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

氮（N）是植物的关键必需营养素。农作物产量主要取决于大量的氮肥投入。目前，每年生产1500万吨氮肥所需的能源消耗约占世界年产量的1%。然而，植物只吸收了一部分施用的肥料，其余的渗入地下水，污染环境，破坏饮用水供应。因此，在各种水平上都希望提高作物吸收和利用氮的效率。为了促进这种效率的提高，我们需要更好地了解N的获取和分配从根到植物的其他部分的细胞基础。然而，我们并不确切地了解根在哪里，何时以及如何获得N或它们如何控制植物中的吸收和分配。这种理解的一个关键障碍是缺乏合适的技术，具有足够的空间和时间分辨率，用于监测氮的获取及其调节。Frommer博士在卡内基科学研究所的实验室最近开创了新工具的工程，这些工具可以监测负责将氮从土壤转移到根部的关键蛋白质的活性。该项目将改进这些新工具，在植物中实施它们，并使用它们直接监测氮的获取。该项目还将整合这一知识和其他公开的植物生物学资源，以生成不同氮制度下根系氮素获取的时空图。该项目还将培养下一代科学家。具体而言，博士后学者和研究生将接受工程和活动探针实施方面的培训，目标是提高植物根系获取肥料的能力，从而减少肥料使用。本科生和高中生将与经验丰富的科学家合作开发一系列数据库，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实验室最近成功地从参考植物拟南芥中设计了用于N-获取的关键转运蛋白作为荧光生物传感器，这些荧光生物传感器具有报告转运蛋白活性的潜力，从而以高的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）设计和优化铵离子、硝酸盐和寡肽转运蛋白的荧光活性传感器，深入研究其作用机制，并用于结构-功能分析;（2）部署荧光传感器来表征与N转运蛋白相互作用的调节蛋白，以通过利用在新资源中鉴定的候选物来深入了解它们的调节，（3）在野生型和突变体植物中实现传感器，并使用定量荧光成像技术测量完整根的单个细胞中的转运活性;（4）设计用于氮形式（即硝酸盐、铵、谷氨酸盐）的荧光传感器，谷氨酰胺和二肽，并将其部署在植物中，以监测体内不同氮形式的动态;（5）将本项目获得的新信息与公共资源中的信息相结合，创建氮吸收及其调控的细胞分辨率根图（由学生自行搭建）。该项目利用最前沿的技术（荧光传感器、定量成像、微流控装置、细胞特异性表达资源），对根中氮素获取的机制进行综合定量分析。该项目由MCB部门的细胞动力学和功能组以及IOS部门的生理和结构系统组共同资助。

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.