Shawn Mansfield

Climate is known to impact the growth of plantation forests in South Africa. To characterize the conditions, this study developed temperature and rainfall models for specific plantations in the Lowveld Escarpment and Highveld forestry regions of South Africa. The local climate models use a finer scale than world climate models, and they improve the understanding of how terrain features impact regional climate and historic tree growth. In addition, the finer scale models give insights into the relationship between the local terrain condition and regional climate. Plantation forests were originally established in South Africa to meet an increasing demand for solid wood products as there was a limited supply from native forests. The majority of the commercial softwood plantations were established with Mexican Pinus patula. Since growing conditions are known to impact tree growth, tree form, and wood quality of P. patula, sample plots were established over a cross-section of plantations in the Lowveld Escarpment and Highveld forestry regions of South Africa that covered an array of geologies and altitudes. Soil, growing days, and temperature were found to have little impact on tree form and wood properties. However, rainfall and specifically, spring rainfall, was found to have a highly significant impact on late wood formation, proportion of juvenile core, and wood density. In addition, tree height was found to be strongly correlated with maximum annual temperature. Veneer derived from Lowveld trees had more splitting, which was largely related to defects. Larger trees also had a greater volumetric heartwood percentage and had a smaller live crown height portion, compared to smaller trees. Highveld trees also had greater net veneer recovery and produced better quality veneer than trees grown on the Lowveld.Annual maximum temperature and tree height had a negative relationship with the recovered lumber properties and dimensional stability. Spring rainfall appears to be the most important factor influencing lumber twist and this was possibly due to the associated larger juvenile core present in the trees.

View record

Among all the stressors on global agricultural systems, soil salinity and salinization is arguably one of the most significant and an ever increasing problem. Traditionally, sodium chloride is the salt employed for studying salt stress in plants, but on the Canadian prairie regions, sulphates are the predominant salts that cause osmotic imbalance and specific ion toxicities, and subsequently induce oxidative damage in plants. Willows (Salix spp.) have been identified as potential candidate plants for multipurpose establishment on saline marginal fields. As a phytoremediation strategy, growing salt tolerant willows could improve biodiversity, quality, and productivity of soil and local ecosystems, while providing biomass production for a series of downstream agricultural and industrial applications.In order to investigate the salt tolerance among willows, and to dissect the ionic effects of different salt ions on plants, two greenhouse trials were conducted with mixed salt treatments in the first trial, as well as sodium and magnesium sulphate treatments in the second trial, with comparable salt levels at moderate and high salinity. A total of 20 commercial hybrid and 16 Canadian native willow genotypes were treated for 8 weeks in the first trial, of which a selection of 12 were further assessed for 12 weeks in the second trial. The genotypes were monitored for growth traits, and gas exchange during the trials. Elemental analyses of leaves and roots were performed via inductively coupled plasma mass spectrometry, and targeted metabolite profiling was used to quantify sugars, organic acids, amino acids, and glutathione in leaves by high performance liquid chromatography and spectrophotometry. Leaf samples of two genotypes, LAR-3, a sodium excluder, and MJW-2, a sodium accumulator, were selected for mRNA sequencing. Overall, it can be concluded that hybrid willows grew better at moderate salinity, while native willows are better suited for higher salinity conditions. Magnesium affected willows more significantly than sodium, largely inhibiting plant growth, mineral balance, photosynthesis, and primary metabolism. In addition, excess sulphate uptake and assimilation induced by sulphate salinity were witnessed. An assessment of growth, physiological, biochemical, and transcriptomic levels uncovered a more comprehensive view of salt responses, strategies, and defence mechanisms in willows.

View record

Woody feedstocks, such as poplar, willow, and eucalyptus, represent abundant and fast-growing sources of lignocellulosic biomass for use in the production of pulp and paper, biofuels, and other bio-based materials. Lignin, a polyphenolic polymer, is the second most abundant chemical constituent of plant secondary cell walls and is typically composed of three canonical monolignols: p-coumaryl, coniferyl, and sinapyl alcohols. Recent efforts to genetically engineer the monolignol biosynthetic pathway have led to significant changes in the content and composition of lignin, highlighting the remarkable metabolic plasticity of this major biosynthetic pathway. Moreover, a wide array of non-traditional monolignols has been found to naturally incorporate into lignins of different plant species, such as the flavonoid tricin found in the lignins of grasses. To investigate the possibility of introducing flavonoids into the lignins of poplar as novel, value-added lignin monomers, I genetically engineered poplar to accumulate flavonoids in lignifying xylem tissue and analyzed the resulting wood chemistry. Chalcone synthase catalyzes the first committed reaction in the production of flavonoid compounds to produce naringenin chalcone which is then isomerized to naringenin. Using a lignin-specific promoter, I have genetically engineered hybrid poplar (Populus alba x grandidentata) to express a chalcone synthase gene (MdCHS3) derived from apple (Malus x domestica). MdCHS3-poplar accumulated naringenin in xylem methanolic extracts and NMR analysis revealed naringenin in the extract-free, cellulase-treated xylem tissue (enzyme lignin). MdCHS3-poplar displayed lower total lignin, an increase in cell wall carbohydrate content, and performed significantly better during saccharification assays compared to wild-type. Building on these promising results, I characterized two flavonoid-modifying enzymes derived from Brachypodium distachyon in vitro: chrysoeriol 5'-hydroxylase (BdCYP75B4) and flavone synthase II (BdCYP93G1), both key enzymes in the production of tricin and O-linked tricin glycosides. I also confirmed that PaxgOMT25, an important O-methyltransferase in monolignol biosynthesis, can participate in tricin biosynthesis. Co-expression of BdCYP75B4 and BdCYP93G1 in MdCHS3-poplar trees resulted in stunted growth and limited plant viability, however analysis of a recoverable low-expressing line revealed accumulation of tricin in xylem methanolic extracts not observable in controls, demonstrating that the successful production of tricin, a high value flavonoid, in poplar xylem is feasible.

