EAGER: Synthesis and Implementation of Click Sugar Analogs for Plant Cell Wall Polysaccharide Imaging

非技术：所有的植物细胞都被由纤维素、半纤维素和果胶组成的富含多糖的细胞壁所包围。虽然植物细胞壁多糖是地球上最丰富的分子，但细胞壁形成的许多方面仍不清楚。例如，半纤维素和果胶在细胞内合成，运输到质膜，并组织形成功能性细胞壁。然而，多糖的运输和组织机制是未知的，主要是因为这些多糖不能通过传统方法可视化。Wallace实验室先前已经证明，一种与“点击”化学相容的代谢结合的单糖类似物可以用来规避这些问题，并荧光标记细胞壁多糖。这项由NSF-EAGER机制支持的研究将建立在这些发现的基础上，通过开发新的探针来可视化植物细胞壁多糖在细胞内和进入细胞壁的过程。通过开发在其原生环境中“观察”细胞壁多糖的技术，这些工具将以现有技术无法实现的基本独特方式促进植物细胞壁多糖合成和运输的研究。该项目的结果将提供对植物细胞壁形成的更深入的了解，这可以用于生产具有量身定制的细胞壁组成的生物质作物，以供人类利用。技术：植物每年吸收1011公吨的二氧化碳，其中大部分被还原的碳被分解到植物细胞壁中，即包围所有植物细胞的富含多糖的细胞外基质。植物细胞壁多糖可分为三类：纤维素、半纤维素和果胶。纤维素是在质膜上合成的，而半纤维素和果胶则是在高尔基体中合成的，通过一个特征不明确的囊泡介导的运输途径传递到质膜上，并沉积到细胞外空间，在那里它们被组织起来形成一个功能性的细胞壁。非纤维素多糖运输和组织的机制细节仍然不清楚，主要是由于缺乏可用的方法来可视化植物细胞壁多糖。Wallace小组先前证明了聚焦炔（FucAl），一种天然存在的细胞壁单糖聚焦物的类似物，通过代谢结合到植物组织中，并且可以使用铜催化的叠氮化物-炔环加成反应用含叠氮化物的荧光团进行荧光标记。这种糖类似物专门报告了聚焦点纳入果胶的位置，并可用于检查天然细胞壁中的果胶动力学。该项目将以这些想法为基础，通过合成和实现与click化学兼容的普通细胞壁糖的新型单糖类似物，并利用这些代谢探针来标记独特的细胞壁多糖。合理设计的含叠氮化物的单糖类似物将被合成，并将被检测是否与细胞壁结合。此外，还将研究标记的多糖、掺入途径、代谢酶和单个糖的反应性。该项目将从根本上改变研究细胞壁多糖运输的方式，并将为非纤维素细胞壁多糖的纳米级结构和运输提供关键见解。此外，该项目将支持植物化学生物学发展领域的高级研究生教育，将通过UNR生物化学系高级论文项目提供高级本科生研究教育，并将支持跨部门化学生态学和化学生物学研讨会。

Nontechnical:All plant cells are surrounded by a polysaccharide-rich cell wall comprised of cellulose, hemicellulose, and pectin. While plant cell wall polysaccharides collectively represent the most abundant molecules on the planet, many aspects of cell wall formation remain unclear. For example, hemicelluloses and pectins are synthesized within the cell, transported to the plasma membrane, and organized to form a functional cell wall. However, the mechanisms of polysaccharide transport and organization are unknown, primarily due to the fact that these polysaccharides cannot be visualized by traditional methods. The Wallace lab has previously demonstrated that a metabolically incorporated monosaccharide analog compatible with "click" chemistry can be used to circumvent these issues and fluorescently label cell wall polysaccharides. This research supported through the NSF-EAGER mechanism will build upon these findings by developing novel probes to visualize plant cell wall polysaccharides as they move throughout the cell and into the cell wall. By developing techniques to "see" cell wall polysaccharides in their native environment, these tools will facilitate the investigation of plant cell wall polysaccharide synthesis and transport in fundamentally unique ways that are unattainable with existing technologies. The results of this project will provide a deeper understanding of plant cell wall formation, which could be utilized to produce biomass crops with tailored cell wall compositions for human utilization. Technical:Plants annually assimilate 1011 metric tons of CO2, and a large portion of the resulting reduced carbon is partitioned into plant cell walls, the polysaccharide-rich extracellular matrices surrounding all plant cells. Plant cell wall polysaccharides can be grouped into 3 categories: cellulose, hemicellulose, and pectin. While cellulose is synthesized at the plasma membrane, hemicelluloses and pectins are synthesized in the Golgi apparatus, delivered to the plasma membrane via a poorly characterized vesicle-mediated trafficking pathway, and deposited into the extracellular space, where they become organized to form a functional cell wall. The mechanistic details of non-cellulosic polysaccharide trafficking and organization remain unclear, primarily due to the lack of available methods to visualize plant cell wall polysaccharides. The Wallace group previously demonstrated that Fucose alkyne (FucAl), an analog of the naturally occurring cell wall monosaccharide fucose, is metabolically incorporated into plant tissue and can be fluorescently labeled with azide-containing fluorophores using the copper-catalyzed azide-alkyne cycloaddition reaction. This sugar analog specifically reports the sites of fucose incorporation into pectin and can be used to examine pectin dynamics in native cell walls. This project will build on these ideas by synthesizing and implementing novel monosaccharide analogs of common cell wall sugars that are compatible with click chemistry, and utilizing these metabolic probes to label unique cell wall polysaccharides. Rationally-designed azide-containing monosaccharide analogs will be synthesized and will be assayed for incorporation into cell walls. Furthermore, the labeled polysaccharides, incorporation pathway metabolic enzymes, and reactivity of individual sugars will be investigated. This project will fundamentally change the way that cell wall polysaccharide trafficking is investigated, and will provide key insights into the nanoscale structure and trafficking of non-cellulosic cell wall polysaccharides. Additionally, this project will support advanced graduate education in the developing field of plant chemical biology, will provide advanced undergraduate research education through the UNR Department of Biochemistry senior thesis program, and will support an inter-departmental chemical ecology and chemical biology colloquium.