Function of the inverted repeat on the chloroplast genome of Nicotiana tabacum

The plastid (chloroplast) genomes of most plants and algae contain a large inverted repeat (IR) region that separates the two single-copy regions of the genome and harbors the ribosomal RNA operon. The functions of the IR, although the subject of much speculation, have remained enigmatic. Here we have addressed the functional significance of the IR region by removing the whole 25.3 kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating tobacco plants that have a substantially reduced plastid genome size. We show that lack of the IR results in a mildly reduced number of plastid ribosomes, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, we discovered that the IR deletion plants contain an increased number of plastid genomes, suggesting that chloroplast genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (i) demonstrate that the IR supports maximum rates of expression of the plastid genome, and (ii) uncover a key mechanism of genome copy number regulation in organelles.