UDP-glucose, the precursor for cellulose biosynthesis, can be produced via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-I-phosphate by UDP-glucose pyrophosphorylase (UGPase). As such, these genes, together with sucrose phosphate synthase (SPS) which recycles fructose (an inhibitor of SuSy), are interesting targets for altering carbon allocation in plants. In an attempt to alter cell wall biosynthesis in plants, targeted overexpression of SuSy, UGPase and SPS independently and in a pyramiding strategy was assessed in tobacco. All lines displayed enhanced growth and biomass production, and in the case of double and triple transgenics, there was an additive effect. Despite the increased growth rates, there was no consistent change in soluble carbohydrate pools. Furthermore, only the triple transgenics had constant changes in structural carbohydrates: with increased hemicellulose content and slight increases in cellulose. Collectively, these results support the role of SPS, SuSy and UGPase in maintaining sink strength, but suggest that the reallocation of carbon to cellulose production in tobacco may not be possible by overexpressing these genes. In contrast, transgenic poplar overexpressing UGPase produced significantly more cellulose than wild-type trees. However, this was accompanied by a severe reduction in growth and the production of a salicylic acid glucoside (SAG) in significant quantities. The UDP-glucose generated by UGPase overexpression appeared to participate in both the synthesis of cellulose and SAG, suggesting that cellulose biosynthesis may be limited by the cellulose synthase complex. Poplar transformed with SuSy and with SuSy x UGPase also had increased cellulose production. The trees were phenotypically normal, with only minor reductions in height growth in some lines. It appears that UDP-glucose may be channelled directly to the cellulose synthase complex by SuSy. The increased cellulose content was associated with an increase in cell wall crystallinity, but there was no change in microfibril angle, confirming the re-allocation to cellulose synthesis was not the result of tension wood formation, again supporting the hypothesis that the cellulose synthase complex is the limiting factor. Clearly, it is possible to alter cellulose deposition in trees by augmenting sucrose metabolism to produce UDP-glucose, the precursor to cellulose biosynthesis.

UDP-glucose, the precursor for cellulose biosynthesis, can be produced via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-I-phosphate by UDP-glucose pyrophosphorylase (UGPase). As such, these genes, together with sucrose phosphate synthase (SPS) which recycles fructose (an inhibitor of SuSy), are interesting targets for altering carbon allocation in plants. In an attempt to alter cell wall biosynthesis in plants, targeted overexpression of SuSy, UGPase and SPS independently and in a pyramiding strategy was assessed in tobacco. All lines displayed enhanced growth and biomass production, and in the case of double and triple transgenics, there was an additive effect. Despite the increased growth rates, there was no consistent change in soluble carbohydrate pools. Furthermore, only the triple transgenics had constant changes in structural carbohydrates: with increased hemicellulose content and slight increases in cellulose. Collectively, these results support the role of SPS, SuSy and UGPase in maintaining sink strength, but suggest that the reallocation of carbon to cellulose production in tobacco may not be possible by overexpressing these genes. In contrast, transgenic poplar overexpressing UGPase produced significantly more cellulose than wild-type trees. However, this was accompanied by a severe reduction in growth and the production of a salicylic acid glucoside (SAG) in significant quantities. The UDP-glucose generated by UGPase overexpression appeared to participate in both the synthesis of cellulose and SAG, suggesting that cellulose biosynthesis may be limited by the cellulose synthase complex. Poplar transformed with SuSy and with SuSy x UGPase also had increased cellulose production. The trees were phenotypically normal, with only minor reductions in height growth in some lines. It appears that UDP-glucose may be channelled directly to the cellulose synthase complex by SuSy. The increased cellulose content was associated with an increase in cell wall crystallinity, but there was no change in microfibril angle, confirming the re-allocation to cellulose synthesis was not the result of tension wood formation, again supporting the hypothesis that the cellulose synthase complex is the limiting factor. Clearly, it is possible to alter cellulose deposition in trees by augmenting sucrose metabolism to produce UDP-glucose, the precursor to cellulose biosynthesis.

