Regulation and Function of Membrane Lipid Hydrolysis in Lipid-Based Signaling of Plants

跨膜信号转导通常涉及产生脂质或脂质衍生信使的磷脂酶的激活。本项目旨在了解植物中主要磷脂酶家族磷脂酶D （PLD）及其脂质产物磷脂酸（PA）介导细胞功能的机制。具体来说，它将确定PLDs和PA在给定信号通路中的分子靶点。待验证的中心假设是PA和PLDs通过与其他细胞信号传导成分相互作用介导细胞信号传导。第一个目标是表征PLD与异三聚体G蛋白（Ga）亚基之间的新型信号相互作用，并阐明这种相互作用的生理功能。第二个目标是定量定义PA与蛋白磷酸酶2C （PP2C）的相互作用，并确定这种相互作用的细胞意义。第三个目的是分析PA与丝裂原活化蛋白（MAP）激酶的结合，以及PA在蛋白激酶-磷酸酶信号复合物形成中的潜在作用。PA/PLD与信号蛋白的相互作用将通过两种互补的方法进行表征，等温滴定量热法和表面等离子体共振。信号复合物中的其他蛋白质和脂质物种将使用基于质谱的方法进行分析。遗传互补和双突变分析将有助于确定信号相互作用和生理意义。这项工作的智力价值在于，它将坚定地建立PLD/PA与关键细胞信号传导成分的直接相互作用，为植物细胞内脂质信使的直接靶点提供具体证据，深入了解脂质和磷脂酶介导细胞反应的机制，并提供有助于定义植物信号转导级联中各种分子网络的关键信息。这些知识将加强对调节植物生长、应激反应和生产力的过程的理解。更广泛的影响本研究的结果将填补目前植物生物学中基于脂质的信号传导知识的关键空白。该项目将为培养新兴、未开发学科的博士后和研究生提供机会。它还将提供一个平台，扩大代表性不足的群体参与研究。少数民族和女性学生将通过各种国家和校园培训项目招收。将通过讲座和研讨会、课堂教学、国家和国际会议以及及时在同行期刊上发表出版物来传播这些资料，以加强科学和教育。将要开发的材料和方法将提供给研究界。这个项目的结果也将对整个社会有潜在的好处，因为所研究的分子相互作用在介导植物对逆境和环境变化的反应中很重要。这些知识有可能应用于开发具有增强抗逆性和生长性能的作物植物。

Signal transduction across membranes often involves the activation of phospholipases that generate lipid or lipid-derived messengers. The goal of this project is to understand the mechanisms by which phospholipase D (PLD), a major phospholipase family in plants, and its lipid product phosphatidic acid (PA) mediate cellular functions. Specifically, it will identify the molecular targets of PLDs and PA in given signaling pathways. The central hypothesis to be tested is that PA and PLDs mediate cell signaling by interacting with other cell signaling components. The first objective is to characterize the novel signaling interaction between PLD and the a subunit of heterotrimeric G protein (Ga) and to elucidate the physiological function of this interaction. The second objective is to quantitatively define the interaction of PA with a protein phosphatase 2C (PP2C) and determine the cellular significance of the interaction. The third objective is to analyze the binding of PA to mitogen-activated protein (MAP) kinases and the potential role of PA in the formation of protein kinase-phosphatase signaling complexes. These interactions of PA/PLD with signaling proteins will be characterized using two complementary approaches, isothermal titration calorimetry and surface plasmon resonance. Other proteins and lipid species in the signaling complexes will be profiled using mass spectrometry-based approaches. Genetic complementation and double mutant analysis will be used to help define the signaling interaction and physiological significance. The intellectual merit of this work is that it will firmly establish the direct interaction of PLD/PA with key cell signaling components, provide concrete evidence for direct targets of intracellular lipid messengers in plants, generate insight into the mechanism by which lipids and phospholipases mediate cellular responses, and provide critical pieces of information that will help define the network of various molecules in plant signal transduction cascades. Such knowledge will enhance the understanding of the processes that regulate plant growth, stress responses, and productivity.BROADER IMPACTSResults of this study will fill critical gaps in current knowledge of lipid-based signaling in plant biology. This project will provide opportunities to train postdoctoral and graduate students in emerging, under-explored disciplines. It will also provide a platform to broaden participation of underrepresented groups in research. Minority and woman students will be recruited through various national and on-campus training programs. The information will be disseminated to enhance science and education through lectures and seminars, in classroom teaching, at national and international meetings, and through timely publications in peer-journals. The materials and methods to be developed will be made available to the research community. Results from this project will also have potential benefits to society at large, because the molecular interactions studied are important in mediating plant response to stress and environmental changes. Such knowledge has the potential to be applicable to developing crop plants with enhanced stress tolerance and growth performance.