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A PROTEOMIC APPROACH TOWARDS IDENTIFYING PROTEINS DIFFERENTIALLY EXPRESSED IN RESPONSE TO GRAVI-STIMULATION IN ARABIDOPSIS ROOTS.  L.-S. Young^1,2 and P.H. Masson¹. ¹Lab of Genetics and ²Plant Breeding and Plant Genetics Graduate Program, Univ of Wisconsin, Madison.

     A proteomic approach was used to identify proteins that are differentially expressed in response to gravi-stimulation in Arabidopsis thaliana roots.

     The apical 2-mm root-tip portion of 600 4-day-old seedlings grown on-agar were subjected to a 3-step sequential extraction protocol based on the differential solubility of proteins (Molloy et al., 1998, Electrophoresis 19:837-844). The root tip proteome was fractionated into suitable sizes for analysis and comparison. Over 1,000 protein spots combined from three sequential extraction procedures were visible by 2-dimensional electrophoresis (2-DE). The overall protein expression pattern was similar before gravi-stimulation and after 12 min of gravi-stimulation, a time that precedes the actual curvature response but follows the presentation time. Interestingly, 12 protein spots increased in abundance after 12 min of gravi-stimulation. These same proteins were all degraded and/or down-regulated after 30 min of gravi-stimulation when the elongation zone had initiated a curvature response.

     This proteomic approach using 2-DE analysis proved highly reproducible in 3 successive experiments. MS identification of those differentially expressed proteins by MALDI-TOF and ESI MS/MS is in progress. Similar comparative 2-DE protein analysis between wild-type, pgm, agr1 and arg1 mutants are also being performed to determine which phase of the gravity signal transduction/transmission response are required for the differential expression. Further reverse genetics and molecular approaches will be used to characterize the individual roles of these proteins in root gravitropism.

     (Supported by NASA: NAG2-1336)