PUB - Publikationen an der Universität Bielefeld

Aim of this work was a comprehensive analysis of the cell wall proteome of Medicago truncatula suspension cell cultures. Three different tasks included the establishment of an optimised protocol for the extraction of the cell wall proteins (CWPs), development of an optimised method to establish the cell wall proteome map of M. truncatula and comparative proteome profiling of CWPs during elicitation with yeast invertase and suppression with Sinorhizobium meliloti lipopolysaccharides (LPS), representing the pathogenic and symbiotic interactions respectively. The CWPs extracted using living cells and/or cell wall fragments were analysed using different techniques. Using a combination of either one-dimensional, preparative isoelectric focussing, two-dimensional gel electrophoresis (2-DE) or LC-MS/MS and MALDI-TOF-MS analysis, established the proteome reference map of M. truncatula CWPs. In addition to establishing methods for proteome analysis of CWPs of the model plant M. truncatula, the presented work highlights the different composition of several protein classes in cell culture. 2D-PAGE analysis for the CWPs extracted from living cells enabled to characterise approximately 48 CWPs. An alternative approach using a preparative free-flow electrophoresis technique for first dimension separation to obtain 20 fractions with proteins differing in their isoelectric points which were subsequently separated by SDS-PAGE was established. This resulted in identification of 59 out of 100 additional proteins, resulting in 107 identified proteins in total. The proteins analyzed were related to defence, oxidative stress, cell wall modification and signal mediation. In order to overcome the limitations caused because of the nature of the CWPs in proteome analysis, a new extraction protocol (using cell wall fragments) and alternate method (LC-MS/MS) to identify more proteins was established. Furthermore CWPs extracted from purified cell wall fragments and using 2D-PAGE or LC-MS/MS approach, 46 and 65 proteins were identified respectively with a total of 111 proteins. Among the identified were the proteins involved in various processes like cell wall modifications, signaling, defence mechanism, membrane transport, protein synthesis & processing, as well as wall construction processes. Also the comparative proteome analysis using yeast elicitor (Invertase) in response to pathogen defence and suppressor using S. meliloti were also established. 2D-PAGE analysis for the CWP samples extracted with elicitor and LPS treated cells resembled the same as the proteome map of the elicitation with invertase alone, with the few upregulated proteins involved in defence and from LPS treated cell wall proteome map there was no significant difference. Using this approach, proteins involved in defence like L-ascorbate peroxidase, specifically targeted proteins to the cell wall during defence response which include glyceraldehydes-3-phosphate dehydrogenase and proteins that play an important role during growth and development were identified. Also some of this defence related proteins are absent in the same gel after elicitation conferring that oxidant protection is regulated by these proteins. For most CWPs the biological functions are yet to be identified. Bioinformatic analysis helps to have a clue to design an experiment, to understand the biochemical and biological functions of these proteins.