Discovery of fiber-active enzymes in Populus wood

Renewable fibers produced by forest trees provide excellent raw material of high economic value for industrial applications. Despite this, the genes and corresponding enzymes involved in wood fiber biosynthesis in trees are poorly characterized. This thesis describes a functional genomics approach for the identification of carbohydrate-active enzymes involved in secondary cell wall (wood) formation in hybrid aspen. First, a 3’ target amplification method was developed to enable microarray-based gene expression analysis on minute amounts of RNA. The amplification method was evaluated using both a smaller microarray containing 192 cDNA clones and a larger microarray containing 2995 cDNA clones that were hybridized with targets isolated from xylem and phloem. Moreover, a gene expression study of phloem differentiation was performed to show the usefulness of the amplification method. A microarray containing 2995 cDNA clones representing a unigene set of a cambial region EST library was used to study gene expression during wood formation. Transcript populations from thin tissue sections representing different stages of xylem development were hybridized onto the microarrays. It was demonstrated that genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of genes without assigned function, were differentially expressed across the developmental gradient. Microarrays were also used to track changes in gene expression in the developing xylem of transgenic, GA-20 oxidase overexpressing hybrid aspens that had increased secondary growth. The study revealed that a number of genes encoding cell wall related enzymes were upregulated in the transgenic trees. Moreover, most genes with high transcript changes could be assigned a role in the early events of xylogenesis. Ten genes encoding putative cellulose synthases (CesAs) were identified in our own Populus ESTdatabase. Full length cDNA sequences were obtained for five of them. Expression analyses performed with real-time PCR and microarrays in normal wood undergoing xylogenesis and in tension wood revealed xylem specific expression of four putative CesA isoenzymes. Finally, an approach combining expression profiling, bioinformatics as well as EST and full length sequencing was adopted to identify secondary cell wall related genes encoding carbohydrate-active enzymes, such as glycosyltransferases and glycoside hydrolases. As expected, glycosyltransferases involved in the carbohydrate biosynthesis dominated the collection of the secondary cell wall related enzymes that were identified.