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

由于许多原因，细胞内酸度（pH）的调节对细胞至关重要。因此，活细胞内pH值的测量对于许多生物过程的研究至关重要。目前可用的用于测量细胞内pH的方法大多使用荧光技术，其具有诸如自发荧光、光毒性、光漂白等的局限性。这些问题在绿色植物组织中可能特别严重。此外，目前可用的pH方法在测量光依赖性pH变化（例如叶绿体中发生的pH变化）时是无用的，因为该方法可以触发要研究的响应。该项目的目标是开发一种测量活细胞pH值的新方法。将采用的实验方法是基于该项目的主要研究者开发的一种技术，该技术允许监测体内蛋白质相互作用，即生物发光共振能量转移（BRET）。BRET避免了荧光方法的问题。该项目将开发一种可遗传编码的、有针对性的比率pH监测方法。因此，可以用这种方法在活的植物和动物细胞中定量pH。这一变革性技术的有用性将通过应用于植物生理学的重要实验问题来证明，包括测量（i）叶绿体中响应光/暗转换的pH值变化，以及（ii）细胞pH值的每日节律。（例如，光/暗）对植物细胞活动的影响，从而使农业适应的新战略成为可能。该项目还将为博士后研究员和研究生学习创新技术开发提供最佳培训环境。

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.