Recombinant Oryza sativa subsp. japonica Expansin-B6 (EXPB6)

What is the molecular structure of rice EXPB6?

Rice EXPB6 likely possesses a two-domain structure similar to other characterized expansins. Based on structural studies of homologous proteins, EXPB6 would consist of a Domain 1 (D1) with a double-ψ β-barrel fold and Domain 2 (D2) with an Ig-like fold. These domains create a planar surface lined with aromatic and polar residues suitable for polysaccharide binding . Domain D1 shows partial conservation of catalytic sites found in family 45 glycosyl hydrolases and the MltA family of lytic transglycosylases, while Domain D2 has structural features similar to type A carbohydrate-binding domains . Unlike bacterial expansins, plant expansins like rice EXPB6 typically contain disulfide bridges that are a highly conserved feature of the D1 expansin fold, contributing to protein stability .

The specific amino acid residues in the binding surface of rice EXPB6 likely determine its substrate specificity and activity. These residues typically include conserved aromatic amino acids (tryptophan, tyrosine, phenylalanine) that form a planar platform suitable for interaction with cell wall polysaccharides. The precise arrangement of these residues varies between different expansin proteins, potentially affecting their binding preferences and biological activity.

What is the primary biological function of EXPB6 in rice development?

EXPB6 functions primarily as a non-enzymatic cell wall-loosening agent that enables cell expansion during plant growth and development. It acts by disrupting non-covalent bonds between cell wall polysaccharides, particularly in the primary cell wall, allowing turgor-driven cell expansion . This function is critical for normal plant development, particularly in rapidly growing tissues.

In rice, EXPB6 likely plays important roles in leaf growth and development, similar to its homolog ZmEXPB6 in maize, which has been shown to be essential for normal leaf growth . The protein acts specifically on the capacity of cell walls to extend, as demonstrated in in vitro assays with recombinant expansin proteins . Unlike hydrolytic enzymes, expansins do not cleave the major structural polysaccharides of the cell wall but rather disrupt the non-covalent bonding between cell wall components, particularly at the interface between cellulose microfibrils and matrix polysaccharides.

How does rice EXPB6 compare functionally to other expansin family members?

Rice EXPB6 belongs to the β-expansin subfamily, which typically shows higher activity on grass cell walls compared to dicot cell walls. This substrate preference relates to differences in cell wall composition between grasses (which have more mixed-linkage β-glucans and less pectin) and dicots .

β-expansins like EXPB6 differ from α-expansins in several ways:

• β-expansins typically show greater activity on grass cell walls, while α-expansins generally have broader specificity

• β-expansins often have more varied sequence diversity compared to the more conserved α-expansin subfamily

• The binding surface features of β-expansins are optimized for interaction with grass cell wall components

Within the β-expansin family, rice EXPB6 likely has specialized functions compared to other members, possibly related to specific developmental stages or stress responses. Comparative studies with ZmEXPB6 from maize suggest that these proteins may have similar functions in growth regulation, particularly under stress conditions .

What are optimal heterologous expression systems for producing active rice EXPB6?

Expression of active plant expansins has historically been challenging in heterologous systems. Based on successful approaches with other expansins, several expression systems can be considered:

E. coli expression system:

• Use of pET vectors with N-terminal His-tags has shown success with other expansins

• BL21(DE3) strains deficient in certain proteases may improve yields

• Optimization of expression conditions: lower temperatures (16-18°C), reduced IPTG concentration (0.1-0.5 mM), and longer induction times (overnight) to promote proper folding

• Codon optimization for E. coli expression may significantly improve yields

Yeast expression systems:

• Pichia pastoris may provide better protein folding environment due to its eukaryotic secretory pathway

• Integration of multiple gene copies can enhance expression levels

• Controlled induction using methanol-inducible promoters allows for optimization of expression conditions

What purification strategies yield highest purity and activity for recombinant rice EXPB6?

Based on successful purification of other expansins, a multi-step purification strategy is recommended:

Immobilized metal affinity chromatography (IMAC):

• Primary capture using Ni-NTA or similar affinity resin with His-tagged EXPB6

• Optimization of imidazole concentrations: low concentration (10-20 mM) in wash buffers to remove non-specific binding, and higher concentration (250-300 mM) for elution

• Addition of reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain disulfide bond integrity

Size exclusion chromatography:

• Secondary purification step to remove aggregates and achieve higher purity

• Buffer optimization to maintain protein stability (typically 25-50 mM Tris or phosphate buffer, pH 6.0-7.5, with 100-150 mM NaCl)

Metal-chelate-affinity chromatography using His-tagged proteins has proven effective for expansin purification, as demonstrated with ZmEXPB6 . Maintaining protein activity during purification requires careful attention to buffer conditions, particularly pH and ionic strength, as these factors can significantly affect expansin stability and activity.

