Understanding how cellulose synthesis occurs is key to understanding the formation of the plant cell wall. This understanding could also be key to modifying cellulose production to permit more efficient extraction of glucose from cellulose for the production of biobased materials. Cellulose biosynthesis is carried out by cellulose synthases; transmembrane multimeric processive glycosyltransferases responsible for polymerizing UDP‐glucose into glucan chains. Thirty‐six glucan chains bind together in parallel to form elementary cellulose microfibrils. Due to the essential nature of cellulose synthases for plant survival and the recalcitrant nature of the cell wall to chemical and enzymatic digestion, the cellulose synthases can be very difficult to analyze by traditional approaches. In an attempt to circumvent some of the issues of studying cellulose synthases, the cellulose synthase genes CESA1 and CESA3, along with the cell wall associated genes COBRA, DET3 and POM1 were recombined into an engineered Autographa californica nucleopolyhedron virus and expressed in Spodoptera fruigiperda ovarian cells. Although recombinant protein could be detected for CESA1 and CESA3, C14‐glucose incorporation on baculovirus infected cell lines have given inconclusive results to the cellulose synthase activity of the CESA1 and CESA3 proteins. With further optimization of the protein expression of CESA1 and optimization of the variability in the C14‐glucose incorporation assays, the baculovirus system may prove a useful tool for studying the cellulose synthases. / UOIT

Pinus patula is the single most important commercial plantation forest tree species in South Africa. It accounts for 52% approximately (700,000 ha) of total commercial plantation area in the country and is utilised for saw logs and pulp and paper production. P. patula is a tropical pine species indigenous to Mexico. Excellent ex situ conservation and range-wide provenance trials have been established for P. patula in South Africa and South America. These highly organised trials provide the opportunity to perform association genetic studies with the long term aim to identify trait linked markers for future molecular improvement of P. patula. In this study, the first gene-based assessment of allelic diversity in P. patula was performed. This pilot study focused on two cellulose biosynthetic genes as representatives of wood formation genes and assayed molecular evolution parameters such as nucleotide diversity, allelic diversity and linkage disequilibrium (LD) in a species-wide reference population of P. patula. Two novel cellulose synthase (CesA) genes were isolated and characterised in P. patula. One of these genes, PpCesA1, is putatively involved in the biosynthesis of secondary cell walls of tissues such as xylem (wood), while the other, PpCesA2 is proposed to be associated with primary cell wall formation in rapidly growing tissue types. The genomic DNA copies of PpCesA1 and PpCesA2 were 6025 bp and 6365 bp in length, respectively. The corresponding cDNA sequences encoded 1083 and 1058 amino acids, respectively, and differed considerably from each other (73% amino acid identity). Both amino acid sequences contained the key domains and motifs characteristic of functional CESA proteins isolated in other higher plants. Phylogenetic analysis revealed that PpCesA1 was most similar (99%) to its putative ortholog in Pinus taeda, PtCesA3, and PpCesA2 was highly similar to a putative ortholog in Pinus radiata, PrCesA2 (99% identity). This phylogenetic analysis supported previous findings that the divergence between the primary and secondary cell wall associated CESA proteins occurred before the divergence of angiosperms and gymnosperms approximately 300 million years ago. A fragment of a putative paralogous gene copy of PpCesA1, named PpCesA1-B was also isolated. The PpCesA1-B gene fragment was found to differ from PpCesA1 by 22 nucleotide polymorphisms and its non-allelic (paralogous) status was confirmed by segregation analysis in P. patula. In order to gain an understanding of molecular genetic variation that might affect wood formation in P. patula, we sequenced multiple allelic variants of PpCesA1, PpCesA1-B and PpCesA2, which we sampled from a species-wide reference population of P. patula. The average levels of nucleotide diversity were found to be low for all three genes (π ≈ 0.0015), which may be a property of functional members of the CesA gene family. As a result of the low nucleotide diversity, only small numbers of pair-wise informative sites were available for LD analysis and the decay in LD could only be studied in PpCesA2 where it was found to decay very rapidly (within 200 bp). Tests of neutrality suggested that the exon sequences of PpCesA1 and PpCesA2 were under significant positive (adaptive) selection. Comparison of levels of nucleotide diversity and selection in different parts of the two genes indicated that the highest levels of adaptive selection occurred in areas where amino acid substitutions could alter protein structure or function. This study provides valuable insights for designing future allele discovery efforts in P. patula with the ultimate goal of developing gene-based markers for the molecular improvement of wood formation in this tree species. / Dissertation (MSc (Genetics))--University of Pretoria, 2009. / Genetics / unrestricted

