Recombinant Arabidopsis thaliana UPF0496 protein At5g66675 (At5g66675)

What is the Recombinant Arabidopsis thaliana UPF0496 protein At5g66675?

The Recombinant Arabidopsis thaliana UPF0496 protein At5g66675 is a full-length transmembrane protein consisting of 412 amino acids that is expressed in vitro using E. coli expression systems. The protein is identified by UniProt number Q8GW16 and is derived from Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology research . This recombinant protein is typically produced with an N-terminal 10xHis-tag to facilitate purification and detection in experimental settings . The full amino acid sequence of this protein includes multiple structural domains that contribute to its putative functions in cellular processes, though its precise biological role remains under investigation.

What are the optimal storage conditions for maintaining At5g66675 stability?

For optimal stability, the recombinant At5g66675 protein should be stored at -20°C for regular use, while extended storage should be at either -20°C or -80°C . Working aliquots can be maintained at 4°C for up to one week to facilitate ongoing experiments . It is important to note that repeated freezing and thawing cycles are not recommended as they can significantly compromise protein integrity and functionality . The shelf life of this protein varies depending on several factors including storage state, buffer composition, storage temperature, and the inherent stability of the protein itself. Generally, the liquid form has a shelf life of approximately 6 months at -20°C/-80°C, while the lyophilized form can remain stable for up to 12 months under the same storage conditions . Researchers should prepare multiple small aliquots during initial preparation to minimize freeze-thaw cycles and maintain protein quality throughout extended research projects.

How does the expression region 1-412 of At5g66675 relate to its functional domains?

The expression region 1-412 represents the full-length protein sequence of At5g66675, which encompasses all functional domains essential for its native activity . This complete sequence contains several key structural elements including potential transmembrane segments as evidenced by its classification as a transmembrane protein . The comprehensive expression of all 412 amino acids ensures that researchers are working with a protein that maintains its natural structural integrity, which is crucial for studying authentic protein-protein interactions, subcellular localization patterns, and functional properties. The full sequence includes regions that likely form extracellular domains, transmembrane helices, and cytoplasmic regions that collectively contribute to the protein's biological function within plant cells.

What are the critical considerations for designing experiments involving At5g66675?

When designing experiments to study At5g66675, researchers must first clearly define their variables and understand how they are related . For instance, when investigating protein function, the independent variable might be experimental conditions (such as stress treatments), while the dependent variable would be protein activity or plant phenotype . A specific, testable hypothesis must be formulated – for example, hypothesizing that At5g66675 plays a role in a particular cellular pathway based on sequence homology or expression patterns . The experimental design should include appropriate controls, account for potential confounding variables, and incorporate randomization where possible to minimize bias .

For transgenic experiments involving At5g66675, considerations should include proper vector selection, promoter choice (native versus constitutive or inducible), and transformation methodology . When studying protein-protein interactions, researchers should select techniques appropriate for membrane proteins, such as split-ubiquitin systems or bimolecular fluorescence complementation. Additionally, researchers must plan for outcome measurement with appropriate sensitivity and specificity, which might include phenotypic analysis, biochemical assays, or transcriptomic profiling depending on the research question . Statistical analysis methods should be determined a priori to ensure sufficient power and appropriate interpretation of results.

How can RNA-seq methodology be applied to understand At5g66675 function in stress responses?

RNA-seq methodology provides a powerful approach to characterize the role of At5g66675 in stress responses by analyzing transcriptome-wide changes associated with its expression or mutation. Based on established protocols in Arabidopsis research, investigators should design experiments comparing wild-type plants with At5g66675 knockout or overexpression lines under various stress conditions . Sample collection should follow a time-course design to capture both early and late transcriptional responses, with careful attention to developmental stage standardization across samples .

The analytical pipeline would begin with quality control of RNA-seq reads, followed by alignment to the Arabidopsis reference genome and quantification of transcript abundance . Differential expression analysis would identify genes affected by At5g66675 modification under stress conditions, while Gene Ontology enrichment analysis would reveal biological processes potentially regulated by this protein . Researchers should validate key differentially expressed genes (DEGs) using quantitative RT-PCR and correlate expression patterns with physiological or biochemical phenotypes . This approach has proven effective in similar studies, such as the comparative RNA-seq analysis of Arabidopsis responses to AtPep1 and flg22, which successfully identified previously overlooked genes involved in plant immunity pathways . The methodology could readily reveal whether At5g66675 regulates specific stress response genes or participates in broader signaling networks during environmental challenges.

What are the most effective transformation techniques for studying At5g66675 in Arabidopsis?

