Recombinant Paspalum notatum Expansin-B (X10S), partial

Advanced Research Questions

• What methodologies are most effective for expressing and purifying functional Recombinant Paspalum notatum Expansin-B?

  Successful expression and purification of functional Recombinant P. notatum Expansin-B requires a strategic approach:

  1. Expression system selection:

     • Bacterial systems (E. coli BL21(DE3)) with specialized vectors containing solubility-enhancing tags

     • Yeast systems (Pichia pastoris) for improved protein folding and post-translational modifications

     • Plant-based expression systems for most native-like processing

  2. Optimization strategies:

     • Codon optimization for the selected expression host

     • Low-temperature induction (16-18°C) to minimize inclusion body formation

     • Co-expression with chaperones to improve folding

     • Periplasmic targeting for proper disulfide bond formation

  3. Purification protocol:

     • Initial capture via affinity chromatography (typically His-tag or GST-tag)

     • Tag removal using specific proteases (TEV or Factor Xa)

     • Ion exchange chromatography for charge-based separation

     • Size exclusion chromatography for final polishing

     • Activity-based validation at each purification step

  4. Refolding strategies (if necessary):

     • Gradual dialysis from denaturing to native conditions

     • On-column refolding during affinity chromatography

     • Pulsed dilution methods with redox agents to facilitate disulfide formation

  This comprehensive approach typically yields 1-5 mg of active protein per liter of culture, sufficient for detailed biochemical and functional analyses.

• How can researchers quantitatively assess the cell wall-loosening activity of Recombinant Paspalum notatum Expansin-B?

  Quantitative assessment of expansin activity requires specialized techniques:

  1. Constant-load extensiometer assays:

     • Preparation of native cell wall specimens (typically heat-inactivated to eliminate endogenous enzyme activity)

     • Application of constant force (20-25g) to induce extension

     • Measurement of extension rate in presence vs. absence of recombinant protein

     • Data analysis using extension rate (μm/min) as primary metric

  2. Stress relaxation measurements:

     • Application of instantaneous strain to cell wall specimens

     • Monitoring of force decay over time (typically 10-30 minutes)

     • Calculation of relaxation half-time (t½) as quantitative measure

     • Comparative analysis with control treatments

  3. Creep rate analysis:

     • Measurement of cell wall extension under constant load over extended periods (1-4 hours)

     • Calculation of creep rate during steady-state phase

     • Dose-response analysis with varying protein concentrations

  4. Biochemical binding assays:

     • Quantification of protein binding to cellulose and other cell wall components

     • Scatchard analysis to determine binding parameters (Kd, Bmax)

     • Correlation of binding with functional activity

  These complementary approaches provide robust quantification of expansin activity under defined conditions, enabling comparative studies between different expansin variants or experimental treatments.

• How does the molecular structure of Paspalum notatum Expansin-B compare with expansins from other grass species?

  Comparative analysis of P. notatum Expansin-B with other grass expansins reveals both conserved features and species-specific adaptations:

  Feature	P. notatum Expansin-B	Zea mays Expansin-B	Oryza sativa Expansin-B
  Domain organization	Typical D1+D2 structure	Similar D1+D2 structure	Similar D1+D2 structure
  Catalytic residues	Highly conserved	Highly conserved	Highly conserved
  Binding domain	More hydrophobic surface	Mixed hydrophobic-polar surface	More polar binding surface
  pH optimum	4.5-5.5	4.0-5.0	4.5-5.5
  Temperature stability	Stable up to 50°C	Stable up to 45°C	Stable up to 55°C
  Glycosylation sites	1-2 predicted sites	2-3 predicted sites	2-3 predicted sites
  Isoelectric point	~9.0-9.5	~8.5-9.0	~8.8-9.3

  These structural differences likely reflect adaptations to the specific environmental conditions and growth patterns of P. notatum, particularly its capacity to thrive in varying seasonal conditions as demonstrated in morphogenetic studies .

• What are the challenges and solutions in designing structure-function studies of Recombinant Paspalum notatum Expansin-B?

