Recombinant Oryza sativa subsp. japonica Expansin-A1 (EXPA1)

How do rice expansins differ structurally and functionally from other plant species?

Rice α-expansins share approximately 55% average amino acid identity between mature proteins, while rice β-expansins share about 51% identity. The sequence identity between α- and β-expansins is much lower at approximately 21% .

Although α- and β-expansins have similar rheological effects on cell walls (inducing creep and stress relaxation), they exhibit different substrate specificities:

• α-expansins work effectively on cell walls in dicots and non-graminaceous monocots

• β-expansins have stronger activity on cell walls of graminaceous monocots (like rice) and only marginal effects on dicot cell walls

This distinction is particularly important when designing experiments with recombinant expansins from different species. Despite these differences, the rice genome contains 34 α-expansin genes, indicating their significant role in rice development .

What are the patterns of EXPA1 expression in rice tissues and how is it regulated?

EXPA1 expression in rice shows specific spatial and temporal patterns:

• Root expression: EXPA1 is expressed in root tissues, particularly in the columella/lateral root cap. It has been implicated in lateral root formation, particularly in pericycle founder cell radial expansion .

• Hormone regulation: EXPA1 expression is regulated by both cytokinin and auxin. Treatment with 5 μM 6-benzylaminopurine (BAP, a cytokinin) transiently upregulates EXPA1 expression 3-4 fold over a 4-hour period. Treatment with 5 μM 1-naphthaleneacetic acid (NAA, an auxin) induces a stronger and more continuous increase, reaching 5-10 fold at 4 hours .

• Developmental regulation: EXPA1 expression is indirectly regulated by auxin through the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) - AUXIN RESPONSE FACTOR (ARF) signaling pathway. Specifically, EXPA1 is a direct target of cytokinin-responsive ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and its homologs ARR10 and ARR12 .

Researchers studying EXPA1 expression should consider these tissue-specific and hormone-dependent patterns when designing experiments, particularly when choosing appropriate tissues and treatment conditions.

What is the genomic context of EXPA1 in rice and how does it compare across rice subspecies?

EXPA1 is one of 58 expansin genes in the rice genome, which are classified into 4 subfamilies: α-expansins (34 members), β-expansins (19 members), expansin-like A (4 members), and expansin-like B (1 member). The expansin genes are distributed across 10 of the 12 rice chromosomes, with several subfamily members forming clusters .

The genomic structure of rice expansin genes varies by subfamily:

• Most α-expansins (including EXPA1) contain 1-2 introns

• β-expansins typically contain 3 introns

• Expansin-like A and B genes contain 4 introns each

This genomic organization provides important context for researchers designing gene expression studies or genetic modifications of EXPA1.

Regarding subspecies differences, significant research has demonstrated that natural variation and introgression between rice subspecies (indica and japonica) has been an important driving force in rice evolution. While specific data on EXPA1 variation between subspecies is limited in the provided search results, other genes show clear patterns of subspecies-specific selection. For example, genes like CTB3 (cold tolerance) have been subject to positive selection in temperate japonica cultivars, with significantly lower nucleotide diversity (π = 0.000239) compared to wild rice (π = 0.001507) .

What are the optimal expression systems and purification methods for producing recombinant EXPA1?

Based on the available data and best practices in recombinant protein production:

Expression systems:
Several expression systems can be used to produce recombinant EXPA1, each with advantages and limitations:

• E. coli expression system:

  • Most commonly used for recombinant EXPA1 production

  • Advantages: High yield, cost-effective, rapid production

  • Limitations: May lack proper post-translational modifications, protein folding issues possible

  • Recommended strain: BL21(DE3) with codon optimization for plant proteins

• Yeast expression systems (Pichia pastoris or Saccharomyces cerevisiae):

  • Advantages: Better protein folding than E. coli, some post-translational modifications

  • Yield typically lower than E. coli but higher than mammalian systems

• Baculovirus expression system:

  • Advantages: Proper folding and post-translational modifications

  • Limitations: More complex and expensive than bacterial systems

Purification strategy:

• Affinity chromatography using His-tag or GST-tag

• Size exclusion chromatography for higher purity

• For properly folded EXPA1, consider using cellulose affinity pulldown, as expansins contain cellulose-binding domains

A typical protocol should aim for ≥85% purity as determined by SDS-PAGE. The final product may be formulated as lyophilized powder or in a suitable buffer solution .

What functional assays are suitable for evaluating recombinant EXPA1 activity?

