Recombinant Spinacia oleracea 30S ribosomal protein S21, chloroplastic (rps21)

Basic Research Questions

• What is the function of 30S ribosomal protein S21 (RPS21) in Spinacia oleracea?

  The 30S ribosomal protein S21 (RPS21) in Spinacia oleracea functions as a critical component of the chloroplastic ribosomal small subunit, participating in the translation machinery within chloroplasts. As part of the 30S subunit, RPS21 contributes to ensuring that mRNA is correctly positioned to maintain the reading frame during protein synthesis and facilitates proper pairing between tRNAs and mRNA codons . To investigate this function experimentally, researchers typically employ ribosome profiling, in vitro translation assays, and structural studies of ribosome-mRNA complexes. Additionally, knockout or knockdown studies can reveal phenotypic consequences of RPS21 disruption, highlighting its essential role in chloroplast translation.

• Where is the RPS21 protein localized within Spinacia oleracea cells?

  RPS21 is primarily localized in the chloroplast of Spinacia oleracea cells, where it functions as part of the translational machinery . This subcellular localization can be experimentally determined through several complementary approaches:

  Localization Method	Technical Approach	Advantages	Limitations
  Fluorescent protein fusion	GFP/YFP tagging of RPS21	Live cell visualization	Tag may affect localization
  Immunogold electron microscopy	Anti-RPS21 antibodies with gold particles	Precise suborganellar localization	Requires tissue fixation
  Subcellular fractionation	Differential centrifugation followed by Western blotting	Biochemical confirmation	Potential cross-contamination
  Chloroplast import assays	In vitro translated protein with isolated chloroplasts	Direct import evidence	Artificial conditions

  The protein contains an N-terminal chloroplast transit peptide that directs its import into the chloroplast, where it becomes incorporated into the 30S ribosomal subunit of the chloroplastic translation apparatus .

• What is the genomic organization of the rps21 gene in Spinacia oleracea?

  The rps21 gene in Spinacia oleracea is encoded in the chloroplast genome. Analysis can be performed using the Spinach Genome DataBase (SOL_r1.1), which provides genomic sequences and annotations for spinach (breeding line 03-009) . The database contains a genome assembly with 287 sequences totaling 935,652,614 bp with an N50 length of 11,296,190 bp . Methodologically, researchers can identify the rps21 gene structure by:

  1. Utilizing the SOL_r1.1a set of predicted genes from the Spinach Genome DataBase

  2. Performing comparative genomic analysis with other Chenopodioideae species

  3. Conducting targeted sequencing of the chloroplast genome region containing rps21

  4. Using bioinformatic tools to identify promoter regions, introns (if present), and regulatory elements

  The genomic context surrounding rps21 is important for understanding its regulation and co-expression with other chloroplast genes.

Experimental Methods and Protocols

• What are the optimal conditions for cloning the rps21 gene from Spinacia oleracea?

  For cloning the rps21 gene from Spinacia oleracea, researchers should implement a methodical approach:

  1. Genomic Template Selection: Extract high-quality chloroplast DNA from young spinach leaves (preferably 6-leaf stage plants, as referenced in pokeweed antiviral protein studies) . Alternative templates include cDNA synthesized from total RNA or commercially available spinach genomic DNA.

  2. Primer Design Strategy:

     • Design primers based on the SOL_r1.1 genome assembly

     • Include restriction enzyme sites compatible with your expression vector

     • Consider codon optimization for the target expression system

     • Add purification tags (His, GST, etc.) if needed

  3. PCR Optimization Protocol:

     Parameter	Recommended Condition	Optimization Range
     Initial denaturation	95°C for 3 min	2-5 min
     Denaturation	95°C for 30 sec	15-45 sec
     Annealing	55-58°C for 30 sec	50-65°C
     Extension	72°C for 1 min/kb	30 sec-2 min/kb
     Final extension	72°C for 10 min	5-15 min
     Cycles	30-35	25-40

  4. Verification Methods: Confirm successful cloning by restriction digestion, PCR verification, and Sanger sequencing to ensure the absence of mutations.

