Novel Biosensors for Monitoring Nitrogen Uptake at the Cellular Level for Improving Fertilizer Utilization by Plants

氮(N)是植物必需的重要营养物质。作物产量在很大程度上取决于大量的氮肥投入。目前，每年生产1500万吨氮肥需要消耗世界年能源产量的1%左右。然而，植物只吸收了施用肥料的一小部分，其余部分渗入地下水，污染环境并破坏饮用水供应。因此，提高氮素的吸收和利用效率的作物植物是可取的在各种水平。为了促进这种效率的提高，我们需要更好地了解从根部到植物其余部分的氮获取和分配的细胞基础。然而，我们并不确切地了解根系在何处、何时以及如何获得氮，以及它们如何控制植物的吸收和分配。这种理解的一个关键障碍是缺乏适当的技术，具有足够的空间和时间分辨率，用于监测氮的获取及其调控。弗罗默博士在卡内基科学研究所（Carnegie Institution for Science）的实验室最近率先开发了一种新型工具，用于监测负责将氮从土壤转移到根系的关键蛋白质的活动。本项目将改进这些新工具，在工厂中实施，并使用它们直接监测氮的获取。该项目还将整合这方面的知识和其他可公开获得的植物生物学资源，生成不同氮素制度下根系氮获取的时空地图。该项目还将培养下一代科学家。具体而言，将培养博士后学者和研究生进行活动探针的工程和实施，目的是提高植物根系获取肥料的能力，从而减少肥料的使用。本科生和高中生将与经验丰富的科学家一起开发一系列数据库，提供有关肥料、营养以及植物如何获得氮的分子细节的信息。这些资源将提供给科学家和公众。Frommer实验室最近成功地设计了从参考植物拟南芥中获取N的关键转运蛋白作为荧光生物传感器，具有报告转运蛋白活性的潜力，从而以高时间和空间分辨率调节N的吸收和分布。本项目的主要目标是：(1)设计和优化铵态、硝态和寡肽转运体荧光活性传感器，深入了解其作用机制，并将其用于结构-功能分析；(2)利用MIND膜蛋白相互作用组数据库中发现的候选蛋白，利用荧光传感器表征与N转运体相互作用的调节蛋白，以深入了解其调控；(3)将传感器植入野生型和突变型植物，利用定量荧光成像技术测量完整根系单个细胞的运输活性；(4)设计硝酸盐、铵、谷氨酸、谷氨酰胺和二肽等氮形态的荧光传感器，并将其部署在植物体内，监测不同氮形态的动态；(5)将本项目获得的新信息与公共资源整合，绘制细胞分辨率氮吸收及其调控根图（由学生自行构建）。该项目利用尖端技术（荧光传感器、定量成像、微流控装置、细胞特异性表达资源），对根系氮获取机制进行综合定量研究。该项目由MCB部门的细胞动力学和功能集群以及IOS部门的生理和结构系统集群共同资助。

Nitrogen (N) is a key essential nutrient for plants. Crop yield depends critically on massive nitrogen fertilizer input. The annual production of 15 million tons of N-fertilizer needed currently consumes ~1% of the world's annual energy production. However, plants absorb only a fraction of the applied fertilizer and the rest leaches into the groundwater, pollutes the environment and damages drinking water supplies. Therefore, increased efficiency of nitrogen uptake and use by crop plants is desirable on a variety of levels. To promote this increase in efficiency, we need a better understanding of the cellular basis of N acquisition and distribution from roots to the rest of the plant. However, we do not understand exactly where, when and how roots acquire N or how they control uptake and distribution in the plant. A key impediment to this understanding is the lack of suitable technologies, with sufficient spatial and temporal resolution, for monitoring N acquisition and its regulation. Dr. Frommer's lab at the Carnegie Institution for Science recently pioneered the engineering of novel tools that monitor the activity of key proteins responsible for moving nitrogen from the soil into the roots. This project will improve these new tools, implement them in plants, and use them to directly monitor N acquisition. The project will also integrate knowledge from this and other publicly available plant biology resources to generate a spatio-temporal map of N-acquisition in roots under different N regimes. This project will also train the next generation of scientists. Specifically, postdoctoral scholars and graduate students will be trained in engineering and implementation of activity probes with the goal of improving the ability of plant roots to acquire fertilizers, thus reducing fertilizer use. Undergraduate and high school students will work with experienced scientists to develop a series of databases providing information on fertilizers, nutrition, as well as molecular detail on how plants acquire N. These resources will be available to scientists and the public.The Frommer lab recently succeeded in engineering the key transporters for N-acquisition from the reference plant Arabidopsis as fluorescent biosensors that have the potential to report transporter activity and thus regulation of N uptake and distribution with high temporal and spatial resolution. Major aims of this project are to: (1) Engineer and optimize the fluorescent activity sensors for ammonium, nitrate and oligopeptide transporters, gain insights into their mechanism and use them for structure-function analyses; (2) Deploy the fluorescent sensors to characterize regulatory proteins that interact with N transporters to gain insights into their regulation by making use of candidates identified in a novel resource, the MIND membrane protein interactome database; (3) Implement the sensors in wild type and mutant plants and measure transport activity in individual cells of intact roots using quantitative fluorescence imaging technology; (4) Engineer fluorescent sensors for nitrogen forms, i.e. nitrate, ammonium, glutamate, glutamine and dipeptides and deploy them in plants to monitor the dynamics of the different nitrogen forms in vivo; (5) Integrate novel information gained in this project with that from public resources to create a cellular resolution root map of nitrogen uptake and its regulation (to be built by students). The project makes use of cutting edge technologies (fluorescent sensors, quantitative imaging, microfluidic devices, cell specific expression resources) to obtain an integrated quantitative view of the mechanisms of N acquisition in roots.This project is funded jointly by the Cellular Dynamics and Function Cluster in the MCB Division and the Physiological and Structural Systems Cluster in the IOS Division.