Disruption of the petB-petD intergenic region in tobacco chloroplasts affects petD RNA accumulation and translation

Translation initiation in chloroplasts is a complex process involving a var iety of cis-elements and trans-acting factors. Many chloroplast mRNAs are p rocessed products of polycistronic primary transcripts, but the functional requirement for processing is mostly enigmatic. Tn tobacco, the petB and pe tD genes, which encode subunits of the cytochrome b(6)/f complex, are trans cribed from the psbB operon, whose primary transcript is processed into pro ducts including di- or tricistronic, but not monocistronic, petB and petD m RNAs. To begin to identify elements important for petB and/or petD translat ion, we generated tobacco chloroplast transformants by inserting selectable aadA marker cassette in the petB-petD intergenic region. The resulting pla nts required sucrose for growth, and their phenotypes depended on the orien tation of the aadA cassette. When aadA was inserted in the same transcripti onal orientation as the psbB operon, petB and petD mRNAs were abundantly pr oduced but aberrant in size, and only 25% of the wild-type amount of the cy tochrome b(6)/f complex accumulated. With the aadA cassette in the opposing orientation, however, very little petD mRNA accumulated, and the cytochrom e b(6)/f complex was undetectable. Polysome analysis suggested that petD mR NAs in both transformants were poorly translated, indicating that the inter genic region contains essential translational elements.