The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants

When expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria.In plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.Since the cloning of the green fluorescent protein (GFP) cDNA and its first heterologous expression in the early 1990s [1,2], the use of intrinsically fluorescent proteins (IFPs) has become one of the most powerful tools in molecular and cell biology. These proteins are applied as reporters in gene expression studies, as indicators of intra-cellular physiological changes, for monitoring dynamics of organelles and proteins, for investigation of protein-protein interactions in vivo and as fusion partners in studies of the subcellular localization of proteins [3,4].Fro