Although the genome sequence of a plant was first reported in 2000, in the ensuing years we have not yet been able to understand fully how these genes act together to allow plants to grow, develop and withstand environmental stresses like drought. Plants (unlike animals) cannot move to avoid the challenges they face in their environment, and new knowledge is needed to allow us to regulate plant growth and sustain improvements in crop yields. This project will help elucidate the molecular mechanisms by which a group of important plant proteins control plant cell growth and development; this information will help us develop methods to maintain the crop yields that are important for our food, fuel, shelter and clothing. This project will provide training for graduate students, and offer educational opportunities and activities for the community, with the basic goal of de-mystifying mass spectrometers and helping the public understand how these instruments can provide amazing sensitivity and power for detecting small amounts of good and bad compounds in our environment. A new high school curriculum called "Global Warming, Campfires and Proteins: What Do They Have in Common?" has been developed and its exercises invite students to experience the ways that scientists explore the unknown as they probe the genetics and biochemistry of plants, and map out the interplay of molecules, plants and humans at the global level.
In the genome of Arabidopsis and other plants, protein kinases represent the largest of all gene families, pointing to a complexity in the phosphoproteome that remains poorly understood. The goal of this project is to increase our understanding of protein kinase mediated signaling by the development and application of mass spectrometric based isotope-assisted quantitative proteomic and computational tools. The biological emphasis is on quantifying and evaluating the in vivo function of rapid protein phosphorylation changes initiated by a newly discovered plant peptide hormone/receptor kinase cognate pair that regulates the rate of proton extrusion and cell expansion. Stable isotope-assisted untargeted "discovery" measurements are performed with a high resolution Orbitrap-based tandem mass spectrometer followed by targeted measurements using chemically synthesized heavy isotope labeled phosphopeptide standards and a triple quadrupole tandem mass spectrometer. Reverse genetic experiments using Arabidopsis plants containing mutations in the phosphorylated amino acids provide final tests of the in planta functions for specific phosphosites. A computational approach will be used to collate data performed under a large number of environmental and genetic perturbations. These network based computational methods of clustering large datasets are aimed at obtaining a more global picture of the protein kinase based signaling pathways. Overall, these experiments will identify networks of key growth regulating signaling pathways, as well as reveal important new insights on the molecular mechanism by which cells expand.