Cellulose is the most important component of paper and pulp products and increased cellulose biosynthesis in commercially important trees like Eucalyptus spp. could greatly benefit paper and pulp industries. Cellulose in plants occurs mostly in the secondary cell walls together with lignin and hemicellulose. It is biosynthesised by membrane-bound rosette-shaped protein complexes. The rosette complexes are believed to be comprised of six sub-units each containing six cellulose synthase (CESA) proteins. The CESA proteins utilise UDP-glucose to polymerize growing glucan chains that coalesce to form cellulose microfibrils. Three distinct CESA proteins form the rosette complexes during primary cell wall formation and three different CESA proteins form complexes during secondary cell wall deposition. The exact means by which the CESA proteins interact within a rosette complex remains unknown. Elucidating rosette protein complex assembly and better characterization of CESA protein activity is required in order to increase cellulose biosynthesis in commercially important trees. Because of the difficulties to characterise genes and proteins in tree species, Arabidopsis thaliana has been used to study xylogenesis. Although it is a herbaceous weed, Arabidopsis has been shown to undergo secondary growth under certain conditions. A literature study of cellulose biosynthesis in plants has highlighted several scientific questions: Will over-expression of a heterologous secondary cell wall CESA protein in Arabidopsis lead to increased cellulose biosynthesis? What effect will the over-expression of a heterologous protein have on the growth and development of Arabidopsis? Will it have an effect on cell wall chemistry in stem tissues? What effect will expression of the transgene have on endogenous Arabidopsis gene expression? The aim of this M.Sc study was to functionally characterise the Eucalyptus secondary cell wall associated cellulose synthase gene, EgCesA1, in Arabidopsis. The EgCesA1 coding sequence was constitutively expressed in wild-type Arabidopsis plants. Three transgenic lines expressing EgCesA1 was generated. Hypocotyl and inflorescence vascular cell wall phenotypes were compared between transgenic and wild-type plants. Chemical analysis of inflorescence tissues were performed to detect changes in monosaccharide and lignin content of transgenic plants compared to wild-type plants. Transcript levels of EgCesA1 and endogenous Arabidopsis genes involved in cell wall biogenesis were quantified and compared between wild-type and transgenic lines. No significant changes in cell wall morphology could be detected, despite small alterations in inflorescence cell wall chemical composition in transgenic plants. Expression of EgCesA1 did not appear to have a statistically significant effect on endogenous gene transcript levels. It was concluded that constitutive expression of a single transgenic CesA gene is insufficient to increase cellulose biosynthesis. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Genetics / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20450号 / 農博第2235号 / 新制||農||1050(附属図書館) / 学位論文||H29||N5071(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 杉山 淳司, 教授 髙部 圭司, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Plant cell walls are complex structures that must not only constrain cellular turgor pressure but also allow for structural modification during the dynamic processes of cell division and anisotropic expansion. Cell walls are composed of highly glycosylated proteins and polysaccharides, including pectin, hemicellulose and cellulose. The primary cell wall polysaccharide is cellulose, a polymer composed of high molecular weight !