Repeated loss of an ancestral multi-isotype tRNA-intron repertoire spared introns linked to anticodon modification during the evolution of green plants

Nuclear tRNA genes of land plants carry introns almost exclusively in tRNA-Tyr(GUA) and elongator tRNA-Met(CAU), whereas more than half of the nuclear tRNA genes of the green alga Chlamydomonas reinhardtii are interrupted. Where along the green lineage this contrast arose, and how the high intron load of algae is built, has not been examined. I investigated the tRNA-intron repertoires of 24 nuclear genome assemblies spanning three outgroups, Prasinoderma, seven chlorophytes, eight streptophyte algae, and five land plants from hornwort to Amborella, using a single detection protocol (tRNAscan-SE 2.0 in eukaryotic mode with the GtRNAdb high-confidence filter), and compared intron sequences and intron-bearing anticodons within and among genomes. Intron load (the fraction of high-confidence nuclear tRNA genes carrying an intron) was 0.28 in Mesostigma viride (introns in 14 isotypes) and 0.46 in Klebsormidium nitens (11 isotypes) but 0.05 in Chlorokybus atmophyticus, 0.07 in Chara braunii, 0.08–0.19 in four Zygnematophyceae, and 0.04–0.05 in all five land plants, where introns occur only in tRNA-Tyr and elongator tRNA-Met. Intron-rich and intron-poor repertoires alternate along the streptophyte backbone, and parsimony requires at least six changes in intron load on the phylogeny. In every genome, introns of different isotypes shared no detectable sequence similarity; similarity was confined to same-anticodon paralogs, and paralogs with identical mature sequences often carried unrelated introns. The introns retained in intron-poor genomes occupy the same anticodons as introns in intron-rich green genomes, and the most widely shared of them (tRNA-Tyr-GUA, tRNA-Ile-UAU, tRNA-Lys-UUU, and tRNA-Leu-CAA) are also intron-bearing in yeast, animals, or both. Retention was highest for the introns that are required for anticodon modification in other eukaryotes: the tRNA-Tyr intron was kept in all 14 intron-poor genomes that have a high-confidence tRNA-Tyr gene, and the tRNA-Ile-UAU intron ranked second, after Tyr-GUA, of 41 ancestrally intron-bearing anticodons (exact permutation P = 0.002 for the modification-linked set); the elongator tRNA-Met intron, with no known modification role, was kept in 12 of 14. Intronless copies in mixed families had mature sequences identical or nearly identical to their intron-bearing paralogs, lay at separate loci, and carried no genomic 3′ CCA, indicating that introns were removed precisely at individual loci rather than by retroposition of processed tRNA or by deletions that damaged the gene. These results show that a multi-isotype tRNA-intron repertoire was ancestral in green plants and was lost independently in Chlorokybus, in the lineage leading to Charophyceae, Zygnematophyceae, and Embryophyta, and several times within Chlorophyta, each time leaving a Tyr/Met core. The high load of intron-rich algae consists of many sequence-independent canonical introns that are retained and propagated by duplication of whole intron-bearing genes. The land-plant pattern was therefore established in streptophyte algae well before the origin of land plants.