What are the critical factors affecting stability and activity of purified rice EXPB6?

Several factors critically affect the stability and activity of purified rice EXPB6:

pH conditions:

• Expansins typically have acidic pH optima (pH 4.5-5.5) for activity

• Storage at slightly higher pH (6.0-7.0) may improve stability

• Activity assays should be conducted at the optimal pH for accurate measurements

Buffer composition:

• Addition of glycerol (10-20%) to storage buffers enhances protein stability

• Inclusion of low concentrations of non-ionic detergents (0.01-0.05% Triton X-100) may prevent aggregation

• Presence of reducing agents helps maintain disulfide bond integrity

Temperature sensitivity:

• Storage at -80°C in small aliquots to minimize freeze-thaw cycles

• Activity typically decreases significantly at temperatures above 40°C

• Flash-freezing in liquid nitrogen before storage may help preserve activity

Protein concentration:

• Higher concentrations may lead to aggregation

• Optimal concentration range of 0.1-1.0 mg/ml for storage

• Dilution immediately before activity assays to appropriate working concentrations

The exogenous application of purified ZmEXPB6 at approximately 0.3 μg/μl has shown biological activity in restoring leaf growth in salt-stressed maize , suggesting that rice EXPB6 might have similar concentration requirements for activity.

What are the standard methods for measuring rice EXPB6 activity in vitro?

Several complementary methods can be used to measure rice EXPB6 activity:

Linear Variable Differential Transducer (LVDT) measurements:

• Gold standard for measuring expansin activity

• Uses frozen-thawed plant cell wall segments under constant force

• Measures extension rate after application of expansin

• High sensitivity allows detection of even weak expansin activity

Cell wall stress relaxation assays:

• Measures changes in mechanical properties of cell walls after expansin treatment

• Quantifies relaxation time and compliance of cell walls

• Provides information on viscoelastic properties affected by expansin

Creep assays:

• Measures time-dependent extension of cell walls under constant load

• Can differentiate between immediate and long-term effects of expansin application

• Useful for comparing activities of different expansin proteins

The LVDT method has been successfully used to measure the activity of ZmEXPB6 on maize cell walls, demonstrating that the protein acts on the capacity of walls to extend . Similar approaches would be applicable to rice EXPB6, potentially with rice cell wall sections as substrates for greater relevance to the protein's natural activity.

What experimental controls are essential for rice EXPB6 activity assays?

Rigorous controls are critical for accurate interpretation of EXPB6 activity assays:

Negative controls:

• Heat-inactivated EXPB6 (typically 95°C for 10 minutes) to confirm activity requires native protein structure

• Buffer-only controls to account for mechanical effects of solution application

• Unrelated proteins of similar size to verify specificity of observed effects

• Non-wall-loosening enzymes to distinguish expansin activity from general protein effects

Positive controls:

• Commercial plant expansins if available

• Well-characterized expansins from other species (e.g., ZmEXPB6 from maize)

• α-expansins as comparative controls to assess relative activity levels

Substrate controls:

• Multiple wall types to assess substrate specificity (grass vs. dicot walls)

• Pre-treatment controls (e.g., different pH, salt concentrations) to understand environmental effects

• Time course measurements to distinguish immediate vs. delayed effects

When testing recombinant ZmEXPB6, control experiments included buffer-only treatments and demonstrated specific effects on cell wall extensibility rather than mechanical weakening . Similar control strategies would be essential for characterizing rice EXPB6 activity.

How can researchers quantify binding affinity of rice EXPB6 to different cell wall components?

Several complementary approaches can quantify binding affinity:

Co-sedimentation assays:

• Incubate purified EXPB6 with insoluble cell wall components

• Centrifuge to pellet the cell wall material

• Measure protein depletion from supernatant by SDS-PAGE or western blotting

• Calculate binding parameters from concentration-dependent binding curves

Surface plasmon resonance (SPR):

• Immobilize purified cell wall polysaccharides on sensor chips

• Flow EXPB6 solutions at various concentrations over the surface

• Measure real-time binding and dissociation kinetics

• Determine association (kon) and dissociation (koff) rate constants and equilibrium dissociation constant (KD)

Isothermal titration calorimetry (ITC):

• Measures heat changes during binding events

• Provides thermodynamic parameters (ΔH, ΔS, ΔG) in addition to binding affinity

• Requires no immobilization or labeling of interaction partners

The binding characteristics of expansins have been studied for bacterial expansin-like proteins, showing that they bind to plant cell walls, cellulose, and peptidoglycan . Similar binding studies with rice EXPB6 would help elucidate its substrate preferences and binding mechanisms.

