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 through the nuclear pore complex into the nucleoplasm. Targeting involves nuclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come primarily from study of proteins carrying 'classical' NLSs, comprising stretches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not entirely satisfactory; they are either technically demanding, are of limited accuracy and statistical rigor, or are unsuitable for in vivo applications. The green-fluorescent protein (GFP) of Aequorea victoria has 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 flow and image cytometry, for the quantitative temporal and spatial analysis of nuclear targeting, and these unique methods are used to provide information concerning the targeting process. Finally, the way in which the chimeric GFP molecules might be employed for the study of various important problems in plant cell and developmental biology is discussed.