Recombinant Arabidopsis thaliana Valine--tRNA ligase (VALRS), partial

What is Valine--tRNA ligase (VALRS) in Arabidopsis thaliana?

VALRS, also known as Valyl-tRNA synthetase (ValRS), is an essential enzyme in Arabidopsis thaliana that catalyzes the attachment of valine to its cognate tRNA. This aminoacylation reaction is critical for protein synthesis, ensuring the correct incorporation of valine into growing polypeptide chains during translation. In Arabidopsis, VALRS belongs to the class I aminoacyl-tRNA synthetase family and plays critical roles in both cytosolic and mitochondrial protein synthesis. The gene encoding VALRS in Arabidopsis is also known as TWN2, which when mutated leads to developmental abnormalities including polyembryony .

What is the structural organization of VALRS in Arabidopsis?

VALRS in Arabidopsis contains several key structural components:

• A core catalytic domain with class I aminoacyl-tRNA synthetase signature motifs

• An N-terminal extension absent in prokaryotic homologs that contains:

  • A mitochondrial targeting sequence at the proximal part

  • An NH2-appended domain distal to the transit peptide

• An amphiphilic helix conserved between yeast and Arabidopsis VALRS

This structural organization facilitates its dual localization to both cytosolic and mitochondrial compartments, with different elements mediating specific functions including tRNA recognition, amino acid activation, and subcellular targeting .

How does VALRS exhibit bifunctionality in Arabidopsis thaliana?

VALRS demonstrates remarkable bifunctionality through several mechanisms:

• Dual subcellular targeting: The same gene encodes both cytosolic and mitochondrial isoforms through alternative translation initiation from two in-frame AUG codons .

• Translation regulation: The context surrounding each initiation codon determines translation efficiency, with the first AUG (producing the mitochondrial form) having a poorer context than the second AUG (producing the cytosolic form) .

• Transcript diversity: Primer extension experiments have revealed multiple transcript ends—some mapping upstream of the first AUG and others between the two AUGs—suggesting complex transcriptional regulation .

• Evolutionary conservation: The bifunctional nature appears to predate the divergence of yeast and Arabidopsis, suggesting strong selective pressure to maintain this arrangement .

What are the implications of the twn2 mutation for understanding VALRS function?

The twn2 mutation provides critical insights into VALRS function beyond protein synthesis:

• Developmental effects: The twn2-1 mutation, caused by a T-DNA insertion in the 5' untranslated region of the valRS gene, results in a defect in early embryogenesis where descendants of the apical cell arrest after one or two divisions of the zygote .

• Cell fate determination: In twn2 mutants, basal cells that normally form the suspensor proliferate abnormally, resulting in multiple embryos, suggesting VALRS influences embryonic cell fate .

• Transcriptional impact: The insertion causes reduced transcription of the valRS gene in reproductive tissues and developing seeds but increased expression in leaves .

• Enhancer interactions: Analysis indicates that enhancer elements inside the first two introns interact with the T-DNA border to cause the altered expression pattern .

These findings support a model in which the apical cell normally suppresses the embryogenic potential of basal cells during early embryo development, with VALRS playing a crucial role in this process.

What expression systems are recommended for recombinant VALRS production?

For optimal expression of recombinant Arabidopsis thaliana VALRS, the following systems have proven effective:

• Baculovirus expression system: This has been successfully employed for VALRS expression, providing high yields of functional protein with proper folding .

• Purification strategies:

  • Affinity chromatography using appropriate tags

  • Ion exchange chromatography as a secondary purification step

  • Size exclusion chromatography for final polishing

What are the optimal storage conditions for recombinant VALRS?

Based on experimental protocols, the following storage conditions are recommended:

• Store at -20°C, or for extended storage, conserve at -80°C .

• Avoid repeated freezing and thawing; working aliquots can be stored at 4°C for up to one week .

• For reconstitution, use deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Addition of 5-50% glycerol (final concentration) is recommended for long-term storage .

How is VALRS targeted to different cellular compartments?

VALRS exhibits sophisticated subcellular targeting mechanisms:

• The protein displays characteristics of cytosolic enzymes but possesses an N-terminal extension relative to prokaryotic homologs .

• The proximal part of this N-terminal extension functions as a mitochondrial-targeting signal .

• Two protein isoforms are generated through alternative use of two in-frame initiation codons:

  • A longer, mitochondrial form translated from the first initiation codon (at reduced levels due to a poor sequence context)

  • A shorter, cytosolic form translated from a second in-phase AUG in a better context for translation initiation .

This dual targeting has been demonstrated through transient expression of GFP fusions in tobacco cells, confirming that a single gene encodes both the cytosolic and mitochondrial forms of VALRS .

