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，测量活细胞内的pH值对许多生物过程的研究至关重要。目前可用的测量细胞内pH值的方法多采用荧光技术，存在自身荧光、光毒性、光漂白等局限性。这些问题在绿色植物组织中尤为严重。此外，目前可用的pH值方法在测量依赖光的pH值变化（如发生在叶绿体中的pH值变化）时是无用的，因为该方法可以触发要研究的反应。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首席研究员开发的一项技术，该技术允许监测体内蛋白质相互作用，生物发光共振能量转移（BRET）。BRET避免了荧光法的问题。该项目将开发一种基因可编码、可靶向、比例pH监测方法。因此，用这种方法可以定量测定活的植物和动物细胞中的pH值。这种变革性技术的实用性将通过应用于植物生理学中的重要实验问题得到证明，包括测量(i)叶绿体中响应光/暗转换的pH变化，以及（ii）细胞pH的日常节律。该项目将提供有关环境（例如，光/暗）对植物细胞活动影响的新信息，从而使农业适应的新战略成为可能。该项目还将为一名博士后和一名研究生提供学习创新技术开发的最佳培训环境。

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.