Recombinant Arabidopsis thaliana UPF0481 protein At3g47200 (At3g47200)

What is UPF0481 protein At3g47200 and what are its basic characteristics?

UPF0481 protein At3g47200 is a full-length protein (476 amino acids) encoded by the At3g47200 gene in Arabidopsis thaliana. The protein belongs to the UPF0481 family of proteins with currently uncharacterized function (UPF stands for Uncharacterized Protein Family). The protein has a UniProt ID of Q9SD53 and can be successfully expressed in E. coli expression systems . Structurally, the protein contains multiple transmembrane domains based on its amino acid sequence, suggesting it may function as a membrane-associated protein. Expression studies indicate it may play roles in plant development processes, particularly in flower development and callus formation .

The complete amino acid sequence is:
MADKTDIISSSSDKASPPPPSAFRNYLSSGSKEPVLLLESAGKESCCIFRVPESFVALNPKAYKPKVVSIGPYHYGEKHLQMIQQHKPRLLQLFLDEAKKKDVEENVLVKAVVDLEDKIRKSYSEELKTGHDLMFMMVLDGCFILMVFLIMSGNIELSEDPIFSIPWLLSSIQSDLLLLENQVPFFVLQTLYVGSKIGVSSDLNRIAFHFFKNPIDKEGSYWEKHRNYKAKHLLDLIRETFLPNTSESDKASSPHVQVQLHEGKSGNVPSVDSKAVPLILSAKRLRLQGIKFRLRRSKEDSILNVRLKKNKLQIPQLRFDGFISSFFLNCVAFEQFYTDSSNEITTYIVFMGCLLNNEDEVTFLRNDKLIIENHFGSNNEVSEFFKTISKDVVFEVDTSYLNNVFKGVNEYTKKWYNGLWAGFRHTHFESPWTFLSSCAVLFVILLTMLQSTVAILSYLNDKKGNGNAAPPPLGLP

What expression patterns does At3g47200 show in different plant tissues?

At3g47200 shows tissue-specific expression patterns that correlate with developmental processes. In Arabidopsis, it has been found to be expressed during various developmental stages. Interestingly, orthologous genes in other plant species show similar patterns - for example, it has been reported to be upregulated during early flower development in Prunus mume (Japanese apricot) .

In Eucalyptus species, a homolog (Eucgr.E04292) shows a distinctive "N-shaped" expression pattern during callus development, with expression levels changing significantly across different callus developmental stages:

This expression pattern suggests the protein may play critical roles in specific phases of callus development, particularly during initial callus formation and the regeneration stage .

What are the recommended methods for producing recombinant At3g47200 protein?

For producing high-quality recombinant At3g47200 protein for research purposes, the following methodological approach is recommended:

• Expression System Selection: E. coli is the preferred expression system for this protein. Using BL21(DE3) or similar strains optimized for recombinant protein expression provides good yields .

• Vector Design: Incorporate an N-terminal His-tag for purification purposes. The tag allows for efficient purification using nickel affinity chromatography .

• Expression Conditions:

  • Induction with IPTG at 0.5-1.0 mM when culture reaches OD600 of 0.6-0.8

  • Post-induction growth at lower temperatures (16-25°C) for 16-20 hours improves soluble protein yield

  • Use rich media such as LB or 2xYT supplemented with appropriate antibiotics

• Purification Protocol:

  • Lysis in Tris/PBS-based buffer containing protease inhibitors

  • Purification using nickel affinity chromatography

  • Secondary purification step (e.g., gel filtration) to increase purity above 90%

  • Final formulation in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Storage Considerations:

  • Store as lyophilized powder for long-term stability

  • For reconstituted protein, add glycerol (recommended final concentration 50%) and store at -20°C/-80°C in small aliquots to avoid freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

How should experimental design be approached when studying At3g47200 function?

