Recombinant Arabidopsis thaliana Uncharacterized protein At5g49945 (At5g49945)

What is the basic annotation information for the At5g49945 protein?

At5g49945 is classified as an uncharacterized protein precursor in Arabidopsis thaliana (Mouse-ear cress). According to UniProt data, it has the accession number Q94CC0 and the UniProt ID Y5994_ARATH. The gene is also referenced by its Ordered Locus Name (At5g49945) and ORF name (K9P8) in the Arabidopsis genome. The protein has been assigned the PRO ID PR:Q94CC0, and is defined as "a protein that is a translation product of the At5g49945 gene in Arabidopsis thaliana."

What approaches can be used to predict the function of At5g49945?

To predict the function of this uncharacterized protein, researchers should employ multiple complementary approaches:

• Sequence-based analysis: Perform BLAST searches against characterized proteins, identify conserved domains, and analyze sequence motifs to predict potential functions.

• Structural prediction: Use tools like AlphaFold2 or RoseTTAFold to predict the 3D structure, which may provide insights into function through structural homology.

• Expression correlation analysis: Analyze transcriptomic datasets (such as those available in GEO, like GSE41779) to identify genes with expression patterns similar to At5g49945, potentially revealing functional relationships or common regulatory mechanisms.

• Protein-protein interaction predictions: Use tools and databases to predict potential interaction partners, which may suggest involvement in specific cellular processes.

• Cellular localization prediction: Predict subcellular localization using algorithms that analyze signal peptides and localization signals.

This multi-faceted approach maximizes the chance of generating meaningful functional hypotheses that can be further tested experimentally.

How should researchers determine if At5g49945 is expressed in response to specific stimuli?

To determine if At5g49945 responds to specific stimuli, researchers should:

• Analyze existing transcriptomic data: Review datasets like GSE41779 which examine Arabidopsis responses to wounding and current injection to determine if At5g49945 shows differential expression under these conditions.

• Design targeted qRT-PCR experiments: Develop primers specific to At5g49945 and measure transcript levels in plants exposed to various stressors (biotic, abiotic) or hormonal treatments.

• Generate reporter lines: Create transgenic Arabidopsis lines with the At5g49945 promoter driving a reporter gene (GFP, GUS) to visualize expression patterns in response to different stimuli.

• Perform time-course experiments: Measure expression at multiple time points after stimulus application to capture both rapid and delayed responses.

• Include appropriate controls: Always include untreated plants and housekeeping genes as references to ensure reliable interpretation of results.

This systematic approach will help determine if At5g49945 is part of specific stress response pathways in Arabidopsis.

What expression systems are most suitable for producing recombinant At5g49945 protein?

The optimal expression system for recombinant At5g49945 depends on research goals and protein characteristics:

When selecting an expression system, consider the presence of the precursor segment noted in the UniProt entry , which suggests the protein may undergo processing that could be critical for function.

What are the optimal methods for purifying recombinant At5g49945?

For effective purification of recombinant At5g49945, researchers should consider this step-by-step approach:

• Affinity tag selection: Since At5g49945 is uncharacterized, fusion with affinity tags like His6, GST, or FLAG is recommended. The optimal tag placement (N- or C-terminal) should be determined empirically, considering the precursor nature of the protein.

• Initial capture: Use affinity chromatography matching the selected tag (e.g., Ni-NTA for His-tagged proteins or glutathione resins for GST fusions).

• Secondary purification: Follow with size exclusion chromatography (SEC) or ion exchange chromatography (IEX) to remove contaminants and aggregates.

• Tag removal: If the tag might interfere with functional studies, incorporate a protease cleavage site between the tag and At5g49945. TEV or PreScission proteases are commonly used for specific cleavage.

• Quality control: Verify purity by SDS-PAGE and confirm identity via Western blot or mass spectrometry. Assess the protein's folding state using circular dichroism (CD) or differential scanning fluorimetry (DSF).

