Analysis of the Apoplast Sub-Proteome During Tomato Fruit Development

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The plant primary cell wall is a highly dynamic and complex structure that plays a critical role in numerous examples of plant growth and development. Despite its importance in regulating cell expansion and differentiation, many aspects of wall synthesis, assembly and disassembly remain poorly understood. For example, wall restructuring during cell expansion is highly regulated and while a number of divergent families of wall-modifying proteins have been identified, the mechanism by which they act in concert to achieve wall loosening in a controlled manner is unknown. Furthermore, many new classes of wall-modifying proteins remain to be identified, or cannot currently be assigned a biochemical function. To understand the process of wall reorganization, an important objective is therefore to identify all of the enzymes involved. The need for a systematic approach to the identification and characterization of cell wall protein populations has recently become a realistic consideration in the form of the developing field of proteomics, or the study of the protein complement of the genome. The research outlined in this proposal describes a proteomics approach to the characterization of protein populations in the cell wall and surrounding apoplast, or cell wall continuum, during tomato fruit development. This will provide valuable insight into biochemical processes occurring in the apoplast/cell wall, including the regulation of wall modification during rapid fruit expansion and ripening. Broad questions to be addressed include:1) What are the qualitative and quantitative characteristics of the protein populations present in the tomato fruit cell wall?2) How do the patterns of apoplastic protein expression change qualitatively and quantitatively during fruit ontogeny?Cell wall protein populations will be separated by high-resolution 2-D gel electrophoresis and individual polypeptides analyzed by mass spectrometry, leading to the identification of the corresponding genes. The proposed research will: (i) provide detailed and unprecedented insight into the patterns of expression and regulation of apoplastic/cell wall proteins; (ii) facilitate the identification of groups of proteins that are required for cell expansion or fruit softening (iii) complement existing research initiatives at Cornell, allowing parallel studies of gene expression at the mRNA and protein levels; (iv) demonstrate the utility of proteomics as a tool to study the biology of the cell wall and apoplast; (v) identify new cell wall proteins with potential practical applications for plant biotechnology.