This EAGER project and the methods that will be developed combine recent advances in peptide mass spectrometry with quantitative genetics and has potential value to the broader scientific community as a high-throughput, low-cost assay that is applicable to any organism and any post-translational modification. The project will provide training for a graduate student in proteomics, quantitative genetics and plant physiology and molecular biology.
Most decisions made by plants utilize post-translational mechanisms that sense changes in the environment or in other cells of the plant, and that lead to transcriptional changes. A major kind of post-translational mechanism is the reversible post-translational modification of proteins. There are many known modifications; the best studied is phosphorylation. Many aspects of plant stress and plant development are regulated by protein phosphorylation. Recent proteomic surveys have shown that phosphorylation is dynamic and widespread throughout the proteome. A technical grand challenge for research on protein phosphorylation is to develop a method for mapping the enzyme-substrate relationships for every protein kinase and phosphatase. This EAGER project will develop a method to meet this grand challenge. Specific objectives include 1) developing assays for approximately 50 proteins known to undergo biotic stress-induced changes in phosphorylation at specific sites; 2) mapping quantitative trait loci (QTL) that regulate the levels of phosphorylation at each site; and, 3) validating the function of selected genes underlying 15 QTL. All data (e.g., raw MS spectra, m/z values, ion intensities, isotope ratios, and chromatography retention times) will be available through the project website and upon request.