• Which expression systems yield the highest recombinant Spinacia oleracea RPS21 protein?

  Selecting the optimal expression system for recombinant Spinacia oleracea RPS21 depends on research objectives. The following systems have been evaluated for chloroplastic ribosomal proteins:

  Expression System	Advantages	Limitations	Yield	Purification Strategy
  E. coli BL21(DE3)	High yield, simplicity	Lacks plant PTMs	5-10 mg/L	IMAC with His-tag
  E. coli ArcticExpress	Better folding at low temp.	Slower growth	3-7 mg/L	IMAC with His-tag
  Plant cell-free systems	Native-like conditions	Higher cost	0.5-2 mg/ml	Affinity chromatography
  Tobacco expression	Plant-specific PTMs	Lower yield	0.1-0.5 mg/g leaf	Immunoprecipitation

  For functional studies requiring proper folding and interaction capability, plant-based expression systems may be preferable despite lower yields. For structural studies requiring large protein quantities, bacterial systems with optimization for chloroplastic proteins are recommended.

Advanced Research Questions

• What protein interactions does Spinacia oleracea RPS21 participate in within the chloroplast ribosome?

  Spinacia oleracea RPS21 participates in multiple protein interactions within the chloroplast ribosome. Based on the pokeweed antiviral protein (PAP) interactome study, RPS21 was identified as a specific interactor with PAP . This suggests RPS21 may have roles beyond its structural function in the 30S ribosomal subunit.

  Methodologically, protein-protein interactions involving RPS21 can be studied through:

  1. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS), as used in the PAP interactome study

  2. Yeast two-hybrid screening with RPS21 as bait against a spinach cDNA library

  3. Bimolecular fluorescence complementation (BiFC) to visualize interactions in planta

  4. Cross-linking coupled with mass spectrometry (XL-MS) to map spatial relationships within the ribosome

  Within the ribosomal complex, RPS21 likely interacts with ribosomal RNA (16S rRNA) and neighboring ribosomal proteins. It may also interact with translation factors during protein synthesis. The interaction with PAP suggests potential involvement in plant defense mechanisms, as PAP has antiviral properties and is implicated in defense responses .

• How does stress affect the expression and function of Spinacia oleracea RPS21?

  Stress responses in plants often involve modulation of chloroplast translation machinery. For RPS21 in Spinacia oleracea, researchers can investigate stress responses through:

  1. Transcriptomic Analysis: RNA-seq data analysis under various stress conditions (drought, salinity, pathogen exposure, temperature extremes)

  2. Proteomics Approach: Quantitative proteomic analysis of chloroplast ribosomal proteins under stress conditions

  3. Translational Efficiency: Ribosome profiling to measure changes in chloroplast translation efficiency during stress

  4. Post-translational Modifications: Mass spectrometry to identify stress-induced PTMs on RPS21

  Research suggests that chloroplastic ribosomal proteins may participate in bacterial-like stress responses within the plastid, potentially altering translation rates of specific chloroplast-encoded genes during stress adaptation . The interaction between RPS21 and defense proteins like PAP further suggests a potential role in biotic stress responses .

• What are the structural differences between Spinacia oleracea RPS21 and its bacterial homologs?

  Structural comparison between chloroplastic RPS21 from Spinacia oleracea and bacterial S21 proteins reveals important evolutionary insights:

  1. Homology Modeling Approach: Using bacterial S21 crystal structures as templates to predict Spinacia oleracea RPS21 structure

  2. Structural Conservation Analysis:

     • The core RNA-binding domain shows structural conservation

     • N-terminal chloroplast transit peptide is unique to the plant protein

     • Specific surface residues show plant-specific conservation patterns

  3. Functional Differences:

     Feature	Bacterial S21	Spinacia oleracea RPS21
     N-terminal region	No transit peptide	Chloroplast targeting sequence
     RNA-binding motifs	Highly conserved	Conserved with plant-specific variations
     Size	Typically 70-75 amino acids	Mature protein similar after transit peptide cleavage
     Electrostatic surface	Basic RNA-binding surface	Similar basic patches with plant-specific differences

  These structural differences may reflect adaptation to the chloroplastic environment and plant-specific translation regulation mechanisms.

Methodological Approaches

• What are the most effective methods for studying RPS21 function in chloroplast translation?

  Multiple complementary approaches can be used to elucidate RPS21 function in chloroplast translation:

  1. In vitro Translation Systems:

     • Develop chloroplast-specific translation systems with purified components

     • Compare translation efficiency with and without RPS21

     • Assess the impact of RPS21 mutations on translation fidelity

  2. Ribosome Profiling:

     • Isolate chloroplast ribosomes and sequence ribosome-protected mRNA fragments

     • Compare wildtype and RPS21-depleted/mutated samples

     • Map translation efficiency changes across the chloroplast transcriptome

  3. Cryo-EM Structural Analysis:

     • Purify chloroplast ribosomes for structural determination

     • Focus on RPS21 position and interactions within the 30S subunit

     • Compare structures at different translation stages

  4. Genetic Approaches:

     • Generate RPS21 knockdown/knockout lines using CRISPR/Cas9

     • Create point mutations in key functional residues

     • Assess phenotypic consequences and changes in chloroplast translation

• How can genome editing be applied to study Spinacia oleracea RPS21 in vivo?

  Genome editing technologies offer powerful approaches to study RPS21 function in vivo:

  1. CRISPR/Cas9 Strategy for Chloroplast Genome Editing:

     • Design sgRNAs targeting the rps21 gene in the chloroplast genome

     • Utilize chloroplast-targeted Cas9 systems

     • Confirm edits through sequencing and protein expression analysis

  2. Experimental Design for Functional Analysis:

     Editing Approach	Target	Expected Outcome	Analysis Method
     Knockout	Full rps21 gene	Translation defects	Chloroplast proteomics
     Domain mutations	RNA-binding motifs	Altered translation	Ribosome profiling
     Promoter modifications	rps21 regulatory region	Expression changes	RT-qPCR, Western blot
     Tag insertion	C-terminus of rps21	Labeled protein	Microscopy, pull-down

  3. Technical Considerations:

     • Chloroplast transformation efficiency in spinach

     • Homoplasmy vs. heteroplasmy of edited chloroplast genomes

     • Phenotypic screening and selection methods

     • Complementation strategies to confirm specificity

  The Spinach Genome DataBase provides valuable resources for designing genome editing approaches, with its high-quality genome assembly (SOL_r1.1) and pseudomolecules (SOL_r1.0_pseudomolecule) .

Applications and Future Directions

• How can structural studies of Spinacia oleracea RPS21 contribute to understanding chloroplast evolution?

  Structural studies of Spinacia oleracea RPS21 provide insights into chloroplast evolution through:

  1. Comparative Structural Biology:

     • Analyze structural conservation between cyanobacterial, chloroplastic, and other bacterial S21 proteins

     • Identify plant-specific structural adaptations in chloroplast ribosomes

     • Map evolutionary pressure on specific residues and domains

  2. Methodological Approaches:

     • X-ray crystallography of isolated RPS21

     • Cryo-EM of intact chloroplast ribosomes

     • Molecular dynamics simulations to analyze structural flexibility

     • Ancestral sequence reconstruction to trace evolutionary trajectory

  3. Evolutionary Implications:
     The study of Spinacia oleracea as a representative of Chenopodioideae (family Amaranthaceae) provides important evolutionary context, as spinach has n = x = 6 chromosomes including sex chromosomes (XY) . Comparative analysis of chloroplast ribosomal proteins across plant lineages can reveal patterns of co-evolution with chloroplast genomes and adaptation to plant-specific translation requirements.

• What are the implications of RPS21's interaction with pokeweed antiviral protein (PAP)?

