Since the majority of proteins studied lack discernable enzymatic or biochemical functions, large-scale characterization of proteins will depend on the identification of molecules that associate with them. The association of a protein with other proteins, small molecules, lipids, or nucleic acids has profound effects on its localization, activity, stability, and cellular function. To elucidate the biochemical activities of the Arabidopsis proteome on a global scale, the project will generate an Arabidopsis thaliana expression collection (ATEC) containing 10,000 Arabidopsis ORFs, fused to tandem affinity purification (TAP) tag. An optimized plant-based expression system will be used to produce and purify these proteins. The proteins will be printed on various surfaces to produce Arabidopsis protein chips which will then be screened for various biochemical assays, including protein-protein interactions, protein-phospholipids interactions, protein-nucleic acid interactions, protein-small molecule interactions, and the identification of substrates for kinases and other enzymes. The reagents and protocols generated will be made available to the scientific community through the project website (http://plants.gersteinlab.org/), the Arabidopsis Biological Resource Center (ABRC) and the Arabidopsis Information Resource (TAIR).
Broader Impacts: The rapid progress in the field of large-scale biology has provided the opportunity to elucidate biological function on a large scale and to construct and understand biological networks. Since the biochemical function of a gene is manifested through its encoded protein, much emphasis has been placed on the development of new tools for proteomics. A protein microarray provides a robust and convenient platform for the simultaneous analysis of thousands of individual protein samples, facilitating the design of sophisticated and reproducible biochemical experiments under highly specific conditions. Therefore, the efforts undertaken in this project will ultimately provide a valuable resource for a variety of applications aimed at the high-throughput study of protein function in Arabidopsis and for the elucidation of complex biological networks. Understanding these networks and how they are controlled is likely to be enabling for application to stress, response, energy production etc. In addition, the methods developed and information gained from this study can also be directly applied to the analysis of other agriculturally and horticulturally important plants. Finally, the project will integrate research and educational training for students and post-doctoral associates in the cutting-edge field of proteomics and will provide a unique opportunity to elevate the awareness and importance of plant proteomics research to minority undergraduate students, public high school students and teachers.
