The objective of this proposal is to develop new tools for the functional identification of new plant genes. It aims at assigning genes to identified proteins In a first step, we propose to focus on a particular set of proteins and choose the plant plasma membrane proteins because (i) functional studies of this interface between the cell interior and external medium are at the forefront of plant cell biology, (ii) less than 5 % of the total number of plasma membrane proteins are represented within databases, and (iii) it will be a challenge to fill this gap in our knowledge by systematic generation of sequence information on plasma membrane proteins and respective DNA sequences. This information must be generated and stored in a centrally available database to provide an important resource for researchers from both academia and industry.
We propose a coordinated effort to construct a fundamental database on Arabidopsis plasma membrane proteins, connecting protein expression and sequence information to available or new EST data. Our action will be based on: the construction of a comprehensive directory of plasma membrane proteins (by 2D PAGE) - the generation of partial amino acid sequence data - the development of new methodologies allowing both to identify rare plasma membrane proteins (by using an original expression cloning procedure) and to assign protein sequence data to partial DNA sequences available in EST databases (by using an innovative gene chips methodology) and the construction of a database accessible either directly or through cross referred existing databases. A first part of the work involving most partners will concern, beside an effort for the standardization of procedures, (i) the analysis of plasma membrane protein expression patterns in various Arabidopsis tissues and under various environmental conditions and (ii) additional characterization of given protein subsets A large part of the project, concerning again most partners, will be devoted to automated protein sequencing using a main preparative material that will be selected from the results obtained at the preceeding step. At the same time, a new procedure using ordered arrays of available ESTs on glass grids will be developped for Arabidopsis by one partner (already running this system for other organisms) to connect protein sequence data to cDNA sequences. In addition, rare plasma membrane proteins will be isolated by one partner using protein expression and insertion into the plasma membrane of mammalian cells according to a procedure this partner recently developped for plasma membrane proteins. Finally, during all the project, one partner will be specifically in charge to construct first the protein directory and then the database that will contain both General Image (GI) data sets and Quantitative and Qualitative Individual Spots (QQIS) data sets.
The final plasma membrane database is expected to constitute a resource of vital importance as a basic directory that increases our knowledge of the Arabidopsis genome, and as a link between other coordinated initiatives from the gene to function.