How does salt stress affect expression and activity of rice EXPB6?

Based on studies with the maize homolog, salt stress likely affects rice EXPB6 in several ways:

Expression regulation:

• Salt stress may downregulate EXPB6 expression at both transcriptional and post-transcriptional levels

• Protein abundance may be significantly reduced in salt-stressed rice tissues

• This reduction likely contributes to growth inhibition under saline conditions

Protein activity modulation:

• Salt stress may alter the cell wall environment, affecting EXPB6 activity

• Changes in apoplastic pH due to stress may impact EXPB6 function

• Ion interactions (particularly Na+ and Cl-) might interfere with protein-substrate binding

Studies with ZmEXPB6 in maize demonstrated that this protein is lacking in growth-inhibited leaves of salt-stressed plants, and exogenous application of purified protein partially restored leaf growth . This suggests that downregulation of expansin proteins is a significant factor in growth inhibition under salt stress, likely applying to rice EXPB6 as well.

Can exogenous application of recombinant rice EXPB6 mitigate stress-induced growth inhibition?

Based on findings with ZmEXPB6 in maize, exogenous application of recombinant rice EXPB6 has potential to partially restore growth in stress-affected plants:

Application methods:

• Foliar application with appropriate surfactants (e.g., 0.1% Silwet) to enhance penetration

• Buffer composition typically includes 25 mM Tris-HCl at pH 6.0-6.5

• Protein concentration of 0.1-0.3 μg/μl has shown effectiveness with related expansins

• Multiple applications may be required for sustained effects

Expected outcomes:

• Partial restoration of leaf growth rates

• Improvements in cell expansion without affecting division rates

• Enhanced recovery from stress-induced growth inhibition

• More significant effects in actively growing tissues

Experiments with ZmEXPB6 showed that exogenous application of the purified protein (0.3 μg/μl) on growth-reduced leaves partially restored leaf growth in salt-stressed maize . Similar approaches with rice EXPB6 could potentially mitigate growth inhibition in rice under stress conditions.

What comparative proteomics approaches can identify stress-related changes in rice EXPB6 levels?

Several complementary proteomics approaches can effectively monitor EXPB6 protein levels:

Two-dimensional gel electrophoresis with Western blotting:

• Separates proteins based on both isoelectric point and molecular weight

• Allows visualization of post-translational modifications and isoforms

• Western blotting with specific antibodies enables precise identification

• Has been successfully used to identify changes in ZmEXPB6 under salt stress

Liquid chromatography-mass spectrometry (LC-MS/MS):

• Higher sensitivity than gel-based methods

• Enables absolute quantification using isotope-labeled standards

• Can detect lower-abundance proteins and post-translational modifications

• Allows for multiplexing with techniques like TMT or iTRAQ labeling

Selected reaction monitoring (SRM) mass spectrometry:

• Targeted approach for specific proteins of interest

• High sensitivity and reproducibility for quantification

• Allows monitoring of specific EXPB6 peptides across multiple samples

• Ideal for time-course experiments tracking stress responses

Gel-based two-dimensional proteomics and two-dimensional Western blotting have been effectively used to analyze ZmEXPB6 abundance against salt stress . These approaches can be adapted for rice EXPB6, potentially with rice-specific antibodies for enhanced detection sensitivity.

How does rice EXPB6 compare structurally and functionally to bacterial expansin-like proteins?

Rice EXPB6 shares fundamental structural elements with bacterial expansin-like proteins but differs in several important ways:

Structural similarities:

Key differences:

• Plant expansins contain disulfide bridges that are absent in bacterial homologs

• Rice EXPB6 likely has an N-terminal extension absent in bacterial proteins

• Activity levels differ significantly, with plant expansins typically showing higher cell wall loosening activity

• Substrate specificity varies, with plant expansins better adapted to plant cell wall components

What are key differences between rice EXPB6 and maize ZmEXPB6 that might affect experimental applications?

Though both are β-expansins from cereal crops, rice EXPB6 and maize ZmEXPB6 likely differ in several aspects:

Sequence and structural differences:

• Variations in key binding residues may affect substrate preferences

• Different post-translational modification patterns could impact activity and stability

• Potential differences in disulfide bond arrangements affecting protein stability

Functional differences:

• Potentially different pH optima reflecting adaptation to different cell wall environments

• Varied thermal stability profiles affecting experimental handling requirements

• Different binding affinities for specific cell wall polysaccharides

• Varied activity levels on different grass cell wall substrates

Regulatory differences:

• Potentially different expression patterns during development

• Varied responses to environmental stresses

• Species-specific regulation mechanisms

ZmEXPB6 has been shown to be downregulated under salt stress conditions, with exogenous application partially restoring leaf growth . Comparative studies between rice EXPB6 and maize ZmEXPB6 would help elucidate their shared and unique functional characteristics.

