protein function

• Special considerations for rice:

  • Account for rice variety-specific transformation efficiency

  • Consider regeneration ability of the selected variety

  • Plan for homozygosity screening in subsequent generations

  • Design strategies for distinguishing edited plants from wild-type

The research on boron tolerance genes demonstrates how targeted genetic modifications can significantly alter stress responses in rice, providing a model for EXPA27 modification strategies .

How should researchers address the challenge of functional redundancy within the expansin gene family?

Addressing functional redundancy within the large expansin gene family requires systematic approaches:

• Comprehensive family analysis:

  • Catalog all expansin genes and their expression patterns

  • Identify co-expressed family members

  • Group expansins by sequence similarity and putative function

  • Map spatial and temporal expression domains

• Multiplex gene editing strategies:

  • Design CRISPR systems targeting conserved regions in multiple family members

  • Create higher-order mutants by crossing single mutants

  • Implement inducible CRISPR systems for temporal control

  • Target shared regulatory elements affecting multiple expansins

• Dominant-negative approach:

  • Express modified expansins that interfere with native protein function

  • Design proteins that compete for binding sites but lack activity

  • Target conserved protein-protein interaction domains

• Tissue-specific approach:

  • Focus on tissues with limited expansin expression

  • Target expansins with unique expression patterns

  • Use tissue-specific promoters for rescue experiments

• Quantitative phenotyping:

  • Implement high-resolution growth analysis to detect subtle phenotypes

  • Measure cell-level responses rather than whole-plant effects

  • Apply stress conditions that may reveal conditional redundancy

  • Develop sensitive assays for cell wall properties