  Structure-function studies of P. notatum Expansin-B present several challenges with corresponding methodological solutions:

  1. Challenge: Determining the contribution of specific residues to function

     • Solution: Site-directed mutagenesis targeting conserved catalytic residues, surface-exposed amino acids, and substrate-binding regions

     • Solution: Alanine-scanning mutagenesis of functional regions followed by activity assays

     • Solution: Construction of chimeric proteins with domains from other expansins to identify region-specific contributions

  2. Challenge: Resolving three-dimensional structure

     • Solution: X-ray crystallography of the recombinant protein, potentially aided by surface entropy reduction mutations

     • Solution: NMR spectroscopy for solution structure determination

     • Solution: Homology modeling based on solved structures of related expansins, validated by experimental data

  3. Challenge: Correlating structure with in vivo function

     • Solution: Transformation of P. notatum with modified expansin genes

     • Solution: Application of recombinant variants to plant tissues with activity monitoring

     • Solution: Complementation studies in expansin-deficient systems

  4. Challenge: Understanding conformational dynamics

     • Solution: Molecular dynamics simulations to model protein-substrate interactions

     • Solution: Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

     • Solution: FRET-based approaches to monitor protein conformational changes during substrate binding

  These approaches collectively enable a comprehensive understanding of structure-function relationships in P. notatum Expansin-B.

• How can gene expression analysis inform our understanding of Paspalum notatum Expansin-B regulation?

  A systematic gene expression analysis approach includes:

  1. Transcriptional profiling:

     • RNA-Seq analysis of P. notatum tissues under various environmental conditions (nitrogen levels, photoperiod, temperature)

     • RT-qPCR validation of expansin expression using gene-specific primers

     • In situ hybridization to localize expansin transcripts in specific cell types

     • Promoter analysis to identify cis-regulatory elements responsive to environmental signals

  2. Experimental design considerations:

     • Time-course sampling to capture dynamic responses (particularly during leaf elongation phases)

     • Comparison between genotypes with different growth habits (upright vs. prostrated)

     • Correlation with observed morphogenetic traits (leaf elongation rate, leaf blade length)

     • Integration with physiological measurements (nitrogen uptake, photosynthetic rate)

  3. Data analysis frameworks:

     • Differential expression analysis to identify key regulatory conditions

     • Co-expression network construction to identify genes coordinated with expansins

     • Correlation analysis with structural traits (tiller density, tiller weight)

     • Hierarchical clustering to identify expression patterns across conditions and genotypes

  This comprehensive approach allows researchers to understand the regulation of P. notatum Expansin-B in relation to growth parameters and environmental responses.

Research Methodology Questions

• What bioinformatic approaches are most effective for identifying and characterizing the complete Expansin-B gene family in the Paspalum notatum genome?

  A comprehensive bioinformatic pipeline would include:

  1. Genome-wide identification:

     • BLAST/HMMER searches using conserved expansin domains against P. notatum genome

     • Gene prediction using specialized tools (AUGUSTUS, MAKER) with expansin-specific training sets

     • Validation of gene models using available transcriptomic data

     • Manual curation to correct potential annotation errors

  2. Structural characterization:

     • Identification of conserved domains using InterProScan, PFAM, or SMART

     • Signal peptide prediction using SignalP

     • Intron-exon structure analysis and comparison with other grass species

     • Identification of potential alternative splicing variants

  3. Phylogenetic analysis:

     • Multiple sequence alignment with characterized expansins from related grasses

     • Construction of maximum likelihood or Bayesian phylogenetic trees

     • Classification into expansin subfamilies (EXPA, EXPB, EXLA, EXLB)

     • Identification of species-specific expansin clades

  4. Promoter analysis:

     • Extraction of promoter regions (1-2kb upstream)

     • Identification of cis-regulatory elements using PlantCARE or PLACE databases

     • Comparative analysis with promoters of characterized expansin genes

     • Prediction of potential regulators using transcription factor binding site analysis

  5. Synteny and evolutionary analysis:

     • Comparison of chromosomal locations and gene organization with related species

     • Identification of tandem and segmental duplications

     • Calculation of Ka/Ks ratios to assess selection pressure

  This systematic approach provides a comprehensive characterization of the expansin gene family in P. notatum and establishes evolutionary relationships with expansins from other grass species.

