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UBC Theses and Dissertations

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UBC Theses and Dissertations

Investigating COBRA's role in regulating cellulose biosynthesis in Arabidopsis thaliana Li, Donglei

Abstract

Plant cell shape is defined by the cell wall. Cellulose microfibrils, which are the major component of the plant cell walls, serve as tension-bearing structures during cell expansion and are important for cells to maintain unidirectional growth and determine cell shape. Cellulose biosynthesis in plants is under tight regulation, it is carried out by cellulose synthases (CESA), arranged in complexes in the plasma membrane. COBRA(COB) was identified as an essential gene for maintaining cell elongation and root anisotropic growth in Arabidopsis (Arabidopsis thaliana). COB is highly expressed in shoot and root elongation zones and is co-expressed with primary cell wall CESAs. Although previous studies have determined COB’s critical role in cellulose synthesis, it is still not clear that the mechanism on how COB is involved in cellulose biosynthesis. Studies in the Wasteneys lab indicate that COB likely plays a role in the regulation of cellulose biosynthesis. With the successful generation of cYFP-conjugated COB (COB-cYFP; COB-mcYFP) and 6x histidine-tagged COB (HisCOB) translational reporter fusion constructs, we were able to detect COB puncta in the cytoplasm and discover that COB undergoes two cleavage events after its secretion to the cell wall, followed by the endocytosis of a polypeptide following cleavage in the cell wall (Chapter 1). In chapter 2, I explored the basis for the cob-1 cellulose deficiency and swollen root phenotype under high sucrose. I used site-directed mutagenesis to re-engineer the HisCOB construct with the cob-1 point mutation, and successfully transformed this new construct (His-cob-1) into the cob-4 background to generate stable transgenic lines. I demonstrated that the His-cob-1 line can be used to purify protein using a gravity column with nickel-conjugated agarose beads, and detected 65 kDa, 48 kDa, and 28 kDa polypeptides from cytosolic extracts. The His-cob-1 cob-4 line is an important resource for future investigations aimed at determining how COB cleavage modulates cellulose production. In chapter 3, I investigated the potential of several candidate enzymes to cleave COB in the extracellular region (Chapter 3). Ultimately, this thesis furthers our understanding of the mechanisms by which COBRA maintains cellulose biosynthesis and unidirectional cell growth during rapid expansion.

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