School of BioSciences - Theses
Permanent URI for this collection
Search Results
1 - 1 of 1
-
ItemBuilding a wall: Developing small molecule biosensors to visualize cell wall biosynthesis and untangling mechanisms underlying nucleotide sugar transport( 2022)The cell wall is one of the main energy sinks in plants constituted of many polysaccharides and glycoproteins. The synthesis of most polysaccharides and the proteins glycosylation occur in the Golgi apparatus. The nucleotide sugars are the precursors of the cell wall building blocks. These molecules are biosynthesized in the cytosol from sugars of the primary metabolism and are transported from the cytosol to the Golgi by the nucleotide sugar transporters (NSTs). The glycosyltransferases (GTs) then consume these nucleotide sugars to produce polysaccharides and the glycoproteins. Although we have a generalized overview, our knowledge of the exact roles of NSTs and nucleotide sugars in regulating the cell wall synthesis is still sparse. The recent determination of the crystal structures of two NSTs, the GDP-D-mannose transporter VRG4 from yeast and the orthologue of CMP-sialic acid transporter from maize indicate that conformational changes occur during the transport process. Therefore, NSTs are excellent candidates to develop fluorescent proteins-based sensors to study the flow of nucleotide sugars. In chapter II, our aim was to generate fluorescent protein- biosensors based on the UDP-XYLOSE TRANSPORTER 1 (UXT1) to follow the import of UDP-D-xylose into the Golgi in vivo as a proxy for xylan and xyloglucan biosynthesis. We show that the UXT1-based FRET sensors maintain the physiological localization of UXT1 in planta, while transporting UDP-D-Xyl in vitro. We also designed ratiometric sensors based on the sfGOMatryoshka. However, these sensors were found to disrupt the Golgi localization of UXT1. In chapter III, we aimed to probe the existence of protein complexes involved in arabinosylation and study the regulation of the arabinosylation pathway. Investigating higher order mutants of the UDP-ARABINOFURANOSE TRANSPORTERs (UAfTs), allowed us to decipher the relative contribution of each of the UAfTs to arabinosylation. These newly generated mutants in combination with other mutants of the arabinosylation pathway led us to propose a mechanistic model to explain a glucose hypersensitivity phenotype in the dark. Phenotypic assessment of nucleotide sugar levels and hypocotyl elongation allowed us to make progress towards dissecting the role of cytosolic UDP-GLUCOSE EPIMERASE 1 and 3 in regulating nucleotide sugar metabolism. Finally, using affinity purification, split-ubiquitin assays and in silico co-expression approaches, we unravel putative NST-GT complexes and suggest that these complexes also involve nucleotide sugar interconverting enzymes.