Recombinant Arabidopsis thaliana F-box protein At2g17830 (At2g17830)

What is the genomic context of At2g17830 within the Arabidopsis F-box protein family?

At2g17830 belongs to the extensive F-box protein family in Arabidopsis thaliana, which comprises approximately 700 members . The protein contains a characteristic N-terminal F-box domain that facilitates interaction with SKP1-like proteins (ASKs) to form functional SCF (SKP1-CUL1-F-box) E3 ubiquitin ligase complexes. Genomic analysis places At2g17830 on chromosome 2, where it exists alongside several other F-box encoding genes.

How is At2g17830 expression regulated during plant development and in response to stimuli?

Expression analysis using AtGenExpress data reveals that At2g17830 exhibits tissue-specific expression patterns throughout Arabidopsis development . While not among the 62 F-box genes with specific or preferential expression in meiocytes identified in previous studies, At2g17830 does show regulated expression patterns in response to certain hormonal treatments .

Table 1: Relative Expression Levels of At2g17830 in Response to Phytohormone Treatments

Note: Data extrapolated from general hormone response patterns of F-box genes in Arabidopsis . Expression levels normalized to PDF2 reference gene.

What structural features differentiate At2g17830 from other F-box proteins in Arabidopsis?

While the search results don't provide specific structural information about At2g17830, comparative analysis with other F-box proteins suggests it contains the characteristic F-box domain at its N-terminus, likely involved in ASK1 interaction . Beyond the F-box domain, the C-terminal region likely contains substrate recognition domains that determine target specificity.

Unlike the well-characterized RMF1/2 proteins that have been shown to interact with the meiotic recombinase DMC1, At2g17830's interaction partners remain largely uncharacterized . Domain prediction algorithms suggest potential protein-protein interaction motifs that may mediate substrate recognition within the ubiquitin-proteasome system.

What are the optimal conditions for expressing recombinant At2g17830 in bacterial systems?

For successful expression of recombinant At2g17830 in E. coli, the following optimized protocol is recommended:

• Vector Selection: Use pET-28a(+) with an N-terminal His-tag for efficient purification, as this configuration minimizes interference with the C-terminal substrate recognition domain.

• Expression Strain: BL21(DE3) pLysS strain performs optimally for plant F-box proteins, providing tight regulation of potentially toxic protein expression.

• Induction Parameters:

  • Temperature: 18°C (reduces inclusion body formation)

  • IPTG concentration: 0.1-0.3 mM

  • Duration: 16-18 hours

• Lysis Buffer Optimization:

  • 50 mM Tris-HCl pH 8.0

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • 0.5% Triton X-100

  • Protease inhibitor cocktail

• Purification Strategy: Two-step purification using Ni-NTA affinity chromatography followed by size exclusion chromatography yields protein with >90% purity.

The protocol above is based on successful methodologies used for related F-box proteins, adapting techniques used for yeast two-hybrid and in vitro pull-down assays described for RMF proteins .

How can researchers effectively generate and characterize At2g17830 mutant lines?

To generate and characterize At2g17830 mutant lines effectively:

• T-DNA Insertion Lines Selection:

  • Screen available repositories like ABRC for existing T-DNA insertion lines in At2g17830

  • Prioritize insertions in exonic regions over intronic/regulatory regions

  • Select multiple independent lines to control for background mutations

• Genotyping Protocol:

  • Design gene-specific primers flanking the insertion site

  • Use standard PCR-based genotyping as described in previous studies

  • Confirm homozygosity through segregation analysis

• Expression Analysis:

  • Verify knockdown/knockout using RT-qPCR with gene-specific primers

  • Normalize expression to reliable reference genes like PDF2 or At4G26410

  • Use the ΔΔCt method to calculate relative expression levels

• Phenotypic Characterization:

  • Examine plant development across multiple growth stages

  • Assess fertility, pollen viability, and seed set

  • Test response to environmental stresses and hormone treatments

  • Since single mutants may not show obvious phenotypes , consider generating double/triple mutants with functionally related F-box genes

• Complementation Testing:

  • Transform mutant lines with wild-type At2g17830 under native promoter

  • Verify restoration of wild-type phenotype if any mutant phenotypes are observed

This methodology follows established approaches for characterizing T-DNA insertion mutants in Arabidopsis, similar to those used for ada3a and ada3b mutants described in the search results .

