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

To study the underlying mechanisms for microbial cellulose biosythesis, a novel compound, CBI28, was used as an inhibitor along with classical genetics and EMS mutagenesis. An EZ-Link Biotin Hydrazide Kit was used to create a CBI28-Biotin conjugate for further studies. Gluconacetobacter xylinus cells were exposed to 10 uM CBI28 to induce cellulose biosythesis inhibition, lysed and small hydrophobic molecules were extracted using methanol and Waters Oasis HLB SPE-Paks. Samples were separated and detected using the Ultra Performance Liquid Chromatograph-Mass Spectrometer/Photo Diode Array. Putative mutants were isolated but did not survive for further study. An ion with the expected mass of a CBI28-Biotin conjugate (552 m/z) was detected but not in sufficiently high concentrations for characterization. Metabolite studies revealed putative metabolites derived from the HLB SPE and methanol extractions with no significant difference in extraction methods. Potential metabolites with masses of ~281.77 m/z and ~79 m/z were detected in CBI28 exposed cells. Further analysis needs to be performed to determine if CBI28 metabolites prevent cellulose production. / UOIT

The cell wall is a multifunctional structure which is implicated in plant growth and development as well as responding to any environmental changes including biotic and abiotic stresses. One of the practical approaches in cell wall integrity studies is the modification of the quality and quantity of particular cell wall components or destroying the specific step in cell wall synthesis pathway using Cellulose Biosynthesis Inhibitors (CBIs). In this case, chemical screen for swollen organ phenotype has proved to be an important technique to identify the genes that are directly or indirectly involved in cellulose biosynthesis. In the present research, a number of synthetic CBIs were obtained through a chemical library screen from Chembridge Company for the root swollen phenotype which is believed to be the response to a defect in cellulose biosynthesis. Therefore, a genome-wide expression profiling based on Affymetrix ATH1 GeneChip arrays (contains 22810 probe sets) were applied to investigate the altered transcriptome of four different CBIs including CBI-15, 18, 22, and 27 and isoxaben in 5 day-old Arabidopsis thaliana seedlings. The results of this project revealed overlapped up and down-regulated genes as well as discriminate responses to each CBI. The most striking modification were found in genes involve in response to the stress as well as cell wall integrity and restructuring. Thus, the identification of regulated genes under CBIs treatment suggests a robust candidate group of genes that likely to be correlated to cell wall biosynthesis. / UOIT

No description available.

Eucalyptus trees are an important source of wood and fibre. The wood (secondary xylem) of this genus is widely used for pulp and papermaking. However, our understanding of the mechanisms which control the wood formation process (xylogenesis) in Eucalyptus and other woody species is far from complete. One reason is that xylogenesis is a very complex developmental process. The major components of wood are lignin and cellulose. Many genes involved in lignin and cellulose biosynthesis have been characterized. For example, Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are two important lignin biosynthesis genes. Plant cellulose is synthesized by cellulose synthase enzymes with the aid of some other proteins, such as sucrose synthase (SuSy) and sucrose phosphate synthase (SPS). Another factor which makes it difficult to analyze the function of Eucalyptus wood formation genes in vivo, is the long generation times of Eucalyptus trees and the difficulty to obtain transgenic Eucalyptus plants. Therefore, in this study, we investigated the use of Arabidopsis thaliana as a model system for functional analysis of wood formation genes. We transformed a lignin and a cellulose biosynthesis gene isolated from Eucalyptus to wild-type and mutant genetic backgrounds of Arabidopsis in order to test our ability to modify the cell wall chemistry of Arabidopsis thaliana using tree genes. The Eucalyptus CCR (EUCCR) gene was transformed into wild-type Arabidopsis (Col-0) and irregular xylem 4 (irx4) mutant plants, in which the homolog of EUCCR is mutated. A Eucalyptus cellulose synthase gene (EgCesA1) was also transformed into irregular xylem 1 (irx1) mutant plants, in which the homolog of EgCesA1 is mutated. Transgenics were only obtained from wild-type Col-0 transformed with EUCCR and from irx1 transformed with EgCesA1. We studied the cell wall chemistry of wild-type Arabidopsis plants overexpressing the Eucalyptus CCR gene. Chemical analysis of inflorescence stems revealed the modification of lignin and cellulose content in transgenic plants. Total lignin content was increased in T2 (5%) and T3 (12%) lines as revealed by micro-Klason lignin and thioglycolic acid quantification methods, respectively. High Pressure Liquid Chromatography (HPLC) analysis revealed that cellulose content was significantly decreased (10%) in T2 transgenic plants. This suggested the reallocation of carbon from cellulose to lignin as a result of overexpression of EUCCR in transgenic plants. Interestingly, thioacidolysis analysis revealed that in T2 plants, monomethoxylated guaiacyl (G) monomer was increased (16%) and bimethoxylated syringyl (S) monomer was decreased (21%). Therefore, the S/G lignin monomer ratio was significant decreased (32%). This implied that EUCCR might be specific to G monomer biosynthesis. The results described above confirmed that Arabidopsis thaliana can be used to model the function of wood formation genes isolated from Eucalyptus. Two novel full-length Eucalyptus sucrose synthase (SuSy) genes, EgSuSy1 and EgSuSy3, and one putative pseudogene, EgSuSy2, were also isolated in this study. Degenerate PCR was used to amplify Eucalyptus SuSy fragments from cDNA and genomic DNA. 3’RACE was used to amplify the 3’ ends of two Eucalyptus SuSy genes. Genome walking was performed to obtain the 5’ ends of EgSuSy1 and EgSuSy2 whereas 5’RACE technology was used to isolate the 5’ end of EgSuSy3. However, 3’RACE analysis failed when we tried to identify the 3’ end of EgSuSy2. Sequencing results from the genome walking product of EgSuSy2 further revealed that the start codon of this gene was missing, and we hypothesize that this is a psuedogene in the Eucalyptus genome. The EgSuSy1 cDNA was 2498 bp in length with an open reading frame of 2418 bp encoding 805 amino acids with a predicted molecular mass of 92.3 kDa. The 2528 bp full-length EgSuSy3 cDNA contained the same length of open reading frame as EgSuSy1, but encoded a polypeptide with a predicted molecular mass of 92.8 kDa. The results of quantitative real-time RT-PCR, phylogenetic analysis and gene structure of the two genes revealed that both genes might be involved in cellulose biosynthesis in primary and secondary cell walls of Eucalyptus. These two genes, EgSuSy1 and EgSuSy3, could therefore be useful targets for genetic engineering of wood properties in Eucalyptus. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