  The interaction between Spinacia oleracea RPS21 and pokeweed antiviral protein (PAP) revealed by co-immunoprecipitation-mass spectrometry has significant implications:

  1. Mechanism of Antiviral Defense:

     • PAP is a ribosome-inactivating protein (RIP) that removes an adenine base from the large ribosomal subunit, halting protein translation

     • The interaction with RPS21 suggests a potential regulatory mechanism for selective translation inhibition

     • This interaction may represent a previously uncharacterized aspect of plant defense pathways

  2. Research Applications:

     • Development of recombinant RPS21 as a tool to study PAP-mediated translation inhibition

     • Engineered RPS21 variants to enhance or block PAP interaction for investigating defense mechanisms

     • Structure-based drug design targeting viral translation in plants

  3. Methodological Validation Approaches:

     • Reverse co-immunoprecipitation followed by Western blot analysis

     • Overexpression studies combining PAP and RPS21 in tobacco leaves

     • Viral resistance assays measuring translation efficiency with modified RPS21

  This interaction highlights the multifunctional nature of ribosomal proteins beyond their structural roles in translation, suggesting RPS21 may participate in plant defense signaling pathways.

Future Research Directions

• What emerging technologies could advance research on Spinacia oleracea RPS21?

  Several cutting-edge technologies show promise for advancing research on Spinacia oleracea RPS21:

  1. Single-molecule techniques:

     • Single-molecule FRET to study RPS21 dynamics during translation

     • Optical tweezers to measure forces during ribosome assembly and function

     • Zero-mode waveguides for real-time visualization of translation

  2. Advanced structural methods:

     • Time-resolved cryo-EM to capture different conformational states

     • Integrative structural biology combining multiple data types

     • AlphaFold2 and similar AI approaches to predict protein structures and interactions

  3. Systems biology approaches:

     • Multi-omics integration to understand RPS21 in the context of chloroplast function

     • Network analysis of ribosomal protein interactions

     • Metabolic flux analysis to link translation to chloroplast metabolism

  4. Synthetic biology applications:

     • Engineered chloroplast ribosomes with modified RPS21 for specialized functions

     • Minimal translation systems incorporating essential components like RPS21

     • Biosensors based on RPS21-interaction networks

• How does the function of RPS21 vary across different developmental stages of Spinacia oleracea?

  The function of RPS21 may vary throughout spinach development, necessitating stage-specific analysis:

  1. Developmental Expression Profiling:

     • Quantitative RT-PCR of rps21 transcript levels across development

     • Western blot analysis of protein abundance in different tissues and stages

     • Ribosome profiling to measure translation activity at different stages

  2. Stage-Specific Studies:

     Developmental Stage	Expected RPS21 Activity	Experimental Approach
     Seed germination	High demand for chloroplast biogenesis	Proteomics of isolated plastids
     Young leaf development	Peak activity for photosynthetic apparatus	Translatomics of expanding leaves
     Mature leaves	Maintenance of chloroplast function	Protein turnover studies
     Reproductive development	Tissue-specific regulation	In situ hybridization
     Stress response	Modified activity	Comparative stress proteomics

  3. Methodological Considerations:
     Research on seed-to-seedling transition in spinach, such as studies on damping-off tolerance , could provide valuable samples for analyzing developmental regulation of chloroplast translation machinery, including RPS21.

• What are the evolutionary implications of RPS21 conservation across plant species?

  Evolutionary analysis of RPS21 across plant species yields important insights:

  1. Phylogenetic Analysis Approach:

     • Sequence collection from diverse plant lineages

     • Multiple sequence alignment and conservation analysis

     • Selection pressure analysis (dN/dS ratios) on different domains

     • Ancestral sequence reconstruction

  2. Comparative Genomics:

     • Analysis of gene synteny around rps21 in different chloroplast genomes

     • Identification of gene transfer events to nuclear genome in some lineages

     • Correlation with chloroplast genome rearrangements

  3. Functional Conservation:
     Spinacia oleracea, as a member of Chenopodioideae within Amaranthaceae , provides an important evolutionary reference point. The conservation of RPS21 function across plant lineages suggests fundamental roles in chloroplast translation, while species-specific variations may indicate adaptation to different ecological niches and photosynthetic requirements.