Recombinant Oryza sativa subsp. japonica Tubby-like F-box protein 7 (TULP7)

What is Tubby-like F-box protein 7 (TULP7) and how is it classified in Oryza sativa?

TULP7 belongs to the family of Tubby-like proteins (TLPs), which are characterized by the presence of a conserved Tubby domain. The Tubby domain was originally named after the TUBBY protein discovered in mice, which binds to phosphatidylinositol 4,5-bisphosphate . In plants such as Arabidopsis, there are 11 Tubby domain-containing proteins, and 10 of these possess the N-terminal F-box domain .

In Oryza sativa (Asian cultivated rice), TULP7 is classified as a member of the Tubby-like protein family within the japonica subspecies, which is one of the two major subspecies of Oryza sativa (the other being indica) . The japonica variety was domesticated in the Yangtze Valley 6,000-9,000 years ago and is characterized by sticky, short-grained rice . The classification of TULP7 follows the standard scientific classification system where Oryza sativa belongs to the genus Oryza in the grass family Poaceae .

What are the structural characteristics of TULP7 in comparison to other Tubby-like proteins?

TULP7 in Oryza sativa japonica shares structural similarities with other plant Tubby-like proteins, particularly those found in Arabidopsis. The protein contains two primary domains:

• The N-terminal F-box domain: This domain enables interaction with SKP-like proteins to form SKP1-Cullin-F-box E3 ligase complexes . This structural feature is critical for protein-protein interactions and potentially for ubiquitination activities.

• The C-terminal Tubby domain: This highly conserved domain facilitates binding to phosphatidylinositol 4,5-bisphosphate in the plasma membrane . The Tubby domain typically consists of a 12-stranded β-barrel that forms a central hydrophobic α-helix.

Unlike mammalian Tubby proteins which have been extensively studied, plant TLPs including TULP7 have distinct structural adaptations that likely reflect their specialized functions in plant cellular processes . The structure-function relationship of TULP7 can be compared to TTLL7 (Tubulin tyrosine ligase-like 7) in mammals, which has specific catalytic domains responsible for its polyglutamylase activity with β-tubulin .

What expression patterns does TULP7 exhibit in different tissues and developmental stages of rice?

TULP7 expression in Oryza sativa japonica varies across different tissues and developmental stages, similar to how TTLL7 in mammals shows tissue-specific expression predominantly in the nervous system . Although specific data for TULP7 is limited in the provided search results, we can infer its expression patterns based on related proteins:

• Tissue distribution: TULP7 likely shows differential expression across vegetative and reproductive tissues, with potentially higher expression in actively dividing cells and developing tissues.

• Developmental regulation: Expression may be upregulated during specific developmental phases, particularly during grain development, as indicated by research on genes affecting grain morphology in rice .

• Stress response: Like other regulatory proteins in rice, TULP7 expression might be modulated in response to various environmental stressors, similar to how phosphate deficiency affects gene expression patterns in rice varieties with differing phosphate efficiency .

Researchers investigating TULP7 expression should consider employing RT-qPCR, RNA-Seq, or in situ hybridization techniques to accurately quantify and localize expression patterns.

How can I express and purify recombinant TULP7 for in vitro studies?

For expressing and purifying recombinant TULP7 from Oryza sativa japonica, researchers can adapt the following methodological approach:

Expression System Selection:
The Escherichia coli Rossetta strain (DE3) has proven effective for expressing plant proteins with complex folding requirements, as demonstrated with other proteins like TTLL7 . For TULP7, consider using this strain with a glutathione S-transferase (GST) fusion tag.

Expression Protocol:

• Clone the TULP7 coding sequence into an expression vector (e.g., pGEX for GST fusion)

• Transform into E. coli Rossetta (DE3) strain

• Induce protein expression with IPTG (0.5-1 mM) at 16-18°C for 16-18 hours to minimize inclusion body formation

Purification Steps:

• Harvest and lyse cells in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, and protease inhibitors

• Purify using glutathione-Sepharose 4B beads as employed for similar proteins

• Remove the GST tag using PreScission protease cleavage at 4°C

• Perform size exclusion chromatography for final purification

Quality Control:
Verify protein purity using SDS-PAGE and Western blot analysis with antibodies against the Tubby domain. Assess protein functionality through phosphatidylinositol binding assays.

The typical yield from 1L bacterial culture is expected to be 2-5 mg of purified protein, though optimization may be necessary based on specific experimental conditions.

What is the role of TULP7 in ubiquitination pathways and how does it affect cellular processes in rice?

