Cette thèse visait à caractériser la composition en polysaccharides des fibres de bois de tension chez le peuplier et établir de possibles corrélations avec les mécanismes de création de la tension. A cette fin, nous avons étudié le bois de jeunes peupliers qui ont poussé sous conditions contrôlées. Nous avons pu déterminer la composition en polysaccharides des couches G isolées ainsi que la structure de ces polysaccharides. L’évolution de cette composition au long de la différentiation du bois de tension et du bois opposé a été évaluée à l’aide de 178 anticorps dirigés contre les principaux polysaccharides pariétales. La fonction de deux protéines à arabinogalactanes avec domaine fascicline-like a été également étudiée à l’aide des anticorps produits à cet effet. La génétique inverse a été également utilisée pour compléter l’étude de la fonction d’une de ces protéines dans la formation du bois de tension. Les résultats obtenus ont mis en évidence une évolution de la composition de la couche G au cours de la différenciation. Des différences entre la composition de la couche S2 des fibres du bois de tension et du bois opposé ont aussi été détectées. Une grande quantité de pectines de type RG-I a été décelé dans la couche G, molécules qui pourraient participer à la formation d’un gel. Le gonflement de ce gel serait responsable de la mise en tension des microfibrilles de cellulose. Cependant, nos travaux ne montrent aucune évidence pour la présence de xyloglucanes dans la couche G. Des indices en faveur de l’implication des FLAs dans la construction de la couche G ont été également trouvés dans cette étude. Ce travail de thèse ouvre des perspectives pour l’identification du déterminisme moléculaire à l’origine de la création de la tension dans le bois de tension. / This work aimed at characterizing the composition in polysaccharides of poplar tension wood fibres and a possible correlation with mechanisms for creating tension. Firstly, we isolated G-layers from tension wood formed in young poplars which grows in a greenhouse under controlled conditions and determine its polysaccharides composition and the structure of these polysaccharides. Therefore, we investigate the polysaccharide distribution during tension and opposite wood fibres differentiation by using 178 antibodies raised against the major wall polysaccharides. The distribution and function of two fasciclin-like arabinogalactan proteins were studied using two antibodies produced to this end and the function of one of the proteins were studied by reverse genetics. This work show an evolution in the polysaccharides composition of G-layer through its differentiation and also differences concerning S2 layer composition between tension and opposite wood fibres. No evidences for the presence of xyloglucans in the G-layer. However, this work shows the presence of a high quantity of RG-I type pectin which may be implicated in a gel-like structure formation which swelling could be responsible for tension creation in cellulose microfibrils. Our results suggest an implication of fasciclin-like arabinogalactan proteins in G-layer construction. Further, this work opens up new perspectives towards identification of molecular basis of tension creation in tension wood.

<p>Lignocellulose is an ubiquitous source of fixed carbon that is presently underexploited for renewable energy technologies. Currently, producing enzyme cocktails that robustly degrade these feedstocks is a significant economic bottleneck. Anaerobic gut fungi native to the digestive tracts of ruminants and hindgut fermenters are widely understudied despite their inherent ability to degrade a significant portion (~50%) of the lignocellulose in herbivorous animals. Challenges in cultivation due to their strict oxygen sensitivity, and the lack of a central repository to maintain axenic stocks substantially impede the progress with anaerobic fungi. Yet, these microbes have evolved elegant strategies and may harbor novel biomass degrading enzymes that could be used to more efficiently hydrolyze lignocellulose. Developing these organisms through characterization and genome engineering will yield significant contributions to the bioenergy community by improving hydrolysis technologies.</p> <p>In this work, we report the isolation of four novel species of anaerobic gut fungi. A more complete characterization of one of our four fungal isolates is investigated, whereby the effects of substrate composition and the corresponding fungal growth rates are compared. I also explore the growth of one of our fungal isolates on transgenic poplar to understand how fungal growth and enzyme secretion adapt to variable lignin composition. Notably, no significant reductions in growth were observed highlighting the ability of anaerobic fungi to degrade diverse feedstocks regardless of lignin composition. I have additionally included preliminary work intended to identify what epigenetic regulational strategies exist for anaerobic fungi, and how they relate to carbohydrate active enzyme expression. We hope to leverage this knowledge to engineer base enzyme cocktails that release significant portions of the fermentable sugars in untreated or mildly treated plant biomass as a means to make bioenergy technologies more efficient.</p>

2,2',3,5',6-pentachlorobiphenyl (PCB95) is a chiral congener of the persistent organic pollutants (POPs) family of PCBs. It has been shown that chiral PCBs can be enantioselectively transformed into hydroxylated metabolites by cytochrome P450 in animals. Previous studies in our group suggested that PCB 95 can be enantioselectively translocated and metabolized in whole poplar plants. In this work, healthy whole poplar plants were hydroponically exposed to PCB95 for 30 days. Two unknown OH-PCB95 metabolites were detected in the roots by HPLC-MS. Different chromatographic and spectrometry techniques, including HPLC-MS, NMR and GC-MS, were tried to determine the structure of the more abundant metabolite of the two. It was identified to be 4'-OH-PCB95 (4'-95) by GC-MS method. The data show that PCB95 can be transformed into at least two hydroxylated metabolites by whole poplar plants, with one of them being 4'-95. Chiral analysis of 4'-95 by HPLC-MS showed slightly more abundance of the second eluting enantiomer E2-4'-95 in the roots, suggesting that the biotransformation of PCB95 to 4'-95 is enantioselective. Comparison with animal studies shows a distinct metabolite profile in whole poplar plants.

The use of ethanol produced from lignocellulosic biomass for transportation fuel offers solutions in reducing environmental emission and the use of non-renewable fuels. However, lignocellulosic ethanol production is still hampered by economic and technical obstacles. For instance, the inhibitory effect of toxic compounds produced during biomass pretreatment was reported to inhibit the fermenting microorganisms, hence there was a decrease in ethanol yield and productivity. Thus, there is a need to improve the bioconversion of lignocellulosic biomass to ethanol in order to promote its commercialization. The research reported here investigated the use of poultry litter biochar to improve the ethanol production from steam-exploded poplar and corn stover. The effect of poultry litter biochar was first studied on Saccharomyces cerevisiae ATCC 204508/S288C growth, and second on the enzyme hydrolysis and fermentation of two steam-exploded biomasses: (poplar and corn stover). The third part of the study investigated optimal process parameters (biochar loading, biomass loading, and enzyme loading) on the reducing sugars production, and ethanol yield from steam-exploded corn stover. In this study, it has been shown that poultry litter biochar improved the S. cerevisiae growth and ethanol productivity; therefore poultry litter biochar could potentially be used to improve the ethanol production from steam-exploded lignocellulosic biomass.

Five experiments were conducted to evaluate tree forage as a feedstuff for small livestock, using the foliage of black locust (Robinia pseudoacacia) and hybrid poplar (Populus spp.). In the first trial, rabbits were fed diets containing 40% alfalfa (control), and 10%, 20% and 40% poplar leaves (PL) from untrimmed or uncoppiced growth, and 10% and 20% PL from trimmed or coppiced growth. At the 20% level, crude protein (CP) was better digested for the coppiced PL than for the uncoppiced (p < .05). No significant difference was evident between treatments in the average daily gain (ADG) or feed efficiency, but dry matter (DM) intake increased for all the PL treatments compared to the control (p < .01). In the second experiment, black locust (BL) leaves were treated by various methods in an attempt to counter the effect of tannins. Rabbits were fed diets containing 50% alfalfa (control), 25% alfalfa and 25% black locust meal (BLM) (BLM control), BLM + 1% polyethylene glycol (PEG), BLM + 1% phytase, and BLM + 0.3% L-methionine and 0.3% choline chloride. In general, nutrient utilization and ADG were better for the control than for the BLM treatments. The addition of PEG increased CP digestibility (p < .01) and ADF (p < .03) compared to the BLM control, partially alleviating the reduced nutrient availability. The addition of L-methionine and choline chloride increased ADF digestibility (p < .02). In the third trial, black locust (BL) bark and other tree products were used to study the possible toxic effects of lectins in BL bark. Rabbits were fed diets containing 25% BL bark, oak sawdust, red alder bark, or red alder sawdust (all diets also included 25% alfalfa). A 50% alfalfa diet served as a control. In general, nutrient digestibilities and ADG were higher for the control than the treatments and, also, higher for the BL bark diet than the alder bark diet. The ADG with the BL bark diet was lower than for the alder bark diet (p < .01), which indicated a possible toxic effect of the BL bark. The fourth trial examined the feeding value of poplar leaves for sheep. Sheep were fed diets containing 50% PL or 50% alfalfa. Nutrient digestibilities were lower for the PL diet (p < .01). In the fifth experiment, BL leaves were fed to sheep and goats in order to determine if goats, being browsers instead of grazers like the sheep, are better equipped to tolerate the anti-nutritive effects from BL forage. Sheep and goats were fed diets composed solely of BL leaves or alfalfa (control). Overall, the nutrient digestibilities were higher for alfalfa than for BL leaves, and there was no difference in terms of digestibility between species. Although the leaves of black locust and poplar contain anti-nutritive factors, it was concluded that the trees have potential as multipurpose trees from which the leaves could be harvested as animal fodder, particularly in temperate areas of the developing world. / Graduation date: 1993

The relationship between an organism’s genome, developmental stage, and environment is complex. The aim of the research presented herein was to provide experimental evidence to contribute to the annotation of the P. trichocarpa genome and to test two major hypotheses addressing the interaction between drought and time of day in A. thaliana and in two hybrid Populus clones. In order to generate data to address these aims, three separate experiments were undertaken. First, all members of the R2R3-MYB family of transcription factors in the P. trichocarpa genome were characterised by phylogenetic analysis and their transcript accumulation patterns across a range of tissues and organs were assessed using whole genome poplar microarrays. Results of this analysis indicated that expansion and diversification of the R2R3-MYB family may have contributed to phenotypic innovation in the Populus lineage. Second, drought-responsive transcriptome adjustments of two hybrid poplar clones, DN34 (P. deltoides X P. nigra) and NM6 (P. nigra X P. maxiomowiczii) were assessed for time-of-day and genotype dependent patterns. For each genotype, each of four time points was characterised by discrete sets of drought-responsive genes. Furthermore, while a number of genes were identified that were responsive to drought in both genotypes, a much larger number of genotype-dependent, drought-responsive transcriptome changes were detected. Finally, the drought-responsive transcriptome adjustments A. thaliana plants were assessed for time-of-day dependent accumulation patterns. Results of this analysis indicate that time-of-day-dependent differences in the drought response were manifest as changes of different magnitudes for a conserved set of genes across the four time points measured. These results emphasise the complex interplay of a plant’s genome, developmental stage, and environment in shaping the observed transcriptome.

Success of hybrid poplar plantations will rely on the efficient management of nutrients and weeds. Relatively little is known about the root uptake characteristics of hybrid poplar and weeds, their belowground interactions and particularly, the quantitative understanding of nutrient uptake using mechanistic models under weed-competing conditions. Therefore, the objectives of this study were to investigate the effects of dandelion and quackgrass on the growth of hybrid poplar, to establish their root uptake characteristics and to quantify their nutrient uptake using the soil supply and nutrient demand (SSAND) model. In a pot study, hybrid poplar stem height, root collar diameter, shoot and root biomass, root length, and N, P and K uptake significantly decreased in the presence of dandelion and quackgrass weeds. Similar weed competition effects on growth of hybrid poplar were also observed in the field at the Pasture and Alfalfa sites where hybrid poplar was grown with and without weeds for 50, 79 and 100 days. In a hydroponic experiment, Imax values for NH4-N, NO3-N, P and K varied significantly among hybrid poplar seedlings and dandelion and quackgrass weed species and was greatest for dandelion followed by hybrid poplar and then quackgrass. The Km values were lowest for quackgrass compared to the other plant species for all of the nutrients. Simulation results from the SSAND model for the pot study showed that N uptake was underpredicted in hybrid poplar by 58 to 73%, depending upon soil type and weed treatment. Incorporation of N mineralization as a model input improve the hybrid poplar N uptake predictions by 24 and 67% in the Pasture and Alfalfa soil, respectively, when grown without weeds. SSAND model underestimated P uptake by 84-89% and overestimated K uptake by 28 to 59% for hybrid poplar depending upon the soil type and weed treatment. In the field, N uptake by hybrid poplar was in close agreement to measured N uptake in the control treatment. N uptake was greatly underestimated for both hybrid poplar and weeds in the weed treatment. Including changing water content greatly improves the N uptake by hybrid poplar and weeds in weed treatments. Results from this study suggest weed control is an essential practice to establish successful hybrid poplar plantations. Also, SSAND model can be an effective tool for predicting the nutrient uptake under two plant species competing environment if all the processes of nutrient supply are adequately described in the model.

As a result of predicted climate change, environmental conditions may make woody plant species such as poplars (Populus spp.) vulnerable unless they are sufficiently adaptable to the new environment. This greenhouse study examined the responses of Hill, Northwest, Okanese and Walker hybrid poplar clones to drought, a potential outcome of a changing climate. Plants were grown from cuttings and subjected to two soil moisture treatments; a well-watered treatment and a drought conditioning treatment in which plants were subjected to cycles of soil moisture deficit. The first study examined growth and gas exchange following treatments, while the second study examined concurrent changes in leaf water potential and gas exchange during a period of increasing soil moisture deficit, following treatments.<p> Hill and Okanese plants had reduced shoot:root ratios, possibly leading to more positive plant moisture balances compared to Northwest and Walker plants. Stomatal characteristics related to steady state gas exchange with Okanese plants having stomata predominantly on lower leaf surfaces, and lower stomatal conductance and Northwest plants having relatively large stomata and increased stomatal conductance. Hill and Okanese plants had the most responsive stomata, which began to close at much higher levels of leaf water potential (-0.45 and -0.54 MPa) than Northwest or Walker plants (-1.03 and -0.88 MPa); however, closure was more gradual in Okanese plants. Drought preconditioning resulted in stomatal closure occurring at higher leaf water potentials in droughted Northwest and Walker plants compared to well-watered plants. Regardless of soil moisture treatment, WUE was highest in Okanese and Walker plants. The drought treatment did however lead to increased WUE in Hill and Northwest plants.<p> Overall, Okanese plants appear to be the best adapted to conditions of reduced soil moisture based on growth and physiological traits, while Northwest and Hill seem better suited to areas where moisture deficits are likely to be less frequent or less severe. Results indicate that variability exists in adaptability of hybrid poplar clones to drought, suggesting that there may also be other hybrid clones that are adaptable to reduced soil moisture conditions, which may merit further investigation.

The relationship between an organism’s genome, developmental stage, and environment is complex. The aim of the research presented herein was to provide experimental evidence to contribute to the annotation of the P. trichocarpa genome and to test two major hypotheses addressing the interaction between drought and time of day in A. thaliana and in two hybrid Populus clones. In order to generate data to address these aims, three separate experiments were undertaken. First, all members of the R2R3-MYB family of transcription factors in the P. trichocarpa genome were characterised by phylogenetic analysis and their transcript accumulation patterns across a range of tissues and organs were assessed using whole genome poplar microarrays. Results of this analysis indicated that expansion and diversification of the R2R3-MYB family may have contributed to phenotypic innovation in the Populus lineage. Second, drought-responsive transcriptome adjustments of two hybrid poplar clones, DN34 (P. deltoides X P. nigra) and NM6 (P. nigra X P. maxiomowiczii) were assessed for time-of-day and genotype dependent patterns. For each genotype, each of four time points was characterised by discrete sets of drought-responsive genes. Furthermore, while a number of genes were identified that were responsive to drought in both genotypes, a much larger number of genotype-dependent, drought-responsive transcriptome changes were detected. Finally, the drought-responsive transcriptome adjustments A. thaliana plants were assessed for time-of-day dependent accumulation patterns. Results of this analysis indicate that time-of-day-dependent differences in the drought response were manifest as changes of different magnitudes for a conserved set of genes across the four time points measured. These results emphasise the complex interplay of a plant’s genome, developmental stage, and environment in shaping the observed transcriptome.

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.