PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants.

The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.

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

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.