Shawn Mansfield

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Mapping xylan biosynthesis in plant Golgi and teaching biology using example answers (2019)

No abstract available.

Functional analysis of KNOTTED-like homeobox and OVATE family proteins involved in secondary cell wall development in Arabidopsis (2018)

No abstract available.

Mitigating the downstream effects of excess soil phosphorus through cultivar selection and increased foliar resorption (2018)

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.

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The cell biology of cellulose deposition in secondary cell walls of protoxylem tracheary elements in Arabidopsis thaliana (2018)

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.

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COBRA-like4 : a GPI-anchored protein functioning as a mediator of cellulose ultrastructure in herbaceous and woody plants (2015)

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.

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Elucidating the function of arabinogalactan proteins during wood formation (2014)

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.

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Pectin methyl esterification functions in seed development and germination (2014)

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.

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Evaluating the role of the raffinose family of oligosaccharides in hybrid poplar (2012)

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.

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Investigating the role of cellulose synthases in the biosynthesis and properties of cellulose in secondary cell walls (2011)

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.

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Function, functional conservation and interactions of the membrane-bound endo-1,4-beta-glucanases orthologous to Korrigan (2010)

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.

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Metabolomic analyses of wood attributes in tree species (2009)

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.

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An investigation of the physiological roles and enzymatic properties of invertases in tobacco and hybrid poplar (2008)

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.

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Modification of cellulose biosynthesis through varied expression of sucrose metabolism genes in tobacco and hybrid poplar (2008)

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.

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Master's Student Supervision (2010 - 2018)
Characterization of two udp glycosyltransferase genes from hybrid poplar (2017)

Glycosyltransferases (GTs) play important roles in plant growth and development. The biological functions of many GTs are unknown. In the present study, two putative GT genes (PopGT1 and PopGT2) were cloned and their biological roles in growth and development of Arabidopsis and hybrid-poplar were investigated. In silico, in vitro, and in vivo methods were used to characterize the two encoded proteins. Phylogenetic analysis, enzyme activity assays, and transcript abundance were studied. In addition, plant growth and development, leaf morphology, stem anatomy, cell wall composition, biomechanical properties, soluble carbohydrate, and phenolic metabolite contents were determined. The results indicated that PopGT1 showed high similarity to tobacco salicylic acid glycosyltransferase, and both PopGT1 and PopGT2 (annotated as AtUGT74F2) were clustered within phylogenetic group L of family-1 GTs (UGTs). In vitro characterization of the two recombinant proteins indicated that PopGT1 glycosylated several flavonoids, showed only trace activities towards cinnamic and indole butyric acid, and accepted UDP-glucose as a sugar donor. The optimum temperature and pH for in vitro PopGT1 activity was 35 ºC and pH 7.5, respectively. PopGT2 showed no enzymatic activity towards any substrates.The two coding sequences (PopGT1 and PopGT2) were cloned in the pSM3 expression vector and over-expressed in Arabidopsis plants to investigate their in vivo functions. Phenotypically, plant height, stem diameter, rosette diameter, and stem number increased significantly in the transgenic plants. In addition, rosette morphology and root gravitropism were altered. Transgenic plants flowered earlier than the control plants. Chemically, cell wall compositions and phenolic metabolite contents changed significantly. In parallel, transgenic trees showed changes in leaf morphology, stem diameter, phloem fibre arrangement, and early bud break. Wood density was reduced revealing a brittle-stem phenotype. Marginal increases in lignin and reductions in cellulose content were apparent. Salireposide content was reduced in the bark of transgenic trees. The results indicated that altering the expression of both genes in Arabidopsis and poplar affected plant growth and development, cell wall composition, phenolic metabolite profiles, and wood biomechanical properties. PopGT1 showed in vivo substrate specificity towards kaempferol and promiscuous in vitro enzyme activity. However, the substrate of PopGT2 remains unclear.

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Identification and characterization of a poplar ECERIFERUM2-LIKE, a clade II BAHD acyltransferase from hybrid poplar (Populus alba × grandidentata) (2017)

