Recombinant Oryza sativa subsp. japonica B3 domain-containing protein Os04g0676600 (Os04g0676600, LOC_Os04g58000)

What is the B3 domain-containing protein Os04g0676600 and what is its significance in rice?

The Os04g0676600 gene encodes a B3 domain-containing protein in Oryza sativa subsp. japonica (rice). B3 domain proteins belong to a plant-specific transcription factor family that plays critical roles in seed development and maturation. These proteins are characterized by their ability to bind to specific DNA sequences, particularly the RY motif (core sequence 5′-CATG-3′), which is found in the promoters of genes involved in seed storage protein and oil accumulation .

The significance of Os04g0676600 lies in its presumed role in regulating gene expression during rice seed development, potentially controlling aspects of nutrient storage and embryo development. Similar B3 domain proteins in other plant species, such as the maize Viviparous1 (Vp1) and Arabidopsis ABI3, have been shown to regulate seed dormancy, germination, and the accumulation of storage compounds .

How are B3 domain proteins classified and what subfamilies exist within rice?

B3 domain proteins in plants are classified into several subfamilies based on their domain architecture and evolutionary relationships. The major subfamilies include:

• ABI3/VP1-type: Contains proteins like maize VP1 and Arabidopsis ABI3

• ARF (Auxin Response Factor)-type: Contains an additional auxin-responsive domain

• RAV (Related to ABI3/VP1)-type: Contains an additional AP2 domain

• REM (Reproductive Meristem)-type: Contains multiple B3 domains

In rice, approximately 51 B3 domain-containing proteins have been identified (by analogy to the maize genome), with varying expression patterns across tissues and developmental stages . Os04g0676600 is believed to belong to the ABI3/VP1-type subfamily based on sequence homology, though specific classification requires detailed phylogenetic analysis that differentiates it from other B3 domain proteins in rice.

What are the known DNA binding motifs for B3 domain proteins in rice?

B3 domain proteins typically bind to specific DNA sequences, with the RY motif (5′-CATG-3′) being the primary recognition site . Research on B3 domain proteins like ZmABI19 in maize has demonstrated their ability to bind to and recognize RY motifs in gene promoters, such as those found in the Opaque2 (O2) promoter .

For Os04g0676600 specifically, while the exact binding sites have not been comprehensively characterized in the provided search results, it likely recognizes similar RY motifs based on the highly conserved DNA-binding properties of B3 domain proteins across plant species. Experimental verification through techniques like electrophoretic mobility shift assays (EMSA) or chromatin immunoprecipitation (ChIP) would be necessary to confirm the exact binding motifs for this specific protein.

What are the optimal systems for recombinant expression of Os04g0676600 protein?

For recombinant expression of Os04g0676600, several expression systems can be considered:

• Bacterial Expression Systems: While E. coli is commonly used for recombinant protein expression due to its simplicity and high yield, plant transcription factors often require post-translational modifications that bacterial systems lack. Nevertheless, for initial structural studies or antibody production, E. coli expression with appropriate solubility tags (MBP, SUMO, etc.) may be sufficient.

• Plant-Based Expression Systems: Transgenic rice or other plant expression systems may provide the most native-like environment for Os04g0676600 expression . The use of rice cells ensures proper folding and post-translational modifications. Methods for transgenic rice production have been well-established, including Agrobacterium-mediated transformation as described in the literature .

• Insect Cell Systems: Baculovirus-infected insect cells offer a eukaryotic environment that can support proper folding and some post-translational modifications of plant proteins.

The choice of expression system should be guided by the intended application, required protein purity, and functional needs. For functional studies requiring properly folded and modified protein, plant-based expression systems would be optimal .

What purification strategies are most effective for isolating recombinant Os04g0676600?

Purification of recombinant Os04g0676600 can be approached with the following strategies:

• Affinity Chromatography: Adding an affinity tag (His, GST, FLAG) to the recombinant protein allows for specific purification. For Os04g0676600, a C-terminal tag might be preferable to avoid interfering with the N-terminal B3 domain.

• Ion Exchange Chromatography: As a DNA-binding protein, Os04g0676600 likely has a positive charge at physiological pH, making cation exchange chromatography a viable option for purification or as a polishing step.

• Size Exclusion Chromatography: This can be used as a final polishing step to separate the target protein from aggregates or degradation products.

• Protein Extraction from Plant Tissues: For native protein extraction from rice, specialized protocols have been developed for isolating proteins from different tissues and organelles . These protocols typically involve tissue homogenization, fractionation, and subsequent purification steps.

