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

种子对开花植物的繁殖很重要，也是人类和动物消费的主要食物来源。 了解种子发育的分子机制有助于保护植物多样性，并确保不断增长的粮食生产需求。 种子发育过程中的快速细胞分裂和组织图案化导致产生各种短寿命的调节蛋白，这些蛋白被及时去除。 这些蛋白质去除途径的改变可导致不育或种子生产的数量和质量的严重降低。 然而，目前对这些短寿命蛋白质的身份以及它们如何被靶向去除知之甚少。 本研究的目的是了解一组特定的蛋白质，称为F-box蛋白，在种子发育过程中调节蛋白质周转的功能。 由此产生的发现可以转化为作物育种，以开发创新的方法来操纵种子生产。 该项目的跨学科性质将为高中，本科和研究生提供广泛的培训机会，包括那些来自科学代表性不足的群体。 此外，该项目还将通过课堂教学、实验室研究指导和推广计划，开发和推广“大数据”支持的教育系统。 由于基因组测序的指数增长，这种培训系统在现代生物学教育中至关重要。 这个项目的教育目标是培养不同的学生，特别是那些来自教育水平低下的阿巴拉契亚地区的学生，培养解决生物学问题的计算思维能力，F-box蛋白家族作为Skp 1-Cullin 1-F-box（SCF）泛素连接酶的底物受体，靶向许多异常和短的活的蛋白质用于泛素化和随后的26 S蛋白酶体降解。 在植物中，该家族发挥着广泛的调控作用，这一点可以通过其在植物基因组中的大量扩增和对该家族中几个成员的遗传分析来证明。 不幸的是，对大多数F-box蛋白的功能知之甚少。 鉴于存在许多保守的细胞分化程序，协调形成一个成熟的种子，该项目的过度的目标是揭示进化保守的F-box蛋白在种子发育中的作用，使用拟南芥作为模型。 首先，将进行RNA-Seq实验和蛋白质组学分析，以确定F-box介导的蛋白质降解在种子发育中的全球影响。 生物信息学的方法，然后将被用来构建动态的F-box成员之间的调控网络，F-box基板，转录因子参与不同的种子发育阶段。 第二，一种新的可诱导的成簇规则间隔短回文重复序列干扰（CRISPRi）方法，结合传统的T-DNA插入突变和过表达策略，将用于揭示胚胎发生中保守的F-box基因的遗传功能。 第三，综合遗传，生物化学和蛋白质组学的方法将被用来微调F-box蛋白参与种子发育的机制作用。 第四，从实验中产生的测序数据将被用于“大数据”支持的生物教育，其中将纳入不同层次的计算思维技能，以培养多样化的学生群体。 这项研究有望为种子生物学和蛋白质泛素化的生物化学提供深入的知识。 该项目中创建的创新材料、方法和算法将对进一步理解蛋白质泛素化在基因表达调控中的作用产生重大影响。该项目由分子和细胞生物科学和整合有机系统联合资助，该奖项反映了NSF的法定使命，并被认为值得支持通过使用基金会的知识价值和更广泛的影响审查标准进行评估。

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.