TULP7 likely functions as part of an E3 ubiquitin ligase complex in rice, similar to the role of TLPs in Arabidopsis. Based on the research findings for Arabidopsis TLPs, we can infer that TULP7 in rice participates in the following processes:

• Substrate Targeting Mechanism: The F-box domain in TULP7 likely mediates interaction with SKP-like proteins to form SKP1-Cullin-F-box E3 ligase complexes . This complex would facilitate the ubiquitination of specific target proteins, marking them for proteasomal degradation.

• Potential Substrates: By analogy with Arabidopsis TLP6, which targets phosphatidylinositol 4-kinase β proteins (PI4Kβs) for ubiquitination and degradation , TULP7 might target similar substrates in rice. These could include enzymes involved in phosphoinositide metabolism, which play crucial roles in membrane trafficking, cell signaling, and cytoskeletal organization.

• Cellular Impact: The ubiquitination activity of TULP7 would regulate the abundance of its target proteins, thereby modulating various cellular processes such as:

  • Signaling pathway regulation

  • Hormone responses

  • Stress adaptation

  • Development regulation, particularly in grain formation

Researchers investigating TULP7's ubiquitination activity should consider immunoprecipitation mass spectrometry approaches to identify interaction partners and potential substrates, similar to methods used for Arabidopsis TLPs .

How does TULP7 contribute to grain morphology and yield in Oryza sativa japonica?

TULP7 may significantly influence grain morphology and yield in rice through several potential mechanisms:

• Cell Proliferation Regulation: Similar to how the TGW2 gene influences grain width and weight by affecting cell proliferation and expansion in glumes , TULP7 might regulate cellular processes that determine grain dimensions.

• Signaling Pathway Integration: TULP7 could function in signaling pathways that control grain development, potentially through its interaction with phosphoinositides and membrane-associated proteins.

• Hormonal Regulation: As a putative component of E3 ubiquitin ligase complexes, TULP7 may regulate the abundance of hormone signaling components that influence grain filling and development.

The impact of TULP7 on grain traits can be experimentally assessed through:

• Genetic Modification Approaches:

  • CRISPR/Cas9-mediated knockout or knockdown

  • Overexpression studies

  • Promoter analysis to understand expression regulation

• Phenotypic Analysis:

  • Detailed grain morphometric measurements

  • Scanning electron microscopy of developing grains

  • Yield component analysis

These approaches would help elucidate whether TULP7 represents a promising gene for improving rice yield, similar to TGW2 which has been identified as a valuable gene for enhancing rice productivity .

What interacting partners of TULP7 have been identified and how do these interactions influence rice development?

While specific interacting partners of TULP7 in rice have not been directly reported in the provided search results, we can extrapolate potential interactions based on related proteins:

Table 1: Potential TULP7 Interacting Partners Based on Homologous Proteins

These interactions would influence rice development through:

• Developmental Timing Control: By regulating the stability of key developmental regulators through ubiquitination

• Tissue Patterning: Through spatial and temporal control of signaling molecule abundance

• Stress Response Integration: By modulating the turnover of stress-responsive factors

For investigating TULP7 interactions, co-immunoprecipitation followed by mass spectrometry analysis would be the recommended approach, similar to the methodology employed for identifying Arabidopsis TLP6 interactors . Yeast two-hybrid screening could also provide complementary data on direct protein-protein interactions.

What are the differences in TULP7 function between phosphate-efficient and phosphate-inefficient rice varieties?

The function of TULP7 likely varies between phosphate-efficient and phosphate-inefficient rice varieties, contributing to their differential responses to phosphate availability:

• Expression Level Differences:

  • In phosphate-efficient varieties like DJ123, TULP7 may show altered expression patterns compared to phosphate-inefficient varieties like Nerica4

  • These expression differences could affect the ubiquitination and turnover of proteins involved in phosphate uptake or utilization

• Substrate Specificity:

  • TULP7 may target different substrates for ubiquitination in phosphate-efficient versus phosphate-inefficient varieties

  • This differential targeting could affect signaling pathways related to phosphate sensing and response

• Root Exudation Influence:

  • If TULP7 affects root development or function, it could contribute to the differences in root exudation patterns observed between contrasting rice lines under phosphate deficiency

  • These differences in exudation could in turn affect phosphate acquisition from the soil

Experimental Approach to Investigate These Differences:

• Compare TULP7 expression levels in contrasting rice varieties (e.g., DJ123 vs. Nerica4) under normal and phosphate-limited conditions

• Perform phosphate uptake and utilization assays in TULP7 overexpression and knockout lines

• Analyze root architecture and exudation patterns in these modified lines

• Use phosphoproteomics to identify differentially phosphorylated proteins that might be TULP7 targets

Such research could reveal whether TULP7 represents a potential target for improving phosphate use efficiency in rice.

What are the optimal conditions for studying TULP7 enzymatic activity in vitro?

For studying the enzymatic activity of recombinant TULP7 in vitro, researchers should consider the following optimized conditions:

Buffer Composition and pH:

• 50 mM Tris-HCl buffer at pH 7.0-7.5

• 8 mM MgCl₂ for enzymatic activity

• 2.5 mM dithiothreitol (DTT) to maintain reducing conditions

• 10% glycerol for protein stability

Temperature and Incubation Time:

• Optimal temperature range: 25-30°C (similar to rice growth conditions)

• Incubation periods: 30-60 minutes for initial activity assessments, extending to 2-4 hours for complete reaction monitoring

Cofactors and Activators:

• ATP requirement: 2 mM ATP as an energy source

• Potential requirement for E1 and E2 enzymes if studying ubiquitination activity

• SKP1 and Cullin proteins to reconstitute SCF complex functionality

Substrate Considerations:

• Potential phosphoinositide substrates (based on the Tubby domain's binding preference)

• Candidate protein substrates based on homology to known targets of related TLPs

• Synthetic peptide substrates containing recognition motifs

Detection Methods:

• For ubiquitination activity: Western blot with anti-ubiquitin antibodies

• For phosphoinositide binding: Lipid overlay assays or surface plasmon resonance

• For protein-protein interactions: Pull-down assays with potential substrates

These conditions should be systematically optimized through factorial experimental designs to determine the precise requirements for TULP7 activity.

What are the most effective genetic modification approaches for studying TULP7 function in rice?

Table 2: Comparative Analysis of Genetic Modification Approaches for TULP7 Functional Studies

For TULP7 specifically, a multi-pronged approach is recommended:

• Tissue-Specific Manipulation:

  • Use tissue-specific promoters (e.g., endosperm-specific for grain development studies)

  • Employ inducible systems for temporal control, particularly for studying developmental roles

• Transformation Protocol:

  • Agrobacterium-mediated transformation of rice callus

  • Selection of transformants using appropriate markers

  • Regeneration of plants under controlled conditions

• Verification Steps:

  • Molecular verification of genetic modifications

  • Expression analysis through RT-qPCR and Western blotting

  • Phenotypic characterization across multiple generations

This integrated approach would provide comprehensive insights into TULP7 function while minimizing artifacts from any single genetic manipulation method.

How can I develop specific antibodies against TULP7 for immunological studies?

Developing specific antibodies against Oryza sativa TULP7 requires a strategic approach:

Antigen Design Strategy:

• Epitope Selection:

  • Analyze the TULP7 sequence for unique regions not conserved in other Tubby-like proteins

  • Consider the F-box domain for specificity against other TLPs

  • Identify surface-exposed regions using structural prediction tools

• Peptide vs. Recombinant Protein Approach:

  • Synthetic peptides (15-20 amino acids) from unique regions offer high specificity

  • Recombinant partial proteins containing unique domains provide better recognition of native protein

Production Protocol:

• For Polyclonal Antibodies:

  • Immunize rabbits with KLH-conjugated synthetic peptides or purified recombinant TULP7

  • Follow a standard 56-day immunization schedule with at least three booster injections

  • Collect serum and purify IgG using protein A/G columns

• For Monoclonal Antibodies:

  • Immunize mice with recombinant TULP7

  • Harvest spleen cells and fuse with myeloma cells

  • Screen hybridomas for specific antibody production

  • Expand and clone positive hybridomas

Validation Methods:

• ELISA against immunizing antigen and related proteins to assess specificity

• Western blot analysis using:

  • Recombinant TULP7

  • Rice tissue extracts

  • TULP7 knockout/knockdown tissues as negative controls

• Immunoprecipitation followed by mass spectrometry to confirm target specificity

• Immunohistochemistry to verify specificity in tissue context

Applications:
The developed antibodies would enable studies of TULP7 expression patterns, protein interactions, subcellular localization, and post-translational modifications in rice tissues under various developmental stages and environmental conditions.

What are the best methodologies for analyzing TULP7's impact on phosphoinositide signaling in rice cells?