How can antibodies against Os04g0676600 be developed for research applications?

Development of antibodies against Os04g0676600 involves several key steps:

• Antigen Preparation: Either the full-length protein or unique peptide sequences can be used. For B3 domain proteins, selecting unique epitopes outside the conserved B3 domain is crucial to avoid cross-reactivity with other family members.

• Antibody Production: Both polyclonal and monoclonal approaches are viable. Polyclonal antibodies can be generated by immunizing rabbits or other suitable animals with the purified recombinant protein or synthetic peptides . For monoclonal antibodies, mouse hybridoma technology would be employed.

• Antibody Validation: Critical validation steps include:

  • Western blotting against recombinant protein and native extracts

  • Immunoprecipitation assays

  • Immunohistochemistry in rice tissues

  • Pre-absorption controls with the immunizing antigen

• Applications: Custom antibodies against Os04g0676600 can be validated for various applications including ELISA, Western blotting, and immunoprecipitation techniques .

For researchers seeking custom antibody development, specialized services like those offered by antibody production companies can generate and validate antibodies against Os04g0676600 with specificity for rice (Oryza sativa subsp. japonica) .

What methods are most effective for studying DNA-binding properties of Os04g0676600?

Several complementary techniques can be employed to characterize the DNA-binding properties of Os04g0676600:

• Electrophoretic Mobility Shift Assay (EMSA): This technique allows visualization of protein-DNA interactions in vitro. For Os04g0676600, synthetic oligonucleotides containing predicted RY motifs (5'-CATG-3') can be labeled and incubated with the purified protein to observe binding . Competition assays with unlabeled DNA probes can confirm binding specificity, as demonstrated with other B3 domain proteins like ZmABI19 .

• Chromatin Immunoprecipitation (ChIP): This in vivo technique identifies genomic binding sites using antibodies against Os04g0676600. ChIP followed by sequencing (ChIP-seq) would provide genome-wide binding profiles, revealing potential target genes regulated by this transcription factor.

• Yeast One-Hybrid Assays: This approach can test if Os04g0676600 can activate transcription when bound to specific DNA sequences, similar to the methodology used for identifying ZmABI19's binding to the O2 promoter in maize .

• DNA Footprinting: This technique precisely maps the nucleotides protected by the protein binding, providing nucleotide-resolution binding information.

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI): These methods quantify binding kinetics and affinity constants for protein-DNA interactions, offering quantitative insights into binding strength.

How can transcriptional activation by Os04g0676600 be measured in vivo?

Measuring the transcriptional activation properties of Os04g0676600 can be approached through several in vivo systems:

• Transient Expression Assays: Using protoplasts isolated from rice cells, a reporter gene (e.g., luciferase) driven by a promoter containing B3 binding sites can be co-transformed with Os04g0676600 expression constructs. Measuring reporter activity provides quantitative data on transcriptional activation.

• Stable Transgenic Plants: Generating rice plants that overexpress or silence Os04g0676600 can reveal its effects on putative target genes. RNA-seq analysis of these transgenic plants compared to wild-type would identify genes regulated by this transcription factor .

• Yeast Systems: A fusion protein containing Os04g0676600 and a GAL4 DNA-binding domain can be tested in yeast strains carrying reporter genes to assess intrinsic activation capabilities, similar to methods used for other B3 domain proteins .

• CRISPR-Cas9 Gene Editing: Creating precise mutations in the Os04g0676600 gene can provide loss-of-function phenotypes that reveal its regulatory roles, following approaches similar to those used to study other transcription factors in rice.

What experimental approaches can determine the role of Os04g0676600 in rice seed development?

To investigate the role of Os04g0676600 in rice seed development, several experimental approaches can be employed:

• Expression Profiling: Analyzing Os04g0676600 expression patterns throughout seed development using RT-qPCR or RNA-seq would establish temporal regulation patterns.

• Genetic Modification:

  • CRISPR-Cas9 Knockout: Creating null mutants of Os04g0676600 to observe phenotypic changes in seed development, dormancy, germination, and storage compound accumulation.

  • Overexpression Lines: Generating transgenic rice overexpressing Os04g0676600 under constitutive or seed-specific promoters.

• Biochemical Analysis of Seeds: Comparing wild-type and mutant seeds for:

  • Protein content and composition (especially storage proteins)

  • Starch and lipid accumulation

  • Hormone levels (particularly ABA)

• Microscopic Analysis: Examining seed structure and development using techniques like scanning electron microscopy.

