Six active phage-type RNA polymerase genes in Nicotiana tabacum

In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum , a natural allotetraploid, identified six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis , uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1 , RpoT2 and RpoT3 ). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum . GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis , Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.