CAREER: genetic approach to identifying proteins necessary for division plane orientation during plant development

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Amanda Joy WrightIOS-1350874CAREER: A genetic approach to identifying proteins necessary for division plane orientation during plant development During plant development, cells divide and grow creating the tissues and organs that make up the adult plant. The process of cell division produces new cells by the cleavage of a pre-existing cell called a mother cell. Mother cells undergo cell divisions with different orientations such as longitudinal or transverse and equal or unequal. Since plant cells are surrounded by rigid cell walls, once they are generated, plant cells are unable to adjust their position relative to neighboring cells. Thus, ensuring that the mother cell undergoes a cell division in the correct orientation is critical for normal plant growth. To fully understand cell division orientation, this proposal lays out a plan to identify the proteins that regulate and execute this process. This research will be conducted in Zea mays (corn), an agriculturally important crop plant. The results of this project will allow for more effective engineering of crops for maximum growth and yield in addition to advancing understanding of a fundamental cellular process. Importantly, previous research has shown that many factors needed for plant cell division are also needed for animal cell division meaning this research is likely to advance our understanding of cell division in many organisms. Finally, during the course of this project, a Cell Biology lab module that allows every biology major at the University of North Texas to participate in a novel scientific investigation will be implemented and evaluated for effectiveness at promoting student learning. The final output of this project will be the identification and characterization of proteins needed for normal plant growth and development. The first objective of the proposal is to characterize the protein disrupted in the Zea mays dcd2 mutant. In this maize mutant, the partitioning cell wall that forms during critical cell divisions in the leaf is misoriented resulting in abnormal cell shapes. The methods that will be used to identify the causative mutation include a combination of positional cloning and RNA-seq while the localization of the DCD2 protein and further characterization of the phenotype will be accomplished by observing fluorescent fusion proteins and immunolocalization. The second objective is to isolate additional maize mutants that form abnormal division planes, roughly map five of the new mutants, and determine the causative mutation in two mutants. The methods that will be used to accomplish this objective include conducting a genetic screen, using RNA-seq to identify the position of the mutations within the genome, and characterizing the mutant phenotype by observing fluorescent fusion proteins and immunolocalization. The third objective is to integrate improvements in science education at the University of North Texas with further characterization of the proteins needed for division plane orientation in plants. To accomplish this objective, students in the Cell Biology Lab will conduct novel research in a classroom setting by conducting a yeast two-hybrid screen to identify proteins that interact with the proteins identified during first two objectives of the project.