• Storage optimization: Determine optimal buffer conditions and storage temperature through stability tests to maintain protein integrity for downstream applications.

This methodical approach should yield pure, functional protein suitable for subsequent structural and functional analyses.

How can researchers verify the correct folding and activity of recombinant At5g49945?

Verifying proper folding and activity of recombinant At5g49945 presents a challenge due to its uncharacterized nature, but several approaches can be employed:

These methods provide complementary information about protein quality and can guide further functional studies despite the current lack of knowledge about At5g49945's specific biological role.

What knockout or knockdown strategies are most effective for studying At5g49945 function in Arabidopsis?

For effective genetic manipulation of At5g49945, researchers should consider these approaches:

• T-DNA insertion lines:

  • Screen existing Arabidopsis T-DNA collections (SALK, SAIL, GABI-Kat) for insertions in At5g49945

  • Verify homozygosity and transcript disruption by PCR and RT-PCR

  • Assess multiple independent lines to control for background mutations

• CRISPR-Cas9 gene editing:

  • Design sgRNAs targeting exons early in the coding sequence

  • Introduce frameshift mutations to ensure complete loss of function

  • Generate multiple independent lines and compare phenotypes

• RNA interference (RNAi):

  • Useful when complete knockout is lethal

  • Design construct targeting unique regions of At5g49945 to avoid off-target effects

  • Consider inducible RNAi systems for temporal control

• Artificial microRNA (amiRNA):

  • Often more specific than traditional RNAi

  • Design following established protocols for Arabidopsis genes

  • Use constitutive or tissue-specific promoters depending on research questions

• Antisense or dominant negative approaches:

  • Express truncated versions of At5g49945 that may interfere with native protein function

  • Particularly useful if At5g49945 functions in protein complexes

Each method has advantages and limitations; the choice depends on research goals, available resources, and preliminary information about gene essentiality.

How should experiments be designed to identify potential interacting partners of At5g49945?

To identify proteins that interact with At5g49945, a comprehensive experimental design should include:

• In vivo approaches:

  • Tandem affinity purification (TAP) tagging: Generate Arabidopsis lines expressing At5g49945-TAP under native or constitutive promoters

  • Co-immunoprecipitation: Develop antibodies against At5g49945 or use epitope-tagged versions

  • Proximity-dependent biotin identification (BioID): Fuse At5g49945 with a biotin ligase to identify nearby proteins

  • Split-reporter systems: Yeast two-hybrid or split-GFP screens against Arabidopsis cDNA libraries

• In vitro approaches:

  • Pull-down assays with purified recombinant At5g49945 as bait

  • Protein arrays to screen for direct physical interactions

  • Crosslinking mass spectrometry to capture transient interactions

• Experimental conditions:

  • Test multiple tissue types and developmental stages

  • Include various stress conditions if At5g49945 is stress-responsive

  • Compare results from plants grown in different environmental conditions

• Data analysis:

  • Use appropriate statistical methods to distinguish true interactions from background

  • Filter against common contaminants in interaction studies

  • Validate top candidates through reciprocal pull-downs or co-localization studies

• Validation experiments:

  • Confirm biological relevance through genetic interaction studies

  • Perform in vitro binding assays to confirm direct interactions

  • Use fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) for in vivo validation

This multi-faceted approach will help build a reliable interactome around At5g49945, providing insights into its cellular function.

What approaches are recommended for investigating At5g49945 subcellular localization?

To definitively determine the subcellular localization of At5g49945, researchers should employ multiple complementary techniques:

• Fluorescent protein fusions:

  • Generate C- and N-terminal GFP/YFP/mCherry fusions under native promoters

  • Introduce these constructs into Arabidopsis via stable transformation

  • Examine localization in multiple tissues and developmental stages

  • Include co-localization with established organelle markers

• Immunolocalization:

  • Develop specific antibodies against At5g49945

  • Perform immunofluorescence microscopy on fixed Arabidopsis tissues

  • Use gold-labeled secondary antibodies for transmission electron microscopy to achieve higher resolution

• Biochemical fractionation:

  • Isolate subcellular fractions (chloroplasts, mitochondria, nucleus, ER, etc.)

