Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plants in vivo

Summary The recent identification of phage‐type RNA polymerases encoded in the nuclear genome of higher plants has provided circumstantial evidence for functioning of these polymerases in the transcription of the mitochondrial and plastid genomes, as demonstrated by sequence analysis andin vitroimport experiments. To determine the subcellular localization of the phage‐type organellar RNA polymerasesin planta, the putative transit peptides of the RNA polymerases RpoT;1 and RpoT;3 fromArabidopsis thalianaand RpoT fromChenopodium albumwere fused to the coding sequence of a green fluorescent protein (GFP). The constructs were used to stably transformA. thaliana. Transgenic plants were examined for green fluorescence with epifluorescence and confocal laser scanning microscopy. Plants expressing the GFP fusions under control of the CaMV35S promoter exhibited a distinct subcellular localization of the GFP fluorescence for each of the fusion constructs. In plants expressing GFP fusions with the putative transit peptides of ARAth;RpoT;1 and CHEal;RpoT, fluorescence was found exclusively in mitochondria, both in root and leaf cells. In contrast, GFP fluorescence in plants expressing the ARAth;RpoT;3‐GFP construct accumulated in chloroplasts of leaf cells and non‐green plastids (leucoplasts) of root cells. By demonstrating targetingin planta, the data add substantial evidence for the phage‐type RNA polymerases fromC. albumandA. thalianato function in the transcriptional machinery of mitochondria and plastids.