View record

Lignin is a phenolic polymer found predominantly in the secondary cell walls of vascular plants where it contributes to water transport, mechanical support, and plant defence. The occurrence, composition, and structure of lignin vary widely between cell types, throughout development, in response to stress, and across plant lineages. In addition to the hydroxycinnamate-derived monolignols, an array of non-canonical monomers occurs in lignin, clearly illustrating that the molecular processes underpinning lignin formation are highly flexible. This innate plasticity enables the rational design of lignins with predetermined structures and physicochemical properties. Given the biological importance of lignin and the industrial significance of plant biomass, this dissertation explores several promising areas of research in cell wall biology, plant biochemistry, and lignin engineering.The shikimate and aromatic amino acid pathways are crucial metabolic gatekeepers that lie upstream of lignin biosynthesis. In this work, chorismate was studied as an important branchpoint metabolite and evidence was uncovered for redundancy of chorismate mutase isomers in poplar (Populus trichocarpa). Next, a lignin engineering strategy that exploits this important branchpoint was developed. Introduction of a bacterial chorismate pyruvate lyase into transgenic hybrid poplar (Populus alba × grandidentata) was used to divert carbon flux away from phenylpropanoid biosynthesis, leading to lignin with more alkali-labile p-hydroxybenzoate pendent groups. In commelinid monocots, p-coumarate occurs analogously as lignin acylations. The discovery of these moieties in the lignin of kenaf (Hibiscus cannabinus), a eudicot species, represents a new example of convergent evolution and provides a useful tool for lignin engineering. Finally, lignification was studied in eastern leatherwood (Dirca palustris) as the woody stems of this native shrub are exceptionally flexible. In addition to an unusual composition and distribution of lignin, this work uncovered a lignin-deficiency in the middle lamella that asks us to revisit unresolved questions in plant cell wall biology.

View record

Lodgepole pine (Pinus contorta Dougl. ex. Loud.) is an important tree species in British Columbia, Canada, both commercially and ecologically. Much of the seed used for reforestation after harvesting and wildfires is produced by seed orchards that rely on an extensive breeding program. Tree breeders require tools to adjust quickly to unforeseen challenges exacerbated by a changing climate and two approaches were examined: mulit-environment trial (MET) analyses and genomic selection. A MET dataset was used to analyse height growth data across five breeding zones using a factor analytic model for estimating additive (co)variance among test sites and the use of molecular markers to predict breeding values for height growth and wood quality traits (average wood density, earlywood density, latewood density, latewood proportion and microfibril angle) was assessed for a subsample of 1,569 trees from four progeny test sites using 19,584 single-nucleotide polymorphism markers. Test sites were clustered into four main breeding zones based on genetic correlations between sites. Climate change projections were applied to the four new zones which suggested that southern zones will expand. Predicted breeding values from Bayesian models (Bayes B and C) and best linear unbiased prediction (BLUP) using a hybrid matrix (H-matrix) were compared to models that used an average relationship matrix (ABLUP) and a realized relationship matrix (GBLUP). Bayesian models had similar prediction accuracies compared to ABLUP and GBLUP when models were confined to a closed population (> 0.74), but accuracy decreased substantially when relatedness was controlled between the training and validation populations (> 0.25). The models also worked well across environments and test cycles and were similar to GBLUP for ranking trees within families. HBLUP was equivalent to ABLUP and GBLUP models for genetic parameters and prediction accuracy, but had very low within-family rank correlations (0.08 average Spearman rank correlation across traits). Bayesian models can be used to predict breeding values and rank trees that have no phenotypic data. In contrast, HBLUP should not be used for ranking trees within families in the absence of phenotypic data, but can effectively improve estimates of variance components and breeding value estimates.