Several methodologies can be employed to assess EXPA1 activity:

• Cell wall extension assays:

  • Measure the ability of EXPA1 to induce creep in isolated cell walls under acidic conditions

  • Methodology: Mount heat-inactivated cell wall specimens in extensometer, apply constant force, measure extension rate before and after EXPA1 addition

• Atomic Force Microscopy (AFM):

  • Quantitative assessment of cell wall mechanical properties

  • Can measure the effect of EXPA1 on Young's modulus of cell walls

  • Studies have shown that EXPA1 overexpression affects cell wall stiffness in Arabidopsis root transition zone

• Brillouin Light Scattering (BLS):

  • Non-invasive in vivo quantitative assessment of cell wall viscoelasticity

  • Particularly useful for time-course experiments

  • Has been validated to detect both cell wall stiffening and softening

  • Example: In Arabidopsis, BLS showed that EXPA1 overexpression increases root cell wall stiffness

• Fourier-Transform Infrared (FT-IR) Spectroscopy:

  • Provides information on cell wall composition changes induced by EXPA1

  • Can identify specific polymer and functional group modifications

  • Example: FT-IR measurements showed that EXPA1 overexpression in Arabidopsis leads to rapid (within 3 hours) demethylesterification of pectin at around 1,730 cm^-1 spectral region

When comparing methodologies, researchers should consider that different techniques may yield complementary information about EXPA1's effects on cell wall properties.

How does EXPA1 overexpression affect cell wall composition and mechanical properties?

Contrary to what might be expected for a cell wall loosening protein, research has shown that EXPA1 overexpression can actually increase cell wall stiffness under certain conditions. This represents a significant finding that challenges our understanding of expansin function.

Cell wall mechanical properties:

• AFM and BLS measurements both showed that EXPA1 overexpression increases cell wall stiffness in Arabidopsis root transition zone

• This stiffening effect is observable at both indentation speeds measured by AFM (order of seconds) and at GHz frequencies through Brillouin technique

• The effect is consistent regardless of whether stiffness is measured on anticlinal or periclinal cell walls, and is observed at both pH 5.8 and pH 4.0

Cell wall composition changes:
FT-IR spectroscopy revealed significant and rapid changes in cell wall composition after EXPA1 overexpression:

• Decreased absorbance at around 1,730 cm^-1 (assigned to ester linkages) indicates lower pectin methylesterification in EXPA1 overexpressors

• This change is detectable as early as 3 hours after induction and becomes more pronounced after 7 days

• The rapid demethylesterification of pectin is linked to altered cell wall viscoelasticity

Transcriptional changes:
RNA-seq analysis of EXPA1 overexpression revealed cascading effects on cell wall-related genes:

• 336 genes upregulated and 287 genes downregulated after 3 hours of EXPA1 induction

• Significantly enriched GO terms included cell wall loosening (GO0009828), modification (GO0009827), and organization (GO0009664)

• Notable upregulation of other expansin genes, especially EXPA2, which reached similar expression levels as the induced EXPA1

• Upregulation of xyloglucan:xyloglucosyl transferases (XTHs), particularly XTH5, XTH7, XTH11, and XTH32

These findings suggest that EXPA1 initiates a complex remodeling of cell wall composition and architecture, rather than simply loosening the cell wall.

What is the role of EXPA1 in root development and how does it interact with plant hormones?

EXPA1 plays a critical role in root development, particularly in lateral root formation:

Role in lateral root initiation:

• EXPA1 functions as an early marker of pericycle founder cell radial expansion

• It is required for proper radial expansion of pericycle cells, which licenses the correct positioning of first anticlinal divisions during lateral root initiation

• Analysis of expa1 mutants demonstrates that EXPA1 is necessary for proper pericycle width, which is crucial for triggering asymmetric pericycle cell divisions

Hormonal regulation:

• EXPA1 expression is regulated by both cytokinin and auxin, key hormones in root development

• Cytokinin: EXPA1 is a direct target of cytokinin-responsive ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and its homologs ARR10 and ARR12

• Auxin: EXPA1 shows stronger response to auxin than cytokinin, with mRNA levels increasing continuously up to 5-10 fold after 4 hours of auxin treatment

• AUXIN RESPONSE FACTOR 5 (ARF5) may directly regulate EXPA1 as indicated by DNA affinity purification sequencing

• EXPA1 expression depends on functional IAA14- or IAA3-dependent signaling but is regulated indirectly by auxin

Cell wall modifications:

• EXPA1-mediated cell wall changes are important for proper positioning of asymmetric cell divisions in lateral root formation

• These changes include localized cell wall loosening that facilitates radial expansion of pericycle founder cells

• Raman spectroscopy of pericycle cell walls in expa1-1 mutants shows altered cell wall composition, providing evidence for EXPA1's role in cell wall remodeling during asymmetric pericycle expansion

These findings highlight the importance of EXPA1 in coordinating hormonal responses with cell wall modifications during lateral root development.