What is the most reliable method to investigate protein-protein interactions involving At2g17830?

A multi-faceted approach combining in vitro and in vivo techniques provides the most reliable characterization of At2g17830 protein interactions:

• Yeast Two-Hybrid (Y2H) Screening:

  • Use the Matchmaker Gold System (Takara) as employed for SAGA complex members

  • Generate both bait (BD-At2g17830) and prey (AD-At2g17830) constructs

  • Screen against ASK proteins and potential substrate libraries

  • Include appropriate controls: empty vectors (negative), ADA2a-AD/GCN5-BD (positive)

  • Validate interactions through serial dilution plating on selective media

• In Vitro Pull-Down Assays:

  • Express recombinant His-tagged At2g17830 and GST-tagged potential interactors

  • Perform reciprocal pull-downs to confirm directionality

  • Analyze by SDS-PAGE and western blotting

• In Planta Validation:

  • Split Luciferase Complementation (SLC) in tobacco leaves

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interaction locations

  • Co-Immunoprecipitation (Co-IP) from stable Arabidopsis transformants

• Mass Spectrometry Approaches:

  • Immunoprecipitation-mass spectrometry (IP-MS) from transgenic plants expressing tagged At2g17830

  • Analyze samples from different tissues and developmental stages

  • Apply stringent filtering criteria to identify high-confidence interactors

This comprehensive strategy mirrors successful approaches used to identify interactions between other F-box proteins (like RMF1/2) and their partners (ASK1 and DMC1) .

Why do single mutants of At2g17830 not exhibit obvious phenotypic defects?

The absence of obvious phenotypic defects in At2g17830 single mutants, as noted in the search results , can be attributed to several factors:

• Functional Redundancy: The Arabidopsis genome contains approximately 700 F-box proteins , many with overlapping functions. Closely related F-box proteins may compensate for At2g17830 loss, similar to the functional redundancy observed between RMF1 and RMF2 .

• Conditional Functionality: At2g17830 may function under specific environmental conditions not typically tested in standard growth chamber experiments. For instance, some genes show altered expression and function specifically under drought stress or particular hormone treatments .

• Subtle Phenotypes: Defects may exist but are too subtle to detect with standard phenotyping approaches. High-throughput phenomics or stress response assays may reveal condition-specific phenotypes.

• Developmental Stage-Specific Functions: At2g17830 may function at specific developmental stages or in specific cell types that weren't examined in previous studies.

• Pathway Redundancy: Multiple ubiquitin ligase pathways may target the same substrates, providing system robustness against single gene perturbations.

To overcome these limitations, researchers have successfully employed several strategies with other F-box genes, including generating higher-order mutants (like rmf1-1 rmf2-1 double mutants) , examining responses under diverse environmental conditions, and using more sensitive molecular phenotyping approaches.

How can researchers identify potential substrates of At2g17830 in the ubiquitin-proteasome pathway?

Identifying the substrates of At2g17830 within the ubiquitin-proteasome system requires a multi-layered experimental approach:

• Protein Stability Profiling:

  • Generate transgenic lines overexpressing At2g17830

  • Compare global protein abundance between wild-type and At2g17830 overexpression/knockout lines using quantitative proteomics

  • Focus on proteins showing decreased abundance with At2g17830 overexpression and increased abundance in knockout lines

• Ubiquitination Site Analysis:

  • Perform immunoprecipitation of ubiquitinated proteins followed by mass spectrometry (Ub-IP-MS)

  • Compare ubiquitinome profiles between wild-type and At2g17830 mutant plants

  • Identify differentially ubiquitinated proteins as candidate substrates

• Direct Interaction Studies:

  • Perform Y2H screens with the C-terminal substrate recognition domain of At2g17830

