Multilevel genomic constraints shape nuclear tRNA gene organization in plants

Abstract

Transfer RNAs (tRNAs) are essential components of the translational machinery. Their abundance and diversity shape decoding capacity and protein synthesis efficiency and accuracy. Because tRNA abundance is encoded in the genome through tDNA copy number, chromosomal organization, and cis-regulatory sequences controlling transcription, these features are expected to influence translational. However, the principles governing nuclear tDNA organization remain poorly understood. Here, we analyzed nuclear tDNA repertoires across 53 photosynthetic eukaryotes spanning major Archaeplastida lineages and secondary endosymbionts, along with seven non-plant eukaryotic outgroups, using comparative genomic approaches at sequence, chromosomal, and genome-wide scales. To support these analyses and enable interactive exploration of tDNA organization, we developed ShinytRNA (https://nebula.ibmp.unistra.fr/shinytRNA/), a web application for genome-wide exploration of tDNA organization. Nuclear tDNA copy numbers vary by more than two orders of magnitude across species, yet the relative representation of tRNA families corresponding to each amino acid remains strikingly conserved across lineages, revealing strong evolutionary constraints on tDNA dosage. In angiosperms, tDNAs show reinforced cis-regulatory features linked to RNA polymerase III transcription, including expanded AT-rich regions, enriched CAA motifs, and extended poly(T) tracts. At the chromosomal scale, tDNAs are predominantly dispersed along chromosome arms, with homogeneous spacing that scales with genome size, while also showing non-random chromosomal distribution, exclusion from centromeric regions, and limited clustering. Together, these patterns reveal conserved yet lineage-specific principles governing nuclear tDNA organization in plants, and highlight how multiple genomic constraints shape the evolution of nuclear tDNA repertoires.