Recombinant Solanum lycopersicum Ubiquitin-40S ribosomal protein S27a (UBI3)

What is Ubiquitin-40S ribosomal protein S27a (UBI3) in Solanum lycopersicum and why is it significant in plant research?

UBI3 in Solanum lycopersicum (tomato) is a fusion protein consisting of ubiquitin and the 40S ribosomal protein S27a. This protein serves dual cellular roles: the ubiquitin component participates in protein degradation pathways through the ubiquitin-proteasome system, while the ribosomal protein component contributes to protein synthesis machinery. The gene belongs to a family of ubiquitin fusion proteins that are highly conserved across plant species, with similar proteins identified in Arabidopsis thaliana and other model plants.

The significance of UBI3 in plant research stems from:

• Its fundamental role in protein homeostasis

• Its utility as a reference gene in expression studies due to relatively stable expression patterns

• The evolutionary conservation of its structure across species

• Its involvement in multiple cellular processes including stress responses and development

Understanding UBI3 provides insights into basic plant cellular mechanisms and potential applications in plant biotechnology and crop improvement.

How is the UBI3 gene commonly utilized as a reference gene in tomato research?

UBI3 serves as an important reference gene in quantitative reverse transcription polymerase chain reaction (qRT-PCR) studies in tomato research . The methodological workflow for utilizing UBI3 as a reference gene typically involves:

• Primer design targeting conserved regions of the UBI3 gene

• Validation of expression stability across experimental conditions

• Implementation of the 2^(-ΔΔCt) method for relative quantification

• Normalization of target gene expression data against UBI3 expression values

Researchers frequently pair UBI3 with other reference genes such as actin to increase reliability of expression normalization, as demonstrated in studies examining tomato responses to pathogens . This multi-reference gene approach helps mitigate potential variations in expression levels that might occur with any single reference gene.

What experimental approaches are used to study UBI3 expression patterns across different tomato tissues?

To investigate UBI3 expression patterns across different tomato tissues, researchers employ several complementary methodologies:

• Tissue-specific qRT-PCR analysis:

  • Comparing UBI3 expression in roots, stems, leaves, flowers, and fruits at different developmental stages

  • Using specific primers targeting the UBI3 gene sequence

  • Analyzing data with appropriate statistical methods to identify significant differences

• RNA-seq transcriptome analysis:

  • Generating comprehensive expression profiles across multiple tissues

  • Quantifying UBI3 transcript abundance relative to other genes

  • Identifying potential tissue-specific splicing variants

• Promoter-reporter fusion assays:

  • Creating UBI3 promoter-GUS or UBI3 promoter-GFP fusions

  • Transforming these constructs into tomato plants

  • Histochemical or fluorescence analysis to visualize tissue-specific activity

• In situ hybridization:

  • Using UBI3-specific probes to detect mRNA in tissue sections

  • Providing spatial resolution of expression patterns within complex tissues

These approaches collectively provide a comprehensive understanding of where and when UBI3 is expressed throughout the tomato plant.

What are the key structural features of the tomato UBI3 promoter and how do they compare to other plant ubiquitin promoters?

Based on research on plant promoters, the tomato UBI3 promoter likely contains several key structural components :

• Core promoter elements:

  • TATA-box: Typically located ~30 bp upstream of the transcription start site (TSS)

  • Initiator (Inr) elements: Following the "YR Rule" with a C or T nucleotide 1 bp upstream and an A or G nucleotide 1 bp downstream of the TSS

  • Y patch motifs: Pyrimidine-rich sequences contributing to transcriptional activity

• Proximal and distal regulatory elements:

  • Enhancer motifs that increase basal transcription levels

  • Potential repressor/silencer elements

  • Cis-regulatory elements responding to specific environmental and developmental signals

When compared with other plant ubiquitin promoters, such as those from Arabidopsis, the tomato UBI3 promoter likely shares conserved motifs. Research indicates that approximately 30-50% of 8-bp promoter motifs are conserved between Arabidopsis and rice, suggesting similar conservation patterns may exist between tomato and other plant species .

How do leader introns influence UBI3 gene expression in tomato plants?