  • Perform Western blot analysis using At5g49945-specific antibodies

  • Include marker proteins for each cellular compartment as controls

• Considerations for At5g49945:

  • Since At5g49945 is described as a precursor protein , track potential processing events

  • Assess whether different experimental conditions affect localization

  • The presence of any predicted signal peptides or targeting sequences should inform experimental design

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed localization

  • Time-lapse imaging to detect dynamic localization changes

  • FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

These approaches will provide robust evidence for the subcellular localization of At5g49945, a critical step in understanding its biological function.

What are the best methods to analyze At5g49945 expression patterns across different tissues and developmental stages?

To comprehensively analyze At5g49945 expression patterns, researchers should implement these methods:

• Transcriptomic analysis:

  • Mine existing Arabidopsis microarray and RNA-seq datasets (such as GSE41779)

  • Use tools like eFP Browser and Genevestigator to visualize expression across tissues and conditions

  • Generate custom RNA-seq data for specific tissues or conditions of interest

• Quantitative RT-PCR:

  • Design gene-specific primers spanning exon junctions

  • Validate primers for specificity and efficiency

  • Sample various tissues and developmental stages

  • Use multiple reference genes for accurate normalization

• Promoter-reporter fusion:

  • Clone the native At5g49945 promoter region (1-2 kb upstream of start codon)

  • Generate transgenic Arabidopsis lines with promoter driving GUS or fluorescent protein expression

  • Perform histochemical GUS staining or fluorescence microscopy across developmental stages

• In situ hybridization:

  • Develop RNA probes specific to At5g49945

  • Perform hybridization on tissue sections from different developmental stages

  • This provides cellular resolution of expression patterns

• Data integration:

  • Compile data in standardized formats for comparison

  • Create comprehensive expression maps across development

  • Correlate expression with known developmental markers or physiological processes

This multi-method approach will provide a detailed spatiotemporal map of At5g49945 expression, guiding hypotheses about its biological function in specific tissues and developmental contexts.

How can researchers identify transcription factors that regulate At5g49945 expression?

To identify transcription factors (TFs) regulating At5g49945, implement this systematic approach:

• Promoter sequence analysis:

  • Extract 1-2 kb upstream of the At5g49945 transcription start site

  • Use plant-specific TF binding site prediction tools (PlantPAN, PLACE, PlantCARE)

  • Look for conserved cis-regulatory elements across related species

  • Prioritize motifs that correlate with observed expression patterns

• Chromatin immunoprecipitation (ChIP) approaches:

  • Perform ChIP-seq with antibodies against specific TFs predicted to bind the promoter

  • Consider ChIP-seq with histone modification antibodies to identify active regulatory regions

  • Use publicly available ChIP-seq datasets to screen for TFs binding near At5g49945

• Yeast one-hybrid (Y1H) screening:

  • Clone promoter fragments as bait

  • Screen against Arabidopsis TF libraries

  • Validate positive interactions with targeted Y1H assays

• Expression correlation analysis:

  • Analyze transcriptomic datasets (like GSE41779) to identify TFs whose expression patterns correlate with At5g49945

  • Focus on TFs that precede At5g49945 expression changes in time-course experiments

• Functional validation:

  • Generate transgenic plants overexpressing candidate TFs

  • Measure At5g49945 expression changes

  • Create TF knockout/knockdown lines and assess At5g49945 expression

  • Perform promoter mutagenesis of predicted binding sites and test activity

• Data integration:

  • Combine evidence from multiple approaches to build a regulatory network

  • Consider conditional regulation under different stresses or developmental stages

This comprehensive approach will help identify the key transcriptional regulators of At5g49945, providing insights into its regulatory network and biological context.