- 1,4-glucan chains. Although cellulose is the most abundant biopolymer on Earth, there is still a lot to learn about its biosynthesis and regulation. This research began by applying a variety of analytical techniques in an attempt to understand differences in cell wall composition and cellulose structure within the plant body, between different plant species and as a result of acclimation by the plant to different environmental conditions. Next, a number of different Arabidopsis thaliana lines possessing mutations affecting cell wall biosynthesis were analyzed for changes in cellulose structure (crystallinity) and biomass saccharification efficiency. One of these mutants, isoxaben resistance1-2 (ixr1- 2), which contains a point mutation in the C-terminal transmembrane region (TMR) of cellulose synthase 3 (CESA3), exhibited a 34% lower biomass crystallinity index and a 151% improvement in saccharification efficiency relative to that of wild-type. The culmination of this research began with a chemical screen that identified the molecule quinoxyphen as a primary cell wall cellulose biosynthesis inhibitor. By forward genetics, a semi-dominant mutant showing strong resistance to quinoxyphen named aegeus was identified in A. thaliana and the resistance locus mapped to a point mutation in the TMR of CESA1. cesa1aegeus occurs in a similar location to that of cesa3ixr1-2, illustrating both subunit specificity and commonality of resistance locus. These drug resistant CESA TMR mutants are dwarfed and have aberrant cellulose deposition. High-resolution synchrotron X-ray diffraction and 13C solid-state nuclear magnetic resonance spectroscopy analysis of cellulose produced from cesa1aegeus, cesa3ixr1-2 and the double mutant shows a reduction in cellulose microfibril width and an increase in mobility of the interior glucan chains of the cellulose microfibril relative to wild-type. These data demonstrate the importance of the TMR region of CESA1 and CESA3 for the arrangement of glucan chains into a crystalline cellulose microfibril in primary cell walls.

The initial aim of this project was to uncover novel cellulolytic organisms or enzymes from the diverse microbial source, activated sludge. Two isolation methods were used; either directly inoculating the sludge material onto filter paper as a carbon source, or using the Evolver� technology as an enrichment device. In both cases, as expected, cellulase activity was evident, however attributing this activity to one species was difficult in either case. This highlighted the complex interrelationships that existed between the many microorganisms present as the cellulosic carbon sources were degraded. In one instance, a Cellvibrio sp. was isolated. This genus of bacteria is known to possess both types of cellulase activity (exo- and endo- acting) and was therefore likely to contribute to the degradation of the cellulose. However, the isolate, once purified, did not display significant cellulolytic ability as compared to the unpurified consortium of microorganisms. Therefore, in each case, microorganisms responsible for the cellulolytic activity were not uncovered. It was suspected that the microorganisms responsible for some of the cellulolytic activity were protists. During the isolation of microorganisms, an Aeromonas sp. bearing the novel phenotype (for this genus) of CMCase activity was isolated. This activity was at first suspected to contribute to the degradation of the filter paper that was seen during isolation. However, tests with the pure isolate suggested that the Aeromonas sp. CMCase was not used for cellulose catabolism. Ironically, the enzyme may instead function in the production of a cellulose-like exopolysaccharide by the bacterium. Part of a cellulose synthase operon was found in the genome of the Aeromonas sp. isolate, including a gene coding for an endoglucanase that gives a predicted molecular weight enzyme similar to the 39 kDa CMCase purified from the bacterium. The CMCase enzyme, operating as part of of a synthetic operon is expected to be important in terms of the biofilm forming ability of this Aeromonas strain. Such capabilities of the bacterium were investigated here, including observing motility behaviour of the organism on agar surfaces. Studying the biofilm forming ability of this genus in general will be important in understanding how the fish and human pathogens persist in aquatic environments

Cellulose is a biopolymer of great relevance in the plant cell walls, where it constitutes the most important skeletal component. Cellulose is also an important raw material in the pulp- and paper, forest, and textile industries, among others. Cellulose biosynthesis in particular, and xylogenesis in general are processes which are currently poorly understood. Yet, research in cellulose synthesis is progressing and different applications of cellulose, mainly cellulose derivatives for e.g. pharmaceuticals and coatings, are constantly emerging. This thesis depicts how cellulose synthase (CesA) genes in Populus were identified and characterized by gene expression- and bioinformatics analyses. Within an EST database of more than 100,000 clones from wood forming tissues of three different Populus taxa, ten CesA genes were identified in Populus tremula x tremuloides. Subsequent gene expression analyses by using microarrays and real-time PCR experiments in woody tissues, revealed distinct regulation patterns among the genes of interest. This enabled proper classification and