What structural features distinguish rice β-expansins from α-expansins?

Several structural features distinguish rice β-expansins like EXPB6 from α-expansins:

Domain arrangement:

Sequence conservation:

• Higher sequence diversity within β-expansin family compared to α-expansins

• Different patterns of conserved residues in binding regions

• Specialized motifs adapted for grass cell wall components in β-expansins

Functional properties:

• β-expansins generally show higher activity on grass cell walls

• Different pH optima between subfamilies

• Varied responses to ions and other environmental factors

The structural differences between α- and β-expansins reflect their evolutionary adaptation to different plant cell wall compositions, with β-expansins like rice EXPB6 specialized for grass cell walls that contain more mixed-linkage β-glucans and less pectin than dicot cell walls .

How can CRISPR-Cas9 genome editing be optimized for studying rice EXPB6 function?

CRISPR-Cas9 approaches offer powerful tools for studying rice EXPB6 function:

Guide RNA design optimization:

• Use of rice-specific promoters for gRNA expression (e.g., U3 or U6 promoters)

• Careful selection of target sites with minimal off-target potential

• Design of multiple gRNAs targeting different exons for higher knockout efficiency

• Verification of guide efficiency using in silico prediction tools specific for rice genome

Cas9 expression strategies:

• Codon-optimized Cas9 for efficient expression in rice

• Use of monocot-optimized promoters (e.g., maize ubiquitin promoter)

• Development of inducible or tissue-specific Cas9 expression systems

• Temperature-optimized Cas9 variants for improved activity in rice

Precise editing approaches:

• Generation of complete knockouts through frameshift mutations

• Creation of specific amino acid substitutions using homology-directed repair

• Development of conditional alleles for temporal control of EXPB6 function

• Domain-specific mutations to dissect structure-function relationships

Transformation and screening methods:

• Optimization of Agrobacterium-mediated transformation for rice calli

• Development of efficient protoplast-based screening systems

• Use of fluorescent reporters for tracking transformation efficiency

• High-throughput screening methods for identifying edited plants

These approaches would enable detailed functional analysis of rice EXPB6, including its role in growth regulation, stress responses, and cell wall modification.

What protein engineering strategies can enhance rice EXPB6 stability and activity?

Several protein engineering strategies can potentially enhance EXPB6 properties:

Stability enhancement:

• Introduction of additional disulfide bridges based on structural models

• Rational design of salt bridges to improve pH and temperature stability

• Optimization of surface charge distribution to reduce aggregation

• Identification and modification of protease-sensitive regions

Activity enhancement:

• Targeted mutations in the binding surface to increase substrate affinity

• Domain swapping with highly active expansins from other species

• Modification of pH optimum through charge distribution alterations

• Engineering of loops involved in substrate recognition

Expression optimization:

• Codon optimization for heterologous expression

• Addition of solubility-enhancing tags or fusion partners

• Modification of N-terminal signal sequences for improved secretion

• Removal of problematic sequence motifs that affect expression

The discovery of bacterial expansins like EXLX1 has opened opportunities for protein engineering, as these proteins can be more readily expressed in heterologous systems than plant expansins . Similar approaches could be applied to rice EXPB6, potentially using structural information from these related proteins as a guide.

What advanced imaging techniques can visualize rice EXPB6 activity on cell walls?

Several cutting-edge imaging techniques can provide insights into EXPB6 activity:

Atomic force microscopy (AFM):

• Enables nanoscale visualization of cell wall structural changes

• Can measure mechanical properties before and after EXPB6 treatment

• Allows for time-lapse imaging of wall loosening processes

• Can be combined with fluorescence microscopy for correlative imaging

Super-resolution microscopy:

• Stimulated emission depletion (STED) microscopy for visualizing labeled EXPB6 localization

• Single-molecule localization microscopy (PALM/STORM) for precise protein tracking

• Structured illumination microscopy (SIM) for enhanced resolution of wall structures

• Expansion microscopy for physical magnification of samples

Live-cell imaging approaches:

• Fluorescently tagged EXPB6 variants to track protein localization

• FRET-based sensors to detect conformational changes during binding

• Correlative light and electron microscopy for ultrastructural context

• Light sheet microscopy for rapid 3D imaging with minimal photodamage

These techniques would provide unprecedented insights into the mechanism of action of rice EXPB6, revealing how it interacts with cell wall components and induces wall loosening at the molecular and nanoscale levels.