UDP-glucose, the precursor for cellulose biosynthesis, can be produced via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-I-phosphate by UDP-glucose pyrophosphorylase (UGPase). As such, these genes, together with sucrose phosphate synthase (SPS) which recycles fructose (an inhibitor of SuSy), are interesting targets for altering carbon allocation in plants. In an attempt to alter cell wall biosynthesis in plants, targeted overexpression of SuSy, UGPase and SPS independently and in a pyramiding strategy was assessed in tobacco. All lines displayed enhanced growth and biomass production, and in the case of double and triple transgenics, there was an additive effect. Despite the increased growth rates, there was no consistent change in soluble carbohydrate pools. Furthermore, only the triple transgenics had constant changes in structural carbohydrates: with increased hemicellulose content and slight increases in cellulose. Collectively, these results support the role of SPS, SuSy and UGPase in maintaining sink strength, but suggest that the reallocation of carbon to cellulose production in tobacco may not be possible by overexpressing these genes. In contrast, transgenic poplar overexpressing UGPase produced significantly more cellulose than wild-type trees. However, this was accompanied by a severe reduction in growth and the production of a salicylic acid glucoside (SAG) in significant quantities. The UDP-glucose generated by UGPase overexpression appeared to participate in both the synthesis of cellulose and SAG, suggesting that cellulose biosynthesis may be limited by the cellulose synthase complex. Poplar transformed with SuSy and with SuSy x UGPase also had increased cellulose production. The trees were phenotypically normal, with only minor reductions in height growth in some lines. It appears that UDP-glucose may be channelled directly to the cellulose synthase complex by SuSy. The increased cellulose content was associated with an increase in cell wall crystallinity, but there was no change in microfibril angle, confirming the re-allocation to cellulose synthesis was not the result of tension wood formation, again supporting the hypothesis that the cellulose synthase complex is the limiting factor. Clearly, it is possible to alter cellulose deposition in trees by augmenting sucrose metabolism to produce UDP-glucose, the precursor to cellulose biosynthesis. / Forestry, Faculty of / Graduate

<p>Photosynthesis captures light from the sun and converts it into carbohydrates, which are utilised by almost all living organisms. The conversion between the different forms of carbohydrates is the basis to form almost all biological molecules.</p><p>The main intention of this thesis has been to study the role of UDP-glucose in carbohydrate synthesis and metabolism, and in particular the genes that encode UDP-glucose pyrophosphorylase (UGPase) and UDP-glucose dehydrogenase (UGDH) in plants and their regulation. UGPase converts glucose-1-phosphate to UDP-glucose, which can be utilised for sucrose synthesis, or cell wall polysaccharides among others. UGDH converts UDP-glucose to UDP-glucuronate, which is a precursor for hemicellulose and pectin. As model species I have been working with both Arabidopsis thaliana and poplar.</p><p>Sequences for two full-length EST clones of Ugp were obtained from both Arabidopsis and poplar, the cDNAs in Arabidopsis correlate with two genes in the Arabidopsis genomic database.</p><p>The derived protein sequences are 90-93% identical within each plants species and 80-83% identical between the two species.</p><p>Studies on Ugp showed that the expression is up-regulated by Pi-deficiency, sucrose-feeding and by light exposure in Arabidopsis. Studies with Arabidopsis plants with mutations in sugar/ starch- and Pi-content suggested that the Ugp expression is modulated by an interaction of signals derived from Pi-deficiency, sugar content and light/ dark conditions, where the signals act independently or inhibiting each other, depending on conditions. Okadaic acid, a known inhibitor of certain classes of protein phosphatases, prevented the up-regulation of Ugp by Pi-deficiency and sucrose-feeding. In poplar, sucrose also up-regulated the expression of Ugp. When poplar and Arabidopsis were exposed to cold, an increase of Ugp transcript content was detected as well as an increase in UGPase protein and activity. In poplar, Ugp was found to be expressed in all tissues that were examined (differentiating xylem, phloem, apical leaves and young and mature leaves).</p><p>By using antisense strategy, Arabidopsis plants that had a decrease in UGPase activity of up to 30% were obtained. In the antisense plants, the soluble carbohydrate content was reduced in the leaves by at least 50%; in addition the starch content decreased. Despite the changes in carbohydrate content, the growth rate of the antisense plants was not changed compared to wild type plants under normal growth conditions. However, in the antisense lines the UGPase activity and protein content in sliliques and roots increased, perhaps reflecting compensatory up-regulation of second Ugp gene. This correlates with a slightly larger molecular mass of UGPase protein in roots and siliques when compared to that in leaves. Maximal photosynthesis rates were similar for both wild type and antisense plants, but the latter had up to 40% lower dark respiration and slightly lower quantum yield than wild type plants.</p><p>Two Ugdh cDNAs from poplar and one from Arabidopsis were sequenced. The highest Ugdh expression was found in xylem and younger leaves. Expression data from sugar and osmoticum feeding experiment in poplar suggested that the Ugdh expression is regulated via an osmoticumdependent pathway.</p>