How does alternative use of initiation codons regulate VALRS expression?

The alternative use of initiation codons in VALRS represents a sophisticated regulatory mechanism:

• Dual initiation sites: The VALRS transcript contains two in-frame AUG codons utilized for translation initiation .

• Context-dependent efficiency: The first AUG, leading to the mitochondrial form, exists in a poor sequence context, resulting in reduced translation efficiency .

• Optimal second start site: The second in-frame AUG exists in a better context for translation initiation, leading to more efficient production of the cytosolic form .

• Transcript heterogeneity: Multiple transcript ends have been identified, suggesting additional complexity in transcriptional regulation .

This mechanism allows precise control of the relative amounts of mitochondrial versus cytosolic VALRS without requiring separate genes.

How does VALRS activity correlate with developmental stages in Arabidopsis?

VALRS activity shows distinct correlations with developmental stages in Arabidopsis:

• Early embryogenesis: VALRS expression is critical during early embryonic development, as demonstrated by the severe defects in twn2 mutants .

• Tissue-specific regulation: The twn2-1 mutation causes reduced transcription in reproductive tissues and developing seeds but increased expression in leaves .

• Adult plant phenotypes: Plants with mutations in VALRS that survive embryogenesis exhibit smaller size, reduced vigor, and severely stunted roots .

• Seed development: A high proportion of seeds in affected plants fail to develop viable embryos, and those that do often contain partially or completely duplicated embryos .

How can mutations in VALRS be leveraged to understand embryogenesis?

Mutations in VALRS provide valuable tools for understanding embryogenesis:

• Cell fate mechanisms: The twn2 mutation reveals how apical cells normally suppress the embryogenic potential of suspensor cells .

• Developmental checkpoints: Altered VALRS expression affects specific stages of embryo development, identifying critical developmental transitions .

• Suspensor function: The abnormal proliferation of suspensor cells in twn2 mutants highlights the developmental plasticity of these cells .

This connection between a tRNA synthetase and embryo development suggests critical roles for regulated protein synthesis in developmental patterning.

How do structural characteristics of VALRS compare between Arabidopsis and yeast?

Comparative analysis of VALRS between Arabidopsis thaliana and yeast reveals important evolutionary insights:

• Conserved domains: Both enzymes share fundamental catalytic domains characteristic of class I aminoacyl-tRNA synthetases .

• N-terminal extensions: Both organisms possess N-terminal extensions relative to prokaryotic homologs .

• Amphiphilic helix: This feature is conserved between yeast and Arabidopsis VALRS, suggesting functional importance in translation .

• Evolutionary implications: The high structural similarities suggest that the acquisition of bifunctionality predates the divergence of these organisms .

The conservation of these features across diverse eukaryotes suggests strong selective pressure to maintain this arrangement, likely due to the efficiency of using a single gene to provide essential functions in multiple cellular compartments.

What comparative analyses can be performed between plant and prokaryotic valyl-tRNA synthetases?

Several comparative approaches offer valuable insights:

• Domain architecture: Plant VALRS contains N-terminal extensions absent in prokaryotic homologs .

• Catalytic core: Analysis of conserved catalytic motifs can reveal fundamental aspects of aminoacylation mechanisms.

• Functional complementation: Testing whether plant VALRS can complement prokaryotic mutants can reveal functional conservation.

• Phylogenetic analysis: Comprehensive phylogenies can trace the acquisition of eukaryote-specific features.

What are the current challenges in characterizing VALRS interactions in plant systems?

Several challenges complicate comprehensive characterization of VALRS interactions:

• Complex targeting: The dual localization complicates identification of compartment-specific interaction partners.

• Multiple functions: Beyond tRNA charging, evidence suggests additional roles in development that may involve distinct protein interactions.

• Tissue-specific regulation: Differential expression suggests tissue-specific regulatory mechanisms that may involve different interaction partners.

• Structural information: Limited structural data about plant VALRS complicates interpretation of interaction studies.

What future research directions might advance understanding of VALRS function?

Promising research directions include:

• Structure-function studies: Determining the three-dimensional structure of plant VALRS would provide insights into its dual functionality.

• Protein interactome analysis: Identifying VALRS interaction partners in different cellular compartments and developmental stages.

• Targeted mutagenesis: CRISPR-based approaches to generate specific mutations in functional domains.

• Single-cell transcriptomics: Analyzing VALRS expression at single-cell resolution during embryogenesis could reveal fine-grained regulatory patterns.

• Comparative genomics: Expanded analysis across diverse plant species could reveal evolutionary patterns in VALRS function and regulation.