Plastid genes transcribed by the nucleus-encoded plastid RNA polymerase show increased transcript accumulation in transgenic plants expressing a chloroplast-localized phage T7 RNA polymerase.
A gene fusion encoding a plastid-targeted bacteriophage T7 RNA polymerase (T7RNAP) under the transcriptional control of the light-regulated promoter and the plastid-targeting signals of a ribulose-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene was introduced into the nuclear genome of Nicotiana tabacum (tobacco). Immunoblot analysis, in vitro transcription assays and protease treatment of isolated chloroplasts revealed that T7RNAP activity was localized within chloroplasts. RNA gel blot analyses showed a substantial increase in transcript abundance for several plastid genes that are normally transcribed by the nucleus-encoded plastid RNA polymerase (NEP) including rpoC1, rpl33, rps18, rps12, and clpP. By contrast, no significant changes were observed in the levels of psbD, 16SrDNA, and ndhA transcripts. These results suggest a possible direct or indirect T7RNAP-mediated enhancement of transcription of a subset of plastid genes that contain NEP promoters. Despite these alterations in plastid transcript levels, the plants showed no visible abberant phenotype.
Magee
Alan M
AM
Plant Molecular Biology Laboratory, Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland.
Kavanagh
Tony A
TA
D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics*; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics; D018087:Plastids / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases