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

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. The transporters are positioned in strategic locations to control the exact level of nitrogen acquisition. Many of the key transporter genes have been identified. Some of the transporters serve as receptors that measure external nitrogen availability to adjust uptake to the availability and the specific needs of the plant. Yet measuring the activity of the transporters in situ in intact roots remains challenging. Thus, still little is known about the regulation of individual transporter activities in specific cells of roots. A key to this understanding is the lack of suitable technologies, with sufficient spatial and temporal resolution, for monitoring N acquisition and its regulation. PI's lab 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 and distribution. 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. The knowledge from this project will available to generate a spatio-temporal map of N-acquisition in roots under different N regimes. Objectives and methods: 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 the 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. 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.