Leader introns can significantly enhance gene expression in plants, as demonstrated in multiple ubiquitin genes . The effects of leader introns on UBI3 expression involve several mechanisms:

• Transcriptional enhancement:

  • Leader introns may contain enhancer elements that promote transcription

  • The process of intron splicing can increase transcription efficiency

  • Intron-mediated enhancement has been observed in multiple plant species

• Post-transcriptional regulation:

  • Improved mRNA stability and nuclear export

  • Enhanced translation efficiency

  • Potential for alternative splicing leading to transcript variants

The presence of leader introns has been shown to enhance gene expression in multiple plant ubiquitin genes, including the Arabidopsis ubiquitin genes (UBQ3, UBQ10, and UBQ11) . Similar enhancement effects have been observed in tobacco Ubiquitin.U4 (Ubi.U4) and tomato (Solanum lycopersicum) ascorbate peroxidase20 (APX20) .

Experimentally, the influence of leader introns can be studied by creating constructs with and without the intron sequence, fused to reporter genes such as GFP or GUS, and comparing expression levels in transgenic plants or transient expression systems.

How does environmental stress impact UBI3 expression and what methodologies best capture these responses?

The ubiquitin-proteasome system plays crucial roles in plant responses to environmental stresses. Methodological approaches to investigate stress impacts on UBI3 expression include:

• Controlled stress exposure:

  • Subjecting tomato plants to precise abiotic stresses (heat, cold, drought, salinity)

  • Inoculating plants with pathogens or herbivores for biotic stress studies

  • Maintaining appropriate controls and replicates

• Multi-level expression analysis:

  • qRT-PCR for transcript level quantification

  • Western blotting for protein abundance measurement

  • Polysome profiling to assess translation efficiency

  • Reporter gene assays to visualize spatial expression patterns

• Time-course studies:

  • Sampling at multiple time points post-stress application

  • Capturing both early and late response phases

  • Analyzing recovery patterns when stress is removed

• Promoter element analysis:

  • Identifying stress-responsive elements in the UBI3 promoter

  • Creating promoter deletion/mutation constructs to test element function

  • Examining transcription factor binding under stress conditions

These methodologies collectively provide a comprehensive understanding of how UBI3 responds to environmental challenges and its potential role in stress adaptation mechanisms.

What are the optimal protocols for recombinant expression and purification of tomato UBI3 protein?

Based on published approaches for similar recombinant proteins, optimal protocols for tomato UBI3 expression and purification would include :

• Expression system selection and optimization:

  • E. coli-based expression (BL21 or similar strains)

  • Fusion with ketosteroid isomerase (KSI) for enhanced expression

  • Inclusion of cleavable His-tag or other affinity tag

  • Insoluble expression approach for high yield

• Expression conditions:

  • IPTG concentration: 0.5-1.0 mM

  • Induction temperature: 25-30°C for soluble expression, 37°C for inclusion bodies

  • Induction duration: 4-6 hours (soluble) or overnight (inclusion bodies)

  • Media supplementation with additional glucose or glycerol

• Purification strategy:

  • Inclusion body isolation if expressed as insoluble protein

  • Solubilization with chaotropic agents (8M urea or 6M guanidine-HCl)

  • Affinity chromatography using Ni-NTA or similar matrices

  • Size exclusion chromatography for final purification

  • Refolding via dialysis or dilution methods

A similar approach for the antimicrobial peptide UBI18-35 yielded approximately 6 mg of purified protein per liter of culture , suggesting this methodology may be effective for tomato UBI3 as well.

What challenges are typically encountered when expressing recombinant tomato UBI3 and how can they be addressed?

The recombinant expression of tomato UBI3 presents several challenges that require specific methodological solutions:

• Protein solubility issues:

  • Challenge: UBI3 may form inclusion bodies when overexpressed

  • Solution: Optimize induction conditions (lower temperature, reduced IPTG concentration)

  • Alternative: Express as fusion with solubility-enhancing partners (MBP, SUMO, Trx)

• Dual-domain protein complexity:

  • Challenge: The fusion nature of UBI3 (ubiquitin + ribosomal protein) may cause structural instability

  • Solution: Express domains separately if studying individual functions

  • Alternative: Include linker optimization if expressing the complete fusion protein

• Post-translational processing:

  • Challenge: Natural UBI3 undergoes proteolytic processing to separate ubiquitin

  • Solution: Design constructs with mutated cleavage sites to maintain fusion protein integrity

  • Alternative: Include protease inhibitors during purification

• Proper folding:

  • Challenge: Achieving native conformation after purification

  • Solution: Implement step-wise refolding protocols with redox buffering

  • Alternative: Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ)

• Purification specificity:

  • Challenge: Separating recombinant UBI3 from endogenous E. coli proteins

  • Solution: Use combination of purification techniques (affinity, ion exchange, size exclusion)

  • Alternative: Include additional washing steps with increased imidazole concentrations

Addressing these challenges systematically can significantly improve the yield and quality of recombinant tomato UBI3 protein.