The floral dip method represents the most widely used and effective transformation technique for studying At5g66675 in Arabidopsis thaliana . This method involves introducing Agrobacterium tumefaciens carrying the desired construct into flowering Arabidopsis plants through simple immersion of inflorescences . For At5g66675 studies, researchers should carefully design constructs that include appropriate regulatory elements, such as native promoters for expression pattern studies or strong constitutive promoters (e.g., 35S) for overexpression analyses . When creating fusion proteins to study subcellular localization or protein interactions, care must be taken to ensure that tags do not interfere with protein folding or membrane insertion.

For successful transformation, researchers should optimize Agrobacterium culture conditions, use plants at the appropriate developmental stage with maximum unopened flower buds, and ensure proper selection of transformants using antibiotic or herbicide resistance markers . When studying transmembrane proteins like At5g66675, it may be beneficial to create chimeric constructs that swap domains between functional and non-functional variants to identify critical regions for protein activity, similar to approaches used in other Arabidopsis studies . Following transformation, researchers should confirm transgene integration through genomic PCR, verify expression levels via RT-PCR or western blotting with antibodies against the protein or its tag, and perform comprehensive phenotypic characterization to elucidate At5g66675 function.

What strategies can optimize heterologous expression of At5g66675 in E. coli systems?

Optimizing heterologous expression of transmembrane proteins like At5g66675 in E. coli requires addressing several challenges specific to membrane protein production. One effective approach involves using specialized expression strains such as C41(DE3) and C43(DE3), which are specifically designed to accommodate toxic or membrane proteins that may otherwise impair host cell growth . These strains contain mutations that allow for better tolerance of membrane protein overexpression. Additionally, researchers should consider employing tightly regulated expression systems to minimize leaky expression, which can be particularly problematic for membrane proteins. The double-lock T7 expression system, requiring both IPTG and pyrimido-pyrimidine-2,4-diamine (PPDA) for induction, provides excellent control over expression timing and intensity .

Codon optimization represents another critical factor for successful expression of plant proteins in bacterial systems. Researchers can address codon bias issues by using E. coli strains supplemented with rare tRNAs, such as BL21(DE3) CodonPlus or Rosetta strains containing pRIL and pRARE plasmids respectively . Alternatively, integration of rare tRNA genes directly into the bacterial chromosome has proven effective in some cases . Expression conditions should be thoroughly optimized, including growth temperature (often lowered to 16-20°C during induction), inducer concentration, and induction duration. For membrane proteins like At5g66675, the addition of specific detergents or lipids to the growth medium may improve proper folding and insertion into bacterial membranes. Finally, fusion partners such as MBP (maltose-binding protein) or SUMO can enhance solubility and facilitate purification, though cleavage sites should be incorporated to allow tag removal if needed for downstream applications.

How can researchers address purification challenges specific to transmembrane proteins like At5g66675?

Purification of transmembrane proteins like At5g66675 presents unique challenges due to their hydrophobic nature and requirement for membrane environments to maintain native conformations. A systematic purification strategy should begin with optimized cell lysis methods that effectively disrupt bacterial membranes while preserving protein structure, such as mechanical disruption combined with carefully selected detergents. The choice of detergent is critical and may require empirical testing of multiple options including non-ionic (e.g., DDM, OG), zwitterionic (e.g., CHAPS), or mild detergents that effectively solubilize membrane proteins while maintaining their functional state .

How can researchers determine the subcellular localization and membrane topology of At5g66675?

Determining the subcellular localization and membrane topology of At5g66675 requires a multi-faceted approach combining both in silico prediction and experimental validation. Researchers should first employ computational tools such as TMHMM, Phobius, or CELLO to predict transmembrane domains and subcellular targeting signals based on the protein's amino acid sequence . These predictions provide a theoretical framework that must be experimentally validated. For experimental determination of subcellular localization, researchers should generate fluorescent protein fusions (e.g., GFP, YFP) with At5g66675, ensuring that the tag does not disrupt targeting signals or protein folding. These constructs should be transformed into Arabidopsis using the floral dip method and examined using confocal microscopy to determine the precise subcellular compartment where At5g66675 resides.

