MCA: Understanding cellulose synthase complex in planta using single molecule methods

Michigan State University, East Lansing MI

Investigators

Abstract

Cellulose is predominantly produced by plants as the main load-bearing component of the plant cell wall to provide strength and play critical roles in plant cell growth and development. As “the most abundant biopolymer on earth,” cellulose also provides a great potential resource for biofuels and renewable biomaterials towards a carbon-negative economy. Despite its importance, the detailed molecular mechanism underlying cellulose biosynthesis in plants remains largely elusive. This project addresses this knowledge gap by applying advanced microscopic imaging methods to capture the dynamic activity of cellulose synthase in living plants, achieved through a synergistic collaboration between a plant biologist and an optical physicist. The research team will exploit and develop new microscopy techniques to allow real-time visualization of cell wall biosynthesis in growing plants, specifically the enzymes that are responsible for cellulose synthesis at the subcellular and the molecular levels. The results will provide new insights into cellulose biosynthesis and critical information required for further rational design and production of bio-based materials. The project also aims to foster collaboration and multidisciplinary team training of young researchers and students in the current education and outreach programs at Michigan State University and South Dakota School of Mines and Technology to explore their interests in plant science and discover their passion in science and engineering. The biosynthesis of cellulose has been described as a spatially and temporally controlled process carried out in the plasma membrane (PM) by a cellulose synthase complex (CSC) containing multiple cellulose synthases (CESAs). In the Arabidopsis genome, ten putative cesa genes are identified and biochemical and genetic studies have revealed that at least three different CESA isoforms at a 1:1:1 ratio are required for cellulose synthesis in planta. However, the architecture of CSC and its dynamic function in synthesizing cellulose has been largely elusive. This project aims to exploit and develop microscopy approaches to correlatively image the assembly and dynamics of CSCs in planta and cellulose microfibril structure in situ. The putative CESA domains that may play critical roles in CSC assembly and trafficking during cellulose biosynthesis have been engineered to express fluorescence protein tags in corresponding cesa knockout backgrounds for in planta imaging using super resolution microscopy. Specifically, the lattice light-sheet (LLSM) and oblique selective plane illumination microscopy systems with enhanced photon efficiency are used for 3D/4D single molecule tracking to improve localization accuracy and deep tissue imaging, and fluorescence resonance energy transfer (FRET) and time correlated single photon counting methods are used to measure CESA-CESA interactions. Furthermore, the 3D/4D trajectory data are analyzed to correlate with other preliminary imaging results, such as ultrastructure of cellulose microfibrils imaged by AFM and physicochemical properties of cell walls imaged by stimulated Raman scattering microscopy. The findings from this project will allow us to test our working hypotheses and formulate future research directions. 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.