Collaborative Research: Reducing complexity in vivo enables investigation of Cellulose Synthase-like D complex formation, trafficking and function

Cellulose has important economic value as a component of wood, paper, and textile fibers. It is produced in the walls of plant cells, where it controls growth and development. The enzymes that make most plant cellulose (CESAs) produce chains of glucose in groups of 18 and bundle them together to form strong, durable microfibrils.

Related enzymes (CSLDs) also produce chains of glucose, but whether these chains are bundled together and how this affects the properties of the final product is unknown. CSLDs are required for normal cell division and growth of certain plant cells that form long filaments, suggesting that special properties of the cellulose produced by CSLDs are important for these processes.

Attempts to study the cellulose produced by CSLDs have been hampered by the abundance of cellulose produced by CESAs. Although most plants cannot survive without CESAs, it is possible to produce moss plants lacking CESAs thereby solely relying on CSLDs for cellulose. These plants will be used to study CSLDs and the structure and properties of the cellulose they produce.

Plant cells regulate their growth and development by controlling the distribution of CESAs and CSLDs in time and space. How cells deliver these enzymes to the right place at the right time will be studied by adding fluorescent tags to CESAs and CSLDs. This research will contribute to our understanding of plant growth and how we might improve the properties of commercial plant fibers. It will also build the scientific workforce by training undergraduate and graduate students.

The aims of this project are to test the hypothesis that Cellulose Synthase-like D (CSLD) proteins form cellulose synthase complexes and synthesize a distinct form of microfibrillar cellulose and to characterize the functional differentiation and cellular targeting of CSLDs to elucidate their roles in deposition of the specialized cell wall domains that support tip growth and cytokinesis. This research is enabled by lines of the moss Physcomitrium (formerly Physcomitrella) patens with all Cellulose Synthase (CESA) genes knocked out.

This unique resource will be used to investigate CSLD activity in vivo without interference from background CESA activity. The first objective is to use the all cesa knockout lines to structurally characterize CSLD-containing membrane complexes by freeze fracture electron microscopy and to isolate and analyze the structure of the cellulose produced by CSLDs.

The second objective is to characterize the roles of CSLDs in protonemal tip growth and cytokinesis by sub-cellular protein localization and mutation analysis. The final objective is to determine cellular trafficking pathways that target CSLDs and CESAs to distinct regions of the plasma membrane using the powerful genetic and live cell imaging tools possible in P. patens.

By defining the division of labor between CESAs and CSLDs, this research will enhance understanding of the synthesis, structure and mechanical properties of the specialized cell wall domains that contribute to the regulation of tip growth and cytokinesis, and how these contribute to whole organism morphogenesis.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.