12001:

The phragmoplastin complex and its role in building the cell plate during cytokinesis in plant cells.

Authors:	Hong, Zonglie(A)Zhang, Zhongming(A)Geisler-Lee, Jane(A)Verma, Desh Pal, S.(A)
Affiliations:	(A): Depts. of Molecular Genetics and Plant Biology, and Plant Biotechnology Center, Ohio State University
Presenter:	Verma, Desh Pal S., verma.1@osu.edu

Cell plate formation is a unique process in plant cells where a new subcellular compartment is developed in the center of the phragmoplast at cytokinesis. This structure is formed by the fusion of Golgi-derived vesicles and is responsible for the synthesis of cell wall components de novo. We have isolated a dynamin-like protein, phragmoplastin, and shown by using immunocytochemistry that this protein is tightly associated with the cell plate vesicles. At the cell plate, vesicles fuse with each other leading to the formation of vesicle-tubule-vesicle (VTV) structures, a process termed as tubulization. The phragmoplastin polymerizes to form oligomers and we have identified two domains in this molecule that are responsible for making a spiral structure which may wrap around the tubules found at the cell plate. We fused soybean phragmoplastin with the green fluorescence protein (GFP) and introduced it into tobacco BY-2 cells to monitor the dynamics of early events in cell plate formation. We also created a GTP domain mutant to determine the role of GTP in transport and function of Phragmoplastin. This data suggest that in order for phragmoplastin to leave Golgi GTP hydrolysis is required. This resembles intracellular endocytosis from transGolgi, a role similar to that of dynamin in endocytosis. Using yeast two hybrid system, we have identified several candidates that may interact with phragmoplastin. One of these, PIP-1 (for Phragmoplastin-Interacting-Protein), is a novel protein with no overall sequence similarity to the known proteins in the databases. PIP-1 contains three zinc-finger motifs near its C terminus that may mediate the interaction with phragmoplastin and other proteins. The other PIP is identified to be a novel glucosyl transferase which may be involved in the transfer of UDP-glucose to the callose/cellulose synthases. We have been also able to immunoprecipitate this complex using phragmoplstin antibodies. Further characterization of the complex containing phragmoplastin may allow us to decipher the mechanism by which cell wall material (callose / cellulose) begins to be deposited at the forming cell plate. A model is developed that explains most of the structural and physiological events associated with the early cell plate formation. Transgenic plants expressing GTP domain mutated phragmoplastin act as dominant negatives with a variety of phenotypes. In the most severe phenotype, the growth of the root and shoot is arrested beyond the first pair of leaves. These plants were used to evaluate the role of phragmoplastin in cell division, and the consequent affects on plant development. Loss in root apical dominance leads to the precocious formation of adventitious root formation from hypocotyl. However, in many of these adventitious roots initial cells failed to continue dividing, instead they differentiated into vessel elements or tracheids. Our ultrastructural study shows phragmoplasts in both transgenic plants seem to be arrested in the later stages of cell plate maturation. The staining of the vesicles at cell plate is altered indicating that the plate begins to mature before its completion. In some cases the cell wall is not fully formed by the time the next cell division initiated. The data suggest that the delayed cell wall maturation may cause altered apoplastic signaling which affects cell identity, meristem identity and other information necessary for plant development. Finally, the questions regarding how cell plate grows and fuses with the parental cell wall and how a switch between the callose and cellulose synthesis occurs may be answered with the help of tools that have become available from this study. Moreover, with the availability of many Arabidopsis cytokinesis and cell plate mutants, a better understanding of this process may be possible.