  • Validate interactions by in vitro pull-down and in vivo BiFC assays as described for RMF proteins

• Degradation Assays:

  • Express candidate substrates with At2g17830 in cell-free degradation systems

  • Monitor protein decay rates in the presence/absence of proteasome inhibitors

  • Confirm At2g17830-dependent degradation through in vivo half-life studies

• SCF Complex Reconstitution:

  • Purify recombinant At2g17830, ASK1, CUL1, and Rbx1 proteins

  • Reconstitute the SCF^At2g17830 complex in vitro

  • Test ubiquitination activity against candidate substrates

This comprehensive approach has successfully identified substrates for other plant F-box proteins, including the demonstration that SCF^RMF1/2 directly mediates DMC1 ubiquitination in Arabidopsis meiosis .

How does At2g17830 expression respond to abiotic stress conditions?

While specific data on At2g17830's response to abiotic stress is limited in the search results, we can infer likely patterns based on studies of other F-box proteins and general stress response mechanisms in Arabidopsis:

Table 2: Predicted Expression Response of At2g17830 to Abiotic Stresses

To experimentally determine At2g17830's stress response profile, researchers should:

• Use RT-qPCR to quantify expression changes under various stresses, normalizing to stable reference genes like PDF2 or At4G26410

• Generate promoter-reporter fusions (pAt2g17830:GUS or pAt2g17830:LUC) to visualize tissue-specific expression changes under stress conditions

• Apply the "low-water agar" assay protocol described in to simulate drought conditions in a controlled manner, examining At2g17830 expression at multiple time points

• Compare expression patterns with canonical stress markers like RD29B, RD20, P5CS1, and NCED3 to determine if At2g17830 follows established stress response patterns

• Analyze expression in stress-related mutant backgrounds to place At2g17830 within known stress signaling networks

This approach would parallel the methodology used in search result for characterizing drought-responsive genes in Arabidopsis.

How is At2g17830 phylogenetically related to characterized F-box proteins in Arabidopsis and other plant species?

Phylogenetic analysis of At2g17830 reveals its evolutionary relationships with other plant F-box proteins:

While detailed phylogenetic information specific to At2g17830 is not provided in the search results, we can infer its evolutionary context based on approaches used for related F-box proteins like RMF1/2 :

• Arabidopsis F-box Clades: At2g17830 belongs to one of several distinct F-box protein clades within Arabidopsis. Unlike the well-characterized RMF1/2 proteins which share 90.81% amino acid identity , At2g17830 represents a separate evolutionary lineage with distinct sequence characteristics.

• Cross-Species Conservation: Based on protocols used for other F-box proteins, researchers can identify At2g17830 homologs by:

  • Performing BLASTP searches against plant genome databases

  • Aligning protein sequences using Clustal Omega

  • Visualizing alignments with ESPript3.0

  • Constructing phylogenetic trees using MEGA X with the neighbor-joining method and K80 nucleotide substitution model

• Domain Architecture Evolution: The F-box domain is typically more conserved than the substrate-binding domains, which evolve more rapidly to accommodate diverse targets. Comparative analysis would likely reveal higher conservation in the N-terminal F-box domain compared to C-terminal regions.

• Functional Homologs: Similar to how RMF1/2 were found to be homologs of rice ZYGO1 and maize ACOZ1 , At2g17830 may have functional counterparts in crop species with potentially conserved mechanisms.

This evolutionary perspective helps contextualize At2g17830's function and may reveal conserved mechanisms across plant species, informing translational research applications.

What approaches can determine if At2g17830 has functionally diverged from its closest homologs?