What experimental approaches can determine if At5g49945 expression changes in response to wound signaling?

Based on the GSE41779 dataset, which includes studies on wound responses in Arabidopsis , researchers can design experiments to investigate At5g49945's response to wounding:

• Targeted expression analysis:

  • Perform qRT-PCR on At5g49945 with a detailed time course (15 min, 30 min, 1h, 3h, 6h, 24h) after wounding

  • Include both local (wounded leaf) and systemic (distal leaves) responses

  • Use established wound-responsive genes as positive controls

• Promoter-reporter studies:

  • Generate transgenic plants with At5g49945 promoter driving luciferase or GFP

  • Monitor reporter activity in real-time after wounding

  • Perform time-lapse imaging to capture spatial and temporal dynamics

• Wound signaling pathway analysis:

  • Apply specific wound-related hormones (jasmonic acid, ethylene) and analyze At5g49945 expression

  • Test At5g49945 expression in wound signaling mutants (jar1, coi1, mpk6)

  • Investigate if electrical signaling affects expression using experimental setups similar to those in GSE41779

• Comparative transcriptomics:

  • Perform RNA-seq comparing wounded vs. unwounded tissues

  • Include samples from different timepoints to capture early and late responses

  • Analyze co-regulated gene clusters to place At5g49945 in specific response networks

• Chromatin state analysis:

  • Perform ChIP for histone modifications (H3K27ac, H3K4me3) at the At5g49945 locus before and after wounding

  • Assess chromatin accessibility changes using ATAC-seq

This comprehensive approach will determine whether At5g49945 participates in wound response pathways, potentially revealing its biological function in plant stress responses.

What are the recommended approaches for determining the three-dimensional structure of At5g49945?

For determining the structure of the uncharacterized At5g49945 protein, researchers should consider this sequential approach:

• Initial structural predictions:

  • Use AlphaFold2 or RoseTTAFold to generate predicted models

  • Compare predictions with similar proteins in structural databases

  • Identify domains and regions of high confidence vs. disordered regions

• X-ray crystallography:

  • Express and purify recombinant At5g49945 as described earlier

  • Perform crystallization screening with commercial kits

  • Optimize promising conditions for diffraction-quality crystals

  • Consider crystallizing individual domains if full-length protein proves challenging

  • Use selenomethionine labeling for phase determination

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Best for smaller proteins or domains (<30 kDa)

  • Label with 15N and 13C for structure determination

  • Provide information on dynamic regions and potential binding sites

• Cryo-electron microscopy (cryo-EM):

  • Particularly useful if At5g49945 forms larger complexes

  • Consider if crystallization proves challenging

  • May provide insights into structural heterogeneity

• Small-angle X-ray scattering (SAXS):

  • Provides low-resolution envelope in solution

  • Useful for validating computational models

  • Can give insights into flexible regions

• Integrative structural biology:

  • Combine multiple structural techniques with computational modeling

  • Use crosslinking mass spectrometry to obtain distance constraints

  • Validate final models with mutagenesis and functional assays

This multi-faceted approach maximizes the chances of obtaining structural information for this uncharacterized protein, guiding subsequent functional studies.

How should researchers design experiments to determine if At5g49945 has enzymatic activity?

