This diploma thesis is focused on microclimatic differences at treelines on north-facing and south-facing slopes and their effect on tree growth. Thesis contains an overview of factors affecting development and timing of xylogenesis. The effects of slope orientation on treeline in relation to exposure effect are mentioned as well. The aim of the practical part of diploma thesis was to determine development, timing and the impact of temperature characteristics on xylogenesis of Norway spurce (Picea abies) on two localities with opposite slope aspect. Locations were situated in the Důl Bílého Labe valley, Krkonoše Mountains. Xylogenesis was studied using sections of micro-cores. Microcores were sampled every 10 days during the growing season. They were subsequently analyzed in dendrochronological laboratory. My results show that influence of air temperature recorded at the start of cambial cell division and the development of enlarging cells in the first part of the growing season. Climatic characteristics of both sites was similar, larger differences in temperatures were recorded at the beginning and at the end of the reference period.

Wood formation (xylogenesis) is a critical developmental process for all woody land plants. As an initial step to understand the molecular basis for temporal and spatial regulation of xylogenesis and the effect of the expression of individual genes on physical and chemical properties of wood, microarray and realtime RTPCR analyses were performed to monitor gene expression during xylogenesis under various developmental and environmental conditions. The specific objectives established for this study were: Objective 1. Microarray analysis of genes preferentially expressed in differentiating xylem compared to other tissues of loblolly pine (see Chapter II); Objective 2. Microarray analysis of seasonal variation in gene expression for loblolly pines (Pinus taeda L.) from different geographical sources (see Chapter III); Objective 3. Realtime RTPCR analysis of loblolly pine AGP and AGPlike genes (see Chapter IV). Based on the results from this study, candidate genes may be further studied for association with significant traits, used for genetic modification of wood properties, or included in future studies to further examine the molecular mechanisms of wood formation.

Pinus radiata is by far the dominant species grown in New Zealand plantations as a renewable source of wood. Several wood quality issues have been identified in the material produced, including the high incidence of compression wood, which is undesirable for end users. At present our understanding of the complex array of developmental processes involved in wood formation (which has a direct bearing on wood quality) is limited. Hence, the forest industry is interested in attaining a better understanding of the processes involved. Towards this goal, and for reasons of biological curiosity, the experiments described in this thesis were carried out to investigate several aspects of xylem cell development. In an in arbor study, changes in the orientation of cortical microtubules and cellulose microfibrils were observed in developing tracheids. Results obtained provide evidence that cortical microtubules act to guide cellulose synthase complexes during secondary wall formation in tracheids. The mechanisms involved in controlling cell wall deposition in wood cells are poorly understood, and are difficult to study, especially in arbor. A major part of this thesis involved the development of an in vitro method for culturing radiata pine wood in which hormone levels, nutrients, sugars and other factors, could be controlled without confounding influences from other parts of the tree. The method developed was used in subsequent parts of this thesis to study compression wood development, and the influence of the hormone gibberellin on cellulose microfibril organisation in the cell wall. Results from the in vitro compression wood experiments suggested that: 1. when a tree is growing at a lean, the developing cell wall was able to perceive compressive forces generated by the weight of the rest of the tree, rather than perceive the lean per se. 2. ethylene, rather than auxin, was involved in the induction of compression wood. Culture of stem explants with gibberellin resulted in wider cells, with steeper cortical microtubules, and correspondingly steeper cellulose microfibrils in the S2 layer of developing wood cells. This observation provides further evidence that the orientation of microtubules guides the orientation of cellulose microfibrils. Overall, the work described in this thesis furthers our knowledge in the field of xylem cell development. The stem culture protocol developed will undoubtedly provide a valuable tool for future studies to be carried out.

Pinus radiata is by far the dominant species grown in New Zealand plantations as a renewable source of wood. Several wood quality issues have been identified in the material produced, including the high incidence of compression wood, which is undesirable for end users. At present our understanding of the complex array of developmental processes involved in wood formation (which has a direct bearing on wood quality) is limited. Hence, the forest industry is interested in attaining a better understanding of the processes involved. Towards this goal, and for reasons of biological curiosity, the experiments described in this thesis were carried out to investigate several aspects of xylem cell development. In an in arbor study, changes in the orientation of cortical microtubules and cellulose microfibrils were observed in developing tracheids. Results obtained provide evidence that cortical microtubules act to guide cellulose synthase complexes during secondary wall formation in tracheids. The mechanisms involved in controlling cell wall deposition in wood cells are poorly understood, and are difficult to study, especially in arbor. A major part of this thesis involved the development of an in vitro method for culturing radiata pine wood in which hormone levels, nutrients, sugars and other factors, could be controlled without confounding influences from other parts of the tree. The method developed was used in subsequent parts of this thesis to study compression wood development, and the influence of the hormone gibberellin on cellulose microfibril organisation in the cell wall. Results from the in vitro compression wood experiments suggested that: 1. when a tree is growing at a lean, the developing cell wall was able to perceive compressive forces generated by the weight of the rest of the tree, rather than perceive the lean per se. 2. ethylene, rather than auxin, was involved in the induction of compression wood. Culture of stem explants with gibberellin resulted in wider cells, with steeper cortical microtubules, and correspondingly steeper cellulose microfibrils in the S2 layer of developing wood cells. This observation provides further evidence that the orientation of microtubules guides the orientation of cellulose microfibrils. Overall, the work described in this thesis furthers our knowledge in the field of xylem cell development. The stem culture protocol developed will undoubtedly provide a valuable tool for future studies to be carried out.

