UDP-glucose 4-epimerase regulation

The staggering structural and developmental complexity of the plant cell wall is matched by an equally complex armoury of genes acting in cell wall carbohydrate biosynthesis and remodelling. Plants possess sophisticated sugar biosynthetic machinery comprising isoforms of nucleotide sugar interconversion enzymes (NSEs) encoded by small gene families - a unique and unexplained feature of plant genomes. The central hypothesis of this proposal states that the control of NSE isoforms regulates glycosylation patterns in response to developmental, metabolic and stress-related cues, thereby linking signalling networks with primary metabolism and the dynamics of the cell wall. Specialized roles for individual NSE isoforms have been proposed, based on genetic evidence and supported by enzyme kinetic and molecular biological studies and cytological observations. Presently the mechanistic basis of carbohydrate biosynthesis control at the level of nucleotide sugar metabolism is largely elusive, control might be exerted both at the level of transcription and at a post-transcriptional or post-translational level. UDP-D-glucose 4- epimerase (UGE), which interconverts UDP-glucose and UDP-galactose, is required for the biosynthesis of a wide variety of biologically and economically important carbohydrates in plants. The family of five Arabidopsis thaliana UGE genes represents the well-characterized paradigm for NSE specialization. Here it is proposed to investigate the mechanistic basis of genetic isoform specialization of plant UGEs and their roles in nucleotide sugar flux control and carbohydrate biosynthesis by directly assessing the contribution of transcriptional vs. post- transcriptional regulation and to establish the significance of protein phosphorylation for isoform function. In a wider context this work will shed light on the regulation of cell wall carbohydrate biosynthesis - a field of increasing importance for human society.

This project resulted in a better understanding of how of growing plants produce cell walls. Plant cells are surrounded by a tough and dynamic cell wall that mainly consists of carbohydrates such as cellulose and pectins as well as complex and highly glycosylated proteins such as the enigmatic arabinogalactan proteins (AGPs) many of which are rich in the sugar D-galactose. To make D-galactose, cells need UDP-glucose-4-epimerase (UGE) and higher plants contain several genes encoding this enzyme. Here it is hypothesized that the biosynthesis of AGPs and other cell wall polymers is regulated by different forms of UGE that can regulate the incorporation of galactose between different cell wall polymers and competing metabolic pathways. To test this hypothesis we constructed a variety of artificial UGE genes and introduced them into plants that lack the gene UGE4 which is essential for the normal formation of AGPs, hemicellulose is also required for normal root growth. We also generated an artificial AGP called FLA4-citrin which we can biochemically quantify and microscopically observe in plants that contain or lack UGE4. We revealed that only UGE2 and UGE4 can direct the formation of FLA4-citrin and hemicellulose and allow normal growth but UGE1 cannot. Despite the high similarity of all UGEs we found that a small region at the end of UGE4 and UGE2 is required for their specific function. On the other hand, UGE1 is modified by addition of phosphate which reduces its ability to produce D-galactose for cell walls and might in this way be reserved for a different function. On a wider perspective this work helps to understand strategies how living organisms find the perfect balance between growth and response to stress.