Multilevel genomic constraints shape nuclear tRNA gene organization in plants

Transfer RNAs (tRNAs) are essential components of the translation machinery. Their abundance and diversity shape decoding capacity as well as the efficiency and accuracy of protein synthesis. 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 the translational system. 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 standardize these analyses and enable interactive exploration of tDNA organization, we developed ShinytRNA (https://nebula.ibmp.unistra.fr/shinytRNA/), a web application for genome-scale analysis of chromosomal 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. Angiosperm tDNAs exhibit coordinated enrichment of cis-regulatory elements involved in RNA polymerase III transcription, including expanded AT-rich upstream regions, positional enrichment of CAA motifs, and extended poly(T) termination stretches. 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 occasional 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.