BAHD acyltransferases belong to a large family of enzymes that are involved in the biosynthesis of a wide variety of biologically diverse and important plant compounds. Poplar makes an assortment of different metabolites and therefore has a large number of putative BAHD-acyltransferases, of which only a few have been characterized to date. In this study, an uncharacterized BAHD acyltransferase, with a putative function in either non-canonical monolignol biosynthesis as the poplar p-hydroxybenzoate-CoA monolignol transferase (pHBMT), or in cuticular wax biosynthesis through very long chain fatty acid (VLCFA) elongation as a poplar ECERIFERUM2-LIKE (CER2-LIKE), was studied. To test the function of Populus alba × grandidentata acyltransferase-like (Pa×gACT-like), transgenic Arabidopsis thaliana and hybrid poplar plants mis-regulating the gene, were generated and subsequently analyzed for changes in both lignin p-hydroxybenzoylation and in the cuticular wax composition. No changes in lignin p-hydroxybenzoylation was observed, negating the possibility of Pa×gACT-like being a p-hydroxybenzoate-CoA monolignol transferase. The introduction of Pa×gACT-like into Arabidopsis caused the accumulation of aliphatic wax components 28 carbons in length (C₂₈), and a reduction in C₃₀ wax components, likely due to competition with the native AtCER2 or slight differences in substrate specificity between Pa×gACT-like and AtCER2. In addition, RNAi-suppression of Pa×gACT-like in poplar resulted in a subtle phenotype showing a trend for accumulation of C₂₈ wax components, suggesting an upstream “block” in VLCFA elongation. The shifts in cuticular wax chain length distribution detected in transgenics support a CER2-LIKE role for Pa×gACT-like. Consistent with this, heterologous gene expression in yeast (Saccharomyces cerevisiae) clearly demonstrated that Pa×gACT-like can participate in the elongation of very long chain fatty acids. Co-expression and tissue-specific expression also support a role in cuticular wax biosynthesis, as 30% of genes co-expressed with Pa×gACT-like have predicted functions in lipid biosynthesis/metabolism. Finally, GUS histochemical staining of Pa×gACT-like Prom::GUS poplar transgenics revealed expression in the epidermis of leaf, petiole, and young stem tissue. This study reveals a function for a previously uncharacterized poplar BAHD acyl transferase as a CER2-LIKE protein that functions in the elongation of VLCFAs for cuticular wax production.

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Investigating the role of sucrose phosphate synthase and hexokinase in carbon sink strength. (2015)

The production, transport and assimilation of organic carbon ultimately drive the growth of plants. In this work, two enzymes, Sucrose Phosphate Synthase (SPS) and Hexokinase (HXK), prominent in their role of carbon production in the form of sucrose at the source, have been examined for their role at the sink, where carbon is assimilated. It has been postulated that the presence of sucrose-forming enzymes in the sink serves a function to reform sucrose from apoplastic cleavage or partake in a “futile” cycle of sucrose cleavage and such that small changes in metabolite enable large changes in sink carbon strength. In order to determine if SPS is involved in carbon sink strength, A. thaliana TDNA insertional lines and P. trichocarpa RNAi stem and developing xylem with decreased SPS transcript expression were analyzed. It was determined that loss of SPS transcript generally increases soluble sugars: sucrose, glucose and fructose, in the leaf and stem as well as starch in the leaf. Structural carbohydrates were generally unaffected and Klason soluble lignin decreased. Similarly, A. thaliana TDNA insertional lines with decreased HXK transcript expression were utilized to determine the role of HXK using stem tissue as a carbon sink model. Soluble sugars mainly increased in the leaf of athxk3 TDNA insertional line whereas starch increased in both leaf and stem of the same line. Interestingly, structural carbohydrate levels of the cell wall were perturbed in HXK TDNA insertional lines. The results were found to be consistent with the postulated roles of SPS and HXK that predict a function in sucrose formation from apoplastic cleavage, which allows for fine-tuning of major intracellular metabolites and adjustment of sink strength.

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The impact of endogenous acetylation on the deconstruction of Populus trichocarpa wood during pretreatment (2015)

Wood is a renewable, sustainable and economic resource. Non-cellulosic wood polysaccharides are acetylated in a species-dependent and spatially-regulated manner. Acetyl groups comprise approximately 5% of the dry weight of poplar wood. Biologically, acetyl groups increase xylan chain solubility and may therefore influence secondary cell wall formation by affecting the association of hemicelluloses with the other cell wall components, such as cellulose, lignin, and the pectic and proteinaceous constituents of the adjacent primary cell wall. In this research, we found that acetyl content correlated positively with lignin (R = 0.28) and negatively with cellulose (R = -0.41) in wood samples from a common garden of over 200 unrelated Populus trichocarpa individuals. During the pretreatment of biomass, acetyl groups are hydrolyzed from wood to form free acetic acid in the reaction media. The present research examined the relationships between wood composition and pretreatment sugar yield of 19 P. trichocarpa genotypes with varying levels of acetylation. The results clearly show a strong correlation (R > 0.77) between acetic acid and polysaccharide dissolution. Fast-reacting xylan had a degree of acetylation of 0.35, while slow-reacting xylan had a degree of acetylation of 0.73. Hydrophobic interactions could explain the negative correlation between lignin content and acetic acid released (R = -0.59). This research highlights the impact acetylation may have on the large-scale industrial utility of plants.

