Trafficking, motility, and regulation of cellulose synthases in plants
Restricted (Penn State Only)
- Author:
- Duncombe, Sydney
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 02, 2023
- Committee Members:
- Enrique Gomez, Outside Unit Member
William Hancock, Outside Field Member
Ying Gu, Major Field Member
Charles Anderson, Chair & Dissertation Advisor
Teh-Hui Kao, Program Head/Chair - Keywords:
- plant cell wall
cellulose
cellulose synthase
hexokinase
cytosolic invertase
confocal microscopy
super-resolution microscopy - Abstract:
- Since cellulose is the most abundant biopolymer on Earth and is widely used as a renewable resource, cellulose biosynthesis is arguably one of the most important plant processes to understand. Plants produce cellulose to form strong but flexible primary cell walls during cell growth and more rigid secondary cell walls in some cells after they reach maturity. In primary cell walls, cellulose sits in a matrix of pectin and hemicellulose that adds strength to the wall and helps adhere cells together via the middle lamella. Modifications to this matrix allow for cellulose reorientation to control anisotropic cell growth. Unlike matrix polysaccharides, cellulose is synthesized directly into the wall via cellulose synthase (CESA) proteins. Here, we used genetics, biochemistry, and light microscopy to analyze the proteins that synthesize cellulose and that help generate the substrate for cellulose synthesis to better understand how the trafficking and behavior of CESAs are regulated. Although much has been discovered about the trafficking and motility of CESAs in recent years, there is still a limited understanding of how plants regulate cellulose biosynthesis-based on carbon availability. Previous work established that the UDP-glucose substrate synthesis pathway for cellulose is important for normal cellulose production and carbon partitioning, but the effects of altered substrate biosynthesis on the CESA proteins responsible for cellulose production is still unknown. Furthermore, while HEXOKINASE1 (HXK1) is a known glucose sensor in this pathway, its effect on the regulation of cellulose biosynthesis has not been studied. We examined mutants in CYTOSOLIC INVERTASE (CINV) genes and HXK1 in the dominant UDP-glucose synthesis pathway to determine how this pathway affects CESA trafficking and behavior. Our data indicate that alterations to the primary UDP-glucose synthesis pathway in Arabidopsis reduce the trafficking of cellulose synthase proteins to the plasma membrane and demonstrate that the glucose sensing and catalytic functions of HXK1 have independent functions in cellulose biosynthesis. Live cell CESA imaging has been an indispensable tool for studying CESA behavior for over a decade. Advancements in our imaging toolset have created higher quality images to help us answer biological questions about cellulose biosynthesis, but modern microscopy techniques have not been used to their full potential in this field. Differences in calculated CESA particle density at the plasma membrane between confocal data and electron microscopy data reveal a gap in our understanding of these proteins in living tissues and how they work to build cellulose into the cell wall. Super-resolution microscopy can push the resolution of light microscopy beyond the diffraction limit of light. Although many super-resolution techniques require long acquisition times that are not compatible with movements of CESAs in the plasma membrane, Structured Illumination Microscopy (SIM) can improve the resolution of light microscopy two-fold and maintain a relatively short acquisition time. We applied SIM to seedlings expressing GFP-CESA3 and a microtubule marker, mCherry-TUA5, to image CESAs and microtubules and found that SIM images showed a higher density of CESA particles at the plasma membrane than confocal microscopy, filling in part of the gap between live cell data and high-resolution electron micrographs. We also observed novel behaviors of GFP-CESA3 particles in SIM timelapse recordings. We describe attempts to create a more robust and less disruptive fluorescent tag for CESA imaging and show results for optimizing CESA imaging in the moss, Physcomitrium patens, to use it as a model system for studying the behaviors of CESAs in living tissues. Together, this work has advanced our understanding of how CESA proteins move in the plasma membrane as they produce cellulose and establishes new techniques and tools as resources for studying these small but mighty cellular machines.