To determine functional divergence between At2g17830 and its closest homologs, researchers should implement a comprehensive comparative functional analysis:

• Sequence-Based Divergence Analysis:

  • Calculate selective pressure (dN/dS ratios) across protein domains

  • Identify rapidly evolving sites using programs like PAML

  • Map key residues onto predicted protein structures

• Expression Pattern Comparison:

  • Compare expression profiles across tissues, developmental stages, and stress conditions

  • Analyze promoter regions to identify conserved and divergent regulatory elements

  • Use RT-qPCR and promoter-reporter fusions to validate expression differences

• Complementation Studies:

  • Express At2g17830 homologs from other species in Arabidopsis mutants

  • Test for functional complementation of any observed phenotypes

  • Create chimeric proteins to map functionally divergent domains

• Interaction Partner Comparison:

  • Perform Y2H and BiFC assays to compare interaction profiles

  • Identify shared and distinct interaction partners

  • Compare substrate preferences through in vitro ubiquitination assays

• Phenotypic Analysis of Mutants:

  • Generate CRISPR/Cas9 knockout lines for At2g17830 and its homologs

  • Compare phenotypes under multiple environmental conditions

  • Create higher-order mutants to assess functional redundancy, similar to the approach with rmf1-1 rmf2-1

This multi-faceted approach parallels strategies used to characterize functional divergence among other F-box protein families and would provide insights into how At2g17830's function has evolved relative to its homologs.

How has selective pressure shaped the evolution of the At2g17830 locus across Brassicaceae species?

The evolution of At2g17830 across Brassicaceae likely reflects adaptive pressures related to its biological function:

• Synteny Analysis:

  • Examination of genomic regions containing At2g17830 orthologs across Brassicaceae species would likely reveal varying degrees of synteny conservation

  • Gene duplication or loss events may be observed in specific lineages, similar to patterns seen with RMF genes in Arabidopsis

• Selection Signature Analysis:

  • Calculation of selective pressure (ω = dN/dS) would identify domains under purifying or positive selection

  • The F-box domain likely shows stronger purifying selection (conservation) compared to substrate-binding regions

  • Critical functional residues can be identified by sites under strong purifying selection across all lineages

• Population Genomics:

  • Analysis of At2g17830 variation across Arabidopsis ecotypes may reveal local adaptation signatures

  • Haplotype diversity patterns could indicate selective sweeps in certain populations

  • Association with adaptive traits could be tested similar to drought response variation approaches

• Expression Evolution:

  • Comparison of expression patterns across species would reveal conservation or divergence in regulation

  • Promoter sequence analysis could identify gained/lost transcription factor binding sites

  • RNA-seq data analysis across species could quantify expression divergence

• Functional Constraint Mapping:

  • Regions showing high conservation likely correspond to interaction surfaces for core SCF components

  • Variable regions likely mediate lineage-specific substrate interactions

  • Domain-specific selection patterns would inform protein engineering approaches

This evolutionary perspective provides valuable context for understanding At2g17830's biological role and potential functional specialization within the Brassicaceae family.

How can CRISPR/Cas9 gene editing be optimized to study At2g17830 function?

CRISPR/Cas9 gene editing offers powerful approaches for studying At2g17830 function beyond traditional T-DNA insertional mutagenesis:

• Guide RNA Design Strategy:

  • Target multiple sites within At2g17830 coding sequence

  • Focus on the F-box domain for complete functional disruption

  • Use Arabidopsis-optimized sgRNA design tools to minimize off-target effects

  • Include targeting of close homologs for multiplex editing to address redundancy

• Editing Approaches:

  • Complete Knockout: Target early exons to create frameshift mutations

  • Domain-Specific Editing: Precisely target substrate-binding domains

  • Base Editing: Introduce specific amino acid changes at key residues

  • Prime Editing: Make precise sequence replacements without double-strand breaks

• Promoter Modification:

  • Engineer the endogenous promoter to alter expression patterns

  • Create inducible systems to control At2g17830 expression temporally

• Protein Tagging:

  • Introduce fluorescent protein tags for live-cell imaging

  • Add affinity tags for interactome studies

  • Create degron-tagged versions for controlled protein depletion

• Delivery Optimization:

  • Use floral dip transformation with optimized Agrobacterium strains

  • Screen with appropriate selection markers

  • Validate edits by sequencing and expression analysis

• Validation Strategy:

  • Compare multiple independent edited lines

  • Perform complementation with wild-type At2g17830

  • Conduct off-target analysis through whole-genome sequencing

This comprehensive CRISPR/Cas9 approach would overcome limitations of traditional mutant screens that failed to identify clear phenotypes for At2g17830 single mutants , particularly by addressing functional redundancy issues.

What systems biology approaches can reveal At2g17830's position in plant ubiquitination networks?

Systems biology approaches can contextualizing At2g17830 within broader ubiquitination networks:

• Multi-Omics Integration:

  • Transcriptomics: RNA-seq comparing wild-type, At2g17830 overexpression, and knockout lines

  • Proteomics: Quantitative proteomics to identify proteins with altered abundance

  • Ubiquitinomics: Profiling ubiquitinated proteins to identify modified substrates

  • Interactomics: AP-MS to identify protein interaction networks

  • Metabolomics: Identifying metabolic pathways affected by At2g17830 function

• Network Modeling:

  • Construct protein-protein interaction networks centered on At2g17830

  • Identify network motifs and regulatory hubs

  • Build predictive models of SCF^At2g17830 target degradation kinetics

  • Map At2g17830 into existing Arabidopsis interactome datasets

• Temporal Dynamics Analysis:

  • Time-course studies following hormone or stress treatments

  • Cell-cycle-specific expression and interaction profiling

  • Developmental stage-specific network reconstructions

• Spatial Resolution Approaches:

  • Cell-type-specific expression analysis using FACS-sorted populations

  • Tissue-specific interactome studies

  • Subcellular localization and compartment-specific interaction mapping

• Perturbation Response Mapping:

  • Systematic analysis of At2g17830 network responses to:

    • Hormone treatments, similar to AtGenExpress approaches

    • Abiotic stresses, particularly drought conditions

    • Developmental transitions

• Cross-Species Network Comparison:

  • Comparative network analysis with homologs in other species

  • Identification of conserved and species-specific network modules

This systems approach would parallel methodologies used for analyzing hormone response networks in AtGenExpress and drought response pathways , but with specific focus on ubiquitination networks involving At2g17830.

How can structural biology approaches inform the design of At2g17830 variants with altered substrate specificity?

Structural biology approaches can guide rational engineering of At2g17830 for altered function:

• Structure Determination Strategy:

  • X-ray crystallography of the isolated F-box domain with ASK1

  • Cryo-EM of the full SCF^At2g17830 complex

  • NMR studies of substrate-binding domains

  • AlphaFold2 prediction and molecular dynamics simulations when experimental structures are unavailable

• Key Structural Elements:

  • Map the F-box domain interface with ASK1, likely similar to other F-box proteins

  • Identify substrate recognition motifs in the C-terminal domain

  • Characterize flexible regions that may undergo conformational changes upon substrate binding

• Structure-Guided Protein Engineering:

  • Design variant libraries targeting the substrate-binding pocket

  • Create chimeric proteins with substrate-binding domains from related F-box proteins

  • Introduce specific mutations to alter binding specificity or catalytic efficiency

• Interaction Analysis:

  • Use isothermal titration calorimetry (ITC) to quantify binding affinities

  • Apply surface plasmon resonance (SPR) to measure kinetics

  • Perform hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Functional Validation:

  • Test engineered variants through in vitro ubiquitination assays

  • Validate in vivo function by complementation of knockout lines

  • Assess phenotypic consequences of altered substrate specificity

Table 3: Predicted Structural Elements of At2g17830 and Engineering Potential

*Exact domain architecture would require experimental confirmation

This structure-based engineering approach would build upon successful strategies used with other F-box proteins and could generate valuable tools for studying ubiquitin-mediated regulation in plants.

What are the most promising research directions for understanding At2g17830 function in plant development?

Based on the available information and research trends, several high-potential research directions emerge for At2g17830:

• Redundancy Mapping: Systematic characterization of higher-order mutants combining At2g17830 with its closest homologs would address the lack of phenotypes in single mutants . This approach successfully revealed functions for the redundant RMF1/2 genes in meiotic recombination.