• Transcriptome Analysis: Performing RNA-seq on developing seeds from wild-type and Os04g0676600 mutant plants to identify downstream genes regulated by this transcription factor, similar to approaches used for other seed development regulators .

• Protein-Protein Interaction Studies: Identifying protein partners of Os04g0676600 using techniques like yeast two-hybrid, co-immunoprecipitation, or proximity labeling to place it within regulatory networks governing seed development.

How can Os04g0676600 be utilized in transgenic rice engineering for improved seed quality?

Utilizing Os04g0676600 in transgenic rice engineering for seed quality improvement requires strategic approaches:

• Promoter Modifications: Modifying the expression pattern of Os04g0676600 by placing it under alternative promoters can potentially enhance seed quality traits. For example, using a strong endosperm-specific promoter could increase expression specifically during grain filling, potentially enhancing storage compound accumulation without affecting vegetative growth .

• Protein Engineering: Creating modified versions of Os04g0676600 with enhanced or altered activity, such as:

  • Chimeric proteins combining domains from other B3 family members

  • Fusion with activation domains to enhance transcriptional activation

  • Site-directed mutagenesis to alter DNA-binding specificity or protein-protein interactions

• Coordinate Expression with Other Factors: Co-expressing Os04g0676600 with other transcription factors involved in seed development could create synergistic effects, as B3 domain proteins often function within transcriptional networks .

• Biotechnological Applications: For recombinant protein production in rice, Os04g0676600 could potentially be engineered as part of a regulatory system controlling expression of valuable proteins, similar to other containment strategies developed for transgenic rice .

• Implementing Safety Features: When developing transgenic rice with modified Os04g0676600, implementing safety features such as the double built-in containment strategy (selective termination method and visual tag technology) would prevent unwanted transgenic rice spreading .

What bioinformatic approaches can predict target genes regulated by Os04g0676600?

Bioinformatic approaches for predicting target genes regulated by Os04g0676600 include:

• Motif-Based Searches: Scanning the rice genome for the RY motif (5'-CATG-3') and related sequences in promoter regions, particularly in genes associated with seed development and storage compound biosynthesis .

• Comparative Genomics: Identifying conserved B3 domain binding sites across related grass species (maize, wheat, barley) can reveal evolutionarily conserved targets.

• Co-expression Network Analysis: Using publicly available RNA-seq data to identify genes whose expression patterns correlate with Os04g0676600 across different tissues and developmental stages.

• Gene Ontology Enrichment: Analyzing potential target genes for enrichment in specific biological processes, particularly those related to seed development, hormone responses, and storage compound synthesis.

• Integration with Chromatin Accessibility Data: Combining motif searches with open chromatin data (ATAC-seq or DNase-seq) from rice seeds to identify accessible binding sites.

• Machine Learning Approaches: Developing predictive models that integrate multiple data types (sequence, expression, chromatin) to rank potential target genes by likelihood of regulation.

How can protein-protein interactions of Os04g0676600 be systematically mapped?

Systematic mapping of Os04g0676600 protein-protein interactions can be approached through complementary techniques:

• Yeast Two-Hybrid (Y2H) Screening: Using Os04g0676600 as bait against a rice cDNA library to identify interacting proteins. This approach has been successful for identifying interactions of other B3 domain proteins .

• Affinity Purification-Mass Spectrometry (AP-MS): Expressing tagged versions of Os04g0676600 in rice cells, followed by purification and mass spectrometry to identify co-purifying proteins.

• Bimolecular Fluorescence Complementation (BiFC): Testing specific protein pairs by expressing fusion proteins in plant cells to visualize interactions in vivo and determine subcellular localization.

• Proximity-Dependent Biotin Identification (BioID): Fusing Os04g0676600 to a biotin ligase that biotinylates nearby proteins, allowing identification of proteins in the same complex or neighborhood.

• Co-Immunoprecipitation (Co-IP): Using antibodies against Os04g0676600 to pull down interacting proteins from rice extracts, followed by Western blotting or mass spectrometry.

• Domain-Based Analyses: Performing interaction studies with individual domains of Os04g0676600 to map specific interaction interfaces.

What are common difficulties in expressing functional recombinant B3 domain proteins and how can they be overcome?

Researchers often encounter several challenges when expressing functional B3 domain proteins like Os04g0676600:

• Poor Solubility: B3 domain proteins frequently form inclusion bodies in bacterial expression systems.

