The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

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Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus. INTRODUCTIONAlthough cellular function often requires maximization of surface area relative to volume, notably in organelles such as the endoplasmic reticulum (ER) and Golgi apparatus, traditional representations of the nucleus depict a rounded structure with little internal organization. Recently, however, the nuclei of animal cells have been found to show considerable spatial and structural organization at the chromosomal level. The discrete and comparatively stable territories that chromosomes occupy within the nucleus are separated by interchromosomal domains through which transcribed RNA and other macromolecules can diffuse (reviewed in Lamond and Earnshaw, 1998). Animal nuclei also sometimes deviate from the characteristic rounded shape. Deviations include a folded, grooved, or notched surface (Majno et al., 1969) and tubular invaginations. Electron microscopy has shown that these nuclear invaginations can penetrate deep into the nucleus and confirmed the presence of the double membrane of the nuclear envelope (Ellenberg et al., 1997;Fricker et al., 1997;Clubb and Locke, 1998). However, revelation of the full nature of nuclear invaginations in living animal cells has required confocal microscopy of fluorescent dyes (Fricker et al., 1997;Lui et al., 1998aLui et al., , 1998b or green fluorescent protein (GFP) fusion proteins targeted to the nuclear envelope (Ellenberg et al., 1997;Broers et al., 1999). The number and nature of invaginations vary from cell type to cell type, ranging from simple invaginations to intricate branched structures that can penetrate into and through the nucleus. Invaginations often associate with nucleoli (Fricker et al., 1997) and appear to be stable (Ellenberg et al., 1997;Broers et al., 1999). Nuclear grooves and invaginations substantially increase the surface area of the ...

SummaryWe show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al., Plant J. 1994, 5, 559±569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green¯uorescent protein coding sequence (mGFP 5 ) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP¯uorescence throughout the plant, indicating that the tTA±Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml ±1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10::mGFP 5 transgene, with a visible ERtargeted GFP signal appearing from 24 to 48 h after de-repression.