Cell walls are a distinct feature of plants and their chemical constituents, cellulose, hemicelluloses and lignin, are economically valuable. Plant fibres rich in cellulose, which mainly resides in their cell wall, are traditionally used in making paper and textiles. The changing global economic situation and environmental concerns have imparted necessity for renewable, but at the same time value added cellulosic materials. The Department of Wood Biotechnology, KTH together with its collaborators, have established EST libraries and performed transcript profiling during wood development in poplar, a tree considered as a model for wood development. The majority of the genes upregulated during cellulose biosynthesis encode proteins with known or predictable functions, such as carbohydrate active enzymes (CAzymes). However, some of them encode proteins with unknown functions. Characterization of these genes will potentially give additional opportunities to modify fibre properties. This thesis describes the discovery and characterization of a highly upregulated gene with a previously unknown function in poplar xylem, here denoted PttMAP20. Following its early discovery by mRNA profiling, the characterization was initiated with a thorough bioinformatic analysis, and the knowledge obtained was used to devise techniques for further functional analysis. Specific antibodies were raised, affinity purified and characterized. The antibodies were used as a tool for screening recombinant expression in E. coli and for the cellular localization of the protein in plant tissues, visualized with confocal and transmission electron microscopy. A purification protocol was developed for the expressed protein, followed by biochemical characterization. Appropriate model systems were used in both in vivo and in vitro studies. Fluorescently labelled protein transiently expressed in tobacco leaves was used for localization studies and the same system was used to characterize the molecular properties of the protein. Phenotypes arising from overexpressing the PttMAP20 gene were traced in the model plant Arabidopsis. All the results obtained so far indicate that PttMAP20 is a novel microtubule associated protein that binds to a cellulose biosynthesis inhibitor, DCB (2,6-dichlorobenzonitrile) and is required during cellulose biosynthesis in secondary cell walls. / QC 20100906

Thesis (Ph. D.)--Institute of Paper Chemistry, 1980. / Includes bibliographical references (p. 48-50).

The acsD gene is a unique gene present in the cellulose biosynthesis operon in G. xylinus. With the use of homologous recombination, the acsD gene disruption mutation was created in the G. xylinus genome. Phenotypic characterization of the acsD gene mutant was investigated with the assistance of light and electron microscopy observations, carboxymethyl cellulose alterations, and lower temperature incubation. The microscopic analysis of the cellulose ribbons secreted from the acsD gene mutant shows that the polymerization and the crystallization components in mutant cells were functional. Observations of the mutant cells after incubation with carboxymethyl cellulose and temperature changes indicate that the arrangements of the pores on the cell surface have been altered. These arrangements led to decreased cellulose secretion capacity of the mutant cells. Successful complementation was achieved by using gene expression plasmids with green fluorescence protein tag in the acsD mutant background. Anti-GFP antibodies were used to determine the in vitro localization of the protein. Using subcellular fractionation and western blotting, the AcsD protein was found to be localized in the periplasm of the cells. Taking all these results together, a new model for bacterial cellulose biosynthesis has been suggested and discussed. / text