Structural variation and evolutionary analysis of chloroplast tRNA molecule in green plants

Plant chloroplasts possess a circular double-stranded genome containing numerous genes with autoregulatory functions. tRNAs, a highly conserved gene family characterized by a typical cloverleaf secondary structure, primarily function in protein synthesis by delivering amino acids to the ribosome. However, the structural features and evolutionary mechanisms of tRNAs in plant chloroplast genomes remain poorly understood, and the potential relationship between minimum free energy (MFE) and chloroplast tRNA evolution has not been systematically explored. In this study, we analyzed tRNA genes in the chloroplast genomes of 122 representative plant species, encompassing 4055 tRNA sequences with lengths ranging from 56 to 93 nt. Each chloroplast genome encoded 26 to 37 tRNA genes. The research found that nucleotide count varied most in the D-loop and variable region, whereas the TΨC-loop was strictly conserved at seven nucleotides with a consensus sequence, U-U-C-x-A-x2. Additionally, we identified four atypical tRNA types: tRNA lacking an acceptor arm, tRNA with a nine-nucleotide anticodon loop, tRNAs with an equal number of nucleotide (7 bp) in both the variable region and the anticodon loop, and tRNA^Ile with a CAU anticodon. Notably, MFE analysis revealed that tRNAs lacking an acceptor stem exhibited higher MFE values, whereas tRNAs with extended loops displayed lower MFE values, suggesting distinct structural stability implications. Phylogenetic analysis indicated that chloroplast tRNAs evolved from multiple common ancestors, exhibited a higher transition rate than transversion rate, and experienced more loss events. These results provide crucial insights into the evolutionary mechanisms and structural characteristics of plant chloroplast tRNAs.