To investigate potential enzymatic activity of the uncharacterized At5g49945 protein, follow this systematic experimental design:

• Initial activity prediction:

  • Analyze sequence for catalytic motifs and conserved residues

  • Perform structural homology searches against known enzyme structures

  • Use enzyme function prediction tools (EFICAz, PRIAM)

  • Consider its classification as a precursor protein, which may indicate processing is needed for activity

• Substrate screening approaches:

  • Design activity assays based on predicted enzyme family

  • Screen substrate libraries relevant to plant metabolism

  • Consider metabolomics approaches comparing wild-type and knockout plants

  • Perform differential scanning fluorimetry with potential substrates to detect binding-induced stability shifts

• Generic activity screening:

  • Test for common enzymatic activities (hydrolase, transferase, oxidoreductase)

  • Screen against panels of model substrates for each enzyme class

  • Consider high-throughput colorimetric or fluorometric assays

• Biochemical characterization:

  • For identified activities, determine:

    • Kinetic parameters (Km, kcat, substrate specificity)

    • pH and temperature optima

    • Cofactor requirements

    • Regulatory mechanisms

• Structure-function analysis:

  • Generate point mutations of predicted catalytic residues

  • Assess activity changes in mutated variants

  • Perform substrate docking in structural models

• In vivo validation:

  • Complement knockout phenotypes with wild-type and catalytically inactive mutants

  • Analyze metabolite profiles in knockout vs. wild-type plants

This comprehensive approach will help determine if At5g49945 possesses enzymatic activity and identify its potential substrates and biochemical function in Arabidopsis.

What methodologies are appropriate for investigating the role of At5g49945 in stress response pathways?

To investigate At5g49945's potential role in stress responses, especially given the context of the wound response studies in GSE41779 , implement these methodologies:

• Stress-response phenotyping:

  • Subject At5g49945 knockout/overexpression lines to multiple stresses:

    • Biotic (bacterial, fungal, insect attack)

    • Abiotic (drought, salt, heat, cold, wounding)

    • Oxidative stress

  • Measure standard physiological parameters (growth, survival, ROS levels)

  • Compare with wild-type under identical conditions

• Transcriptional profiling:

  • Perform RNA-seq comparing knockout vs. wild-type under stress conditions

  • Analyze for differential regulation of known stress-responsive pathways

  • Identify co-regulated gene networks

• Protein interaction studies under stress:

  • Compare At5g49945 interactome before and after stress application

  • Focus on interactions with known stress signaling components

  • Investigate post-translational modifications induced by stress

• Subcellular localization changes:

  • Monitor potential relocalization of At5g49945-GFP fusion under stress

  • Track protein stability and turnover rates during stress responses

• Biochemical activity assays:

  • Assess if enzymatic activity (if identified) changes under stress conditions

  • Test if stress-related molecules directly interact with At5g49945

• Genetic interaction studies:

  • Generate double mutants with known stress response pathway components

  • Look for enhanced or suppressed phenotypes indicating pathway connections

• Electrophysiological measurements:

  • Given the electrical signaling context in GSE41779 , measure membrane potential changes in response to wounding

  • Compare wild-type and knockout responses

This systematic approach will reveal whether At5g49945 plays a direct role in stress signaling pathways and will help position this uncharacterized protein within the broader stress response network of Arabidopsis.

What data analysis approaches are recommended for interpreting large-scale datasets involving At5g49945?

For effective analysis of large-scale datasets involving At5g49945, researchers should implement these analytical strategies:

• Transcriptomic data analysis:

  • Normalize RNA-seq or microarray data using appropriate methods (RPKM, TPM, or RMA)

  • Identify differentially expressed genes in At5g49945 mutants compared to wild-type

  • Perform Gene Ontology enrichment and pathway analysis

  • Use tools like WGCNA (Weighted Gene Co-expression Network Analysis) to identify co-regulated modules

• Proteomic data analysis:

  • For interaction studies, filter against common contaminants using CRAPome database

  • Apply appropriate statistical methods to distinguish true interactors

  • Perform network analysis to identify protein complexes and functional clusters

  • Compare interactome data across different conditions

• Metabolomic integration:

  • Analyze metabolite profiles from At5g49945 mutants

  • Correlate metabolite changes with transcriptomic alterations

  • Map affected pathways using tools like MetaboAnalyst

• Multi-omics data integration:

  • Use integrative platforms like Cytoscape for network visualization

  • Apply machine learning approaches to identify patterns across datasets

  • Create predictive models for At5g49945 function

• Comparative genomics:

  • Analyze At5g49945 orthologs across plant species

  • Correlate evolutionary conservation with functional importance

  • Identify species-specific adaptations

• Visualization strategies:

  • Create custom data visualizations highlighting At5g49945's position in networks

  • Develop interactive visualization tools for exploring complex datasets

This systematic approach to data analysis will help extract meaningful biological insights about At5g49945 from diverse high-throughput datasets, revealing its functional context within Arabidopsis cellular networks.

