Evolution of a domain conserved in microtubule-associated proteins of eukaryotes

The microtubule network, the major organelle of the eukaryotic cytoskeleton, is involved in cell division and differentiation but also with many other cellular functions. In plants, microtubules seem to be involved in the ordered deposition of cellulose microfibrils by a so far unknown mechanism. Microtubule-associated proteins (MAP) typically contain various domains targeting or binding proteins with different functions to microtubules. Here we have investigated a proposed microtubule-targeting domain, TPX2, first identified in the Kinesin-like protein 2 in Xenopus. A TPX2 containing microtubule binding protein, PttMAP20, has been recently identified in poplar tissues undergoing xylogenesis. Furthermore, the herbicide 2,6-dichlorobenzonitrile (DCB), which is a known inhibitor of cellulose synthesis, was shown to bind specifically to PttMAP20. It is thus possible that PttMAP20 may have a role in coupling cellulose biosynthesis and the microtubular networks in poplar secondary cell walls. In order to get more insight into the occurrence, evolution and potential functions of TPX2-containing proteins we have carried out bioinformatic analysis for all genes so far found to encode TPX2 domains with special reference to poplar PttMAP20 and its putative orthologs in other plants

Functional genomics of wood degradation and biosynthesis

Forest biotechnology is a fast emerging field of research. The application of biotechnological tools will enhance the quality of the forest products. The resultant value added and environmentally sustainable products are an absolute necessity in the future. The study of wood biosynthesis and degradation will result in enormous knowledge resources, which can be used for exploiting wood properties. This thesis addresses questions representing both wood degradation and biosynthesis. The wood degrading fungus Phanerochaete chrysosporium is expression profiled with the microarray technology. The objective is to understand the expression pattern of the extracellular carbohydrate active enzymes (CAZymes) secreted by the organism. The data obtained increases our understanding of gene expression upon growth on cellulose. Wood biosynthesis is studied with the model wood forming tree species, Populus. The plentiful data resources from the expression profiling during wood formation in Populus are used as the platform of this work. One of the wood specific genes, PttMAP20, previously with an unknown function is studied in this thesis. The immunolocalisation of PttMAP20 with specific antibodies is demonstrated. The putative microtubule-targeting domain of the protein is demonstrated microscopically and by using a biochemical binding assay.QC 2010121

Functional genomics of wood degradation and biosynthesis

Forest biotechnology is a fast emerging field of research. The application of biotechnological tools will enhance the quality of the forest products. The resultant value added and environmentally sustainable products are an absolute necessity in the future. The study of wood biosynthesis and degradation will result in enormous knowledge resources, which can be used for exploiting wood properties. This thesis addresses questions representing both wood degradation and biosynthesis. The wood degrading fungus Phanerochaete chrysosporium is expression profiled with the microarray technology. The objective is to understand the expression pattern of the extracellular carbohydrate active enzymes (CAZymes) secreted by the organism. The data obtained increases our understanding of gene expression upon growth on cellulose. Wood biosynthesis is studied with the model wood forming tree species, Populus. The plentiful data resources from the expression profiling during wood formation in Populus are used as the platform of this work. One of the wood specific genes, PttMAP20, previously with an unknown function is studied in this thesis. The immunolocalisation of PttMAP20 with specific antibodies is demonstrated. The putative microtubule-targeting domain of the protein is demonstrated microscopically and by using a biochemical binding assay.QC 2010121

Biochemical characterization of family 43 glycosyltransferases in the Populus xylem: challenges and prospects

Wood formation is a biological process of great economical importance. Genes active during the secondary cell wall formation of wood fibers from Populus tremulaxtremuloides were previously identified by expression profiling through microarray analyses. A number of these genes encode glycosyltransferases (GTs) with unknown substrate specificities. Here we report heterologous expression of one of these enzymes, PttGT43A, a putative IRREGULAR XYLEM9 (IRX9) homologue. Expression trials in Pichia pastoris and insect cells revealed very low levels of accumulation of immunoreactive PttGT43A, whereas transient expression in Nicotiana benthamiana leaves by Agrobacterium infiltration (agroinfiltration) using a viral vector produced substantial amounts of protein that mostly precipitated in the crude pellet. Agroinfiltration induced weak endogenous xylosyltransferase activity in microsomal extracts, and transient PttGT43A expression further increased this activity, albeit only to low levels. PttGT43A may be inactive as an individual subunit, requiring complex formation with unknown partners to display enzymatic activity. Our results suggest that transient co-expression in leaves of candidate subunit GTs may provide a viable approach for formation of an active xylan xylosyltransferase enzymatic complex