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 IMPACTS Results 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.
