SGER: Monitoring Cytoplasmic and Intraorganellar pH in Plants with a Novel BRET Reporter

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Regulation of intracellular acidity (pH) is vital to cells for many reasons. The measurement of pH inside living cells is therefore crucial to the study of many biological processes. The currently available methods for measuring intracellular pH mostly use fluorescence technologies that have limitations such as autofluorescence, phototoxicity, photobleaching, etc. These problems can be particularly serious in green plant tissue. Additionally, the currently available pH methods are useless when measuring light-dependent pH changes (such as those occurring in the chloroplast) because the method can trigger the very responses that are to be studied. The goals of this project is to develop a new method for measuring pH in living cells. The experimental approach that will be employed is based on a technique developed by the Principal Investigator of the project that allows for monitoring of in vivo protein interactions, Bioluminescence Resonance Energy Transfer (BRET). BRET avoids the problems of fluorescence methods. The project will develop a genetically encodable, targetable, ratiometric pH monitoring method. Therefore, pH can be quantified in living plant and animal cells with this method. The usefulness of this transformative technology will be demonstrated by application to important experimental questions in plant physiology, including the measurement of (i) pH changes in the chloroplast in response to light/dark transitions, and (ii) daily rhythms of cellular pH.Broader impacts.This project will lead to new information about the impact of the environment (for example, light/dark) on cellular activities in plants, thereby enabling new strategies for agricultural adaptation. The project will also provide an optimal training environment for a postdoctoral fellow and a graduate student to learn innovative technology development.