View record

Sucrose synthase (SuSy) is one of the two enzyme families catalyzing the first step of sucrose utilization. It has been reported to serve different functional roles during plant growth and development, including supplying carbon for plant respiration, modulating sink strength and phloem loading, and facilitating the biosynthesis of starch and cell wall polymers. In a widely accepted model of cellulose biosynthesis, sucrose synthase is proposed to be tightly associated with the plasma membrane-localized cellulose synthase (CesA) complex and serves to channel carbon from photoassimilate (sucrose) directly to cellulose biosynthesis. Although many studies support this model, direct evidence of true interaction between SuSy and CesA is still lacking. As such, the primary objective of this thesis was to investigate the proposed model of cellulose biosynthesis using Arabidopsis thaliana as a model. The spatiotemporal localization of each of the six Arabidopsis SuSy proteins was investigated via live-cell imaging in a series of tissues including stems, roots, petioles, and siliques. Surprisingly, no single isoform of SuSy was detected in xylem, the major site of cellulose deposition in Arabidopsis plants. In contrast, SuSy1 and SuSy4 were expressed exclusively in phloem companion cells, SuSy5 and SuSy6 were confined to sieve elements, while SuSy2 and SuSy3 showed elevated expression in developing seeds. Thus, a new sus1/sus4/sus5/sus6 quadruple mutant was generated and examined for a putative cellulose/cell wall phenotype, however, the plants showed no obvious growth defect. This can be explained by the activity of invertase (INV) which may compensate for the lack of SuSy activity in phloem tissue. Furthermore, the effect of phosphorylation on SuSy4 activity and membrane association was examined. Results showed that both phosphomimetic and phosphoresistant SuSy4 were largely localized to the cytoplasm of companion cells, similar to that of the native SuSy4. When subjected to flooding, only SuSy4 phosphomimetic transgenic lines exhibited obvious reductions in soluble sugar and starch content. Collectively, these findings suggest a need to reconsider the established and largely accepted model of cellulose biosynthesis in Arabidopsis, and implicate SuSy in biological events related to phloem loading and carbon allocation.

View record

Secondary cell walls (SCWs) containing the hemicellulose xylan are essential for normal plant growth and development. Great strides have been made to identify the many Golgi-localized biosynthetic enzymes that work in concert to make xylan, however, we still understand little about how these critical proteins and their product are organized in the Golgi to facilitate synthesis and trafficking. To address this question, I characterized the Arabidopsis Golgi in cells producing SCWs using a combination of confocal and transmission electron microscopy (TEM). This analysis indicates that the number of Golgi stacks increases significantly with the onset of SCW synthesis, and that during this process the randomly distributed Golgi stacks work together to produce and secrete xylan. Furthermore, nanoscale characterization of Golgi structure revealed significant increases in Golgi diameter, swelling of the cisternal margins, and secretory vesicle size. Loss of the xylan-biosynthetic enzyme IRREGULAR XYLEM 9 (IRX9) resulted in a dramatic increase in cisternal fenestration and a decrease in swollen margins, but did not affect the number or size of Golgi. Finally, immunogold labelling was used to map IRX9-GFP and xylan to different regions of Golgi cisternae, indicating that xylan is abundant in the outer margins of trans-cisternae, IRX9-GFP is abundant in an inner margin of medial-cisternae, and both are absent from cisternal centers. This new concentric circle model of Golgi organization has expanded our understanding of Golgi structure and function and has implications for Golgi function in other cell types and organisms.The second part of this thesis explores problem-solving instruction in undergraduate cell biology classes, by testing how different teaching techniques affect student attitudes and performance. These results demonstrate that worked examples can be effective teaching techniques for cell biology problem-solving, with lower-performing students seeing greater benefits. Furthermore, providing worked examples did not ameliorate student desires for answer keys to practice problems. This work can be used to guide the appropriate level of instructional support for students of different expertise levels in future courses, and across curricula.