  • Solution: Use solubility-enhancing fusion tags (MBP, SUMO, TRX), lower expression temperature (16-20°C), or specialized strains (e.g., Arctic Express, Rosetta).

  • Alternative: Consider plant-based expression systems which provide a more native environment for proper folding .

• Proteolytic Degradation: Transcription factors are often susceptible to proteolysis.

  • Solution: Include protease inhibitors throughout purification, optimize buffer conditions (pH, salt), and consider using protease-deficient expression strains.

• DNA Contamination: B3 domain proteins bind DNA, leading to contamination during purification.

  • Solution: Include DNase treatment, increase salt concentration (300-500 mM NaCl) during lysis and early purification steps, and consider heparin chromatography as a DNA-mimetic affinity step.

• Loss of DNA-Binding Activity: Recombinant proteins may fold improperly, losing functional activity.

  • Solution: Verify activity through EMSAs or reporter assays, optimize purification to maintain native conditions, and consider refolding protocols if necessary.

• Low Expression Yields: Transcription factors often express at low levels.

  • Solution: Optimize codon usage for the expression host, use strong inducible promoters, and scale up culture volumes.

How can phenotypic contradictions in Os04g0676600 functional studies be reconciled?

When facing contradictory results in functional studies of Os04g0676600, consider the following reconciliation approaches:

• Genetic Background Effects: Different rice varieties may show different phenotypic responses to Os04g0676600 manipulation.

  • Resolution: Always include appropriate controls from the same genetic background and consider testing in multiple varieties.

• Functional Redundancy: Other B3 domain proteins may compensate for Os04g0676600 loss in single mutants.

  • Resolution: Generate double or triple mutants targeting related B3 domain genes, or use inducible RNAi to bypass developmental compensation.

• Dosage Effects: Different expression levels may lead to different or even opposite phenotypes.

  • Resolution: Create an allelic series with varying expression levels and quantify protein levels in each line.

• Temporal/Spatial Expression Differences: The timing and location of expression manipulations can significantly affect outcomes.

  • Resolution: Use tissue-specific and developmentally regulated promoters for precise expression control.

• Environmental Influences: Seed development is sensitive to environmental conditions, which can mask or enhance genetic effects.

  • Resolution: Conduct experiments under controlled conditions and include environmental variables (temperature, humidity, light) as factors in experimental design.

• Methodology Differences: Different phenotyping methods may assess different aspects of seed development.

  • Resolution: Use multiple, complementary phenotyping approaches and standardize protocols between studies.

How can off-target effects be minimized when studying Os04g0676600 function through genetic modification?

Minimizing off-target effects in genetic studies of Os04g0676600 requires careful experimental design:

• CRISPR-Cas9 Guide RNA Design:

  • Strategy: Design multiple guide RNAs targeting Os04g0676600 with minimal predicted off-targets.

  • Validation: Sequence the entire gene and potential off-target sites in edited plants.

  • Controls: Generate independent lines with different guide RNAs to confirm consistent phenotypes.

• RNAi Approaches:

  • Strategy: Design RNAi constructs targeting unique regions of Os04g0676600 mRNA.

  • Validation: Confirm specificity through RNA-seq or qRT-PCR of related genes.

  • Controls: Use multiple independent RNAi lines with different levels of knockdown.

• Overexpression Studies:

  • Strategy: Use native or tissue-specific promoters rather than constitutive promoters when possible.

  • Validation: Create an expression series with varying levels to distinguish physiological from artificial effects.

  • Controls: Include proper empty vector controls and monitor expression of related genes.

• Complementation Tests:

  • Strategy: Introduce the wild-type Os04g0676600 into mutant backgrounds to confirm phenotype rescue.

  • Validation: Quantify the degree of phenotypic rescue correlated with expression levels.

• Conditional Expression Systems:

  • Strategy: Use inducible promoters to control timing and level of expression.

  • Advantage: Allows temporal control to distinguish direct from indirect effects.

• Safety Features in Transgenic Rice:

  • Strategy: Implement containment strategies such as those described for recombinant protein production .

  • Examples: Include selectively terminating features (e.g., bentazon sensitivity through CYP81A6 suppression) and visual markers for transgenic identification .

How might integration of multi-omics data enhance our understanding of Os04g0676600 function?

Integration of multi-omics approaches offers powerful insights into Os04g0676600 function:

• Genomics + Transcriptomics: Combining ChIP-seq data (genomic binding sites) with RNA-seq of mutants or overexpression lines can identify direct target genes by correlating binding events with expression changes.

