Recombinant Solanum lycopersicum 60S ribosomal protein L38 (RPL38)

What is RPL38 and what is its role in tomato ribosomes?

RPL38 is a component of the 60S large ribosomal subunit in Solanum lycopersicum. As with other ribosomal proteins, it plays crucial roles in ribosome assembly and function. Ribosomal proteins in tomato, like in many plant species, often exist as multiple co-orthologs, with the majority of ribosomal proteins encoded by two or three co-ortholog genes . The primary function of RPL38 is to contribute to the structural organization of the 60S ribosomal subunit and participate in protein synthesis.

In plants, ribosomal proteins like RPL38 can have additional specialized functions beyond their canonical roles in translation. These may include tissue-specific expression patterns that support developmental processes or stress responses. The presence of multiple co-orthologs suggests possible functional specialization, where different RPL38 variants might participate in specialized ribosomes with distinct translational capacities.

How is RPL38 gene expression regulated in different tomato tissues?

Expression analysis reveals that most ribosomal protein genes in tomato, including those encoding RPL38, are highly expressed in both vegetative and reproductive tissues. Studies using next-generation sequencing approaches such as RNA-seq and Massive Analysis of 3′-cDNA Ends (MACE) have shown that ribosomal protein genes generally have higher transcript abundance in leaves compared to anthers .

For investigating tissue-specific expression patterns of RPL38, the following methodological approach is recommended:

• Isolate total RNA from various tomato tissues (leaves, stems, roots, flowers, fruits at different developmental stages)

• Perform qRT-PCR using RPL38-specific primers

• Normalize expression data against stable reference genes

• Compare relative expression levels across tissues and developmental stages

Most RP genes are expressed in multiple tissues, but tissue-specific expression can be observed for a subset of RPs, suggesting specialized functions in different developmental contexts .

What approaches are used to clone and express recombinant S. lycopersicum RPL38?

The cloning and expression of recombinant S. lycopersicum RPL38 typically follows these methodological steps:

Gene Isolation and Cloning:

• Design primers based on the RPL38 coding sequence from tomato

• Amplify the target gene using RT-PCR from total RNA extracted from tomato tissue

• Clone the amplified fragment into an appropriate expression vector

• Verify the insert by sequencing to confirm correct orientation and sequence integrity

Expression Systems:

• Bacterial expression (E. coli BL21 or derivatives)

  • Optimize codon usage if necessary

  • Use T7 or similar strong promoter systems

  • Include affinity tags (His6, GST) for purification

• Yeast expression (P. pastoris, S. cerevisiae)

  • Consider for improved folding of eukaryotic proteins

  • Use inducible promoters (e.g., GAL1, AOX1)

• Plant-based expression

  • Transient expression in N. benthamiana

  • Stable transformation in model plants

Protein Purification Strategy:

• Affinity chromatography using attached tags

• Ion exchange chromatography

• Size exclusion chromatography for final polishing

• Verification by SDS-PAGE and Western blotting

Based on studies of ribosomal proteins, expression levels can be optimized by adjusting induction conditions, temperature, and duration to minimize inclusion body formation .

How does the sequence of tomato RPL38 compare with orthologs in other species?

Comparative sequence analysis of ribosomal proteins across plant species reveals high conservation, which is expected for proteins involved in the fundamental process of translation. For tomato RPL38, sequence comparison with other plant species would likely show:

Table 1: Predicted Sequence Identity of S. lycopersicum RPL38 with Other Species

Ribosomal proteins typically show higher sequence conservation in functional domains involved in rRNA binding and intersubunit interactions. The number of RPL38 co-orthologs may vary between species, with multiple co-orthologs often present in plant genomes . For instance, in rats, L38 has been characterized as a protein of 69 amino acids with a molecular weight of 8,081 Da .

What methods are used to study RPL38 expression under different conditions?