For membrane topology determination, researchers can employ protease protection assays, where microsomes containing At5g66675 are treated with proteases in the presence or absence of membrane-disrupting detergents. Domains exposed to the cytosol will be digested without detergent treatment, while lumenal domains remain protected until membranes are solubilized. Another effective approach involves the use of self-associating split fluorescent proteins, where complementary fragments are fused to different domains of At5g66675. Fluorescence will only occur when both fragments are located on the same side of the membrane. Additionally, immunolocalization using antibodies against either native At5g66675 or its epitope tag in conjunction with markers for different cellular compartments can provide high-resolution information about its precise location within the cell. These combined approaches will generate a comprehensive understanding of both where At5g66675 resides in the cell and how it is oriented within the membrane.

How does recombination in Arabidopsis affect genetic studies of At5g66675 function?

The recombination landscape in Arabidopsis thaliana has significant implications for genetic studies of At5g66675 function, particularly in crossing experiments and segregation analysis. Research has shown that Arabidopsis plants typically carry only one or two crossovers per chromosome pair, resulting in the inheritance of very large, non-recombined genomic fragments from each parent . This limited recombination means that phenotypes attributed to At5g66675 mutations might actually result from closely linked genes that are co-inherited due to physical proximity on the chromosome. Furthermore, studies have documented segregation distortion between parental alleles in over half of examined Arabidopsis populations, where certain allelic combinations are under-represented due to various genetic factors .

When designing genetic studies of At5g66675, researchers must account for these recombination patterns through several strategies. First, multiple independent mutant alleles or transgenic lines should be characterized to confirm that observed phenotypes are specifically due to At5g66675 disruption rather than linked mutations. Second, complementation tests, where the wild-type At5g66675 gene is reintroduced into mutant backgrounds, provide essential validation that phenotypes are directly attributable to the gene of interest. Third, researchers should map the genomic location of At5g66675 relative to known recombination hotspots, particularly considering that recombination frequencies in Arabidopsis consistently increase adjacent to centromeres . Finally, awareness of population-specific recombination patterns is crucial, as these patterns vary between different Arabidopsis accessions . By implementing these strategies, researchers can navigate the complexities of Arabidopsis recombination landscapes to achieve reliable genetic characterization of At5g66675 function.

How can RNA-seq data be integrated with proteomics to characterize At5g66675 function in plant immunity?

Integration of RNA-seq data with proteomics represents a powerful approach to comprehensively characterize At5g66675 function in plant immunity. This multi-omics strategy provides insights at both transcriptional and translational levels, capturing the complex regulatory networks in which At5g66675 may participate. To implement this approach, researchers should first conduct RNA-seq analysis comparing wild-type and At5g66675 mutant plants under both basal conditions and following treatment with immunity elicitors such as flg22 or AtPep1, similar to the methodology described in recent Arabidopsis immunity studies . This transcriptomic profiling will identify differentially expressed genes (DEGs) directly or indirectly regulated by At5g66675, providing initial insights into its potential immune signaling roles.

In parallel, researchers should perform quantitative proteomics using techniques such as iTRAQ or TMT labeling coupled with mass spectrometry to identify proteins whose abundance changes in response to At5g66675 mutation or immune elicitation. Particular attention should be paid to membrane proteome analysis, given At5g66675's transmembrane nature . The integration of transcriptomic and proteomic datasets can reveal correlations between mRNA and protein abundance changes, post-transcriptional regulation mechanisms, and protein interaction networks. Pathway enrichment analysis across both datasets can identify biological processes affected by At5g66675, similar to how recent studies identified previously overlooked immunity genes like PP2-B13 and ACLP1 . Functional validation of key findings should include assays measuring specific immune outputs such as reactive oxygen species production, ethylene biosynthesis, or resistance to pathogens like Pseudomonas syringae . This integrated approach will provide a comprehensive understanding of At5g66675's role in plant immunity by capturing both transcriptional reprogramming and protein-level changes that occur downstream of this transmembrane protein.

What statistical approaches are appropriate for analyzing phenotypic data from At5g66675 mutant studies?

When analyzing phenotypic data from At5g66675 mutant studies, researchers should implement robust statistical approaches that account for biological variability while maintaining sufficient statistical power. For continuous phenotypic measurements (e.g., growth parameters, protein expression levels, stress response metrics), parametric tests such as Student's t-test (for two-group comparisons) or ANOVA (for multiple group comparisons) are appropriate if data meet assumptions of normality and homoscedasticity. When these assumptions are violated, non-parametric alternatives like Mann-Whitney U or Kruskal-Wallis tests should be employed. For all statistical tests, researchers must correct for multiple comparisons when analyzing multiple phenotypes simultaneously, using methods such as Bonferroni correction or False Discovery Rate (FDR) control.

How can researchers differentiate between direct and indirect effects when studying At5g66675 function?