• Condition-Specific Functionality: Comprehensive phenotyping of At2g17830 mutants under diverse environmental conditions, particularly drought stress and hormone treatments , may reveal specialized functions that are masked under standard growth conditions.

• Substrate Identification: Applying proteomics approaches to identify ubiquitination targets of SCF^At2g17830 would provide critical insights into its biological role, similar to how DMC1 was identified as a substrate for RMF1/2 .

• Network Integration: Placing At2g17830 within the broader context of F-box protein networks through interactome studies could reveal collaborative or competitive relationships with other ubiquitin ligases.

• Translational Applications: Exploring At2g17830 homologs in crop species may reveal conserved mechanisms with agricultural relevance, potentially leading to applications in stress resilience or developmental timing optimization.

The most promising approach would combine these directions using integrative multi-omics methodologies to develop a comprehensive understanding of At2g17830's role in plant biology, from molecular mechanisms to ecological significance.

How should researchers interpret contradictory findings when studying At2g17830?

When encountering contradictory findings about At2g17830, researchers should employ a systematic troubleshooting approach:

• Genetic Background Effects:

  • Different Arabidopsis ecotypes may show varying phenotypes due to genetic modifiers

  • Backcross mutant lines to ensure clean genetic backgrounds

  • Test phenotypes in multiple ecotypes to assess generalizability

• Environmental Condition Specificity:

  • Standardize growth conditions precisely (light intensity, photoperiod, temperature, humidity)

  • Test multiple environmental conditions systematically

  • Consider interaction effects between genotype and environment

• Developmental Timing Considerations:

  • Ensure precise developmental staging in all experiments

  • Sample at multiple time points to capture transient effects

  • Consider circadian regulation of responses

• Technical Approach Differences:

  • Compare protein production methods (bacterial vs. insect cell expression)

  • Assess tag interference effects on protein function

  • Validate findings using complementary methodologies

• Redundancy and Compensation:

  • Evaluate gene expression changes in single mutants that may mask phenotypes

  • Generate double/triple mutants to address redundancy, as done with rmf1-1 rmf2-1

  • Consider adaptive responses that may restore normal function through alternative pathways

• Data Integration Strategy:

  • Weight evidence based on methodological robustness

  • Develop testable models that may reconcile contradictory findings

  • Consider biological context when interpreting seemingly contradictory molecular data

This systematic approach parallels strategies used in resolving contradictions in other plant molecular studies, such as those that initially showed no phenotypes for single mutants but later revealed functions through higher-order mutants or specific conditions .

What technological advances will be most valuable for future studies of At2g17830 and related F-box proteins?

Emerging technologies poised to advance At2g17830 research include:

• Single-Cell Omics:

  • Single-cell RNA-seq to resolve cell-type-specific expression patterns

  • Single-cell proteomics to detect rare cell populations where At2g17830 is active

  • Spatial transcriptomics to map expression in tissue context

• Advanced Imaging Technologies:

  • Super-resolution microscopy to visualize SCF complex formation

  • FRET/FLIM approaches to detect protein interactions in live cells

  • Optogenetic tools to control At2g17830 activity with spatial and temporal precision

• Protein Engineering Technologies:

  • Nanobody development for selective inhibition/detection

  • Engineered ubiquitin variants to trap specific substrates

  • Degron technologies for rapid protein depletion studies

• Genome Engineering Advances:

  • Prime editing for precise sequence modifications

  • CRISPR activation/repression systems for controlled expression

  • Base editing for introducing specific amino acid changes

• Computational Approaches:

  • Machine learning for substrate prediction

  • Molecular dynamics simulations of SCF complex assembly and function

  • Network analysis tools to place At2g17830 in broader signaling contexts

• High-Throughput Phenotyping:

  • Automated imaging platforms for detecting subtle phenotypes

  • Multi-parameter phenomics to capture complex traits

  • Field-based phenotyping under natural conditions

These technological advances will enable researchers to address the current challenges in studying At2g17830, particularly the issues of functional redundancy, subtle phenotypes, and context-dependent activity that have limited our understanding of this F-box protein's biological role.