Global Measurement of Turnover of Plant Proteins

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PI: Jerry D. Cohen (University of Minnesota) CoPI: William M. Gray (University of Minnesota) Collaborators: Gary L. Nelsestuen (University of Minnesota) and David Angulo (DePaul University)Scientific objectives and approaches: The objectives of the research are to develop robust methods for stable isotope labeling of plant proteins to determine absolute rates of turnover (the half-life) for a wide number of different proteins under changing developmental and environmental conditions and to study the effects of specific mutations on rates of protein turnover. Several different methods for labeling plant proteins, including general methods based on growing plants on 2H2O, in the presence of 13CO2, or on 15N-ammonium nitrate, as well as methods where a specific stable isotope labeled amino acid, sugar or other amino acid precursor will be used to label plant proteins. Loss of stable isotope from specific proteins after a pre-labeling period will be used to determine the suitability of each method for further studies. Changes to normal plant function due to the labeling method and procedures will be monitored by measurement of gene expression changes by microarray analyses. The turnover for proteins known to be degraded at different rates and use of mutants altered in their pathways for protein degradation will be used to test the suitability of the procedures developed. Expected broader impacts: This project will support and train postdoctoral scientists and graduate students who will be exposed to a unique multidisciplinary approach to functional proteomics. International activities will be encouraged through exchange visits by the graduate students and by hosting reciprocal visits. A training program will be established in the second year to transfer knowledge of new methods as they are developed, with a particular targeted effort to reach institutions that are either traditionally minority-serving and/or those with significant minority enrollment. Finally, the project will work towards developing a unique approach to global protein profiling and generating the resources that will be widely applicable and serve as an enabling technology to the plant research community. Data can be accessed at the project website to be developed and methods and protocols will be deposited and accessed at TAIR.