• How should researchers design experiments to investigate the relationship between nitrogen fertilization and Expansin-B activity in Paspalum notatum?

  A robust experimental design would include:

  1. Field or greenhouse setup:

     • Randomized complete block design with split-plot arrangement

     • Main plots: Nitrogen rates (e.g., 0, 100, 200, 300 kg N ha-1)

     • Sub-plots: P. notatum genotypes representing different growth habits (prostrated, intermediate, upright)

     • Multiple replications (minimum 3) to account for environmental variation

  2. Application schedule:

     • Split applications of nitrogen fertilizer throughout the growing season

     • Timing aligned with key phenological stages

     • Control treatments receiving equivalent water applications

  3. Sampling protocol:

     • Time-course sampling at defined intervals after fertilization

     • Specific tissue collection (actively elongating leaf tissue, leaf growth zones)

     • Flash-freezing in liquid nitrogen to preserve RNA and protein integrity

     • Sample processing under controlled conditions

  4. Analytical measurements:

     • Morphogenetic parameters: leaf elongation rate, leaf blade length, tiller weight

     • Expansin gene expression: RT-qPCR with gene-specific primers

     • Protein quantification: Western blotting with anti-expansin antibodies

     • Expansin activity: Cell wall extension assays using native cell walls

  5. Data analysis:

     • ANOVA with appropriate post-hoc tests for morphological data

     • Correlation analysis between N rates, expansin expression, and growth parameters

     • Regression analysis to establish dose-response relationships

     • Path analysis to determine direct and indirect effects

  This approach enables quantitative assessment of nitrogen fertilization effects on expansin-mediated growth processes in P. notatum.

• What are the key considerations for developing genetically modified Paspalum notatum lines with altered Expansin-B expression?

  Developing transgenic P. notatum with modified expansin expression requires attention to several critical factors:

  1. Transformation strategy:

     • Agrobacterium-mediated transformation of embryogenic callus

     • Biolistic particle delivery for direct DNA transfer

     • CRISPR/Cas9-based genome editing for precise modifications

     • Selection of appropriate promoters (constitutive vs. tissue-specific)

  2. Genetic constructs:

     • Overexpression cassettes using strong constitutive promoters (e.g., CaMV 35S, maize Ubiquitin)

     • RNAi or antisense constructs for downregulation

     • CRISPR guide RNAs targeting specific expansin genes

     • Reporter genes for transformation monitoring (GFP, GUS)

  3. Selection and regeneration:

     • Optimized tissue culture conditions for callus induction

     • Appropriate selection markers (herbicide or antibiotic resistance)

     • Efficient plant regeneration protocols

     • Hardening and acclimatization procedures

  4. Molecular characterization:

     • PCR confirmation of transgene integration

     • Copy number determination by qPCR or Southern blotting

     • Expression analysis by RT-qPCR and Western blotting

     • Heritability assessment across generations

  5. Phenotypic evaluation:

     • Morphogenetic analysis (leaf elongation rate, leaf blade length, tiller dynamics)

     • Growth responses to environmental factors (nitrogen, photoperiod, temperature)

     • Comparison with non-transformed controls under field conditions

     • Assessment of reproductive development and seed production

  These considerations ensure successful development and characterization of P. notatum lines with modified expansin expression for functional studies.

• How can researchers correlate the biochemical properties of Recombinant Paspalum notatum Expansin-B with observed phenotypic traits?