Differentiating between direct and indirect effects of At5g66675 function presents a significant challenge in functional genomics research. To address this challenge, researchers should implement a multi-layered experimental approach that systematically narrows down causal relationships. Time-course experiments represent a crucial first step, as they can establish the temporal sequence of molecular and physiological changes following At5g66675 perturbation. Early responses (minutes to hours) are more likely to represent direct effects, while later responses (days) often reflect downstream consequences. Inducible expression or repression systems for At5g66675 provide temporal control that helps distinguish immediate from secondary effects.

Protein-protein interaction studies using techniques adapted for membrane proteins (such as membrane yeast two-hybrid or proximity labeling approaches) can identify direct interaction partners of At5g66675, establishing potential direct regulatory targets . For transcriptional effects, chromatin immunoprecipitation followed by sequencing (ChIP-seq) of transcription factors differentially regulated in At5g66675 mutants can help establish direct regulatory connections. Pharmacological approaches that inhibit protein synthesis (cycloheximide) or specific signaling pathways can help determine whether observed effects require de novo protein synthesis or intermediate signaling steps, further distinguishing direct from indirect effects.

Genetic approaches are equally valuable, including epistasis analysis where double mutants combining At5g66675 mutation with mutations in putative downstream factors are examined for phenotypic suppression or enhancement. Finally, systems biology approaches integrating transcriptomic, proteomic, and metabolomic data can help reconstruct regulatory networks and identify the most proximal effects of At5g66675 perturbation. By combining these complementary approaches, researchers can build a hierarchical model of At5g66675 function that clearly delineates direct molecular interactions from their indirect physiological consequences.

What emerging technologies could advance our understanding of At5g66675 function?

Emerging technologies across multiple disciplines offer exciting opportunities to deepen our understanding of At5g66675 function. CRISPR/Cas9-based approaches beyond simple gene knockouts, such as base editing or prime editing, could enable precise modification of specific amino acids within At5g66675 to test structure-function hypotheses without completely eliminating the protein. CRISPRi and CRISPRa systems adapted for plants would allow tunable repression or activation of At5g66675 expression, providing insights into dosage-dependent functions. Single-cell RNA-seq techniques, increasingly applicable to plant tissues, could reveal cell type-specific expression patterns and responses to At5g66675 manipulation that might be masked in whole-tissue analyses .

For protein-level characterization, advanced structural biology techniques such as cryo-electron microscopy are becoming more accessible for membrane protein structure determination, potentially revealing the three-dimensional architecture of At5g66675 . Proximity labeling techniques like BioID or TurboID could identify proteins that transiently interact with At5g66675 in living cells, overcoming limitations of traditional co-immunoprecipitation approaches for membrane proteins. Optogenetic tools could allow spatiotemporal control of At5g66675 activity, enabling precise dissection of its function in specific subcellular compartments or developmental contexts. Mass spectrometry imaging could map the spatial distribution of metabolites altered in At5g66675 mutants, providing insights into its metabolic functions.

Long-read sequencing technologies facilitate improved genome assembly and transcriptome analysis, potentially revealing previously uncharacterized splice variants or genetic diversity in At5g66675 across Arabidopsis ecotypes . Finally, advanced phenotyping platforms using machine learning for image analysis could detect subtle morphological phenotypes in At5g66675 mutants that might escape human observation. Integration of these technologies promises to comprehensively characterize At5g66675 function across molecular, cellular, and organismal scales.

How might comparative studies across plant species enhance our understanding of At5g66675 function?

Comparative studies across diverse plant species represent a powerful approach to elucidate the evolutionary conservation and functional significance of At5g66675. Researchers should begin by identifying orthologs of At5g66675 in model and crop species through phylogenetic analysis, focusing particularly on the UPF0496 protein family to which At5g66675 belongs . Sequence comparison across species can reveal highly conserved domains likely critical for protein function, as well as species-specific variations that might relate to specialized adaptations. Expression pattern analysis of orthologs across different plant lineages can provide insights into conservation or divergence of regulatory mechanisms and potential functions.

Functional complementation experiments represent a crucial approach, where At5g66675 orthologs from diverse species are introduced into Arabidopsis At5g66675 mutants to test if they can restore wild-type phenotypes. The degree of functional rescue can indicate the conservation of biochemical activity across evolutionary distance. Conversely, heterologous expression of Arabidopsis At5g66675 in other plant species can reveal whether its function is maintained in different genetic backgrounds. Comparative analysis of protein-protein interaction networks across species can identify conserved interaction partners that likely participate in fundamental processes versus species-specific interactors that might contribute to specialized functions.