Arabidopsis 2010: An Isotope-Assisted Quantitative Phosphoproteomics Approach to AtHK1-Mediated Osmosignaling in Arabidopsis thaliana

作为固着生物的智力优势植物已经进化出多种机制来感知和响应许多不同的环境输入，包括非生物条件(例如。光、水的可用性)和生物因素(如昆虫、病原体)。这个项目将通过定义向植物细胞传递环境信号所涉及的特定分子步骤来促进我们对植物反应机制的理解。利用模式植物拟南芥和先进的质谱学技术(同位素辅助定量磷酸蛋白质组学)，该项目将识别被磷酸化修饰的蛋白质，作为重要信号通路的一部分。最初的重点将是ATHK1，一种质膜组氨酸激酶，作为一种受体来感知和响应植物细胞水分供应的变化(干旱)。这种蛋白质是几十个组氨酸激酶家族中的一个，它们启动对植物激素和环境参数变化的短期和长期反应。组氨酸激酶的作用至少部分是通过三种不同蛋白质中组氨酸和天冬氨酸残基之间的一系列磷酸转移来实现的。这些修饰(磷酸化)最终导致转录、新陈代谢、细胞分裂和分化的大规模变化。除了组氨酸和天冬氨酸的磷酸化，对干旱反应的遗传研究还暗示了一种钙依赖的信号级联反应，其中包括丝氨酸/苏氨酸蛋白激酶。目前还没有出现明确的模型来将这些化学上不同的蛋白质磷酸化系统整合到对水可获得性的总体反应中。在本项目中，我们将利用植物内代谢非放射性重同位素标记技术和串联质谱仪进行定量的磷蛋白质组测定，并结合表现出各种干旱相关表型的突变体，来全面描述拟南芥干旱信号通路的组成。以前得到支持的研究已经开发和改进了使用同位素辅助质谱学定量的稳健的定量蛋白质组学技术。这些先进的方法将能够识别对干旱等重要环境变化做出一致反应的磷酸蛋白质组。定量蛋白质组学测量将同时提供对特定于水分传感的反应的洞察，并为植物群落成员应用这项技术定义一个有用的范例。更广泛的影响众所周知，干旱是向世界人口提供充足粮食的最严重限制。该项目将确定植物用来感知和响应水供应变化的关键蛋白质修饰，并进一步开发和提供复杂的质谱学技术。为了教育公众和我们未来的领导人，为什么研究需要如此昂贵的设备和技术，威斯康星州大学生物技术中心主任PI和外展工作人员在为广大社区提供教育机会和活动方面有着既定的记录。在目前的资助期内，将针对“测量分子”这一一般领域制定一项新计划。其目标是揭开质谱仪的神秘面纱，并展示它们如何提供惊人的灵敏度和力量来检测我们环境中的少量好的和坏的化合物。该节目将首先讨论这些仪器的常见用途，如在机场筛查爆炸物，以及在火星上报告外星分子。其目的也是开发和使用高中和本科水平的课堂练习，以获得新一代质谱仪显微镜的实践经验，包括之前通过NSF MRI拨款获得的MALDI-TOF/TOF。最终，通过这些活动，希望向公众灌输对先进技术的理论和应用的更好理解，这些技术正日益根深蒂固地植根于日常生活。

INTELLECTUAL MERIT As sessile organisms plants have evolved multiple mechanisms to sense and respond to many different environmental inputs, including abiotic conditions (eg. light, water availability) and biotic factors (eg. insects, pathogens). This project will advance our understanding of plant response mechanisms by defining the specific molecular steps involved in transmitting environmental signals to plant cells. Using the model plant Arabidopsis thaliana and advanced techniques in mass spectrometry (isotope-assisted quantitative phosphoproteomics) this project will identify proteins that are modified by phosphorylation as part of important signaling pathways. The initial emphasis will be on AtHK1, a plasma membrane histidine kinase that acts as a receptor for sensing and responding to changes in water availability (drought) to plant cells. This protein is one of a family of several dozen histidine kinases that initiate short- and long-term responses to changes in plant hormones and environmental parameters. Histidine kinases act, at least in part, by a series of phosphotransfers between histidine and aspartyl residues within three different proteins. These modifications (phosphorylations) ultimately result in large-scale changes in transcription, metabolism, cell division and differentiation. In addition to histidine and aspartate phosphorylation, genetic studies of drought responses have implicated a calcium-dependent signaling cascade that includes a serine/threonine protein kinase. No clear model has emerged for integrating these chemically distinct protein phosphorylation systems in the overall response to water availability. In this project, quantitative phosphoproteome measurements via in planta metabolic labeling with non-radioactive heavy isotopes and tandem mass spectrometry will be used, together with mutants displaying various drought-related phenotypes, to comprehensively delineate components of the drought signaling pathway in Arabidopsis thaliana. Previously supported research has developed and refined robust quantitative proteomics technologies using isotope-assisted quantitation by mass spectrometry. These advanced methods will enable identification of groups of phosphoproteins that act in concert in response to important environmental changes, such as drought. The quantitative proteomic measurements will simultaneously provide insights into the response specific for water sensing as well as define a useful paradigm for applying this technology by members of the plant community. BROADER IMPACTS It is well recognized that drought represents the most severe limitation to providing an adequate supply of food for the world population. This project will identify key protein modifications that plants use to sense and respond to changes in water availability, and further develop and make available sophisticated mass spectrometry technology. In order to educate the public and our future leaders on why such expensive equipment and technology are required for research, the PI, Director of the UW Biotechnology Center, and an outreach staff have an established record of providing educational opportunities and activities for the community at large. In the current grant period, a new program will be developed aimed at the general area of "Measuring Molecules". The goal is to demystify mass spectrometers and demonstrate how they provide amazing sensitivity and power for detecting small amounts of good and bad compounds in our environment. The program will start with a discussion of the familiar uses for these instruments, such as at airports to screen for explosives, and on the planet Mars, for reporting on extraterrestrial molecules. The intention is to also develop and use classroom exercises at the high school and undergraduate level, for hands on experience with a new generation of mass spectrometer microscopes, including a MALDI-TOF/TOF that was obtained previously with an NSF MRI grant. Ultimately, through these activities there is hope to instill in the public a better appreciation for the theory and application of advanced technologies that are becoming ingrained in daily life.