  Establishing correlations between biochemical properties and phenotypic traits requires a multi-level experimental approach:

  1. Biochemical characterization:

     • Determination of kinetic parameters (optimal pH, temperature dependence)

     • Substrate specificity using different cell wall preparations

     • Binding affinity to cell wall components (cellulose, hemicellulose)

     • Stability under various environmental conditions

  2. In vitro to in vivo transition:

     • Application of purified recombinant protein to plant tissues

     • Microscopic analysis of treated tissues for cell expansion effects

     • Comparison of effects across different genotypes and tissue types

     • Dose-response studies to establish quantitative relationships

  3. Genetic correlation studies:

     • Selection of P. notatum genotypes with varying growth characteristics

     • Sequencing of expansin genes to identify natural variants

     • Association analysis between sequence polymorphisms and growth traits

     • Correlation analysis between expansin expression levels and phenotypic variables

  4. Integrative data analysis:

     • Principal component analysis to identify relationships between biochemical and phenotypic variables

     • Path analysis to determine direct and indirect effects

     • Hierarchical clustering to identify genotypes with similar biochemical and phenotypic profiles

     • Regression models to quantify relationships between variables

  Parameter	Measurement Method	Expected Correlation with Phenotype
  pH optimum	Activity assays across pH range	May correlate with adaptation to soil pH conditions
  Temperature stability	Thermal inactivation assays	Could relate to seasonal growth patterns
  Binding affinity	Isothermal titration calorimetry	Potentially correlates with tissue-specific growth rates
  Specific activity	Standard extension assays	Likely correlates with leaf elongation rate
  Expression level	RT-qPCR	Expected to correlate with growth during active phases

  This multi-faceted approach enables meaningful correlations between molecular properties and observed phenotypic traits.

• What methods are most effective for studying the role of Paspalum notatum Expansin-B in reproductive development?

  Investigating expansin roles in reproductive development requires specialized approaches:

  1. Expression profiling:

     • Tissue-specific RNA extraction from vegetative vs. reproductive tillers

     • RT-qPCR quantification of expansin expression during reproductive transition

     • In situ hybridization to localize expansin transcripts in developing inflorescences

     • Comparison between genotypes with different reproductive tiller densities

  2. Histological and microscopic analysis:

     • Tissue sectioning and staining to visualize developmental changes

     • Immunolocalization of expansin proteins in reproductive structures

     • Confocal microscopy to track cell expansion patterns

     • Quantitative morphometry of reproductive structures

  3. Manipulative experiments:

     • Application of recombinant expansin to developing reproductive structures

     • Transgenic modification of expansin expression with reproductive-specific promoters

     • RNAi or CRISPR-based silencing of expansin genes

     • Correlation of nitrogen fertilization effects on reproductive tiller density with expansin expression

  4. Environmental manipulation:

     • Photoperiod treatments to induce or delay flowering

     • Nitrogen treatment effects on reproductive development (significant increases in reproductive tiller density observed after N-fertilization)

     • Temperature regime effects on reproductive transition

     • Correlation of environmental responses with expansin expression patterns

  These approaches can reveal the specific contributions of Expansin-B to the reproductive development of P. notatum, particularly in the context of the significant increase in reproductive tiller density (262.5%) observed after nitrogen fertilization .

• How should researchers approach the development of expansin inhibitors to study Paspalum notatum growth regulation?

  A systematic approach to developing and utilizing expansin inhibitors includes:

  1. Inhibitor design strategies:

     • Structure-based design using homology models of P. notatum Expansin-B

     • Screening of chemical libraries against recombinant protein

     • Peptide-based inhibitors targeting the catalytic domain

     • Antibody-based inhibition approaches

  2. In vitro validation:

     • Activity assays to determine IC50 values

     • Binding studies to confirm direct interaction

     • Specificity testing against different expansin isoforms

     • Stability assessment under physiological conditions

  3. Application methodologies:

     • Vacuum infiltration for leaf tissues

     • Micropipette application to specific growth zones

     • Hydroponic supplementation for root studies

     • Foliar spraying for whole-plant effects

  4. Phenotypic analysis:

     • Quantification of leaf elongation rates before and after inhibitor application

     • Microscopic analysis of cell expansion patterns

     • Comparative analysis across different genotypes

     • Dose-response studies to establish quantitative relationships

  5. Validation experiments:

     • Comparison with genetic knockdown approaches

     • Rescue experiments with recombinant protein

     • Combination with environmental treatments (nitrogen, photoperiod)

     • Time-course studies to determine reversibility of effects

  This comprehensive approach enables functional validation of expansin roles in P. notatum growth processes through specific inhibition of protein activity.