Poster at the Cold Spring Harbor Laboratory Meetings (1996), Cold Spring Harbor, Optical Imaging of gene expression and signaling in living cells. Best poster award. To determine if the Green Fluorescent Protein (GFP) can be used as a sub cellular marker in plant cells in vivo we transformed suspension cells of Nicotiana tabacum NTI and tobacco Petite Havana plants with a constitutively expressed GFP without (35S35SAMV-GFP and 35S-modGFP4) and with a mitochondrial localization signal (35S35SAMV-coxIVGFP). Green fluorescence is visible by eye when NTI calli expressing modGFP4 or the coxIVGFP fusion protein are exposed to UV light. Confocal or two photon laser scanning microscopy reveals a distinct sub-cellular localization of fluorescence in cells expressing GFP or coxIVGFP. In NTI cells expressing GFP or modGFP4 fluorescence is distributed throughout the cytoplasm but seems to be excluded from compartments that are surrounded by selective membranes. GFP is not visible in vacuoles or nucleoli and is excluded from round spaces in the cytoplasm that seem to be organelles such as leucoplasts or amyloplasts. In contrast, fluorescence in cells expressing the coxIVGFP fusion protein is restricted to particles of about 1 to 2 mm in diameter that are visible only in the cytoplasm, but not in vacuoles or the nucleoplasm. The coxIVGFP fusion protein co-localizes with the mitochondria-specific dye MitoTrackerTM CMTM ROS-H2 showing that the coxIVGFP is targeted specifically to mitochondria. GFP-Fluorescence in mitochondria permits the direct observation of mitochondria in plant cells in vivo and the three-dimensional reconstruction of the distribution of mitochondria in cells. Preliminary results from transgenic tobacco plants expressing mGFP4 and coxIVGFP will be shown and discussed.
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