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Investigating the role of sucrose synthase in Arabidopsis thaliana and hybrid poplar (2013)

Sucrose synthase (SuSy) cleaves sucrose to produce fructose and UDP-glucose, the latter serving as the precursor for cellulose biosynthesis. Sucrose synthase often exists as many isoforms in plants; for example, Arabidopsis has six isoforms. Along with it role in routine metabolism, sucrose synthase is also important during stress conditions. For example, during hypoxia plants can accumulate reduced substances, often to phytotoxic levels, as well as displaying energy deprivation stemming from the reduced levels of oxygen. Given that SuSy is more energetically efficient than invertase it therefore plays important roles in combatting hypoxic stress. This thesis describes transgenic studies involving functional complementation of sus1/sus4 Arabidopsis mutants, as well as overexpression of exogenous SuSy in wild-type Arabidopsis plants under hypoxia to determine if the Populus trichocarpa (Pt) SuSy1 and 2 genes are functionally conserved orthologs of the Arabidopsis SuSy1 and SuSy4 isoforms. Real-time PCR and microscopy were employed quantify and observe the YFP reporter, respectively. Changes in Arabidopsis plant stem height under hypoxia documented abnormalities in plant growth. Under hypoxia, sus1/sus4 Arabidopsis plants displayed slowed stem growth, while all transgenic lines (complemented sus1/sus4 and over-expressed wild-type plants) grew normally. Histochemical staining of stem cross sections, demonstrated that the sus1/sus4 mutants subject to hypoxia displayed thickened xylem cell walls, and possessed increased in cell wall glucose content and hemicellulose-derived carbohydrates. Together, the findings suggest the conserved functionality of the PtSuSy1 and PtSuSy2 orthologs. Real-time PCR was employed to determine the transcript abundance of PtSuSy1 and PtSuSy2 in poplar in several tissues (phloem, cambium, and leaf) collected over 24 hours and a complete growing season. Examination of SuSy in the three tissues revealed a minor role for SuSy1 and SuSy2 in source tissues as levels of SuSy expression were consistently lower in both diurnal and annual samples. In all samples, PtSuSy1 was consistently more highly expressed than PtSuSy2 suggesting a more essential role in sucrolysis during active growth. The role of SuSy in sucrolysis was further reinforced as the expression of SuSy in both diurnal and annual tissues was constantly associated with plant growth during which UDP-glucose would be in high demand.

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Wood quality and growth characterization across intra- and inter-specific aspen hybrid clones (2013)

Trembling aspen (Populus tremuloides Michx) is Canada’s most abundant poplar species; it is native, found nationwide, and is an ecologically and economically important hardwood species. As the demand for raw material continues to rapidly increase, there is an incentive to improve tree quality and growth rates through breeding, particularly in fast-growing species suitable for the Canadian landscape. Hybridization is considered one of the best options to accelerate tree productivity and improve wood quality. Two aspen species showing particular promise for hybridization with trembling aspen are European aspen (P. tremula) and Chinese aspen (P. davidiana) because their native climates are similar to that of western Canada. In 2003, poplar clones were planted in Athabasca, Alberta from the following species crosses: open pollinated (OP) P. tremuloides (NN), OP P. davidiana (CC), P. tremula × P. tremula (EE), P. tremula × P. tremuloides (EN), and P. tremuloides × P. davidiana (CN). In, November 2010, growth measurements and core samples were taken from the clones. Productivity was quantified through analysis of stem volume. Wood quality attributes were quantified via an assessment of fibre length, fibre width, coarseness, wood density, microfibril angle, total cell wall carbohydrate and lignin content, and lignin composition. Comparisons of the mean values for each attribute were made between crosses using generalized linear model least squares means tests. The NN cross had lower volume growth than the CC and EE crosses. The EN and CN crosses had greater volume than the NN cross. The NN cross had shorter fibre length, but greater syringyl-guaiacyl ratio (S:G) relative to the CC cross. It also had lower density and S:G compared to the EE cross. The EN cross had longer, wider fibres and a greater carbohydrate concentration compared to the NN cross. The CN cross had greater fibre length and lignin concentration, but lower S:G compared to the NN cross. The results indicated that the inter-specific crosses were more desirable wood sources than the pure P. tremuloides cross. Specifically, the P. tremula x P. tremuloides cross showed the best potential to improve future generations of aspen on the Canadian landscape.