Pinus radiata is the dominant species of the plantations forests in New Zealand. The forest industry in New Zealand is heavily dependant on it. However, Pinus radiata can develop wood quality flaw called 'intra-ring checking'. The checks or splits appear in wood during kiln drying and usually affect the earlywood region of the wood. It lowers value of appearance grade timber leading to huge economic loses for the forest industry. This thesis presents a study that was undertaken as a part of ongoing collaborative work that is being carried out to understand wood quality issues in Pinus radiata, with a vision of improving its wood quality. This study was a part of that effort and was conducted with an aim to gain an insight into intra-ring checking, and the process of xylogenesis in Pinus radiata. The investigations for this study were carried out in two steps. The first step was to understand intra-ring checking. The location of intra-ring checking was determined by observing the checks using various microscopy techniques. Scanning electron microscopy confirmed that checking was as an intercell failure that usually occurs at the cm1/S1 boundary. A comparative study was also conducted to see if the checked wood had some inherent properties that made it more susceptible to checking. It was found that checking could be influenced by tracheid geometry and cell wall thickness. If the wood had large tracheids with thin walls, it was more likely to develop checks during drying. Lignin distribution in the cell wall layers was also seen to play an important role in checking. Lower lignin levels and disruption in the pattern of lignification of the cell wall layers increased the tendency of the wood to develop checks. Similarly, it the tracheids have larger pits then their tendency to check increases. Structural features that disrupt the uniformity of the interlocking pattern of the tracheid such as rays and resin canals could also play a role in checking. Checked wood tends to have more surface area occupied by ray tissue. However, resin canals do not seem to be directly involved in checking, though their arrangement could indicate disturbances during xylogenesis. The second step was to understand the process of xylogenesis in Pinus radiata especially with respect to the influence of auxin and boron on it. Nutrient and organ culture methods were manipulated and successfully used to study xylogenesis. An exhaustive comparative study was carried out to observe and measure selected wood properties. Microscopy and image analysis revealed that auxin and boron changes in the medium led to the alterations in the cell division, expansion and lignification. However, the analysis of the measurements and the observations displayed complex 'between-tree' and 'within-culture variations'. Clear trends did not emerge from the analysis hence, a confident conclusion on the association between auxin, boron and lignification could not be drawn from this organ culture study. The study has added to the knowledge about checking and wood properties associated with it. A new tool of organ culture had been established that can hlep future research on the process of xylogenesis in Pinus radiata.

