A study of the molecular pathways of wood formation using peptide mass spectrometry

Abstract: Wood consists essentially of secondary plant cell walls, composed of a cellulose and hemicellulose network impregnated with lignin. It is formed via the development of derivatives of a thin layer of cells (the vascular cambium) into xylem cells through cell division, expansion, secondary wall formation, lignification and, finally, programmed cell death. Throughout the formation of the resulting, complex matrix (wood) diverse proteins are involved. Hence, biosynthesis of wood is tightly regulated at the molecular level, primarily by transcriptional regulation, together with various post-transcriptional processes. In the studies this thesis is based upon, a key technique for studying proteins, liquid chromatography coupled to mass spectrometry, was used to elucidate pathways of wood formation. First, proteins involved in the central transcriptional unit Mediator, regulating most essential processes in plants, were identified in arabidopsis. Mediator subunits that most likely provide secondary cell wall activity are differentiated paralogous subunits located in the tail part of the complex. Point mutations in the Mediator tail subunit, Med5#2 affect secondary cell wall lignification. Moreover, control of xylem expansion may be indirectly regulated by Med25. A strategy to integrate transcript-, protein- and metabolite-data was then developed, using data acquired from analyses of poplar mutants with perturbed wood development. The results showed that lignin biosynthesis is heavily affected at both transcript and protein levels in the mutant with the strongest phenotypic deviations. Interestingly, when transcript levels are decreased, the protein levels are increased. Finally, the front of cell wall synthesis at poplar plasma membranes was dissected. A high coverage of proteins known to be involved in the cell wall synthesizing machinery and associated components was captured, as well as several potential new ones.