Organisms share the common problem of how to create multicellular diversity and to form beginning from a single cell. To accomplish this task, the processes by which cells divide, grow, and acquire new identities and functions must be coordinated with the activities and identities of other cells. Short-range interactions among plant cells have been shown to play key roles in establishing cellular identity, patterns of cell morphogenesis, and multicellular pattern formation. However, little is known about how the cellular machinery to perform these interactions is assembled and regulated, or how cell-cell interaction affects subcellular organization and morphogenesis. Researchers in the Ehrhardt lab are pursuing the problem of cell-cell interaction and its role in cellular and multicellular development in the model plant Arabidopsis thaliana. The scientists are taking a multidisciplinary approach--including genetics, protein engineering, and confocal microscopy--with an emphasis on observation and manipulation of live cells and their components.

The ability to introduce a fluorescent molecule--the jellyfish green fluorescent protein (GFP)--into cells as an expressed genetic sequence is enabling new experimental approaches to cell and developmental biology. The Ehrhardt lab is exploiting GFP to create new tools for visualization of plant cell structure and behavior, and to identify genes of functional interest. For example, many proteins that are involved in cell-cell and cell-wall interaction are expected to localize to the cell periphery or to sub-domains of the cell periphery. By using GFP, combined with the ease with which Arabidopsis can be transformed, it is now possible for the researchers to design and conduct large-scale screens for genetic information that confers specific subcellular localization properties. In a collaborative project with Sean Cutler, a GFP::cDNA fusion library was created and transformed en mass into Arabidopsis. Individual transgenic seedlings were then screened for GFP localization patterns with confocal microscopy. The screen identified a large number of genetic tags that confer distinct subcellular localization patterns to GFP, including tags for many major organelle systems and markers that show dynamic patterns of relocalization within the cell. These tags comprise a cellular toolkit that has generated a variety of projects being pursued at Carnegie and by labs at other institutions. (See http://deepgreen.stanford.edu for images, movies, and more information about this screen and investigations that are being pursued with the cell tags.)

Among the variety of tags that were recovered is a marker that localizes to the cell periphery and accumulates in discreet foci at cell-cell contact sites, structures that are likely to be cell-cell channels, or plasmodesmata. Plasmodesmata are the only points where most plant cells come into direct contact with each other without an intervening cell wall. They are hypothesized to play important roles in cell-cell interaction, including the possible exchange of informational macromolecules involved in development. Very little is known about the components that make up plasmodesmata, including the genes that encode the components, how the plasmodesmata are assembled at particular cell junctions, or how communication through them may be regulated. The researchers hope to exploit the new GFP tag as an entry point to pursue these questions.

Confocal imaging of GFP is proving to be a robust method for visualizing cell behavior, including behavior related to morphogenesis. The group is employing their collection of GFP markers to study the mechanism of cytokinesis in Arabidopsis shoot cells. Plant cells divide very differently from animal cells. In plants, a new cell partition is initiated as an internally assembled structure, a phragmoplast, before it is fused to the parental cell membrane. It is during phragmoplast development that some plasmodesmal channels are initiated. Confocal imaging of GFP is opening a new window on cell division by giving researchers the ability to visualize three-dimensional aspects of cytokinesis as it occurs in living cells. This technique provides access to cells that were previously difficult to observe, such as those in the shoot epidermis and cortex, permitting the researchers to address questions about how these cells perform cytokinesis in their native multicellular context. These observations are revealing that cell-plate development in shoot cells is highly polarized with respect to the plane of division, a pattern of development that is not reflected in widespread models of plant-cell cytokinesis and which has mechanistic implications for the process of cell-plate formation. The variety of GFP markers that the group has isolated is also allowing them to extend questions about cytokinesis to the behavior of discreet organelles and cell compartments.

Important patterns of cell-cell interaction are established during embryogenesis. However, the Arabidopsis embryo has been challenging to study as a living system. Using their GFP tools, the Ehrhardt group is developing methods to image living embryos. The methods have allowed the researchers to obtain three-dimensional images of the entire cellular structure of live early embryos. These techniques will offer new opportunities to define the cellular phenotypes of mutations that affect the development of the embryo, including cytokinetic mutants and plasmodesmal mutants that the Ehrhardt group is attempting to isolate with aid of the GFP marker.

A greater understanding of plant development will give biologists insight into the diverse mechanisms that have evolved to solve the problem of multicellular life. This information may one day help scientists to engineer crop plants that make more efficient use of limiting resources and are better optimized to produce food and novel products.

Figure 1. Computer reconstruction of living Arabidopsis cells expressing a cDNA::GFP fusion protein that accumulates at locations on the cell periphery that are likely cell-cell channels, or plasmodesmata. These sites are visible as bright spots in this image.

Figure 2. Confocal time series showing cytokinesis of an Arabidopsis hypocotyl cell. The cytosol and nuclei are visualized with a cDNA::GFP fusion protein that moves into and then out of the nucleus over the course of nuclear mitosis. Chromosomes at metaphase are visible as dark spots in a bright volume of cytosol in frame 1. The new cell plate, visible as a thin line in the center of frames 2-6, is built in a polarized fashion starting at one side of the cell and extending to the opposite side.

Figure 3. Three dimensional reconstruction of a living Arabidopsis embryo visualized with a cDNA::GFP fusion protein that highlights the plasma membrane of each cell.

Figure 4. An onion skin cell simultaneously expressing two different GFP fusion proteins, one targeted to the nucleus and the other to the plasma membrane. Use of two spectral variants of GFP allows the two fusion proteins to be discriminated from each other. The onion cell was ballistically transformed using gold particle projectiles. In the merged image, darkfield illumination highlights cell outlines.