How do functions of EXPA1 compare with other expansin family members in rice?

Rice contains a diverse expansin gene family with distinct expression patterns and functions:

Expression patterns of rice expansins:

Functional comparisons:

These differences highlight the functional specialization within the expansin family despite their structural similarities.

What are the evolutionary differences in EXPA1 between japonica and indica rice subspecies?

While the search results do not provide specific information about EXPA1 evolutionary differences between japonica and indica subspecies, we can draw insights from patterns observed in other genes:

General patterns of subspecies differentiation:

• Rice subspecies (japonica and indica) show significant genomic differentiation that affects various traits

• Introgression between subspecies has been an important driving force in rice evolution

• Many genes show evidence of selection in specific subspecies, particularly genes related to adaptation to different environments

Examples from other genes:

• Cold tolerance genes like CTB3 and CTB5 show clear evidence of positive selection in temperate japonica

• CTB3 shows significantly lower nucleotide diversity in temperate japonica (π = 0.000239) compared to wild rice (π = 0.001507)

• CTB5 also shows reduced nucleotide diversity in temperate japonica (π = 0.0080) compared to indica (π = 0.0658) and wild rice (π = 0.0925)

• These patterns suggest that genes important for adaptation to cooler environments have been selected in temperate japonica

Implications for EXPA1 research:
For researchers studying EXPA1 across rice subspecies, it would be valuable to:

• Compare sequence variations of EXPA1 between japonica and indica

• Examine expression patterns in different genetic backgrounds

• Test functional conservation through complementation studies

• Assess whether EXPA1 shows evidence of selection in specific subspecies

Such comparative studies could reveal whether EXPA1 has undergone subspecies-specific adaptation and could identify valuable genetic resources for crop improvement.

How can recombinant EXPA1 be used to study cell wall biomechanics in different plant tissues?

Recombinant EXPA1 provides a powerful tool for investigating cell wall properties:

Methodological approaches:

• Ex vivo application to isolated cell walls:

  • Apply purified recombinant EXPA1 to isolated cell walls

  • Measure mechanical properties before and after treatment

  • Can be combined with various pH conditions to study pH-dependent activity

  • Useful for comparison across different plant tissues and species

• In vivo inducible expression systems:

  • Create transgenic plants with dexamethasone (Dex)-inducible EXPA1 expression

  • Allows precise temporal control of EXPA1 overexpression

  • Enables study of both short-term (3 hours) and long-term (7 days) effects

  • Can be combined with cell wall analysis techniques described below

• Complementation studies:

  • Express recombinant EXPA1 in expansin mutant backgrounds

  • Test functional conservation across species or subspecies

  • Example: Complementation of Arabidopsis expa1 mutants with rice EXPA1

Analytical techniques:

• Atomic Force Microscopy (AFM):

  • Measures Young's modulus (stiffness) of cell walls at nanoscale resolution

  • Can detect spatial heterogeneity in cell wall properties

  • Works at indentation speeds of seconds

  • Studies have shown EXPA1 overexpression increases stiffness in Arabidopsis root transition zone

• Brillouin Light Scattering (BLS):

  • Non-invasive in vivo quantitative assessment of viscoelasticity

  • Measures at GHz frequencies

  • Has been validated to detect both cell wall stiffening and softening

  • Complements AFM measurements

• Fourier-Transform Infrared (FT-IR) Spectroscopy:

  • Provides information on cell wall composition

  • Can identify specific polymer modifications

  • Example: EXPA1 overexpression leads to decreased pectin methylesterification

• Raman Spectroscopy:

  • Offers detailed chemical information about cell wall polymers

  • Has been used to detect altered cell wall composition in expa1 mutants

These approaches can be combined to provide comprehensive insights into how EXPA1 influences cell wall properties across different tissues and developmental contexts.

What are the contradictory findings regarding EXPA1 function and how might they be reconciled?

Several seemingly contradictory findings about EXPA1 function have emerged from research:

Contradictory finding #1: Cell wall loosening vs. stiffening

• Traditional view: Expansins are cell wall loosening agents that promote cell expansion

• Contradictory observation: EXPA1 overexpression increases cell wall stiffness in Arabidopsis root transition zone

• Possible reconciliation:

  • EXPA1 may trigger a compensatory response that leads to cell wall stiffening

  • The effect may be context-dependent (tissue type, developmental stage)

  • EXPA1 may trigger pectin demethylesterification, which can lead to either stiffening or softening depending on the pattern

Contradictory finding #2: Growth promotion vs. inhibition

• Traditional view: Expansins promote cell expansion and growth

• Contradictory observation: EXPA1 overexpression leads to root growth arrest through shortening of the root apical meristem

• Possible reconciliation:

  • Precise control of expansin levels is needed for normal growth

  • Overexpression disrupts the balance of cell wall modifications

  • Different tissues may respond differently to EXPA1

Contradictory finding #3: Direct vs. indirect effects

• Question: Does EXPA1 directly modify cell walls or act through transcriptional cascades?