How can researchers validate the structural integrity and functionality of purified recombinant UBI3?

Validating the structural integrity and functionality of purified recombinant UBI3 requires a multi-faceted approach:

• Structural validation:

  • SDS-PAGE and Western blotting to confirm size and immunoreactivity

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Mass spectrometry for accurate mass determination and detection of modifications

  • Limited proteolysis to probe domain structure and accessibility

• Functional assays for ubiquitin domain:

  • In vitro ubiquitination assays with E1, E2 enzymes

  • Binding assays with ubiquitin-binding domain proteins

  • Thermal shift assays to measure protein stability

• Functional assays for ribosomal protein S27a domain:

  • RNA binding assays

  • In vitro translation assays to assess incorporation into ribosomes

  • Interaction studies with ribosomal assembly factors

• Comparative analysis:

  • Comparing properties with native UBI3 isolated from tomato

  • Assessing function against homologous proteins from other species

  • Testing complementation of mutants in model systems

These validation steps ensure that the recombinant protein maintains both structural and functional characteristics of native UBI3, making it suitable for downstream applications in research.

How can the tomato UBI3 promoter be utilized in plant biotechnology and genetic engineering?

The tomato UBI3 promoter offers multiple applications in plant biotechnology based on the understanding of plant promoter function :

• Gene expression control:

  • Driving constitutive expression of transgenes across tissues

  • Creating tissue-specific expression by combining core elements with tissue-specific enhancers

  • Developing stress-inducible expression systems by incorporating stress-responsive elements

• Synthetic biology applications:

  • Creating synthetic promoter variants with enhanced or modified activity

  • Developing regulatory circuits with predictable expression patterns

  • Computational modeling to predict and optimize promoter performance

• Practical biotechnology applications:

  • Developing disease-resistant tomato varieties by driving defense gene expression

  • Enhancing nutritional content through controlled metabolic engineering

  • Improving stress tolerance by regulating stress-response pathways

  • Modifying fruit quality, ripening, or shelf-life characteristics

The research indicates that synthetic core promoters can be designed by inserting core promoter motifs (TATA-box, Inr, Y patch) into appropriate nucleotide backgrounds . When applied to the UBI3 promoter, this approach could generate promoter variants with specifically tailored expression characteristics for various biotechnological applications.

What methodologies are most effective for studying UBI3's role in protein degradation pathways in tomato?

Investigating UBI3's role in protein degradation pathways requires sophisticated experimental approaches:

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated gene editing for precise UBI3 modification

  • RNA interference (RNAi) for transient or stable knockdown

  • Virus-induced gene silencing (VIGS) for tissue-specific silencing

  • Overexpression studies using constitutive or inducible promoters

• Biochemical analysis methods:

  • Ubiquitination assays to measure UBI3 contribution to the ubiquitin pool

  • Co-immunoprecipitation to identify UBI3-interacting proteins

  • In vitro reconstitution of ubiquitin-dependent degradation

  • Proteasome activity assays in plants with altered UBI3 expression

• Advanced imaging techniques:

  • Fluorescent protein tagging for subcellular localization

  • Fluorescence resonance energy transfer (FRET) for protein interaction studies

  • Live-cell imaging to track protein degradation dynamics

  • Super-resolution microscopy for detailed subcellular analysis

• Systems biology approaches:

  • Quantitative proteomics to identify proteins with altered stability

  • Phosphoproteomics to examine effects on signaling networks

  • Transcriptomics to identify genes responding to altered UBI3 function

  • Network analysis to map UBI3-dependent degradation pathways

These complementary approaches provide comprehensive insights into UBI3's specific roles in protein degradation and cellular homeostasis.

How does UBI3 function in tomato defense responses against pathogens and what experimental designs best elucidate these mechanisms?