View record

The formation of plant secondary cell walls requires a complex network of transcriptional regulation, culminating in a coordinated suite of biosynthetic genes depositing walls, in a spatial and temporal fashion. The transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is a Class II KNOTTED1-like homeobox (KNOX2) gene, that acts as a negative regulator of secondary cell wall biosynthesis in interfascicular fibers. Previously, members of Ovate Family Proteins (OFP1 and OFP4), were shown to interact with KNAT7 to negatively regulate wall formation. However, the function of other closely related KNOX2 and OFP genes in secondary wall formation remains unclear. Herein, I showed that knat3knat7 double mutants possess an enhanced irregular xylem (irx) phenotype relative to single mutants, and decreased interfascicular fiber cell wall thickness. Additionally, unlike the increased lignin content characteristic of knat7 mutants, knat3knat7 had no change in lignin content, while the monomeric lignin composition was substantially reduced relative to the wild-type plants. In contrast, KNAT3 overexpression resulted in thicker interfascicular fiber secondary walls, suggesting a positive regulation of KNAT3 in wall development.A thorough examination of OFP mutants showed that none of the single mutants revealed any wall defects, including ofp4, which was previously shown to interact with KNAT7. However, they do display leaf phenotypes. In contrast, plants overexpressing OFP isoforms consistently exhibited cell swelling, disordered microtubules, and dark-grown de-etiolated phenotypes, resembling phenotypes common to brassinosteroid deficient mutants. Using yeast two-hybrid and bimolecular fluorescence complementation assays, I identified two genes that interacted with OFP4, NAP1;1 and NAP1;2, members of the Nucleosome Assembly Protein 1 (NAP1) family. Higher-order, loss-of-function NAP1 and OFP mutants also exhibit altered cotyledon shape and a reduced cotyledon width:length ratio. The kidney-shaped cotyledon phenotype apparent in OFP4 overexpressing plants was suppressed in the nap1;1 nap1;2 nap1;3 triple mutant background. Together, my research suggests that in addition to KNAT7, KNAT3 also contributes to cell wall deposition, and that a complex network of positive and negative regulation governed by KNOX2 proteins regulates secondary wall formation. Moreover, the complex of OFP4 and NAP1 plays a significant role in the cotyledon development.

View record

Elemental phosphorus has been categorized as a non-renewable resource that is crucial to global food security. This is largely due to the transport of phosphorus being dependent on aqueous transfer and therefore an inherent inability to return to upstream ecosystems. It is only through mining and transport of rock phosphate that agricultural land remains productive. Simultaneously, due to agricultural over-fertilization, phosphorus has been characterized as a pollutant in aquatic environments. Diffuse source run-off from high phosphorus soils continues to contribute to downstream eutrophication decades after nutrient management practices have been put into place. A potential solution involves planting high biomass-producing tree species along riparian areas. Trees belonging to the Salicaceae are ideal candidates as they have a wide geographical distribution in Canada and broad-scale applications, ranging from fibre production to biofuel feedstock to uses in phytoremediation. The objective of this thesis was to identify a commercially available tree genotype, be it poplar or willow, well suited for widespread planting in agricultural areas to limit nutrient enrichment of riparian ecosystems. Phenotypic differences in phosphorus storage and allocation were analyzed using ICP-AES and HPLC. Poplar varieties Tristis and Northwest demonstrated the highest capacity for luxury uptake with an estimated 3.7 – 3.9 mg P g-¹ when 2.2 mM soluble phosphate (100N:70P) was applied, with no measurable metabolic consequences. However, the majority of phosphorus was stored in leaves as phosphate and subsequently returned to the environment as autumnal senescence progressed. This led to the exploration of factors limiting phosphate translocation and resorption. Expression of an exogenous phosphate H⁺/H₂PO₄- symporter in poplar led to a small, but significant increase in phosphate resorption and a pronounced increase in sulfate resorption, leading to further questions surrounding anion efflux from the vacuole and the role of the tonoplast in limiting nutrient translocation. If resorption proficiency could be increased under the high nutrient loads found in productive lands, poplar genotypes with luxury consumption could be bred for improved resorption and used to reduce phosphorus entry into riparian ecosystems. Extrapolation of this information to crop species could lead to reduced fertilizer application and improved nutrient management of perennial production systems.

View record

Cellulose is the most abundant polymer in nature and is a major component of both primary and secondary cell walls in plants. The cellulose produced in these different walls are synthesized by completely independent sets of non-redundant CELLULOSE SYNTHASE (CESA) enzymes. In the last decade, live cell imaging techniques have answered a number of fundamental questions regarding CESA dynamics and organization in the primary cell wall. However, attempts to repeat these experiments in cells producing secondary cell walls has been met with limited success due to the fact that cells forming secondary walls are deep inside plant organs. The development of an inducible system driving the ectopic expression of the master regulator for protoxylem tracheary element development, VASCULAR RELATED NAC-DOMAIN7 (VND7), has generated a valuable biological tool to track secondary cell wall synthesis via live-cell imaging. With these tools, I was able to directly visualize secondary cell wall-specific CESA complexes moving around the plasma membrane, and to quantify that they move at a significantly faster rate than primary cell wall-specific complexes. Additionally, bundling of the underlying cortical microtubules causes the densities of the CESA complexes to be much higher during secondary wall synthesis than during primary wall synthesis, giving a possible explanation for the rapid and abundant development of these walls. Analysis of the transition from primary to secondary cell wall production revealed that primary wall-specific CESAs are selectively targeted into distinct pre-vacuolar compartments for degradation to the lytic vacuole, while secondary cell wall-specific CESAs accumulate. Finally, cesa mutants were investigated to explore the effects of the loss of each of the three CESAs involved in secondary cell wall cellulose synthesis on both the wall patterning and localization of their interacting partners. While the loss of a CESA causes significant defects in secondary cell wall cellulose patterning, the loss of CESA7 specifically resulted in the complete loss in patterning, indicating a possible role for CESA7 in anchoring the CESA complexes to the underlying cortical microtubules. Taken together, these results refine our model of how plant cells coordinate their cellulose synthesis machinery during secondary cell wall production.

