The objectives of the research are to develop robust methods for stable isotope labeling of plant proteins for the purpose of determination of absolute rates of turnover (the half-life) for a wide number of different proteins under changing developmental and environmental conditions, as well as for the determination of 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 of13CO2 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.