Based on research showing UBI3's use as a reference gene in pathogen studies , investigating its role in defense responses requires specialized experimental designs:

• Pathogen challenge studies:

  • Inoculating tomato plants with diverse pathogens (bacteria, fungi, viruses)

  • Time-course analysis of UBI3 expression during infection progression

  • Comparison between compatible (susceptible) and incompatible (resistant) interactions

  • Analysis in plants with altered immune signaling pathways

• UBI3 manipulation approaches:

  • Creating UBI3 knockdown/knockout lines via CRISPR or RNAi

  • Developing UBI3 overexpression lines

  • Engineering chimeric UBI3 variants with modified functions

  • Pathogen challenge assays with these modified plants

• Protein-level investigations:

  • Identifying defense-related proteins targeted for UBI3-mediated ubiquitination

  • Examining how pathogen effectors interact with or modify UBI3 function

  • Assessing changes in UBI3 processing during immune responses

  • Monitoring UBI3 subcellular localization during infection

• Integrated multi-omics:

  • Transcriptome analysis of defense responses in UBI3-modified plants

  • Proteome analysis focusing on ubiquitinated proteins during infection

  • Hormone profiling to connect UBI3 function with defense signaling networks

The research on endophytic Beauveria bassiana in tomato provides methodological frameworks for studying induced resistance that could be applied to investigate UBI3's role in defense signaling .

How does tomato UBI3 compare structurally and functionally with UBI3 orthologs in other plant species?

Comparative analysis of tomato UBI3 with orthologs from other plant species reveals important evolutionary insights:

• Sequence and structural conservation:

  • The ubiquitin domain shows extremely high conservation (>95% identity) across plant species

  • The ribosomal protein S27a domain displays moderate conservation (70-85% identity)

  • Domain arrangement and linker regions show species-specific variations

  • Post-translational modification sites may differ between species

• Functional comparative analysis:

  • Core functions in protein degradation and ribosome biogenesis are conserved

  • Species-specific adaptations may relate to environmental challenges

  • Regulatory mechanisms show greater divergence than protein structure

  • Expression patterns may reflect species-specific developmental programs

• Evolutionary considerations:

  • UBI3 represents an ancient fusion protein conserved across eukaryotes

  • Duplication events have created paralogs in some plant lineages

  • Selection pressure maintains critical functional domains

  • Diversification occurs primarily in regulatory regions

Plant transcriptional regulation shows significant conservation, with approximately 30-50% of 8-bp promoter motifs conserved between Arabidopsis and rice , suggesting similar patterns may exist in regulatory elements controlling UBI3 expression across species.

What can comparative genomics approaches reveal about UBI3 evolution across the Solanaceae family?

Comparative genomics approaches provide valuable insights into UBI3 evolution within Solanaceae:

• Sequence-based evolutionary analysis:

  • Multiple sequence alignment of UBI3 from diverse Solanaceae species

  • Phylogenetic tree construction to trace evolutionary relationships

  • Calculation of selection pressures (Ka/Ks ratios) across protein domains

  • Identification of lineage-specific accelerated evolution

• Genomic context analysis:

  • Examination of synteny and gene order conservation around UBI3

  • Analysis of intron-exon structure variation across species

  • Identification of transposable element insertions affecting UBI3 regulation

  • Detection of gene duplication or loss events

• Promoter evolution investigation:

  • Comparison of cis-regulatory elements across Solanaceae species

  • Identification of conserved transcription factor binding sites

  • Analysis of promoter structural variations (insertions, deletions, rearrangements)

  • Correlation of regulatory element conservation with expression patterns

• Functional divergence assessment:

  • Complementation studies across species

  • Domain swapping experiments to test functional equivalence

  • Expression pattern comparison in equivalent tissues across species

These approaches collectively illuminate how UBI3 has maintained core functions while potentially adapting to specific ecological niches or domestication processes within Solanaceae.

What methodological considerations are important when comparing UBI3 expression patterns across different plant species?

When comparing UBI3 expression patterns across plant species, researchers must address several methodological challenges:

• Experimental design considerations:

  • Selection of truly equivalent tissues and developmental stages

  • Standardization of growth conditions to minimize environmental variables

  • Appropriate timing of sampling based on species-specific developmental rates

  • Inclusion of multiple biological replicates to account for genetic variation

• Technical methodology standardization:

  • Optimization of RNA extraction protocols for different plant tissues

  • Design of species-specific primers with equivalent efficiency

  • Selection of appropriate reference genes for each species

  • Normalization strategies that account for genomic differences

• Data analysis and interpretation challenges:

  • Development of statistical approaches appropriate for cross-species comparisons

  • Methods to account for differences in baseline expression levels

  • Visualization techniques that highlight both conservation and divergence

  • Integration of expression data with other -omics datasets

• Evolutionary context consideration:

  • Accounting for differences in ploidy levels between species

  • Consideration of whole-genome duplication events in the lineage

  • Recognition of potential neofunctionalization or subfunctionalization

  • Interpretation of expression differences in light of adaptive significance