View record

Cellulose biosynthesis is a dynamic and specialized cellular process with multiple layers of organization. This abundant, vital polymer is synthesized by cellulose synthase complexes (CSCs) localized at the plasma membrane. Cellulose chains are extruded into the apoplast, and rapidly self-assemble into microfibrils. The mechanisms controlling organization of the product, cellulose microfibrils, are still unclear. The GPI-anchored protein COBRA (COB), localized at the outer leaflet of the plasma membrane, is required for normal cellulose deposition in primary cell walls. A closely related protein, COBRA-LIKE4 (COBL4), is required for secondary cell cellulose organization. Loss-of-function, in Arabidopsis cobl4 mutants originally called irregular xylem 6 (irx6), results in reduced cellulose content, cellulose of lower crystallinity, and thinner secondary cell walls. To better understand COBL4 function, I investigated the chemical and ultrastructural properties of novel irx6-2 and irx6-3 alleles of Arabidopsis. I followed this up by demonstrating functional conservation between COBL4 in woody (Populus trichocarpa) and herbaceous (Arabidopsis) species. A fluorescently labelled poplar COBL4, PtCOB4a, was co-localized with secondary cell wall thickenings in an inducible Arabidopsis protoxylem experimental system. To further refine our understanding the molecular role of COBL4, AtCOBL4 was over-expressed in hybrid poplar, in a secondary cell wall specific manner. Increased AtCOBL4 abundance did not significantly alter cell wall derived glucose content compared to control plants; this was confirmed by the absence of a significant increase in α-cellulose. The ultra-structural characteristics of deposited cellulose, specifically cellulose DP and cellulose crystallinity, were significantly increased in a number of over expression lines relative to control trees. These findings confirm COBL4 as a protein involved in organizing cellulose biosynthesis in plants. The increased cellulose DP and subsequent proportion of crystalline cellulose suggest that COBL4, in part, affects cellulose biosynthesis efficiency. To further resolve the role that cellulose ultrastructure plays in limiting intrusive tip growth of fibre cells, we measured xylary fibre lengths of AtCOBL4 overexpression poplar lines. Overexpression lines had on average shorter fibres than wild-type trees. This demonstrates that increased DP and the overall structural organization of cellulose, mediated by AtCOBL4, may be sufficient to restrict intrusive growth of fibre cells.

View record

Arabinogalactan proteins (AGPs) are cell wall proteins with abundant glycosylation, belonging to the large, multi-gene hydroxyproline-rich glycoprotein (HRGP) family. It has been reported that AGPs may contribute to cell expansion, xylem cell differentiation and secondary cell wall deposition. However, the roles of specific AGP in wood developmental processes have never been thoroughly elucidated. Therefore, the objective of this thesis was to investigate the functional role(s) of three AGPs in wood cell wall development. Specifically, the lysine-rich AGP18; a classical AGP, AGP9; and an AGP peptide, AGP14 were studied, because they demonstrated high gene expression levels in the developing xylem of Populus trichocarpa during transcriptome re-sequencing initiatives. Based on the phenotypic changes observed when PtAGP18 was down-regulated in transgenic poplar trees and Arabidopsis atagp18 T-DNA mutant analyses, I showed roles for AGP18 in fiber cell shape and fiber secondary cell wall formation (Chapter 2). Moreover, the poplar PtAGP18 was able to complement the Arabidopsis atagp18 T-DNA mutants which displayed altered fiber shape and cell wall thickness, indicating that these two genes are functionally equivalent (Chapter 2). Analysis of the growth of Arabidopsis hypocotyls cultivated in darkness revealed that AGP18 is involved in cell expansion (Chapter 2). In parallel, I showed that the AGP9 affects xylem vessel differentiation and vessel cell expansion (Chapter 3). A role for AGP9 in cell expansion was also demonstrated with Arabidopsis agp9 mutant hypocotyls grown in the dark (Chapter 3). Furthermore, AGP14 appears to contribute to cell wall formation in poplar (Chapter 4). Taken together, the functional characterization of these AGPs suggests that AGP18 and AGP9 play roles in the development of fibers and vessels, respectively. However, further research is needed to delineate the exact cellular and molecular mechanisms through which AGPs contribute to secondary xylem development.

