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Chancellor's Award - Nathan Jayne - Project Description
- 1. Endosidin 7: A Possible New Herbicide and Unique Tool to Study Cell Plate Formation Today, the field of biotechnology is rapidly expanding, especially in areas like biofuels and genetically modified crops. Contrary to popular belief, we still do not fully understand all of the mechanisms of plant growth. As we push the boundaries of plant biology, the elucidation of such mechanisms becomes increasingly important. The Drakakaki lab is studying the formation of the cell plate, the precursor to the cell wall, during cytokinesis of plant cells. Vesicles carrying the materials for the cell plate are known to congregate along the plane of division and eventually fuse in order to form the cell plate. However, the mechanisms of vesicle trafficking during cell plate formation and maturation into the cell wall have yet to be uncovered (Worden et. al 2012). Unfortunately, a classical genetics approach, using mutations in genes to study the process of cell plate formation and cytokinesis, leads to lethal mutations. In order to perturb cell plate formation in a non-lethal manner, a small molecule, Endosidin 7 (ES7), is used by the Drakakaki lab. Discovered in an extensive chemical screen, ES7 disrupts cell plate maturation and the deposition of callose, a polysaccharide synthesized during cytokinesis that offers the delicate cell plate stability (Drakakaki et. al 2011; Park et al., 2014). At lower concentrations (5?M), ES7 disrupts only a fraction of cell plates, enabling plant growth and providing a model to study the effects of ES7 in the cells as well as the overall effect on the plant. If the concentration is increased further, cytokinesis is disrupted such that the plant is prevented from successfully germinating. This growth inhibition effect highlights ES7 as a potential herbicide in addition to its use as a tool to study vesicle trafficking and cell plate formation (Park et al., 2014). In order to investigate the effects of ES7 on the macroscopic as well as subcellular level, the Drakakaki lab observes the effect of ES7 on Arabidopsis thaliana, a small flowering plant that has been extensively studied. We believe ES7 binds to a particular protein, finding this protein target of ES7 can lead to greater understanding of cell plate maturation and vesicular trafficking. a forward genetics ES7- tolerance screen was used to help identify the target protein. Briefly, this involves treating A. thaliana seeds with ethane methyl sulfonate (EMS -a mutagen) to induce a variety of mutations in the genome. These mutant seeds are then grown on varying concentrations of ES7 and compared to a control to select for plants tolerant to ES7 (those with longer root length compared to control on the chemical). Thus far, I have helped identify and confirm three mutants that have shown significant tolerance to ES7 via root length analysis. Although these mutants appear to be tolerant using root length as a proxy, sub-cellular phenotypes are subtle. These phenotypes can be observed using confocal microscopy. In order to visualize the cell plate during cytokinesis, a transgenic line is used in these experiments, expressing a fluorescent protein YFP-RABA2A. This protein is a small GTPase localized specifically at the cell plate during cytokinesis. Exciting the fluorophore on this protein will allow me to obtain 3D images of cell plates disrupted by ES7. Additionally, a callose specific stain, aniline blue fluorochrome, is used to observe callose phenotypes in live cells of the mutants. Using the Zeiss 710 confocal microscope, I conducted subcellular analysis on these tolerant lines. The callose phenotype in the mutants was subtle at best, showing no callose at the cell plate in ES7 treated seedlings. This may have been due to low amounts of callose that is too low for detection with aniline blue fluorochrome or the root length tolerance is not due to a restoration of cell plate callose but rather through another mechanism.
- 2. I will continue by backcrossing and outcrossing the mutant lines and selecting for ES7 tolerant progeny, in order to remove extraneous EMS mutations. These tolerant seedlings will be screened via root length assay and cell file organization by confocal microscopy. After confirming tolerance, I will create a mapping population to use for sequencing and identification of the target of ES7. The molecular mechanisms of vesicle trafficking at the cell plate will be elucidated by analyzing the target of ES7. Understanding this key step in plant cell division will be vital as the bioenergy industry surges forward, pushing the boundaries of plant growth. Citations Drakakaki, G., Robert, S., Szatmari, A.-M., Brown, M.Q., Nagawa, S., Van Damme, D., Leonard, M., Yang, Z., Girke, T., Schmid, S.L., et al. (2011). Clusters of bioactive compounds target dynamic endomembrane networks in vivo. Proceedings of the National Academy of Sciences of the United States of America 108, 17850-17855. Park, E., Diaz-Moreno, S.M., Davis, D.J., Wilkop, T.E., Bulone, V., and Drakakaki, G. (2014). Endosidin 7 Specifically Arrests Late Cytokinesis and Inhibits Callose Biosynthesis, Revealing Distinct Trafficking Events during Cell Plate Maturation. Plant Physiology (Rockville) 165, 1019-1034. Worden, N., Park, E., and Drakakaki, G. (2012). Trans-Golgi Network-An Intersection of Trafficking Cell Wall Components. Journal of Integrative Plant Biology 54, 875-886.