To investigate RPL38 expression under various conditions, researchers can employ several complementary approaches:

Transcriptional Analysis:

• Quantitative RT-PCR (qRT-PCR)

  • Design gene-specific primers for RPL38

  • Use reference genes appropriate for the conditions being tested

  • Analyze relative expression using ΔΔCt or similar methods

• RNA-Seq and MACE Analysis

  • Perform global transcriptome profiling

  • Analyze RPL38 expression patterns across tissues or treatments

  • Compare expression of different RPL38 co-orthologs if present

Protein-Level Analysis:

• Western blotting

  • Use specific antibodies against RPL38

  • Quantify protein levels under different conditions

• Proteomics

  • Mass spectrometry-based quantification

  • Analysis of post-translational modifications

Reporter Systems:

• Promoter-reporter constructs

  • Clone the RPL38 promoter region upstream of GUS or GFP

  • Generate stable transgenic plants

  • Analyze reporter expression patterns

Expression analysis of ribosomal proteins in tomato has revealed differential expression patterns in various tissues, with most ribosomal proteins showing high expression in actively growing tissues . These methods can be adapted to study RPL38 expression under different developmental stages, stress conditions, or pathogen infections.

How does RPL38 contribute to specialized ribosomes and selective translation in tomato?

Recent research has uncovered the concept of "specialized ribosomes," where variations in ribosomal protein composition can affect the translation of specific mRNAs. For investigating RPL38's role in selective translation in tomato, the following methodological approach is recommended:

Experimental Strategy:

• Genetic Manipulation:

  • Generate RPL38 knockdown/knockout lines using CRISPR/Cas9

  • Create overexpression lines with tagged RPL38 variants

  • Develop lines expressing mutated forms of RPL38

• Translatomic Analysis:

  • Perform ribosome profiling (Ribo-seq) to identify differentially translated mRNAs

  • Compare transcriptome (RNA-seq) with translatome (Ribo-seq) data

  • Identify mRNAs specifically affected at the translational level

  • Analyze 5′ UTR features of RPL38-dependent mRNAs

• Structural Studies:

  • Perform cryo-EM analysis of ribosomes with and without RPL38

  • Identify structural changes affecting mRNA recruitment

  • Map RPL38 position relative to the mRNA channel

Expected Findings:
RPL38 might affect translation of specific subsets of mRNAs, potentially those involved in development, tissue specification, or stress responses. The presence of multiple RPL38 co-orthologs in plants suggests possible functional specialization, where different variants participate in ribosomes with distinct translational preferences .

What experimental approaches can identify RPL38 interaction partners in tomato ribosomes?

Understanding the interaction network of RPL38 within the ribosomal complex requires specialized approaches:

In Vivo Interaction Analysis:

• Affinity Purification Coupled with Mass Spectrometry:

  • Express tagged RPL38 in tomato cells

  • Perform gentle lysis to maintain interactions

  • Purify RPL38 complexes and identify interacting proteins by MS

  • Compare results under different cellular conditions

• Proximity Labeling Approaches:

  • Fuse RPL38 with BioID or APEX2

  • Identify proteins in close proximity through biotinylation

  • Purify biotinylated proteins and analyze by MS

Structural Analysis:

• Crosslinking Mass Spectrometry (XL-MS):

  • Crosslink assembled ribosomes

  • Identify protein-protein interaction sites

  • Map the 3D interaction network

• Cryo-Electron Microscopy:

  • Determine high-resolution structures of tomato ribosomes

  • Map RPL38 position and interactions

  • Compare with structures from other species

RNA-Protein Interactions:

• RNA Immunoprecipitation (RIP):

  • Immunoprecipitate RPL38-containing complexes

  • Identify associated rRNAs and mRNAs

  • Map interaction sites through sequencing

• CLIP-seq Approaches:

  • Perform crosslinking immunoprecipitation

  • Identify direct RNA-binding sites at nucleotide resolution

These approaches would help create a comprehensive map of RPL38 interactions within the ribosomal complex and potentially identify non-canonical interactions that might contribute to specialized ribosome function .

How do environmental stresses affect RPL38 expression and function in tomato?

Environmental stresses likely modulate RPL38 expression and function as part of the plant's stress response. A comprehensive methodology to investigate this includes:

Expression Analysis Under Stress:

• Stress Treatment Series:

  • Expose tomato plants to various stresses:

    • Abiotic: drought, salt, heat, cold, nutrient deficiency

    • Biotic: bacterial, fungal, viral pathogens

  • Sample tissues at multiple time points

• Transcript Analysis:

  • Perform qRT-PCR for RPL38

  • Compare expression of different RPL38 co-orthologs

  • Correlate with known stress-responsive genes

Translational Regulation:

• Polysome Profiling:

  • Separate polysomes on sucrose gradients

  • Analyze distribution of RPL38 mRNA across fractions

  • Compare stressed vs. non-stressed conditions

• Ribosome Profiling:

  • Analyze translational efficiency changes under stress

  • Identify stress-specific translation patterns

Functional Analysis:

• Phenotypic Comparison:

  • Compare wild-type and RPL38-modified plants under stress

  • Measure physiological parameters (growth, photosynthesis, etc.)

