Understanding the diversity, biochemistry, and biological role of endo-glucanase 16 enzymes in the context of plant evolution

A defining feature of plants, the plant cell wall is an essential structure that provides mechanical support, cell shape, and protection from pathogens and the environment. Cell walls are dynamic structures that are composed of a network of crystalline cellulose microfibrils, amorphous polysaccharides, and structural proteins. The synthesis, maintenance, and modification of the plant cell wall polysaccharides are carried out by carbohydrate active-enzymes (CAZymes). Xyloglucan endotransglycosylase/hydrolase (XTH) are secreted cell wall enzymes that restructure the cell wall matrix glycan, xyloglucan, thereby facilitating cell wall remodelling and growth. The plant endo-glucanase 16 (EG16) enzymes were recently identified as a unique clade that is structurally and functionally intermediate between the bacterial mixed-linkage β(1,3)/(1,4)-glucanases and the plant XTHs in glycoside hydrolase family 16 (GH16). Correspondingly, EG16 members were broadly specific towards the cell wall polysaccharides mixed-linkage β-glucan (MLG) and xyloglucan, as well as soluble cellulosic substrates. Although EG16 activity and protein structure have been demonstrated, their biological role is unknown. This thesis employs a multi-disciplinary approach to the characterization of the newly designated EG16 group. A comprehensive census of the publicly available genome and transcriptomes showed that EG16 orthologs were found in all plant clades spanning nearly 500 million years of evolution. Notably, the presence of EG16 orthologs observed in the charophyte green algae species that are the most phylogenetically distant from angiosperms, support that EG16-like genes are ancestors to extant EG16 and XTH genes. EG16 substrate specificities, primary hydrolysis site, and digest products are consistent between vastly different plant species. Elucidation of the biological role of EG16 in the moss Physcomitrella patens and a hybrid poplar species, two different distantly related land plants, show gene expression in young tissues and leaderless cell wall localization. In P. patens, EG16 deletion yielded larger plants that senesced earlier than wild-type, whereas, in poplar, downregulation did not affect growth. This thesis serves as a foundation for the EG16 clade and informs their evolutionary history, conserved activity, cell wall localization, and involvement in growth. The research opens the door to investigate unexplored topics such as the EG16 specific secretion mechanism and their role in MLG-containing cell walls.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat