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

The differentiation of plant vascular tissue is regulated by plant hormones and transcription factors. One of the key plant hormones involved in this process is auxin. Auxin signals are mediated by auxin response factor transcription factors (ARFs). These transcription factors are involved in the perception of auxin signals and the subsequent activation or deactivation of suites of downstream genes. Based on its mutant phenotype, one of the most interesting members of this family is the ARF MONOPTEROS (MP). This thesis investigates the role played by MP in secondary vascular differentiation, as well as taking a look at other molecular aspects of secondary vascular differentiation, with a focus on the model plants Arabidopsis thaliana and poplar (Populus trichocarpa and hybrid poplar). A dexamethasone inducible RNAi silencing strategy was developed, and transgenic Arabidopsis lines produced. When silencing was induced in these lines from germination, a phenotype closely resembling the mp mutant was observed. When MP silencing was induced in bolting stems, early senescence, as well as a dramatic reduction in interfascicular fibre production was observed, and these stems were thinner and less rigid than empty vector controls. RNA from these stems was isolated and used in a global transcript profiling microarray experiment. This experiment showed that several auxin-related genes, as well as several transcription factors, were differentially regulated in response to MP silencing. Because Arabidopsis is not a typical woody plant, further investigation into the role played by MP in wood formation was done using the model tree poplar. A BLAST search of a poplar xylem EST database identified a single promising partial sequence. Based on this sequence information, a poplar MP homolog was isolated and named PopMP1. The full-length sequence of this gene demonstrated remarkable structural conservation when compared with that of Arabidopsis. Subsequent complete sequencing of the poplar genome revealed a second copy of the MP gene in poplar and named PopMP2. Expression profiling across a range of tissues suggests that subfunctionalization has occurred between the two copies. Overexpression transgenic lines for PoptrMP1 were developed. AtHB8 is known to be regulated by MP in Arabidopsis, and a poplar HB8 homolog was upregulated in the transgenic lines. However, no obvious physical phenotype in these lines was apparent. To investigate the transcriptome-wide changes associated with initiation of cambium formation in poplar stems, a global transcript profiling experiment was performed. Out of 15400 genes tested, 2320 met an arbitrary cutoff of >1.3 fold and p-value <0.05 and were labeled differentially expressed (DE). These included several transcription factors and showed remarkable similarity to analogous data from Arabidopsis. The conclusions drawn from this thesis support the hypothesis that MP plays roles in later development, and do not rule out the possibility that MP is directly involved in wood development. The data reported also offer a large number of candidate for further investigation into the genetic control of wood development.

The differentiation of plant vascular tissue is regulated by plant hormones and transcription factors. One of the key plant hormones involved in this process is auxin. Auxin signals are mediated by auxin response factor transcription factors (ARFs). These transcription factors are involved in the perception of auxin signals and the subsequent activation or deactivation of suites of downstream genes. Based on its mutant phenotype, one of the most interesting members of this family is the ARF MONOPTEROS (MP). This thesis investigates the role played by MP in secondary vascular differentiation, as well as taking a look at other molecular aspects of secondary vascular differentiation, with a focus on the model plants Arabidopsis thaliana and poplar (Populus trichocarpa and hybrid poplar). A dexamethasone inducible RNAi silencing strategy was developed, and transgenic Arabidopsis lines produced. When silencing was induced in these lines from germination, a phenotype closely resembling the mp mutant was observed. When MP silencing was induced in bolting stems, early senescence, as well as a dramatic reduction in interfascicular fibre production was observed, and these stems were thinner and less rigid than empty vector controls. RNA from these stems was isolated and used in a global transcript profiling microarray experiment. This experiment showed that several auxin-related genes, as well as several transcription factors, were differentially regulated in response to MP silencing. Because Arabidopsis is not a typical woody plant, further investigation into the role played by MP in wood formation was done using the model tree poplar. A BLAST search of a poplar xylem EST database identified a single promising partial sequence. Based on this sequence information, a poplar MP homolog was isolated and named PopMP1. The full-length sequence of this gene demonstrated remarkable structural conservation when compared with that of Arabidopsis. Subsequent complete sequencing of the poplar genome revealed a second copy of the MP gene in poplar and named PopMP2. Expression profiling across a range of tissues suggests that subfunctionalization has occurred between the two copies. Overexpression transgenic lines for PoptrMP1 were developed. AtHB8 is known to be regulated by MP in Arabidopsis, and a poplar HB8 homolog was upregulated in the transgenic lines. However, no obvious physical phenotype in these lines was apparent. To investigate the transcriptome-wide changes associated with initiation of cambium formation in poplar stems, a global transcript profiling experiment was performed. Out of 15400 genes tested, 2320 met an arbitrary cutoff of >1.3 fold and p-value <0.05 and were labeled differentially expressed (DE). These included several transcription factors and showed remarkable similarity to analogous data from Arabidopsis. The conclusions drawn from this thesis support the hypothesis that MP plays roles in later development, and do not rule out the possibility that MP is directly involved in wood development. The data reported also offer a large number of candidate for further investigation into the genetic control of wood development.

<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>

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. The wood degrading fungus Phanerochaete chrysosporium 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. Wood biosynthesis is studied with the model wood forming tree species, Populus. 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, PttMAP20, 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. / QC 20101217

Proper development of skeletal muscle occurs through a highly complex process where activation and repression of genes are essential. Control of this process is regulated by timely and spatial expression of specific transcription factors (TFs). Six1 and Six4 are homeodomain TFs known to be essential for skeletal muscle development in mice. Using the C2C12 cell line, a model for skeletal muscle differentiation, I used a functional genomics approach, employing siRNA specific to both these TFs, to characterize their role in skeletal myogenesis. To identify the genes that are regulated by both these TFs, gene expression profiling by microarray of cells treated with siRNA against Six1 and/or Six4 was performed. The knock-down of these TFs caused lower expression of markers of terminal differentiation genes in addition to an impairment of myoblast fusion and differentiation. Interestingly, transcript profiling of cells treated with siRNA against myogenin revealed that several of the Six1 and Six4 target genes are also regulated by myogenin. Through a combination of bioinformatic analyses it was also found that specific knock-down of Six4 causes an up-regulation of genes involved in mitosis and the cell cycle. In summary, these results show that Six1 and Six4 can both independently regulate different genes, but can also cooperate together with other TFs where they play an important role in the proper regulation of skeletal myogenesis.

Proper development of skeletal muscle occurs through a highly complex process where activation and repression of genes are essential. Control of this process is regulated by timely and spatial expression of specific transcription factors (TFs). Six1 and Six4 are homeodomain TFs known to be essential for skeletal muscle development in mice. Using the C2C12 cell line, a model for skeletal muscle differentiation, I used a functional genomics approach, employing siRNA specific to both these TFs, to characterize their role in skeletal myogenesis. To identify the genes that are regulated by both these TFs, gene expression profiling by microarray of cells treated with siRNA against Six1 and/or Six4 was performed. The knock-down of these TFs caused lower expression of markers of terminal differentiation genes in addition to an impairment of myoblast fusion and differentiation. Interestingly, transcript profiling of cells treated with siRNA against myogenin revealed that several of the Six1 and Six4 target genes are also regulated by myogenin. Through a combination of bioinformatic analyses it was also found that specific knock-down of Six4 causes an up-regulation of genes involved in mitosis and the cell cycle. In summary, these results show that Six1 and Six4 can both independently regulate different genes, but can also cooperate together with other TFs where they play an important role in the proper regulation of skeletal myogenesis.