Function, functional conservation and interactions of the membrane-bound endo-1,4-beta-glucanases orthologous to Korrigan

• Main Author: Maloney, Victoria Jane
• Language: English
• Published: University of British Columbia 2010
• Online Access: http://hdl.handle.net/2429/24242

Plant endoglucanses (E.C. 3.2.1.4) encompass multi-gene families across several plant clades, all belonging to the glycosyl hydrolase 9 (GH9) family. One class of GH9 enzymes is unique in that all members possess sequences that encode an N-terminal membrane-anchoring domain. This class of enzymes, termed membrane-bound endo-1,4-beta-glucanases, is the focus of this thesis. The most extensively studied enzyme was first discovered in Arabidopsis and was given the name KORRIGAN (KOR) because of the dwarfed phenotype and cellulose deficiency apparent in plants exhibiting KOR gene mutations. Research has principally focused on Arabidopsis and other non-tree species and the possible role that the enzyme might play in primary cell wall development and cellulose synthesis. However, very little research with KOR has been conducted on trees and secondary cell wall development. Consequently, I investigated the effects of mis-regulating KOR in hybrid poplar and white spruce. I was able to demonstrate that the down-regulation of the hybrid poplar KOR gene increases the crystallinity of the secondary cell wall cellulose and affects the relationship between cellulose and the hemicellulose cell wall components. Concurrently, we were the first to isolate and characterize the KOR gene and suppress KOR gene activity in white spruce. Expression of white spruce KOR in Arabidopsis kor1-1 mutants demonstrated that the gene is able to rescue the mutant phenotype, providing evidence for functional equivalence. Additionally, suppression of the gene in white spruce reduced growth and cellulose content. Since KOR has been demonstrated to be required in cells undergoing cellulose synthesis, we investigated whether or not the KOR protein and the cellulose synthase complex (CSC) interact. Although we were not able to provide evidence for any KOR-protein interaction, we were able to disprove the hypothesis that KOR interacts with CesA7, a member of the secondary cell wall CSC. Collectively, the expression, functional characterization, and interaction data suggest that KOR does not function in direct contact with the CSC, but rather that it plays a role in the later stages of cell wall development, presumably in the relaxation of the stresses around the cellulose microfibril or in the separation of putative cellulose macrofibrils.