  • Assess stress tolerance and recovery

• Proteomic Analysis:

  • Quantify changes in the proteome under stress

  • Identify differentially translated proteins

Based on studies of pathogenesis-related (PR) proteins in tomato, gene expression can be significantly induced by pathogen infection and treatments with salicylic acid (SA) and methyl jasmonate acid (MeJA) . Similar regulatory mechanisms might affect RPL38 expression during stress responses.

What role might RPL38 play in tomato development and reproduction?

To investigate RPL38's potential role in tomato development and reproduction, researchers can employ the following methodology:

Expression Analysis During Development:

• Tissue-Specific Expression:

  • Sample tissues throughout development:

    • Vegetative: shoot apex, expanding leaves, mature leaves

    • Reproductive: flower buds, anthers, pollen, ovaries, developing fruit

  • Perform qRT-PCR or RNA-seq analysis

  • Create expression maps across developmental stages

• In Situ Hybridization:

  • Develop RPL38-specific probes

  • Analyze spatial expression patterns in developing tissues

  • Focus on meristems and reproductive structures

Functional Analysis:

• Genetic Manipulation:

  • Generate tissue-specific or inducible knockdown/knockout lines

  • Create reporter lines to visualize expression in vivo

  • Analyze developmental phenotypes

• Cellular Analysis:

  • Examine cell division rates in meristems

  • Analyze pollen development and fertility

  • Assess embryo and seed development

Based on research findings, ribosomal proteins and ribosome biogenesis factors often show tissue-specific expression patterns in tomato, with some expressed preferentially in reproductive tissues like anthers . This suggests potential specialized roles in reproduction and development, particularly in rapidly dividing tissues.

How can CRISPR/Cas9 genome editing be optimized for studying RPL38 function in tomato?

CRISPR/Cas9 offers powerful tools for studying RPL38 function through precise genetic modification. The following methodological approach is recommended:

Guide RNA Design and Optimization:

• Target Selection:

  • Identify conserved functional domains in RPL38

  • Design multiple sgRNAs targeting different regions

  • Use tomato-optimized CRISPR design tools

  • Consider targeting multiple co-orthologs if present

• Construct Development:

  • Use tomato-optimized Cas9 variants

  • Select appropriate promoters (constitutive or tissue-specific)

  • Include selectable markers for transformation

Transformation and Screening:

• Agrobacterium-Mediated Transformation:

  • Optimize transformation protocols for tomato cultivar

  • Use cotyledon or hypocotyl explants

  • Establish efficient regeneration system

• Mutation Detection:

  • PCR-based screening approaches

  • T7E1 or Surveyor assays for mutation detection

  • Deep sequencing to identify precise modifications

Functional Analysis Strategies:

• Complete Knockout Approach:

  • Target conserved regions to disrupt protein function

  • Create frameshift mutations

  • Target multiple co-orthologs simultaneously

• Domain-Specific Modifications:

  • Create precise deletions or substitutions

  • Target specific functional domains

  • Generate point mutations in key residues

• Promoter Modifications:

  • Alter expression patterns

  • Introduce inducible elements

  • Create reporter fusions

Since ribosomal proteins are often essential, conditional approaches might be necessary to study RPL38 function if complete knockouts prove lethal .

What are the best methods for studying RPL38's role in tomato disease resistance?

To investigate potential roles of RPL38 in tomato disease resistance, researchers can follow this methodological framework:

Expression Analysis During Pathogen Challenge:

• Pathogen Infection Series:

  • Inoculate tomato plants with key pathogens:

    • Bacterial: Ralstonia solanacearum, Pseudomonas syringae

    • Fungal: Fusarium spp., Alternaria spp.

