CHARACTERIZATION OF THE TARGETED NUCLEAR ACCUMULATION OF GFP WITHIN THE CELLS OF TRANSGENIC PLANTS

The soluble proteins of the nucleoplasm are synthesized on cytoplasmic ribosomes. Proteins larger than about 40 kDa are post-translationally targeted to the nucleus via energy-dependent processes, passing throu gh the nuclear pore complex into the nucleoplasm. Targeting involves n uclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come prima rily from study of proteins carrying 'classical' NLSs, comprising stre tches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not en tirely satisfactory; they are either technically demanding, are of lim ited accuracy and statistical rigor, or are unsuitable for in vivo app lications. The green-fluorescent protein (GFP) of Aequorea victoria ha s recently emerged as a versatile marker for transgenic expression in vivo. Conditions under which GFP gene fusions can be employed for the analysis of nuclear targeting in plant protoplasts have been described . This study demonstrates for the first time the nuclear targeting of chimeric GFP molecules in transgenic tobacco. This is accompanied by a description and evaluation of novel analytical methods, involving flo w and image cytometry, for the quantitative temporal and spatial analy sis of nuclear targeting, and these unique methods are used to provide information concerning the targeting process. Finally, the way in whi ch the chimeric GFP molecules might be employed for the study of vario us important problems in plant cell and developmental biology is discu ssed.