View record

Homogalacturonan pectin domains are synthesized in a highly methyl esterified form and can be de-methyl esterified by the cell wall enzyme Pectin Methyl Esterase (PME). The prevalent model for PME mode of action indicates that when PMEs act on a stretch of adjacent galacturonic acid glycosides, they may strengthen the cell wall but when PMEs act on non-adjacent galacturonic acid glycosides they may loosen the cell wall. PME activity can be regulated in planta by the proteinaceous inhibitor, PMEI. I used PME and PMEI to study the importance of methyl esterification in seed development and germination. As a means to identify PMEs involved in seed coat mucilage I identified 7 PMEs expressed in the seed coat. The PME gene HIGHLY METHYL ESTERIFIED SEED (HMS) is highly expressed at 7 Day Post Anthesis (DPA) both in the seed coat and the embryo. Using a hms-1 mutant, I showed that HMS is required for normal levels of PME activity and methyl esterification in the seed, mucilage extrusion and proper embryo cell expansion, rigidity and morphogenesis between 4 and 10 DPA. The mucilage extrusion defect is a secondary effect of the function of HMS in the embryo. I hypothesize that HMS is required for cell wall loosening in the embryo to allow for cell expansion during the accumulation of storage reserves. To evaluate the importance of methyl esterification in germination my collaborators and I first showed that PME activity changed during the different stages of germination: it first increased before testa rupture and decreased during endosperm rupture. Treatment with the hormone abscisic acid (ABA) to increase dormancy prolonged PME activity in the seeds. Inversely when we negatively regulated PME activity in the A. thaliana seed with the overexpression of a PMEI (OE PMEI5), we generated larger seeds with bigger cells. These seeds germinated faster both in presence or absence of ABA. Therefore we hypothesize that the PME(s) inhibited by PMEI5 establishes stronger cell walls that restrict germination. This thesis clearly demonstrates that PME activity is important in the regulation of seed cell wall methyl esterification impacting embryo growth and germination.

View record

The raffinose family of oligosaccharides (RFO) function as transport carbohydrates in the phloem, storage compounds in sink tissues, and as putative metabolic agents that combat plant stresses. Research on the RFO pathway has focused on seed biology and plants that transport raffinose as their primary photoassimilate. In contrast, few studies have explored this pathway in woody species. As such, this thesis investigated the fundamental function of the RFO enzymes in hybrid poplar, with emphasis on galactinol synthase (GolS), an enzyme key to the pathway. Phylogenetic comparisons of Populus Go1S with other known GolS suggest a putative role for these enzymes in stress response. Protein analysis of two heterologously expressed isoforms demonstrated that they are true Go1S; with Pa×gGolSI possessing a broader pH and temperature range than Pa×gGolSII. Expression patterns also revealed that the Pa×gGolSII transcript abundance varied seasonally. Together, the results suggest that Pa×gGolSI may be involved in basic metabolic activities, while Pa×gGolSII is likely involved in seasonal mobilization of carbohydrates. To further elucidate the in-planta Go1S function, transgenic trees with mis-regulated GolS were generated. Two AtGolS3 over-expression (OE) transgenic lines showed effects on growth, while other lines appeared normal and possessed marginally modified cell wall characteristics. The extreme over-expressers were severely stunted and had cell wall traits characteristic of tension wood. AtGolS3-OE lines showed reduction in the microfibril angle, increase in cell wall crystallinity and possessed higher cellulose, and lower mannose and lignin contents. Interestingly, although galactinol and raffinose contents increased dramatically, they were not more tolerant to abiotic stress under the conditions tested. These results suggest that the over-expression of GolS and its product galactinol may serve as a molecular signal that initiates different metabolic changes for combating stress, culminating in the formation of tension wood. Additionally, over expression of raffinose synthase (RFS) resulted in increased biomass and total cellulose content. However, it does not appear to have a similar signalling role. Collectively, this research opens new insight about functions of the RFO in poplar, with the participation of GolS in stress signalling and consequent tension wood formation, and the importance of RFS to carbon allocation and growth.

View record

Cellulose synthases are the enzymes responsible for the production of cellulose in plant cell walls. Mutations in any one of the Arabidopsis cellulose synthase (CesA) AtCesA4, AtCesA7, and AtCesA8 genes cause plants to develop collapsed xylem as a result of reduced cellulose content, demonstrating their critical role in secondary cell wall biosynthesis. A thorough characterization of the growth, cell wall properties, and cellulose ultrastructure of the AtCesA4irx⁵-¹, AtCesA7irx³-¹, and AtCesA8irx¹-¹ mutants, presented herein, is the first report of the changes to cellulose microfibril angle, cell wall crystallinity, and cellulose degree of polymerization (DP) in these mutants. This study suggests that the non-redundant functions of individual CesAs may be related to CesA-specific thresholds required for the formation of a cellulose synthesizing complex (CSC), and CesA-specific roles in regulating crystallinity and DP. Additionally, the results illustrate the importance of a fully formed CSC in regulating cellulose microfibril angle. By identifying and characterizing three new CesA genes from spruce (Picea glauca), PgCesA1, PgCesA2, and PgCesA3, which are homologous to the Arabidopsis AtCesA8, A4, and A7 and the Populus trichocarpa PtiCesA8-A, A4, and A7-A genes, respectively, the degree of functional conservation among AtCesA homologs was explored. Expression of PgCesA1 or the PtiCesAs in AtCesAirx plants rescued the collapsed xylem phenotype, thus demonstrating for the first time that orthologs of AtCesA4, A7, and A8 have conserved functions. Lastly, in planta techniques were used to measure interactions between AtCesAs to investigate if specific and consistent interactions exist. The results suggest that CesA8 and A4 can form homodimers in planta, and that there might be weak or transient interactions between AtCesA7-A4 and AtCesA7-A8. Collectively, the results presented suggest, indirectly, an unequal ratio of CesA subunits (AtCesA4:A7:A8) is required for proper cellulose biosynthesis, and that each CesA likely has a unique function which ultimately affects cellulose properties such as cell wall crystallinity and DP. Our conclusions shed new light on the role of CesAs in cellulose biosynthesis in secondary cell walls and elicit questions about the current model of CSC form and function.

