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UBC Theses and Dissertations

Engineering cell wall carbohydrate composition in Arabidopsis thaliana seed mucilage as a means to understand the plant cell wall McGee, Robert Thomas


The seed coat epidermal cells of Arabidopsis thaliana synthesize and secrete large quantities of mucilage, a specialized secondary cell wall which is released from mature seeds upon hydration. The goal of this thesis was to study the biological and biochemical consequences of modifying the structure of cell wall carbohydrates using cell wall degrading enzymes as a tool. To develop seed mucilage as a model, promoters that would only drive expression of genes encoding cell wall degrading enzymes within the mucilage-producing epidermal cells were utilized. In order to demonstrate their ability to promote gene expression sufficiently to modify mucilage, the seed coat-specific promoters from the following genes; TESTA-ABUNDANT2 (TBA2), PEROXIDASE36 (PER36), and MUCILAGE-MODIFIED4 (MUM4), were fused upstream of MUCILAGE-MODIFIED2 (MUM2), a gene that encodes a known mucilage modifying enzyme, which were transformed into a mum2 mutant background (Chapter 3). All three promoters were shown to be able to drive sufficient expression of MUM2, in a spatial and temporal pattern to manipulate mucilage composition and complement the mum2 phenotypes. The strongest of the three promoters, TBA2p, was then used to examine the ability of three previously uncharacterized MUM2 homologs; BGAL11, BGAL16 and BGAL17, to complement the mum2 extrusion and cell wall compositional phenotypes (Chapter 3). It was found that cytological and biochemical complementation of mum2 varied and correlated with the amino acid sequence similarity of the homologous gene products to MUM2. Consistent with the fact that the pectin rhamnogalacturonan-I (RG-I) is the major component of seed mucilage, transgenic plants expressing genes encoding RG-I degrading enzymes driven by TBA2p produced seeds with very little mucilage and greatly reduced levels of sugars comprising RG-I (Chapter 4). Unexpectedly, modifications to a minor component of seed mucilage, homogalacturonan (HG), using HG-degrading enzymes driven by TBA2p, resulted in reduced cell adhesion, no mucilage pocket formation and cell death in developing seed coat epidermal cells (Chapter 5), highlighting HG’s essential role in the cell wall. In summary, the data represented in this thesis has demonstrated the feasibility of manipulating cell wall carbohydrate composition using a genetic engineering approach to explore the relationships between structure and function within the plant cell wall.

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