Characterization of novel genetic alleles of cellulose synthase

Abstract

Cellulose is the most abundant polymer on earth. Despite this, relatively little is known about the proteins and biochemical mechanisms governing cellulose biosynthesis. Only in the past 10 years since the sequencing of the Arabidopsis thaliana genome have we begun to unravel the full complexity of this process in plants. A chemical genetics screen using the cellulose biosynthesis herbicide flupoxam was undertaken to identify potentially novel proteins involved in this process. Despite falling short of identifying these new players, six novel mutations were identified on Cellulose Synthase (CESA) 1 and 3 leading to flupoxam resistance. Two of these mutations were positionally cloned using Next-Generation Mapping (NGM) technology, while the others were identified using sequencing. The mutations led to both decreased cellulose crystallinity as well as enhanced cellulose hydrolysis. Future research will fully characterize using additional enzymatic hydrolysis methods and gas chromatography. The second aspect of the project involved the development of a membrane yeast two hybrid (MbyTH) for use with primary cell wall cellulose synthases. Paired interaction studies determined that CESA1 interacts strongly with itself. Also, CESA3 interacts strongly with itself. However, interaction between CESA1 and 3 was weak. It is possible that a tertiary protein is required in order for this interaction to occur. Also, a weak interaction between CTL1/Pom1 (Chitinase –like protein 1) was observed with CESA3. An interaction trap using either CESA1 or 3 as bait may elucidate additional proteins involved in the process of cellulose synthesis.