View record

Plant endoglucanses (E.C. 3.2.1.4) encompass multi-gene families across several plant clades, all belonging to the glycosyl hydrolase 9 (GH9) family. One class of GH9 enzymes is unique in that all members possess sequences that encode an N-terminal membrane-anchoring domain. This class of enzymes, termed membrane-bound endo-1,4-beta-glucanases, is the focus of this thesis. The most extensively studied enzyme was first discovered in Arabidopsis and was given the name KORRIGAN (KOR) because of the dwarfed phenotype and cellulose deficiency apparent in plants exhibiting KOR gene mutations. Research has principally focused on Arabidopsis and other non-tree species and the possible role that the enzyme might play in primary cell wall development and cellulose synthesis. However, very little research with KOR has been conducted on trees and secondary cell wall development. Consequently, I investigated the effects of mis-regulating KOR in hybrid poplar and white spruce. I was able to demonstrate that the down-regulation of the hybrid poplar KOR gene increases the crystallinity of the secondary cell wall cellulose and affects the relationship between cellulose and the hemicellulose cell wall components. Concurrently, we were the first to isolate and characterize the KOR gene and suppress KOR gene activity in white spruce. Expression of white spruce KOR in Arabidopsis kor1-1 mutants demonstrated that the gene is able to rescue the mutant phenotype, providing evidence for functional equivalence. Additionally, suppression of the gene in white spruce reduced growth and cellulose content. Since KOR has been demonstrated to be required in cells undergoing cellulose synthesis, we investigated whether or not the KOR protein and the cellulose synthase complex (CSC) interact. Although we were not able to provide evidence for any KOR-protein interaction, we were able to disprove the hypothesis that KOR interacts with CesA7, a member of the secondary cell wall CSC. Collectively, the expression, functional characterization, and interaction data suggest that KOR does not function in direct contact with the CSC, but rather that it plays a role in the later stages of cell wall development, presumably in the relaxation of the stresses around the cellulose microfibril or in the separation of putative cellulose macrofibrils.

View record

Metabolomics is an emerging field in functional plant biology that attempts to relate patterns in the molecular intermediates and products of metabolic pathways with genetic, gene expression, environmental and phenotypic traits - at the whole-tissue and/or whole-organism level. There is enormous potential for metabolomics tools to be applied in the study of tree species, and the demand for widespread application is promoting an ongoing evolution and refinement of newly-developed techniques. This body of research addresses the application of broad-scale, non-targeted metabolomics to questions of wood formation and quality in tree systems. Overall, it was shown that variation in metabolite profiles from developing xylem tissue was indeed correlated with the strength of specific phenotypic traits. Frequently, the strength of these relationships was such that phenotypic severity could be predicted accurately on the basis of metabolite profile data alone. The specific correlative patterns and metabolite/trait pairings observed in each study provided insight into the biological mechanisms by which these traits arise. Studies of secondary xylem development were conducted on breeding populations of Douglas-fir and radiata pine, as well as genetically modified hybrid poplar. In the Douglas-fir families studied, environment-induced variation in growth rate, fibre morphology and wood chemistry were correlated with metabolite profiles from developing xylem; metabolites involved in carbohydrate and lignin biosynthesis were primarily implicated in these relationships. Similarly, in juvenile trees from a series of radiata pine families, correlations were observed between metabolite profiles of developing xylem and the internal checking wood defect, a known heritable trait. In a different approach, two poplar hybrids, each modified separately with two exogenous gene constructs related to lignin biosynthesis, provided controlled model systems in which to investigate the interaction between genotype, metabolite profiles of developing xylem, and physico-chemical wood traits. Wood traits and metabolite profiles alike were altered by the genetic modifications, and it was found that the metabolic impact of the transgenes was not confined to pathways that were directly coupled to lignin biosynthesis. In fact, the scarcity of lignin-related metabolites in profiles from either the wild-type or modified genotypes suggested that metabolite channelling phenomena operate in the lignin biosynthetic pathway. Moreover, the analyses demonstrated that transgene-induced gradients in phenotypic traits could be associated with similar gradients within broad-scale metabolite profiles, and also that the wood-forming metabolisms of different poplar hybrids can respond similarly to the influences of genetic manipulation, at a global level. To conclude, the demonstrated associations between genotype, the metabolism of wood formation, and wood phenotype, as revealed by metabolite profiles, confirm the value of non-targeted metabolomics as a systems biology approach to understanding and modeling growth and secondary cell wall biosynthesis in trees.

