Defining the regulatory role of protein O-fucosylation during pollen-pistil communication

植物繁殖负责产生新的植物胚胎及其周围富含淀粉或油的组织（种子/果实），这些组织在世界范围内用作重要的食物和能源。 此外，在田间环境中繁殖隔离生长的植物以防止转基因和非转基因变体之间的基因流动变得越来越重要。 华莱士实验室最近证明，一组称为蛋白质O-岩藻糖基转移酶（POFT）的蛋白质将糖附着在目标蛋白质上以调节其行为，在植物繁殖过程中发挥作用。 华莱士实验室将使用先进的生物化学和遗传学方法来剖析POFT在植物繁殖中的贡献，并确定受这些酶调节的蛋白质，以阐明POFT如何促进植物繁殖，以及如何操纵这些酶来优化这一过程。 此外，该项目将支持来自不同和代表性不足背景的研究生，本科生和高中生的研究培训。 该项目还将支持基于网络的视频协议的开发，该协议以免费的方式传达用于进行这项研究的先进方法。长期目标是从机制上了解催化蛋白质O-岩藻糖基化的酶，它们的蛋白质靶点，以及植物系统中这种修饰的程度。 指导这项研究的中心假设是POFT岩藻糖基化靶蛋白，这些蛋白控制植物细胞壁多糖生物合成、细胞粘附和细胞间通讯。 具体目标1试图通过采用糖核苷酸水解测定来了解哪些糖核苷酸是推定的POFT的底物，从而了解拟南芥推定的POFT的酶学和糖核苷酸底物特异性。 具体目标2将采用生物素邻近标记和先进的定量蛋白质组学分析，以确定这些POFT的推定蛋白质糖基化底物。 具体目标3将利用亲和捕获方法和先进的蛋白质组学分析，以广泛地识别拟南芥蛋白质组中的蛋白质，这些蛋白质经历翻译后蛋白质O-岩藻糖基化，从而提供以这种方式修饰的蛋白质的目录。 这项研究的统一目标是阐明蛋白质O-岩藻糖基化在植物系统中的作用，特别关注植物有性生殖，并了解这些翻译后修饰如何启动不同的过程，例如细胞粘附和细胞-该奖项反映了NSF的法定使命，并通过利用基金会的知识价值和更广泛的影响进行评估，被认为值得支持审查标准。

Plant reproduction is responsible for the generation of new plant embryos with their surrounding starch or oil rich tissues (seeds/fruits) which are used worldwide as important food and energy sources. Additionally, it is becoming increasingly important to reproductively isolate plants growing in a field setting to prevent gene flow between transgenic and non-transgenic variants. The Wallace lab has recently demonstrated that a group of proteins called Protein O-FucosylTransferases (POFTs), which attach sugars onto target proteins to regulate their behavior, play a role in this plant reproduction process. The Wallace lab will use advanced biochemical and genetic methods to dissect the contribution of POFTs in plant reproduction and identify the proteins that are being regulated by these enzymes to elucidate how POFTs contribute to plant reproduction and how these enzymes could be manipulated to optimize this process. Additionally, this project will support the research training of graduate, undergraduate, and high school students from diverse and under-represented backgrounds. This project will also support the development of web-based video protocols that communicate the advanced methods used to approach this research in a freely available manner. The long-term goal is to mechanistically understand the enzymes that catalyze protein O-fucosylation, their protein targets, and the extent of this modification in plant systems. The central hypothesis guiding this research is that POFTs fucosylate target proteins that govern cell wall polysaccharide biosynthesis, cell adhesion, and cell-cell communication in plants. Specific Aim 1 seeks to understand the enzymology and sugar nucleotide substrate specificity of Arabidopsis putative POFTs by employing sugar nucleotide hydrolysis assays to understand which sugar nucleotides are substrates for putative POFTs. Specific Aim 2 will employ biotin proximity labeling and advanced quantitative proteomic analysis to identify putative protein glycosylation substrates of these POFTs. Specific Aim 3 will utilize affinity capture methods and advanced proteomic analysis to broadly identify proteins in the Arabidopsis proteome that are subject to post-translational protein O-fucosylation, thus providing a catalog of proteins that are modified in this manner. The unifying goal of this research is to elucidate the role of protein O-fucosylation in plant systems, with a particular focus on plant sexual reproduction, and to understand how these post-translational modifications actuate diverse processes, such as cell adhesion and cell-cell communication.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.