TRTech-PGR: Spatiotemporal Mapping of Protein Life in Plant Cell Signaling, Trafficking, and Development with the Next-Generation Tandem Fluorescent Timers

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Visualizing individual proteins in organisms based on their property is an essential analytical process for studying the function of cellular proteins. Upon biogenesis, proteins undergo a series of maturation and transport processes as well as turnover with a unique rate, which are often regulated by developmental and environmental cues. The longevity of proteins often determines the amplitude of output signals, which determines the biological outcomes. Because protein maturation, transport, and turnover could occur in cell-specific manner and at specific subcellular domains, linking age information and location information of proteins facilitate understanding the observation. However, to date, no standard tools to visualize the age of proteins in living plant tissues have been available. This project will develop a series of plant-optimized, genetically coded sensor proteins, i.e., tandem fluorescent timer proteins (tdFT), which change colors based on the protein ages. The operation principle of tdFTs is simple and relies on differential maturation time between green and red fluorescent proteins connected in tandem, and requires only conventional fluorescent microscopy set up. Difficulty in identifying optimum configurations specific to each application has been prohibitive for the broad application of tdFT technology in plants. The project will generate a toolbox of validated, user-friendly tdFT vectors for plant purposes. The developed tdFT will be deployed to address fundamental questions in plant cell biology, including transport and turnover of a cell wall biosynthesis protein and immunity signaling proteins and cell lineage tracking during stomatal development.This project aims to develop analysis pipelines for non-destructive visualization of spatiotemporal protein life in plants and examine intracellular transport/turnover dynamics of key traffic/signaling proteins in vivo. In living cells, dynamic spatiotemporal distributions of nascent and aged proteins are integral parts of cellular signaling. Conventional fluorescence-tagging strategies, which are prevalently used for the research community, typically provide an endpoint profile of protein distribution but lack the resolution in temporal dynamics of protein maturation and trafficking. Essential regulatory processes attributed to protein maturation and various post-translational modifications can only be imaged by time-sensitive fluorescent tags. tdFTs produce time-specific fluorescent signatures based on differential maturation times of GFP and RFP. These are an intrinsic property of fluorescent proteins, which do not require special instruments other than conventional epifluorescence or confocal microscopes. To make tdFT technology widely available for plant research, this project will develop a series of tdFT with various time ranges. Each tdFT will be validated using a transient expression system in Arabidopsis and other plant species. Characterized tdFT will be used to address fundamental questions in plant biology, such as protein stability upon complex formation during plant immune response, membrane protein turnover in the secretory system, and cell lineage analysis during stomatal development.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.