View record

Plant invertases (EC 3.2.1.26) represent a multi-gene family of β-fructofuranosidases that perform integral roles in several biochemical processes. The central importance of this family of enzymes to plant growth and development has made them a primary target of investigation in plant biology. Research has principally focused on sink-source interactions, and the potential to increase sink capacity in several economically important crop species, including potato and tomato. However, studies exploring the impacts of invertase mis-regulation on cellulose and lignin, the two most abundant biopolymers on earth, had not been conducted. Consequently, we investigated the effects of overexpressing yeast-derived invertases in tobacco and hybrid poplar. Transgenic tobacco expressing the yeast-derived invertases showed reduced height and interference in sink-source metabolism. In addition, some transgenic lines showed significant changes in cellulose and lignin content, providing evidence that sink capacity can be altered via the overexpression of this class of enzyme. In contrast, hybrid poplar expressing foreign invertase genes showed no visible phenotype, with only minor changes to the structural polymers cellulose and lignin, suggesting the mechanism of carbohydrate transport differs between tobacco and hybrid poplar. However, there was evidence for post-translational modification of the foreign invertases in hybrid poplar, which may also explain the difference in phenotypes observed. We suggest that the yeast-derived invertases may not be the most effective target to alter sink biopolymers, and that mis-regulating endogenous invertases may be a more suitable alternative. Consequently, we identified three cell-wall invertase genes in hybrid poplar and investigated their spatial and temporal expression profiles during the complete first year of growth. In addition, we heterologously expressed and characterized two hybrid poplar cell-wall invertase genes involved in vegetative growth. Collectively, the expression and functional characterization data suggest that one floral-specific and two vegetative cell-wall invertases exist in hybrid poplar. Of the two vegetative cell-wall invertases, one (PaxgINV1) appears to be involved in processes relating to dormancy, while the other (PaxgINV2) appears to be involved in phloem unloading and the seasonal reallocation of carbohydrate. We therefore hypothesize that PaxgINV2 may be a suitable target for future mis-regulation studies aimed at altering sink capacity.

View record

UDP-glucose, the precursor for cellulose biosynthesis, can be produced via thecatalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation ofglucose-I-phosphate by UDP-glucose pyrophosphorylase (UGPase). As such, thesegenes, together with sucrose phosphate synthase (SPS) which recycles fructose (aninhibitor of SuSy), are interesting targets for altering carbon allocation in plants.In an attempt to alter cell wall biosynthesis in plants, targeted overexpression ofSuSy, UGPase and SPS independently and in a pyramiding strategy was assessed intobacco. All lines displayed enhanced growth and biomass production, and in the caseof double and triple transgenics, there was an additive effect. Despite the increasedgrowth rates, there was no consistent change in soluble carbohydrate pools.Furthermore, only the triple transgenics had constant changes in structuralcarbohydrates: with increased hemicellulose content and slight increases in cellulose.Collectively, these results support the role of SPS, SuSy and UGPase in maintainingsink strength, but suggest that the reallocation of carbon to cellulose production intobacco may not be possible by overexpressing these genes.In contrast, transgenic poplar overexpressing UGPase produced significantlymore cellulose than wild-type trees. However, this was accompanied by a severereduction in growth and the production of a salicylic acid glucoside (SAG) in significantquantities. The UDP-glucose generated by UGPase overexpression appeared toparticipate in both the synthesis of cellulose and SAG, suggesting that cellulosebiosynthesis may be limited by the cellulose synthase complex.Poplar transformed with SuSy and with SuSy x UGPase also had increasedcellulose production. The trees were phenotypically normal, with only minor reductionsin height growth in some lines. It appears that UDP-glucose may be channelled directlyto the cellulose synthase complex by SuSy. The increased cellulose content wasassociated with an increase in cell wall crystallinity, but there was no change inmicrofibril angle, confirming the re-allocation to cellulose synthesis was not the result oftension wood formation, again supporting the hypothesis that the cellulose synthasecomplex is the limiting factor.Clearly, it is possible to alter cellulose deposition in trees by augmenting sucrosemetabolism to produce UDP-glucose, the precursor to cellulose biosynthesis.

View record