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

The recent identification of phage-type RNA polymerases encoded in the nucl ear genome of higher plants has provided circumstantial evidence for functi oning of these polymerases in the transcription of the mitochondrial and pl astid genomes, as demonstrated by sequence analysis and in vitro import exp eriments. To determine the subcellular localization of the phage-type organ ellar RNA polymerases in planta, the putative transit peptides of the RNA p olymerases RpoT;1 and RpoT;3 from Arabidopsis thaliana and RpoT from Chenop odium album were fused to the coding sequence of a green fluorescent protei n (GFP). The constructs were used to stably transform A. thaliana, Transgen ic plants were examined for green fluorescence with epifluorescence and con focal laser scanning microscopy. Plants expressing the GFP fusions under co ntrol of the CaMV35S promoter exhibited a distinct subcellular localization of the GFP fluorescence for each of the fusion constructs. In plants expre ssing GFP fusions with the putative transit peptides of ARAth;RpoT;1 and CH Eal;RpoT, fluorescence was found exclusively in mitochondria, both in root and leaf cells. In contrast, GFP fluorescence in plants expressing the ARAt h;RpoT;3-GFP construct accumulated in chloroplasts of leaf cells and nongre en plastids (leucoplasts) of root cells. By demonstrating targeting in plan ta, the data add substantial evidence for the phage-type RNA polymerases fr om C. album and A. thaliana to function in the transcriptional machinery of mitochondria and plastids.