Genetically Encoded Fluorescent Biosensors Enable Noninvasive Real-Time Visualization of Nitrate Dynamics in Intact Living Plants

Nitrate (NO3−) serves as a pivotal molecule with dual functions in nutrient supply and signaling during plant growth and development. Precise monitoring of its spatiotemporal dynamics in planta is therefore essential for dissecting the regulatory mechanisms underlying plant nitrogen metabolism. However, conventional nitrate detection methods suffer from inherent limitations, including destructive sampling, insufficient spatiotemporal resolution, and an inability to achieve real-time whole-plant monitoring. Here, we report a genetically encoded nitrate biosensor, designated NitNRCL1, constructed using a split firefly luciferase complementation system. Functional validation in both prokaryotic and eukaryotic systems demonstrates that NitNRCL1 responds to changes in nitrate availability and generates stable chemiluminescent signals in bacteria and diverse plant species. Importantly, NitNRCL1 enables non-invasive, real-time, and whole-plant monitoring of nitrate levels in living plants. Using NitNRCL1, we successfully imaged the spatiotemporal dynamics of nitrate signaling in Arabidopsis thaliana. Collectively, our findings establish NitNRCL1 as a robust and novel tool for investigating nitrate transport, signaling, and metabolic pathways in plants. This biosensor advances our mechanistic understanding of plant nitrate biology and provides a technical foundation for breeding nitrogen-use-efficient crops and developing precision fertilization strategies.