Photosynthesis captures light from the sun and converts it into carbohydrates, which are utilised by almost all living organisms. The conversion between the different forms of carbohydrates is the basis to form almost all biological molecules. The main intention of this thesis has been to study the role of UDP-glucose in carbohydrate synthesis and metabolism, and in particular the genes that encode UDP-glucose pyrophosphorylase (UGPase) and UDP-glucose dehydrogenase (UGDH) in plants and their regulation. UGPase converts glucose-1-phosphate to UDP-glucose, which can be utilised for sucrose synthesis, or cell wall polysaccharides among others. UGDH converts UDP-glucose to UDP-glucuronate, which is a precursor for hemicellulose and pectin. As model species I have been working with both Arabidopsis thaliana and poplar. Sequences for two full-length EST clones of Ugp were obtained from both Arabidopsis and poplar, the cDNAs in Arabidopsis correlate with two genes in the Arabidopsis genomic database. The derived protein sequences are 90-93% identical within each plants species and 80-83% identical between the two species. Studies on Ugp showed that the expression is up-regulated by Pi-deficiency, sucrose-feeding and by light exposure in Arabidopsis. Studies with Arabidopsis plants with mutations in sugar/ starch- and Pi-content suggested that the Ugp expression is modulated by an interaction of signals derived from Pi-deficiency, sugar content and light/ dark conditions, where the signals act independently or inhibiting each other, depending on conditions. Okadaic acid, a known inhibitor of certain classes of protein phosphatases, prevented the up-regulation of Ugp by Pi-deficiency and sucrose-feeding. In poplar, sucrose also up-regulated the expression of Ugp. When poplar and Arabidopsis were exposed to cold, an increase of Ugp transcript content was detected as well as an increase in UGPase protein and activity. In poplar, Ugp was found to be expressed in all tissues that were examined (differentiating xylem, phloem, apical leaves and young and mature leaves). By using antisense strategy, Arabidopsis plants that had a decrease in UGPase activity of up to 30% were obtained. In the antisense plants, the soluble carbohydrate content was reduced in the leaves by at least 50%; in addition the starch content decreased. Despite the changes in carbohydrate content, the growth rate of the antisense plants was not changed compared to wild type plants under normal growth conditions. However, in the antisense lines the UGPase activity and protein content in sliliques and roots increased, perhaps reflecting compensatory up-regulation of second Ugp gene. This correlates with a slightly larger molecular mass of UGPase protein in roots and siliques when compared to that in leaves. Maximal photosynthesis rates were similar for both wild type and antisense plants, but the latter had up to 40% lower dark respiration and slightly lower quantum yield than wild type plants. Two Ugdh cDNAs from poplar and one from Arabidopsis were sequenced. The highest Ugdh expression was found in xylem and younger leaves. Expression data from sugar and osmoticum feeding experiment in poplar suggested that the Ugdh expression is regulated via an osmoticumdependent pathway.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sulfated galactans (agarans and carrageenans) are accumulated in the cell wall of various red algae (Rhodophyta) species. These polysaccharides are of commercial importance in the food, pharmaceutical and biotechnology industries due to their unique physicochemical properties. Although having received significant research attention over the last 20 years, events regarding their biosynthesis have not been elucidated. Aiming for the identification of galactosyltransferase (GalT) genes involved in sulfated galactan biosynthesis, cDNA expression libraries were constructed from the prolific agar-producing South African red seaweed Gelidium pristoides (Turner) Kützing and screened by functional complementation of UDP-galactose 4-epimerase deficient mutants (E. coli and S. cerevisiae). Regretfully, no GalTs were identified. The study however yielded the first UGE enzyme described for a red seaweed. Southern hybridization indicated the presence of two UGE copies and confirmed the gene originated from G. pristoides. Bioinformatic analysis of G. pristoides UGE shows amino acid sequence homology to known UGEs from various organisms. The enzyme was shown to be functional in E. coli crude extracts and showed affinity for UDP-D-galactose, similar to other UDP-galactose 4-epimerases. Further, the isolated G. pristoides UGE (GpUGE) was biochemically characterized and its kinetic parameters determined. We found that there was no kinetic difference between this enzyme and previously described UGE enzymes except enhanced activity in the presence of exogenously added NAD+. The UDP-galactose 4-epimerase (UDP-glucose 4-epimerase, UGE, EC 5.1.3.2) is an essential Leloir pathway enzyme facilitating the catalytic inter-conversion between UDP-D-glucose and UDP-D-galactose. UDP-D-galactose is the nucleotide sugar required by galactosyltransferases for the production of red algae sulfated galactans. UGE is suspected as being responsible for supplying UDP-D-galactose for the synthesis of sulfated galactans. In planta monitoring of GpUGE transcript levels with respect to dark and light cycling indicated high expression of the enzyme at night, while expression diminished during the day. The occurrence of increased nocturnal UGE expression correlates with floridean starch breakdown at night. Evidence for hydrolysis of floridean starch is also reflected in obtained G. pristoides transcriptome sequence data. In red algae, floridean starch degradation coincides with sulfated galactan production. The detection of starch hydrolysis enzyme transcripts alongside increased expression of GpUGE suggests the enzyme plays a role in supplying UDP-Dgalactose for sulfated galactan production. As far as we know, this the first report of sequencing and biochemical characterization of a UGE from red seaweed.

<p>Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures<i> </i>and growing<i> </i>plants<i>. </i>The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated.</p><p>A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb.</p><p>Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis.</p><p>Expression of the bacterial UGDH in <i>E. coli</i> resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. </p><p>Overexpression of UGD1, one of four <i>A. thaliana</i> UGDH genes, in <i>A. thaliana,</i> resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.</p>

<p>The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins.</p><p>In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells.</p><p>The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-<i>β</i>1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation.</p><p>The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.</p>

The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins. In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells. The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-β1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation. The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.

Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated. A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb. Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis. Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.