2007 Annual Report 1a.Objectives (from AD-416) The primary objective of the project is to characterize the quantitative and qualitative dynamics of the cell wall proteome and cell wall phosphoproteome in ripening fruit and in leaves following elicitation of defense responses. The value of this research will be in the expansion of our knowledge base concerning the molecular biology of fruit development and disease resistance. Specifically we hope to gain a better understanding of the pathways involved with a view towards developing the “know how” necessary to manipulate these processes to improve the quality and safety of the nations food supply. Associated 425 Log #31496. 1b.Approach (from AD-416) We will use both 2 dimensional difference gel electrophoresis (DIGE) and a shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique termed iTRAC (isobaric Tag Relative Absolute Protein Quantitation) to profile changes in protein expression as a function of both fruit ripening and the elicitation of the defense response. Furthermore, we will carry out a preliminary characterization of the cell wall phosphoproteome using immobilized metal affinity chromatography and nanoLC-MS/MS. We will contrast the observed changes in the secretome of ripening wild type tomato fruit with corresponding changes in the wall-associated transcriptome of wild type and ripening impaired mutant fruit to create a publicly accessible tomato secretome database. 3.Progress Report This report documents research conducted under a Non Funded Cooperative Agreement between ARS and Cornell University. Additional details of research can be found in the report for the in-house associated project 1907-21000-025-00D "Genomics approaches for improving nutritional quality of food crop species.” Additional staff was hired (one postdoctoral associate, one technician) to carry out the tasks that attach to this project. Methods to isolate, resolve, identify and quantify the proteins that characterize the tomato secretome have been developed and continue to be refined. These involve electrophoretic separations using Difference Gel Electrophoresis (DIGE) and two dimensional “shotgun” chromatography separations involving a multiplexed isotope coding quantification strategy (iTRAQ). Protein identification is accomplished using mass spectrometry carried out on proteolytic digests of the isolated proteins. Mass spectrometers equipped with both matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) sources have been evaluated and have been found to be complementary. Preliminary investigations have begun to characterize the extracellular phosphor-proteome and glycol-proteome. Gene expression profiling of tissues used in proteomics analysis has also been initiated (note that this component of the project is not scheduled/funded to begin until fall of 2007). This project is in all respects on schedule and on budget. Quarterly project meetings were held over the course of the year to insure project activities remained on track, problems were addressed and all project leaders were current on activities occurring in cooperating laboratories.