• Proteomics + Interactomics: Integrating protein interaction networks with proteome-wide studies of developing rice seeds can place Os04g0676600 within functional protein complexes and pathways .

• Metabolomics + Transcriptomics: Correlating metabolite profiles with gene expression changes in Os04g0676600 mutants can reveal impacts on metabolic pathways, particularly those related to seed storage compounds.

• Epigenomics + Transcriptomics: Analyzing how Os04g0676600 affects or is affected by chromatin modifications and accessibility can uncover epigenetic regulatory mechanisms.

• Phenomics + Genomics: High-throughput phenotyping of seed traits in populations with Os04g0676600 variants can identify subtle phenotypic effects and genetic interactions.

• Systems Biology Integration: Computational models integrating all data types can predict network-level effects of Os04g0676600 perturbation and guide experimental validation.

The integration of proteomic approaches is particularly relevant, as described in research on rice proteins, where detailed protocols for isolation, separation, and identification of proteins from different tissues have been established .

What potential applications exist for biotechnological modification of Os04g0676600 in crop improvement?

Biotechnological applications of Os04g0676600 modification offer several promising directions for crop improvement:

• Enhanced Seed Quality: Modifying Os04g0676600 expression or activity could potentially alter seed composition for improved nutritional value, similar to how other transcription factors like Opaque2 have been used to enhance protein content in maize .

• Stress Resistance: If Os04g0676600 is involved in ABA signaling pathways (like its homologs in other species), its modification could enhance seed dormancy or stress resistance during germination.

• Yield Enhancement: Optimizing the timing and level of Os04g0676600 expression could potentially increase seed size or number by affecting resource allocation during grain filling.

• Biotechnology Platforms: Os04g0676600 promoter elements or the protein itself could be incorporated into sophisticated gene regulatory systems for recombinant protein production in rice, building upon existing strategies for transgenic rice bioreactors .

• Germination Control: Precise control of Os04g0676600 could help develop rice varieties with optimized germination properties for different agricultural contexts.

• Biocontainment Strategies: Incorporating Os04g0676600 modifications into existing biocontainment approaches could enhance safety features for transgenic rice, potentially by linking its expression to selective markers or termination systems .

When developing these applications, considerations should include implementation of appropriate biocontainment strategies to prevent unintended spreading of transgenic material, such as the double built-in containment approach using herbicide sensitivity and visual markers .

How can CRISPR-Cas9 gene editing be optimized for functional studies of Os04g0676600?

Optimizing CRISPR-Cas9 gene editing for Os04g0676600 functional studies requires several strategic considerations:

• Guide RNA Design:

  • Target Selection: Design gRNAs targeting conserved regions within the B3 domain to maximize functional disruption.

  • Specificity: Use algorithms like CRISPOR or Cas-OFFinder to minimize off-target effects.

  • Efficiency: Select guides with high predicted on-target efficiency based on nucleotide composition and secondary structure.

• Delivery Methods:

  • Protoplast Transformation: For preliminary validation of gRNA efficiency.

  • Agrobacterium-Mediated Transformation: For stable genome editing, following established protocols for rice transformation .

  • Ribonucleoprotein (RNP) Delivery: For DNA-free editing to avoid integration of foreign DNA.

• Editing Strategies:

  • Knockout: Using paired gRNAs to delete critical regions.

  • Base Editing: For introducing specific amino acid changes without double-strand breaks.

  • Prime Editing: For precise insertions or replacements to study specific domain functions.

• Validation Approaches:

  • T7 Endonuclease I or Surveyor Assays: For initial detection of editing events.

  • Sanger Sequencing: To confirm specific mutations.

  • Next-Generation Sequencing: For comprehensive analysis of editing outcomes and potential off-targets.

• Functional Analysis Pipeline:

  • Expression Analysis: RT-qPCR and Western blotting to confirm gene/protein disruption.

  • Phenotypic Characterization: Focused on seed development, dormancy, and storage compound accumulation.

  • Molecular Profiling: RNA-seq and metabolite analysis to identify affected pathways.

• Advanced Applications:

  • Multiplexed Editing: Simultaneously targeting Os04g0676600 and related B3 domain genes to address functional redundancy.

  • Inducible CRISPR Systems: Using chemical or light-inducible Cas9 expression for temporal control of editing.

  • CRISPR Activation/Repression: Using catalytically inactive Cas9 fused to activators or repressors for modulating Os04g0676600 expression without editing.