Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology, biochemistry and ultrastructure

The plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The res ulting off-white mutant phenotype is identical for inactivation of the diff erent genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a p oorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The r po(-) material is capable of synthesising pigments and lipids, similar in c omposition but at lower amounts than the wild-type. Western analysis demons trates that plastids contain nuclear-coded stroma and thylakoid polypeptide s including terminally processed lumenal components of the Sec but not of t he Delta pH thylakoid translocation machineries. Components using the latte r route accumulate as intermediates. In striking contrast, polypeptides inv olved in photosynthesis encoded by plastid genes could not be detected by W estern analysis, although transcription of plastid genes, including the rm operon, by the plastid RNA polymerase of nuclear origin is found as expecte d. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo(-) plastids contain functional ribos omes. The detection of the plastid-encoded ribosomal protein Rpl2 is consis tent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid poly merase types into the entire cellular context, are considerably more comple x than presently assumed.