• Evidence for direct action: EXPA1 can induce cell wall creep in isolated cell walls

• Evidence for indirect action: EXPA1 overexpression rapidly alters expression of numerous cell wall-related genes

• Possible reconciliation:

  • EXPA1 may have both direct enzymatic effects and indirect signaling roles

  • Initial cell wall modifications by EXPA1 could trigger mechanosensitive responses that alter gene expression

  • The observed effects may represent a complex feedback loop between direct cell wall modifications and transcriptional responses

Methodological considerations for resolving contradictions:

• Use multiple complementary techniques to measure cell wall properties

• Conduct time-course experiments to distinguish primary from secondary effects

• Compare different tissues and developmental stages

• Use both loss-of-function and gain-of-function approaches

• Consider dose-dependent effects by using inducible expression systems with varying induction levels

These approaches may help reconcile apparently contradictory findings and develop a more comprehensive understanding of EXPA1 function.

What emerging technologies could advance our understanding of EXPA1 function in rice?

Several cutting-edge technologies hold promise for elucidating EXPA1 function:

• CRISPR-Cas9 gene editing:

  • Create precise mutations in EXPA1 and related expansins

  • Generate allelic series with varying levels of EXPA1 function

  • Simultaneously target multiple expansins to overcome functional redundancy

  • Modify native promoters to alter expression patterns

• Live-cell imaging of cell wall dynamics:

  • Fluorescent protein-tagged cell wall components

  • Super-resolution microscopy to visualize nanoscale changes in cell wall architecture

  • FRET-based biosensors to detect cell wall mechanical properties in living cells

  • Single-molecule tracking of fluorescently labeled EXPA1 to determine its dynamics and interactions

• Multi-omics approaches:

  • Integrate transcriptomics, proteomics, and metabolomics data

  • Cell type-specific or single-cell RNA-seq to resolve tissue heterogeneity

  • Spatial transcriptomics to map EXPA1 and related gene expression patterns at high resolution

  • Comparative genomics across rice varieties to identify natural variation in EXPA1 sequence and function

• Advanced biomechanical measurements:

  • Micro-indentation to measure tissue-level mechanical properties

  • Cellular force microscopy to measure turgor pressure and cell wall elasticity

  • Acoustic microscopy for non-invasive measurement of mechanical properties

  • Machine learning approaches to analyze complex biomechanical data

These technologies could provide unprecedented insights into how EXPA1 functions in the context of rice development and adaptation.

What genetic engineering strategies utilizing EXPA1 might improve agronomic traits in rice?

Based on current understanding of EXPA1 function, several genetic engineering strategies could be explored:

• Root system architecture improvement:

  • Controlled expression of EXPA1 to enhance lateral root development

  • Similar to EXPA8 overexpression, which improved root system architecture with longer primary roots and more lateral roots

  • Potential benefits: Enhanced nutrient and water uptake, improved drought tolerance

  • Strategy: Use root-specific or inducible promoters to control timing and location of expression

• Stress tolerance enhancement:

  • Targeted modification of EXPA1 expression under specific stress conditions

  • Potential application for cold tolerance improvement based on patterns observed in other genes

  • Research suggests subspecies-specific adaptations and introgression between japonica and indica play important roles in stress adaptation

  • Strategy: Identify naturally occurring EXPA1 variants with enhanced stress response properties

• Cell wall composition engineering:

  • Modulate EXPA1 activity to alter cell wall properties for specific applications

  • Target improved biofuel production through altered cell wall digestibility

  • Modify lignin-to-polysaccharide ratios, as observed in EXPA8 overexpressors

  • Strategy: Combine EXPA1 modification with other cell wall-related genes

• Controlled growth regulation:

  • Use EXPA1 to modulate growth responses to environmental conditions

  • Fine-tune hormone responses by manipulating EXPA1 expression

  • Potential for creating semi-dwarf varieties with improved lodging resistance

  • Strategy: Develop switchable expression systems responsive to environmental cues

Important considerations for these strategies include:

• Potential trade-offs between improved traits and other aspects of plant performance

• Tissue-specific and developmental timing of expression

• Interactions with other expansins and cell wall-modifying enzymes

• Subspecies-specific responses and adaptations

Research suggests that careful modulation of EXPA1 expression or activity, rather than simple overexpression, would likely be most effective for crop improvement applications.