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SUMMARY The function of transfer RNAs (tRNAs) depends on enzymes that cleave primary transcript ends, add a 3′ CCA tail, introduce post‐transcriptional base modifications, and charge (aminoacylate) mature tRNAs with the correct amino acid. Maintaining an available pool of the resulting aminoacylated tRNAs is essential for protein synthesis. High‐throughput sequencing techniques have recently been developed to provide a comprehensive view of aminoacylation state in a tRNA‐specific fashion. However, these methods have never been applied to plants. Here, we treatedArabidopsis thalianaRNA samples with periodate and then performed tRNA‐seq to distinguish between aminoacylated and uncharged tRNAs. This approach successfully captured every tRNA isodecoder family and detected expression of additional tRNA‐like transcripts. We found that estimated aminoacylation rates and CCA tail integrity were significantly higher on average for organellar (mitochondrial and plastid) tRNAs than for nuclear/cytosolic tRNAs. Reanalysis of previously published human cell line data showed a similar pattern. Base modifications result in nucleotide misincorporations and truncations during reverse transcription, which we quantified and used to test for relationships with aminoacylation levels. We also determined that the Arabidopsis tRNA‐like sequences (t‐elements) that are cleaved from the ends of some mitochondrial messenger RNAs have post‐transcriptionally modified bases and CCA‐tail addition. However, these t‐elements are not aminoacylated, indicating that they are only recognized by a subset of tRNA‐interacting enzymes and do not play a role in translation. Overall, this work provides a characterization of the baseline landscape of plant tRNA aminoacylation rates and demonstrates an approach for investigating environmental and genetic perturbations to plant translation machinery. 

Plant mitochondrial genomes (mitogenomes) experience remarkable levels of horizontal gene transfer, including the recent discovery that orchids anciently acquired DNA from fungal mitogenomes. Thus far, however, there is no evidence that any of the genes from this interkingdom horizontal gene transfer are functional in orchid mitogenomes. Here, we applied a specialized sequencing approach to the orchid Corallorhiza maculata and found that some fungal-derived tRNA genes in the transferred region are transcribed, post-transcriptionally modified, and aminoacylated. In contrast, all the transferred protein-coding sequences appear to be pseudogenes. These findings show that fungal horizontal gene transfer has altered the composition of the orchid mitochondrial tRNA pool and suggest that these foreign tRNAs function in translation. The exceptional capacity of tRNAs for horizontal gene transfer and functional replacement is further illustrated by the diversity of tRNA genes in the C. maculata mitogenome, which also include genes of plastid and bacterial origin in addition to their native mitochondrial counterparts. 

Eukaryotic nuclear genomes often encode distinct sets of translation machinery for function in the cytosol vs. organelles (mitochondria and plastids). This raises questions about why multiple translation systems are maintained even though they are capable of comparable functions and whether they evolve differently depending on the compartment where they operate. These questions are particularly interesting in plants because translation machinery, including aminoacyl-transfer RNA (tRNA) synthetases (aaRS), is often dual-targeted to the plastids and mitochondria. These organelles have different functions, with much higher rates of translation in plastids to supply the abundant, rapid-turnover proteins required for photosynthesis. Previous studies have indicated that plant organellar aaRS evolve more slowly compared to mitochondrial aaRS in eukaryotes that lack plastids. Thus, we investigated the evolution of nuclear-encoded organellar and cytosolic aaRS and tRNA maturation enzymes across a broad sampling of angiosperms, including nonphotosynthetic (heterotrophic) plant species with reduced plastid gene expression, to test the hypothesis that translational demands associated with photosynthesis constrain the evolution of enzymes involved in organellar tRNA metabolism. Remarkably, heterotrophic plants exhibited wholesale loss of many organelle-targeted aaRS and other enzymes, even though translation still occurs in their mitochondria and plastids. These losses were often accompanied by apparent retargeting of cytosolic enzymes and tRNAs to the organelles, sometimes preserving aaRS–tRNA charging relationships but other times creating surprising mismatches between cytosolic aaRS and mitochondrial tRNA substrates. Our findings indicate that the presence of a photosynthetic plastid drives the retention of specialized systems for organellar tRNA metabolism.