Apesar da importância econômica e ambiental que a madeira representa como fonte natural e renovável de energia e fibras, pouco é conhecido sobre os processos celulares, moleculares e bioquímicos envolvidos com a sua formação. Usando metodologias proteômicas como 2D-PAGE e espectrometria de massas foi iniciada a análise do proteoma do caule de Eucalyptus grandis em diferentes estádios de desenvolvimento (5 meses, 3 anos e 6 anos). O presente trabalho baseou-se especificamente na idade de cinco meses. As plantas tiveram suas folhas, raízes e cascas removidas e seus caules foram macerados em almofariz com nitrogênio líquido e as proteínas extraídas pelo método de extração fenólica. As proteínas foram separadas por eletroforese bidimensional em fitas IPG com gradiente de pH imobilizado linear de 4-7 na primeira dimensão e gel de poliacrilamida (12,5%) na segunda dimensão. A coloração dos géis foi realizada com coomassie G250. Foram detectados 438 spots e um total de 168 spots foram retirados do gel, digeridos com tripsina e submetidos ao sequenciamento por espectrometria de massas através do sistema LCMS/ MS. O sequenciamento por MS apresentou uma eficiência de 72,02% possibilitando a identificação de 121 spots, enquanto que 35 (20,83%) não apresentaram homologia com nenhuma base de dados. Entre as proteínas identificadas 22 foram representadas por mais de um spot, podendo indicar a ocorrência e eventos provenientes do splicing alternativo, modificações pós-traducional, variações alélicas de uma mesma proteína ou degradação da amostra. Entre os spots analisados, 22,02% estão relacionados com a produção de energia, (17,86%) metabolismo, (13,69%) processes celulares, (0,60%) transporte, (8,33%) componentes estruturais, (5,36%) metabolismo macromolecular, (4,17%) proteínas putativas, (20,83%) não apresentaram homologia com nenhuma base de dados e (7,14%) não demonstraram resultado. A comparação realizada entre o volume de 59 proteínas e os seus respectivos transcritos demonstrou que não existe correlação entre mRNA e as proteínas do caule. O método possibilitou uma rápida e precisa separação e identificação das proteínas do caule de Eucalyptus grandis que são diferencialmente expressas durante a fase de crescimento de cinco meses. / The process of wood formation is an important economical factor for the forestry industry and it is also of ecological importance, although little is known about the proteins involved in wood formation. The sequencing, identification and analysis of proteins provides such information of wood formation. Using proteomics techniques such as two-dimensional gel electrophoresis and mass spectrometry we have started a proteomic analysis of wood formation in Eucalyptus grandis at different stages of development (5 months, 3 and 6 years old). This work presents data related to the stage of 5 months. Using high resolution 2DE with linear pH gradient ranging from 4 to 7, a total of 438 spots were detected. However, only 168 spots were analyzed by LC ESIMS/ MS and 121 were identified (72.02%) while 35 (20.83%) presented no homology in the database used. Overall, 22 proteins appeared as multiple spots and accounted for most of the proteins found in the group. This observation may reflect post-translation modification, alternative splicing events, isozyme variation, allelic variation of the same protein, but also protein degradation. Over the 168 spots analysed, (22.02%) play a role in energy, (17.86%) metabolism, (13.69%) cellular processes, (0.60%) transport, (8.33%) structural components, (5.36%) macromolecular metabolism, (4.17%) putative protein, (20.83%) no homology and (7.14%) no result. For 59 proteins, the spot volume was compared with their respective transcript with mRNAs extracted from wood forming tissue. The method provided a faster and accurate tool for separation and identify of protein which are differentially expressed under different stages of development in Eucalyptus grandis.

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

<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>

Pinus radiata is the dominant species of the plantations forests in New Zealand. The forest industry in New Zealand is heavily dependant on it. However, Pinus radiata can develop wood quality flaw called 'intra-ring checking'. The checks or splits appear in wood during kiln drying and usually affect the earlywood region of the wood. It lowers value of appearance grade timber leading to huge economic loses for the forest industry. This thesis presents a study that was undertaken as a part of ongoing collaborative work that is being carried out to understand wood quality issues in Pinus radiata, with a vision of improving its wood quality. This study was a part of that effort and was conducted with an aim to gain an insight into intra-ring checking, and the process of xylogenesis in Pinus radiata. The investigations for this study were carried out in two steps. The first step was to understand intra-ring checking. The location of intra-ring checking was determined by observing the checks using various microscopy techniques. Scanning electron microscopy confirmed that checking was as an intercell failure that usually occurs at the cm1/S1 boundary. A comparative study was also conducted to see if the checked wood had some inherent properties that made it more susceptible to checking. It was found that checking could be influenced by tracheid geometry and cell wall thickness. If the wood had large tracheids with thin walls, it was more likely to develop checks during drying. Lignin distribution in the cell wall layers was also seen to play an important role in checking. Lower lignin levels and disruption in the pattern of lignification of the cell wall layers increased the tendency of the wood to develop checks. Similarly, it the tracheids have larger pits then their tendency to check increases. Structural features that disrupt the uniformity of the interlocking pattern of the tracheid such as rays and resin canals could also play a role in checking. Checked wood tends to have more surface area occupied by ray tissue. However, resin canals do not seem to be directly involved in checking, though their arrangement could indicate disturbances during xylogenesis. The second step was to understand the process of xylogenesis in Pinus radiata especially with respect to the influence of auxin and boron on it. Nutrient and organ culture methods were manipulated and successfully used to study xylogenesis. An exhaustive comparative study was carried out to observe and measure selected wood properties. Microscopy and image analysis revealed that auxin and boron changes in the medium led to the alterations in the cell division, expansion and lignification. However, the analysis of the measurements and the observations displayed complex 'between-tree' and 'within-culture variations'. Clear trends did not emerge from the analysis hence, a confident conclusion on the association between auxin, boron and lignification could not be drawn from this organ culture study. The study has added to the knowledge about checking and wood properties associated with it. A new tool of organ culture had been established that can hlep future research on the process of xylogenesis in Pinus radiata.