CAREER: The Role of F-box-Mediated Protein Degradation in Seed Development

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Seeds are important for the propagation of flowering plants and are also the primary source of food for human and animal consumption. Understanding the molecular mechanisms underpinning seed development can help protect plant diversity and secure the ever-increasing demand for food production. Rapid cell division and tissue patterning during the process of seed development result in the production of various short-lived regulatory proteins that are removed in a timely manner. Alterations to these protein removal pathways can cause sterility or a severe reduction in the quantity and quality of seed production. However, currently little is known about the identity of these short-lived proteins and how they are targeted for removal. The goal of this research is to understand the functions of a specific group of proteins, termed the F-box proteins, in regulating protein turnover during seed development. The resulting discoveries can then be translated into crop breeding for developing innovative approaches to manipulate seed production. The interdisciplinary nature of this project will provide a wide array of training opportunties for high school, undergraduate, and graduate students, including those from groups under-represented in science. In addition, this project will develop and promote a "big data" - enabled educational system through classroom teaching, research mentoring in the laboratory, and outreach programs. This training system is critical in modern biological education due to the exponential increase in genomic sequencing. The educational goal of this project is to train diverse students, particularly those from educationally underserved Appalachian areas, to develop computational thinking skills in solving biological questions, thus empowering them to pursue a STEM-based career.The F-box protein family serves as the substrate receptor in Skp1-Cullin 1-F-box (SCF) ubiquitin ligases that target many abnormal and short-lived proteins for ubiquitylation and subsequent degradation by the 26S proteasome. In plants, this family plays a wide range of regulatory roles as suggested by its large expansion in plant genomes and genetic analyses of several members in the family. Unfortunately, little is known about the functions of most F-box proteins. Given the presence of many conserved cellular differentiation programs that coordinate to form a mature seed, the over-arching goal of this project is to uncover the role of evolutionarily-conserved F-box proteins in seed development, using Arabidopsis thaliana as a model. First, RNA-Seq experiments and proteomic analyses will be undertaken to define the global influence of F-box-mediated protein degradation in seed development. Bioinformatic approaches will then be used to construct dynamic regulatory networks between active F-box members, F-box substrates, and transcription factors involved in different seed developmental stages. Second, a novel inducible clustered regularly interspaced short palindromic repeat interference (CRISPRi) approach, combined with conventional T-DNA insertion mutations and over-expression strategies, will be used to uncover the genetic functions of conserved F-box genes in embryogenesis. Third, comprehensive genetic, biochemical, and proteomic approaches will be utilized to fine tune the mechanistic roles of F-box proteins involved in seed development. Fourth, the sequencing data generated from the experiments will be adopted in "big data" - enabled biological education, in which different levels of computational thinking skills will be incorporated to train a diverse group of students. This research is anticipated to contribute in-depth knowledge to both seed biology and the biochemistry of protein ubiquitylation. Innovative materials, methods, and algorithms created in the project will have substantial impacts on furthering the understanding of the role of protein ubiquitylation in gene expression regulation.The Project is jointly funded by Molecular and Cellular Biosciences and Integrative Organismal Systems, with additional support provided by the Directorate's Rule of Life Venture Fund.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.