characterization of the secondary cell wall related CesA genes, in particular. Bioinformatic analyses of the genome sequence of Populus trichocarpa further provided a complete picture of the number of putative CesA genes retained after several duplication events during tree evolution. In contrast to the previously reported set of ten 'true' CesA genes in many other plant species, the genome of P. trichocarpa encodes 18 putative proteins, which could be assembled into nine groups according to their sequence similarities. Interestingly, studies in the EST database suggested that paralogs within at least two groups have corresponding orthologs in P. tremula x tremuloides, which are furthermore transcribed. This implies that at least some of the duplicated genes have remained functional, or may have acquired a modified function. By focusing on the CesA genes associated with secondary cell wall formation, cellulose synthesis was also studied in poplar cell suspension cultures. Selection of CesA enriched material was performed by determining expression intensities of the CesA genes using RT-PCR, whereupon membrane protein extraction was initiated. CesA proteins are part of large cellulose synthesizing complexes in the plasma membrane. Subsequent proteomic approaches comprised partial purification of these cellulose synthesizing complexes from protein enriched culture material and in vitro cellulose synthesis experiments. De novo synthesized material was successfully characterized and the acquired yields were as high as 50% cellulose (compared to previously reported yields of 30% in other plant systems) of the total in vitro synthesized product. Elevated CesA gene expression levels can thus be correlated to increased protein activity in poplar cell suspension cultures. In addition, antibodies raised against CesA antigens were used in Western blot analyses comprising samples along the protein extraction- and purification procedure. Proteins with corresponding molecular weight to the theoretical 120kDa of CesA proteins were recognized by a range of different specific antibodies. The study demonstrates that poplar cell suspension cultures can provide a valuable model system for studies of cellulose synthesis and different aspects of xylogenesis. / QC 20101005

<p>Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus <em>Phytophthora </em>are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus <em>Saprolegnia</em>, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen <em>Phytophthora infestans</em> and the fish pathogen <em>Saprolegnia monoica</em>.</p><p> </p><p>A new Oomycete <em>CesA</em> gene family was identified. It contains four homologues designated as <em>CesA1, CesA2, CesA3</em> and <em>CesA4</em>. The gene products of <em>CesA1, 2</em> and <em>4 </em>contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete <em>CesA </em>genes. <em>CesA3</em> is the dominantly expressed <em>CesA </em>homologue in the mycelium of both <em>S. monoica</em> and <em>P. infestans</em>, while <em>CesA1</em> and<em> CesA2</em> are up-regulated in virulent life stages of <em>P. infestans</em>. <em>CesA4</em> was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole <em>CesA</em> gene family in <em>P. infestans</em> lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on <em>SmCesA</em> gene expression, which was accompanied by an increased b-glucan synthase activity <em>in vitro</em>. In addition, these inhibitors slowed down the growth of the mycelium from <em>S. monoica</em>. Zoospores from <em>P. infestans</em> treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the <em>CesA</em> gene family.</p><p>Altogether these results show that at least some of the <em>CesA1-4</em> genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by <em>P. infestans</em>.</p><p> </p>

Made available in DSpace on 2014-07-29T15:16:29Z (GMT). No. of bitstreams: 1 Marcela Mendes Salazar.pdf: 1545192 bytes, checksum: 56aea77af0ca027e7443fc376eaa381f (MD5) Previous issue date: 2006-12-01 / Cellulose is the world s most abundant polymer, being the main constituent of plant biomass. Genes from CesA family, that encodes the cellulose synthase enzyme, was identified in several plant species, especially in Arabidopsis thaliana, in which three of them (AtCesA4, AtCesA7 e AtCesA8) were associated with secondary cell wall cellulose production. Eucalyptus species represent an important target of genetic improvement studies, since it is the most planted forest genus in the world, beyond deserving a special place in the international market of cellulose and paper. The molecular breeding technology enables that gene characterization can be applied to forest species genetic improvement programs with the aim to improve the quality and productivity of its products. In this work, 320 Eucalyptus ESTs, separated in four genes related with cellulose