Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen. First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod. A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient. Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis. Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our ownPopulusESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes. Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified. Key words:Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase

Hemicellulose is a valuable component of agro-food industrial wastes. Although there are some potential usage areas of hemicelluloses such as drug manufacture, encapsulation, and emulsification in food processing plants, they are not extensively utilized. In this study, effective extraction methods of the hemicelluloses from agro-food industrial wastes (corn peels and sugar beet pulp) and their application as coating material for banana fruits were investigated. Firstly, the effects of raw material type, particle size, and extraction conditions (alkaline concentration, temperature, time) and methods (direct alkaline extraction, alkaline extraction after component removal, and acidic extraction method) on hemicellulose yields were investigated. Sugar beet pulp and corn wastes were used as raw material. Alkali extraction resulted in 40.2% pure extracts while acidic hydrolysis gave only 27.4% purity in the same extraction conditions. The optimal extraction conditions were found as 30&deg / C temperature, 10% NaOH, and 24 h time (64.3%). It was also observed that removal of constituents such as fat, protein, starch, and soluble sugar increased the purity of hemicellulose from 40.2 to 58.2% at the same conditions. After finding optimum hemicellulose yield for practicle use, it was used as edible coating material to prevent darkening of banana fruits and any quality losses. On the fourth day of storage at 4 &ordm / C, there was no detected fungal decay of coated bananas / however, 20% of uncoated bananas (control) were infected. Moreover, the control group lost 5.1% of total weight but coated samples with 1%, 1.5%, and 2% hemicellulose (HC) lost 3.6%, 3.3%, and 3.1% of their total weight, respectively. Hemicellulose coating also protected firmness of bananas (701.1 gf for coated and 509.6 gf for uncoated samples at the end of 4th day). Color was another important quality parameter and it was showed that lightness and yellowness of coated bananas were preserved with very little losses at the end of storage period while control samples turned brown at an unacceptable level. The results indicated that using both low alkaline concentration and low temperature allows to recover higher quality extracts. In addition, hemicellulose showed a characteristic of a good edible coating material for banana in order to preserve their visual acceptance and other quality parameters.

Le bois est un bio polymère qui se distingue des autres matériaux industriels (polymères, métaux) par son origine végétale. Dérive d'un tissu cellulaire, il est hygroscopique, anisotrope, instable dimensionnellement et vulnérable aux attaques biologiques (insectes et champignons). Le procédé de rétification du bois consiste en une pyrolyse ménagée (entre 180 et 250°C) sous atmosphère contrôlée et réduit de façon notable l'hygroscopie, l'instabilité dimensionnelle et la biodégradabilité du matériau traité. Cette étude a mis en évidence des différences de sensibilité thermique entre les feuillus et les résineux. Ces derniers peuvent présenter des taux de résines plus ou moins importants qui influent sur le comportement du matériau au cours de la rétification. Les départs gazeux observes sont attribués à une destruction importante des hemicelluloses et une modification de la lignine. Le bois traité thermiquement est plus hydrophobe que le matériau de départ en raison d'une destruction de groupes hydroxyles des hemicelluloses. La présence d'acide acétique, issu de la dégradation des hemicelluloses à haute température, permet de catalyser les réactions de condensation de la lignine, mais pourrait également contribuer à la dégradation mécanique du matériau. Une étude menée sur le matériau massif a démontré que la structure cristalline de la cellulose n'est pratiquement pas affectée par le traitement thermique. L'augmentation de la résistance aux attaques fongiques semble être due à des réactions de condensation de la lignine. La quantité de composés extractibles (essentiellement des phénols) augmente suite au traitement thermique. Le paramètre fondamental du procédé de rétification est la température de traitement du matériau. Les analyses de la structure chimique du bois, avant et après traitement par spectrométrie drift, mettent en évidence un seuil pour une température de rétification de 240°C.

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types.

No description available.

No description available.

There is an increasing trend in the world for using renewable sources of fuels and chemicals due to the continuous depletion of fossil fuel reserves besides the environmental issues related with the exploitation of these resources. Lignocellulosic biomass is seen as the most promising candidate to be used instead of fossil sources because of its availability, relatively low price and less competition with food and feed crops. In this study, corn cobs, a lignocellulosic agricultural waste, were subjected to alkaline pretreatment for the co-production of glucose and hemicellulose based films with a multi-product approach in order to diversify the product range and to increase the revenues of the process. The pretreatment applied to lignocellulosic agricultural waste has a significant impact on the quantities and properties of the products that can be produced from the lignocellulosic feedstock upon pretreatment. Within the context of this study, the parameters utilized during the alkaline pretreatment of corn cobs were investigated in terms of their effect on the amount of glucose obtained through the enzymatic v hydrolysis of the cellulosic portion and on the mechanical properties of the films obtained through the solvent casting of the hemicellulosic portion of corn cob. The pretreatment parameters including the alkaline type and concentration, addition and type of boron compound as well as the duration of pretreatment, were optimized with respect to the amounts and the properties of the products. Following the pretreatments conducted with 24 % KOH and 1% NaBH4, which were the initial pretreatment parameters in the study, a glucose yield of 22 % and a tensile energy to break of 2.1 MJ/m3 were obtained. Upon the optimization of the pretreatment procedure, the optimum pretreatment conditions were determined as 5 % NaOH, 1 % NaBH4 and 3 hours and a glucose yield of approximately 31% and a tensile energy to break of around 1.7 MJ/m3 were obtained.

<p>Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen.</p><p>First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod.</p><p>A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient.</p><p>Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis.</p><p>Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our own<i>Populus</i>ESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes.</p><p>Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified.</p><p><b>Key words:</b>Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase</p>

<p>Forest biotechnology is a fast emerging field of research. The application of biotechnological tools will enhance the quality of the forest products. The resultant value added and environmentally sustainable products are an absolute necessity in the future. The study of wood biosynthesis and degradation will result in enormous knowledge resources, which can be used for exploiting wood properties. This thesis addresses questions representing both wood degradation and biosynthesis.</p><p>The wood degrading fungus <i>Phanerochaete chrysosporium</i> is expression profiled with the microarray technology. The objective is to understand the expression pattern of the extracellular carbohydrate active enzymes (CAZymes) secreted by the organism. The data obtained increases our understanding of gene expression upon growth on cellulose.</p><p>Wood biosynthesis is studied with the model wood forming tree species, <i>Populus</i>. The plentiful data resources from the expression profiling during wood formation in Populus are used as the platform of this work. One of the wood specific genes, <i>PttMAP20</i>, previously with an unknown function is studied in this thesis. The immunolocalisation of PttMAP20 with specific antibodies is demonstrated. The putative microtubule-targeting domain of the protein is demonstrated microscopically and by using a biochemical binding assay. </p>

Thesis (Ph. D.)--Institute of Paper Chemistry, 1968. / Includes bibliographical references (p. 91-96).