When designing experiments to investigate At3g47200 function, a systematic approach following the principles of rigorous experimental design is essential:

• Define Clear Research Questions and Hypotheses:

  • Formulate testable hypotheses about At3g47200 function

  • Identify specific independent variables (e.g., expression levels, mutations) and dependent variables (e.g., phenotypic changes, interaction partners)

• Variable Control:

  • Identify potential confounding variables in your experimental system

  • Control for extraneous variables that might affect results, particularly when working with plant developmental processes

• Experimental Treatments:

  • Design manipulations of At3g47200 expression systematically (e.g., knockout, overexpression, point mutations)

  • Consider using inducible expression systems to control timing of expression

• Statistical Considerations:

  • Ensure adequate biological and technical replicates

  • Plan appropriate statistical tests for data analysis in advance

  • Consider power analysis to determine sample size requirements

• Complementary Methodologies:

  • Combine protein-level studies with transcriptomic approaches

  • Use both in vitro biochemical assays and in vivo functional studies

  • Consider evolutionary approaches by studying orthologs in other species

What approaches are recommended for studying At3g47200's role in callus development?

Given the evidence suggesting At3g47200's involvement in callus development, the following methodological approaches would be most effective:

• Temporal Expression Analysis:

  • Perform high-resolution time-course experiments during callus induction and development

  • Use qRT-PCR to validate expression patterns observed in transcriptomic studies

  • Compare expression in high-potential vs. low-potential callus development systems

• Genetic Manipulation Strategies:

  • Create knockout and overexpression lines in model plants

  • Use CRISPR-Cas9 for precise gene editing

  • Employ inducible expression systems to manipulate expression at specific developmental stages

• Co-expression Network Analysis:

  • Perform WGCNA (weighted gene co-expression network analysis) to identify genes with similar expression patterns

  • Look for enriched biological processes within co-expression modules

  • Identify potential transcription factors regulating At3g47200 expression

The data from Eucalyptus studies showing differential expression during callus development provides a foundation for comparative studies:

This differential expression during callus development suggests At3g47200 and its orthologs may function within developmental signaling networks, potentially interacting with plant hormone and MAPK signaling pathways known to regulate callus formation .

How can protein-protein interaction studies be designed to identify At3g47200 binding partners?

To effectively identify and characterize At3g47200 binding partners, a multi-method approach is recommended:

• Yeast Two-Hybrid (Y2H) Screening:

  • Design bait constructs carefully, considering potential membrane association

  • For transmembrane proteins like At3g47200, use modified Y2H systems such as split-ubiquitin Y2H

  • Screen against Arabidopsis cDNA libraries or specific candidate interactors

• Co-Immunoprecipitation (Co-IP):

  • Use anti-His antibodies for tagged recombinant protein

  • Perform reciprocal Co-IPs to confirm interactions

  • Consider crosslinking approaches for transient interactions

• Bimolecular Fluorescence Complementation (BiFC):

  • Design fusion constructs with split fluorescent proteins

  • Use appropriate subcellular localization controls

  • Perform in planta to maintain native cellular environment

• Proximity Labeling:

  • Fuse At3g47200 with BioID or TurboID for proximity-dependent biotinylation

  • Identify neighboring proteins via streptavidin pulldown and mass spectrometry

  • Particularly useful for membrane-associated proteins

• Surface Plasmon Resonance (SPR) or Microscale Thermophoresis (MST):

  • Use purified recombinant At3g47200 protein

  • Determine binding kinetics and affinities for confirmed interactors

  • Validate and quantify interactions identified through screening methods

What methodologies are appropriate for investigating potential epigenetic regulation of At3g47200?

Given the evidence of epigenetic regulation in Arabidopsis gene clusters, particularly for defense-related genes , investigating epigenetic control of At3g47200 requires specific methodologies:

• DNA Methylation Analysis:

  • Perform bisulfite sequencing of the At3g47200 promoter and gene body

  • Use methylation-sensitive PCR to assess specific regulatory regions

  • Compare methylation patterns across developmental stages and in response to environmental stresses

• Chromatin Immunoprecipitation (ChIP):

  • Analyze histone modifications associated with At3g47200 (e.g., H3K4me3, H3K27me3)

  • Identify transcription factors binding to the At3g47200 promoter

  • Investigate chromatin remodeling factors associated with the locus

• Genetic Approaches:

  • Examine At3g47200 expression in epigenetic mutants (e.g., ddm1, met1)

  • Create reporter constructs to monitor expression changes

  • Consider the genomic context, as R-gene clusters often show complex epigenetic regulation

• ATAC-Seq Analysis:

  • Assess chromatin accessibility at the At3g47200 locus

  • Compare accessibility patterns across developmental stages

  • Correlate with expression data to establish functional relationships

What are the challenges in comparing At3g47200 orthologs across different plant species?

Comparing At3g47200 orthologs across plant species presents several methodological challenges:

• Sequence Divergence:

  • Sequence similarity may be limited to functional domains

  • Design degenerate primers targeting conserved regions for identification

  • Use profile-based searches rather than simple BLAST for distant orthologs

• Functional Equivalence Testing:

  • Test functional complementation by expressing orthologs in Arabidopsis mutants

  • Compare expression patterns using promoter-reporter fusions

  • Assess protein localization patterns across species

• Evolutionary Rate Variation:

  • Account for differences in evolutionary rates when comparing orthologs

  • Use appropriate phylogenetic models that accommodate rate heterogeneity

  • Consider synteny and genomic context for ortholog identification

• Expression Pattern Comparison:

  • Normalize expression data across species and experimental platforms

  • Use comparable developmental stages when comparing expression

  • Consider how tissue culture responses may differ between species

How should contradictory data regarding At3g47200 function be addressed?

When faced with contradictory data about At3g47200 function, implement the following methodological approach:

• Critical Evaluation of Methodologies:

  • Assess differences in experimental design, conditions, and plant materials

  • Consider statistical power and appropriate controls in each study

  • Evaluate whether differences might be due to ecotype or environmental variation

• Replication Studies:

  • Design experiments that directly address contradictions

  • Use multiple methodologies to test the same hypothesis

  • Include positive and negative controls to validate experimental systems

• Reconciliation Framework:

  • Consider whether contradictory results reflect context-dependent functions

  • Investigate potential post-translational modifications or interaction partners

  • Examine spatial and temporal specificity of effects

• Meta-analysis Approach:

  • Systematically review all available evidence

  • Weight findings based on methodological rigor

  • Identify patterns that explain apparent contradictions

What novel methodologies should be considered for future At3g47200 functional studies?

Future research on At3g47200 would benefit from these cutting-edge methodological approaches:

• Single-Cell Transcriptomics:

  • Analyze expression at single-cell resolution during development

  • Identify cell-specific functions and regulation

  • Map expression in spatial context within developing tissues

• Cryo-EM Structure Determination:

  • Resolve the three-dimensional structure of At3g47200

  • Identify functional domains and potential interaction surfaces

  • Guide structure-based functional predictions

• Optogenetic Control:

  • Develop light-inducible At3g47200 expression systems

  • Control protein activity with temporal and spatial precision

  • Study acute vs. chronic effects of protein function

• Synthetic Biology Approaches:

  • Create minimal synthetic systems to test At3g47200 function

  • Design protein variants with enhanced or modified functions

  • Test hypothesized functions through domain swapping experiments

• Systems Biology Integration:

  • Incorporate At3g47200 into plant developmental models

  • Predict phenotypic outcomes of genetic perturbations

  • Identify emergent properties from network analyses

How can high-throughput phenotyping technologies be applied to At3g47200 functional analysis?

High-throughput phenotyping offers powerful approaches for At3g47200 functional analysis:

• Automated Imaging Systems:

  • Track growth and developmental phenotypes over time

  • Quantify subtle morphological differences between genotypes

  • Measure responses to environmental variables systematically

• Metabolomic Profiling:

  • Identify metabolic changes associated with At3g47200 manipulation

  • Link protein function to specific biochemical pathways

  • Detect biochemical phenotypes that precede visible phenotypes

• Multi-omics Integration:

  • Combine transcriptomic, proteomic, and metabolomic datasets

  • Build predictive models of At3g47200 function

  • Identify emergent patterns not visible in single datasets

• Environmental Response Phenotyping:

  • Systematically test responses to biotic and abiotic stresses

  • Identify conditional phenotypes under specific conditions

  • Quantify resilience and recovery after stress exposure