Reverse genetics in the <i>Arabidopsis</i> chloroplast genome identifies <i>rps16</i> as a transcribed pseudogene

The plastid (chloroplast) genomes of seed plants contain a conserved set of ribosomal protein genes. The rps16 gene represents an exception: It has been lost from the plastid genomes of gymnosperms and several lineages of angiosperms, and may have undergone pseudogenization in a few other lineages, including members of the Brassicaceae family. Here we report a reverse genetic approach to test the annotated rps16 gene in the Arabidopsis plastid genome for functionality. Employing the recently developed plastid transformation technology for the model plant Arabidopsis, we have deleted the putative rps16 gene from the Arabidopsis plastid genome. We report that the resulting transplastomic plants display wild-type-like growth and photosynthetic performance under a wide range of conditions. Moreover, genome-wide analyses of chloroplast transcript levels and ribosome footprints revealed unaltered plastid translational activity in Δrps16 mutants compared with wild-type plants. We conclude that the annotated rps16 gene in the plastid genome of Arabidopsis is a transcribed pseudogene that has been replaced in evolution by a nuclear gene copy that supplies functional S16 protein to chloroplasts.