To comprehensively analyze TULP7's impact on phosphoinositide signaling in rice cells, researchers should employ multiple complementary approaches:

• Biochemical Phosphoinositide Quantification:

  • Extract lipids using acidified chloroform-methanol methods

  • Separate phosphoinositide species using thin-layer chromatography

  • Quantify using mass spectrometry (LC-MS/MS)

  • Compare levels in wild-type versus TULP7-modified plants

• Fluorescent Biosensor Approaches:

  • Generate transgenic rice expressing phosphoinositide-specific biosensors (e.g., PH domains fused to fluorescent proteins)

  • Use confocal microscopy to visualize phosphoinositide dynamics in living cells

  • Measure temporal changes in response to stimuli in different genetic backgrounds

• Enzyme Activity Assays:

  • Measure kinase/phosphatase activities involved in phosphoinositide metabolism

  • Assess whether TULP7 directly affects these enzymatic activities

  • Determine if TULP7 influences the turnover of phosphoinositide metabolizing enzymes, similar to how Arabidopsis TLP6 regulates PI4Kβ protein levels

• Protein-Lipid Binding Analysis:

  • Use protein-lipid overlay assays to determine TULP7's binding specificity

  • Employ liposome binding assays to quantify binding under physiological conditions

  • Perform mutagenesis studies to identify critical residues for phosphoinositide binding

• Downstream Signaling Analysis:

  • Phosphoproteomics to identify changes in signaling cascades

  • Transcriptomics to assess altered gene expression profiles

  • Metabolomics to evaluate impact on metabolic pathways

These methodologies would provide a comprehensive understanding of how TULP7 influences phosphoinositide signaling pathways that potentially regulate important agronomic traits in rice.

How should researchers interpret phenotypic data from TULP7-modified rice lines?

When interpreting phenotypic data from TULP7-modified rice lines, researchers should implement the following analytical framework:

• Comprehensive Phenotyping Approach:

  • Evaluate multiple traits across developmental stages (germination, vegetative growth, flowering, grain filling)

  • Assess both macroscopic traits (plant height, tiller number, panicle architecture) and microscopic features (cell size, cell number)

  • Pay particular attention to grain characteristics (width, weight, filling rate) given the potential role of TULP7 in grain development

• Statistical Analysis Guidelines:

  • Use appropriate experimental designs (e.g., randomized complete block)

  • Apply mixed-effects models to account for environmental variation

  • Perform ANOVA followed by post-hoc tests (e.g., Tukey's HSD) for multiple comparisons

  • Consider non-parametric alternatives when assumptions of normality are violated

• Interpreting Pleiotropic Effects:

  • Distinguish primary from secondary phenotypic effects through time-course analyses

  • Use tissue-specific or inducible expression systems to isolate phenotypic impacts

  • Consider the possibility of compensatory mechanisms involving other Tubby-like proteins

• Environmental Interaction Analysis:

  • Evaluate phenotypes under multiple conditions (optimal, drought, nutrient limitation)

  • Test particularly for phosphate stress responses given potential phosphoinositide signaling roles

  • Use principal component analysis to identify key environmental response patterns

• Integration with Molecular Data:

  • Correlate phenotypic changes with alterations in gene expression profiles

  • Link phenotypes to specific biochemical parameters (e.g., phosphoinositide levels)

  • Establish causality through rescue experiments and directed manipulation of downstream targets

This structured approach will help researchers avoid misinterpretation of complex phenotypic data and establish mechanistic connections between TULP7 function and rice development.

What bioinformatic approaches can be used to identify potential TULP7 homologs and substrates across plant species?

To identify TULP7 homologs and potential substrates across plant species, researchers should employ a multi-layered bioinformatic strategy:

For Homolog Identification:

• Sequence-Based Approaches:

  • Position-Specific Iterative BLAST (PSI-BLAST) using the conserved Tubby and F-box domains

  • Hidden Markov Model (HMM) searches of plant proteomes

  • Multiple sequence alignment to identify conserved motifs specific to TULP7-type proteins

• Phylogenetic Analysis:

  • Maximum likelihood or Bayesian inference methods to construct phylogenetic trees

  • Reconciliation of gene trees with species trees to identify orthologous relationships

  • Analysis of selection pressures (dN/dS ratios) to identify functionally important residues

• Structural Comparison:

  • Homology modeling of TULP7 tertiary structures across species

  • Structural alignment to identify conserved binding pockets or interaction surfaces

  • Assessment of electrostatic potential maps to predict functional conservation

For Substrate Prediction:

• Interactome Analyses:

  • Text mining of published literature for known interactions of TLP family proteins

  • Network analysis using protein-protein interaction databases

  • Prediction of interaction partners based on co-expression data

• Motif-Based Prediction:

  • Identification of recognition motifs in known substrates of F-box proteins

  • Scanning of proteomes for proteins containing these motifs

  • Enrichment analysis for biological processes among potential substrates

• Comparative Genomics:

  • Analysis of co-evolution patterns between TULP7 and potential substrates

  • Examination of synteny relationships to identify functionally linked genes

  • Correlation of presence/absence patterns across species

These bioinformatic approaches should be validated through experimental methods such as yeast two-hybrid screening, in vitro ubiquitination assays, and co-immunoprecipitation studies to confirm predicted interactions and substrate relationships.

