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UBC Theses and Dissertations
Elucidating the role of COBRA in plant development in Arabidopsis thaliana Pang, Karlson
Cellulose is the most abundant biopolymer on Earth and was integral for evolution of land plants. Cellulose microfibrils are one of the primary components of plant cell walls, and are critical in maintaining anisotropic growth. These microfibrils are synthesized at the plasma membrane by cellulose synthases (CESAs). Alterations in cellulose biosynthesis, such as mutations in CESAs, can cause defects ranging from decreased unidirectional growth to embryonic lethality. COBRA (COB) is an essential gene in Arabidopsis thaliana important for cellulose deposition and maintaining unidirectional growth, and is highly co-expressed with primary cell wall CESAs. Despite the importance of COB in cellulose biosynthesis, there is still little known about its role or function. This is primarily due to the lack of tools, such as reporter fusion constructs, to assess COBRA’s localization, trafficking, and function. While a cYFP-conjugated COB reporter fusion construct (COB-cYFP) was recently made available, this construct could not fully complement the cob-4 null mutant, casting doubt on any results obtained. However, using construct as a base I was able to improve its ability to complement cob-4 in addition to generating plant lines that are more optimal for live-cell imaging (Chapter 3). In addition to improving the COB-cYFP construct, I also generated a 6x histidine-tagged COB construct (HisCOB) that was able to fully complement the cob-4 null mutant (Chapter 4). Using HisCOB, I was able to demonstrate that COB undergoes at least 2 cleavage events after its secretion to the apoplast, and that this cleaved peptide is ultimately endocytosed. Furthermore, I show evidence that the abundance of COB is too low for it to be a structural component of the cell wall as previously hypothesized, and instead COB likely plays a role in signaling and regulation of cellulose biosynthesis. Finally, cob mutants were generated and investigated to identify potential COB functional domains that were previously uncharacterized (Chapter 5). I identified a region of the COB protein that may contain the primary cleavage site that allows COB to be endocytosed, and demonstrate the importance of the cellulose-binding domain for function. In addition, I provide evidence that COB likely functions as a homodimer.
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