How can researchers resolve contradictory findings about At5g49945 function?

When faced with contradictory findings regarding At5g49945 function, researchers should implement this systematic reconciliation approach:

• Critical evaluation of experimental designs:

  • Compare methodological differences between contradictory studies

  • Assess statistical power and sample sizes

  • Evaluate the specificity of tools used (antibodies, probes, primers)

  • Consider differences in experimental conditions (growth conditions, developmental stages)

• Technical validation:

  • Repeat key experiments using standardized protocols

  • Use multiple independent techniques to address the same question

  • Employ more sensitive or specific methods when possible

  • Include appropriate positive and negative controls

• Genetic validation:

  • Use multiple alleles or mutant lines to confirm phenotypes

  • Perform complementation tests with the wild-type gene

  • Create rescue lines with specific domains to narrow down functional regions

  • Consider genetic background effects and use backcrossed lines

• Context-dependent function assessment:

  • Test if At5g49945 has different functions in different tissues or developmental stages

  • Investigate environmental or stress-dependent roles

  • Consider potential redundancy with related genes

  • Explore if contradictions arise from different protein isoforms or post-translational modifications

• Collaborative resolution:

  • Establish direct collaboration between labs with contradictory findings

  • Exchange biological materials and protocols

  • Perform joint experiments with team members from both groups

  • Publish reconciliation studies explaining the source of contradictions

This structured approach helps resolve contradictory findings about At5g49945, advancing understanding of its true biological function while addressing the complexities of experimental biology.

What methodologies effectively integrate At5g49945 research findings into broader plant biological knowledge?

To integrate At5g49945 research into broader plant biology knowledge, implement these methodologies:

• Pathway and network integration:

  • Map At5g49945 to known biological pathways in Arabidopsis

  • Use protein-protein interaction data to position At5g49945 in cellular networks

  • Identify central pathways affected in At5g49945 mutants

  • Create visual network models showing connections to established processes

• Cross-species comparison:

  • Identify orthologs of At5g49945 in crop species and model plants

  • Compare function, expression, and regulation across species

  • Assess if functional knowledge can be transferred to agriculturally important plants

• Physiological context mapping:

  • Connect molecular findings to whole-plant phenotypes

  • Relate At5g49945 function to established physiological processes

  • Develop models explaining how At5g49945 contributes to plant adaptation

• Database submission and annotation:

  • Update gene and protein databases with new functional information

  • Submit structures to PDB and models to appropriate repositories

  • Ensure comprehensive Gene Ontology annotation

  • Contribute to community annotation efforts like TAIR

• Ontology development:

  • Utilize standardized plant ontology terms to describe At5g49945 phenotypes

  • Map experimental results to existing ontology frameworks

  • Propose new ontology terms if needed for novel functions

• Synthetic biology applications:

  • Explore if At5g49945 knowledge can be applied in synthetic pathways

  • Assess potential for engineering improved plant traits

  • Design rational modifications based on structure-function insights

• Review and perspective publications:

  • Publish review articles positioning At5g49945 findings in broader contexts

  • Develop perspective pieces suggesting integrative research directions

  • Create comprehensive models incorporating At5g49945 into current knowledge frameworks

This integrative approach ensures that findings about the previously uncharacterized At5g49945 protein contribute to advancing plant biology as a whole, rather than remaining isolated observations.