production in secondary cell wall, using AtCesA4, AtCesA7 e AtCesA8 genes as reference. For these genes, primers were designed in order to screen an Eucalyptus BACs library. An emphasis was given to genes that are orthologs to AtCesA7 from Arabidopsis thaliana for witch it was sequenced a 1197 base pairs region from the BACs library and this served as a support to expression level studies of this genes in different species/tissues, showing that this is preferentially expressed in xylem and weakly expressed in leaves. The expression level of this gene was higher in E. urophylla than in other species studied. Sequences from approximately 500bp was obtained from different Eucalyptus species and in these, with one intron between two exons, the amount of SNPs in the intron (4), as waited, was higher than that found in exons (1 in each), although the intron nucleotide diversity index (&#960;) (0,0824) were less than in the exon (0,2029). In this manner, one expects that this work can contribute for one better understanding of the mechanisms involved in biosynthesis and regulation of the cellulose pathway in Eucalyptus species, as well as subsidize genetic mapping studies and linkage disequilibrium analysis for this genus. / A celulose é o polímero mais abundante do planeta, sendo o principal constituinte da biomassa das plantas. Genes da família CesA, que codificam a enzima celulose sintase foram identificados em diversas espécies de plantas, especialmente em Arabidopsis thaliana, na qual três deles (AtCesA4, AtCesA7 e AtCesA8) foram correlacionados com a produção de celulose na parede celular secundária. Espécies de Eucalyptus constituem um importante alvo de estudos de melhoramento genético, já que é o gênero florestal mais plantado no mundo, além de merecer destaque no mercado internacional de papel e celulose. A tecnologia de melhoramento molecular possibilita que a caracterização de genes de interesse possa ser aplicada a programas de melhoramento genético de espécies florestais visando o aumento da qualidade e produtividade dos seus produtos. Neste trabalho, 320 ESTs de Eucalyptus, divididos em quatro genes, responsáveis pela produção de parade celular secundária foram anotados, utilizando-se como referência os genes AtCesA4, AtCesA7 e AtCesA8. Para estes genes, primers foram desenhados a fim de que uma triagem em uma biblioteca de BACs de Eucalyptus fosse realizado. Uma maior ênfase foi dada ao gene ortólogo ao gene AtCesA7 de Arabidopsis thaliana para o qual foi seqüenciado uma região de 1197 pares de base à patir da biblioteca de BACs e esta serviu de base para estudo do nível de expressão desse gene em diferentes espécies/tecidos, revelando que este é preferencialmente expresso em tecidos de xilema e fracamente expresso em folhas, o que corrobora com a atuação desses genes em paredes celulares secundárias. O nível de expressão desse gene foi maior em E. urophylla que em outras espécies estudadas. Seqüências de aproximadamente 500 pb também foram obtidas à partir de diferentes espécies de Eucalyptus e nestas, contendo um íntron flanqeado por dois éxons, observou-se que a quantidade de SNPs identificados no íntron (4), como esperado, foi maior do que aquela encontrada nos éxons (1 em cada), embora o valor estimado para o índice de diversidade nucleotídica (&#960;) relativo ao íntron (0,0824) tenha sido menor do que aquele estimado em um dos éxons (0,2029). Desse modo, espera-se que esse trabalho possa contribuir para uma melhor compreensão dos mecanismos envolvidos na biossíntese e regulação da via de celulose em espécies de Eucalyptus, bem como subsidiar estudos de mapeamento genético e análise de desequilíbrio de ligação nesse gênero.

Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus Phytophthora are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus Saprolegnia, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen Phytophthora infestans and the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified. It contains four homologues designated as CesA1, CesA2, CesA3 and CesA4. The gene products of CesA1, 2 and 4 contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete CesA genes. CesA3 is the dominantly expressed CesA homologue in the mycelium of both S. monoica and P. infestans, while CesA1 and CesA2 are up-regulated in virulent life stages of P. infestans. CesA4 was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole CesA gene family in P. infestans lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on SmCesA gene expression, which was accompanied by an increased b-glucan synthase activity in vitro. In addition, these inhibitors slowed down the growth of the mycelium from S. monoica. Zoospores from P. infestans treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the CesA gene family. Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by P. infestans. / QC 20101110