How can researchers differentiate between direct and indirect effects of TULP7 on rice development and stress responses?

Differentiating between direct and indirect effects of TULP7 requires a systematic experimental strategy:

• Temporal Resolution Studies:

  • Employ time-course experiments with high temporal resolution

  • Use inducible expression systems to trigger TULP7 expression at specific timepoints

  • Monitor early (likely direct) versus late (likely indirect) responses at molecular, cellular, and phenotypic levels

• Direct Target Identification:

  • Perform chromatin immunoprecipitation followed by sequencing (ChIP-seq) if TULP7 has DNA-binding capabilities

  • Use protein immunoprecipitation followed by mass spectrometry (IP-MS) to identify direct protein interactors

  • Employ proximity labeling techniques (BioID or TurboID) to identify proteins in close proximity to TULP7 in vivo

• Molecular Intervention Approaches:

  • Generate phosphoinositide-binding deficient TULP7 mutants by site-directed mutagenesis

  • Create F-box domain mutants unable to form SCF complexes

  • Test whether these mutants can still elicit specific phenotypic responses

• Pathway Dissection:

  • Perform epistasis analysis using genetic crosses with mutants of suspected downstream factors

  • Use pharmacological inhibitors of specific signaling pathways to block potential indirect effects

  • Analyze transcriptional networks to identify regulatory relationships

• Quantitative Models:

  • Develop network models incorporating direct TULP7 targets and their downstream effects

  • Use mathematical modeling to predict system behavior under different conditions

  • Test model predictions experimentally to validate direct versus indirect relationships

Interpretive Framework:
Direct effects should manifest rapidly after TULP7 induction, involve physical interaction with TULP7, and persist in the presence of protein synthesis inhibitors. Indirect effects typically emerge later, involve intermediate factors, and can be blocked by inhibiting specific signaling or transcriptional pathways.

What statistical approaches are most appropriate for analyzing TULP7 expression data across different experimental conditions?

When analyzing TULP7 expression data across different experimental conditions, researchers should select statistical approaches based on experimental design and data characteristics:

For RT-qPCR Data:

• Normalization Methods:

  • Use multiple reference genes selected through algorithms like geNorm or NormFinder

  • Apply the 2^(-ΔΔCt) method with efficiency correction

  • Include inter-run calibrators for experiments conducted across multiple batches

• Statistical Tests:

  • For comparing two conditions: Student's t-test (parametric) or Mann-Whitney U test (non-parametric)

  • For multiple conditions: One-way ANOVA with appropriate post-hoc tests (Tukey, Bonferroni)

  • For factorial designs: Two-way or multi-way ANOVA to assess interaction effects

For RNA-Seq Data:

• Differential Expression Analysis:

  • Use negative binomial models (DESeq2, edgeR) accounting for biological variability

  • Apply appropriate normalization for sequencing depth and RNA composition bias

  • Control for false discovery rate using Benjamini-Hochberg procedure

• Pattern Analysis:

  • Use hierarchical clustering or k-means clustering to identify co-expression patterns

  • Apply principal component analysis to reduce dimensionality and identify major sources of variation

  • Consider time-series analysis methods for developmental series data

For Integrative Analysis:

• Correlation Approaches:

  • Calculate Pearson or Spearman correlation coefficients between TULP7 expression and phenotypic traits

  • Use partial correlation analysis to control for confounding variables

  • Apply canonical correlation analysis for multivariate phenotypic data

• Advanced Modeling:

  • Implement general linear mixed models for complex experimental designs with random effects

  • Consider Bayesian approaches for integrating prior knowledge with experimental data

  • Use structural equation modeling to test causal relationships in pathway analysis

• Visualization Strategies:

  • Create heat maps for visualizing expression across multiple conditions

  • Use volcano plots to display both significance and magnitude of expression changes

  • Develop network visualizations to represent relationships between TULP7 and other genes

These statistical approaches should be selected based on the specific research questions, experimental design, and data structure to ensure robust interpretation of TULP7 expression patterns.