    • Viral: Tomato yellow leaf curl virus

  • Sample tissues at multiple time points post-infection

• Expression Profiling:

  • Monitor RPL38 expression using qRT-PCR

  • Compare with known defense-related genes

  • Analyze expression in resistant vs. susceptible varieties

Functional Analysis:

• Genetic Manipulation:

  • Generate RPL38-modified plants

  • Challenge with pathogens

  • Assess disease progression and symptoms

  • Quantify pathogen growth

• Molecular Analysis:

  • Measure defense hormone levels (SA, JA, ethylene)

  • Analyze expression of defense marker genes

  • Examine callose deposition and ROS production

• Translational Control Analysis:

  • Investigate translational changes during infection

  • Identify defense-related mRNAs regulated by RPL38

  • Examine protein synthesis rates during defense responses

Based on studies of pathogenesis-related proteins in tomato, pathogen infection can induce significant changes in gene expression and protein production . RPL38 might play a role in modulating translation of defense-related transcripts during infection, potentially contributing to resistance mechanisms.

What bioinformatic tools and approaches are most effective for analyzing RPL38 evolutionary conservation?

For comprehensive evolutionary analysis of RPL38, the following bioinformatic approach is recommended:

Sequence Retrieval and Alignment:

• Database Mining:

  • Retrieve RPL38 sequences from multiple plant species

  • Include representatives across evolutionary distances

  • Search for co-orthologs within each species

• Multiple Sequence Alignment:

  • Use MUSCLE, MAFFT, or T-Coffee for alignment

  • Manually inspect and refine alignments

  • Identify conserved domains and variable regions

Evolutionary Analysis:

• Phylogenetic Tree Construction:

  • Use maximum likelihood or Bayesian methods

  • Test multiple evolutionary models

  • Perform bootstrap analysis for branch support

  • Visualize with tools like FigTree or iTOL

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to detect selection

  • Perform site-specific selection analysis

  • Identify residues under positive or purifying selection

Structural Analysis:

• Homology Modeling:

  • Generate 3D models using AlphaFold2 or similar tools

  • Map conserved residues onto structural models

  • Analyze conservation of interaction surfaces

• Comparative Structural Analysis:

  • Compare RPL38 structure across species

  • Identify structural features unique to plants

  • Map evolutionary conservation onto structures

This comprehensive approach would provide insights into RPL38 evolution, potential functional adaptations, and structurally important regions across plant species .

How can RNA-seq and ribosome profiling be combined to study RPL38-dependent translation in tomato?

To investigate RPL38-dependent translation, an integrated approach combining RNA-seq and ribosome profiling is recommended:

Experimental Design:

• Genetic Materials:

  • Wild-type tomato plants

  • RPL38-modified plants (knockdown, knockout, or overexpression)

  • Consider multiple tissues or developmental stages

• Sample Preparation:

  • Synchronized growth conditions

  • Proper tissue sampling and preservation

  • Minimum 3-4 biological replicates per condition

Transcriptome Analysis (RNA-seq):

• Library Preparation:

  • Poly(A) selection or rRNA depletion

  • Strand-specific library preparation

  • Include spike-in controls for normalization

• Data Analysis:

  • Quality control and preprocessing

  • Mapping to tomato reference genome

  • Transcript quantification

  • Differential expression analysis

Translatome Analysis (Ribosome Profiling):

• Ribosome Footprint Preparation:

  • Freeze tissues in liquid nitrogen

  • Pulverize and extract in translation buffer

  • Treat with nuclease to digest unprotected RNA

  • Isolate 80S monosomes

  • Purify and sequence ribosome-protected fragments

• Data Analysis:

  • Map ribosome footprints to transcriptome

  • Calculate translation efficiency for each transcript

  • Analyze triplet periodicity to confirm active translation

  • Identify differentially translated mRNAs

Integrated Analysis:

• Translation Efficiency Calculation:

  • Compare ribosome footprint abundance to mRNA abundance

  • Identify mRNAs with altered translation independent of transcript levels

• Feature Analysis:

  • Examine 5′ UTR features of RPL38-dependent mRNAs

  • Look for conserved sequence or structural elements

  • Analyze codon usage patterns

• Pathway Analysis:

  • Identify biological processes affected at translational level

  • Compare with transcriptional responses

  • Look for coordinated regulation of functional gene groups

This integrated approach would provide comprehensive insights into the role of RPL38 in modulating translation of specific mRNAs in tomato .