Comparative analysis of plastid transcription profiles of entire plastid chromosomes from tobacco attributed to wild-type and PEP-deficient transcription machineries

Abstract

Transcription of plastid chromosomes in vascular plants is accomplished by at least two RNA polymerases of different phylogenetic origin: the ancestral (endosymbiotic) cyanobacterial-type RNA polymerase (PEP), of which the core is encoded in the organelle chromosome, and an additional phage-type RNA polymerase (NEP) of nuclear origin. Disruption of PEP genes in tobacco leads to off-white phenotypes. A macroarray-based approach of transcription rates and of transcript patterns of the entire plastid chromosome from leaves of wild-type as well as from transplastomic tobacco lacking PEP shows that the plastid chromosome is completely transcribed in both wild-type and PEP-deficient plastids, though into polymerase-specific profiles. Different probe types, run-on transcripts, 5' or 3' labelled RNAs, as well as cDNAs, have been used to evaluate the array approach. The findings combined with Northern and Western analyses of a selected number of loci demonstrate further that frequently no correlation exists between transcription rates, transcript levels, transcript patterns, and amounts of corresponding polypeptides. Run-on transcription as well as stationary RNA concentrations may increase, decrease or remain similar between the two experimental materials, independent of the nature of the encoded gene product or of the multisubunit assembly (thylakoid membrane or ribosome). Our findings show (i) that the absence of photosynthesis-related, plastome-encoded polypeptides in PEP-deficient plants is not directly caused by a lack of transcription by PEP, and demonstrate (ii) that the functional integration of PEP and NEP into the genetic system of the plant cell during evolution is substantially more complex than presently supposed.