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Wood quality of trembling aspen (Populus tremuloides Michx) and white spruce (Picea glauca (Moench) Voss) in the boreal mixedwood forest (2013)

The boreal forest is the most widespread forest type in Canada, with a large percentage represented by mixedwood forests of white spruce (Picea glauca (Moench) Voss) and trembling aspen (Populus tremuloides Michx). It serves as not only a vast ecological reserve, but also as the supply source for forest-based industries. A better understanding of the interactions between the different species and their affects on productivity and wood quality traits helps create a more efficient industry that better utilizes the available resources, and concurrently preserves as much forest of ecological reserves and societal vistas. In this study, three sites composed of trembling aspen and white spruce, with varying compositions (one composed of mainly aspen, one of mainly spruce, and a mixed site with both species) were compared to determine how the presence of one species affects the growth and wood quality traits of the other. Four main wood quality traits were examined: wood density, microfibril angle (MFA), fibre traits (fibre length, fibre width and fibre coarseness) and cell wall chemistry. Along with site comparisons, social classes were determined for each site in an attempt to provide a more in-depth comparison across sites.Wood density showed very little variation among sites for both species, with only significant variations occurring between social classes. The aspen site showed statistically lower MFAs than the aspen from the mixed site, however, no differences were observed between the spruce from the mixed and spruce sites. Fibre length, width and coarseness were higher in the pure species sites for both trembling aspen and white spruce. In terms of cell wall composition, there were no differences in carbohydrate contents across sites for both species. Lignin content did vary, with the aspen site possessing higher lignin content than the mixed site, while for spruce the spruce site showed a lower lignin content. Overall, the use of social classes did not refine the characterization of site, producing similar results to those obtained when comparing trees by site, regardless of class.

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Role of two Populus trichocarpa endo-beta-1,4-glucanases and their Arabidopsis orthologs in plant cell wall development (2012)

Plant cell walls are imperative to the normal growth and development of plants as they serve many functions, including protecting the protoplast and providing rigidity to the stem. In this study two poplar and Arabidopsis endoglucanases, which have been hypothesized to play a role in secondary cell wall development, were examined. The Class B endoglucanases, PtGH9B5 and AtGH9B5, are secreted enzymes that have a predicted GPI anchor, while the Class Cendoglucanases, PtGH9C2 and AtGH9C2, are also predicted to be secreted but contain acarbohydrate-binding module (CBM). The poplar endoglucanases were up-regulated inArabidopsis using a 35S promoter as well as the Arabidopsis CesA8 promoter, respectively.Additionally, Arabidopsis t-DNA insertion lines of each Arabidopsis gene were analyzed, and anRNAi construct was created to down-regulate AtGH9C2 in Arabidopsis. All of the transgenicplant lines were examined for changes in cell morphology and patterning, growth anddevelopment, cell wall crystallinity, microfibril angle, and proportion of cell wall carbohydrates.Mis-regulation of PtGH9B5/AtGH9B5 resulted in changes in glucose and xylose content,suggesting that this endoglucanase may be involved in regulating the amount of celluloseand/or xylans in the developing secondary cell wall. Furthermore, mis-regulation ofPtGH9C2/AtGH9C2 resulted in a change in crystallinity, which was inversely correlated with a change in plant height and rosette diameter. This suggests that this endoglucanase may beinvolved in modifying cell wall crystallinity at the time of primary growth cessation and/or earlysecondary cell wall development. Together, these results support the role of these endoglucanases in secondary cell wall development, though their exact enzymatic function remains to be discovered.

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Effects of drought stress on metabolite profiles of hybrid and pure lines of Populus spp. (2010)

Drought stress is perhaps the most commonly encountered abiotic stress plants experience in the natural environment, and is one of the most important factors limiting plant productivity. In the studies described in this text, drought-stressed poplar trees (Populus spp.) were analyzed for their metabolite content using the technique of untargeted metabolite profiling by employing gas chromatography coupled to mass spectrometry (GC/MS). The purpose of these analyses was to characterize the metabolite profile of poplar trees under drought stress in order to assess relative drought resistance and to investigate what mechanisms might be employed in the ability to resist drought. To do this, three independent experiments were carried out, each employing different poplar tree species and drought application protocols. For all three experiments, metabolite profiling identified key metabolites that increased or decreased in relative abundance upon exposure to drought stress. Overall, amino acids, the antioxidant phenolic compounds, catechin and kaempferol, and the osmolytes raffinose and galactinol exhibited increased levels under drought stress, whereas metabolites involved in photorespiration, redox regulation, and carbon fixation showed decreased levels under drought stress. One clone in particular, Okanese, in common with Experiments #1 and #2 described in this thesis, displayed unique responses to the imposed drought conditions. This clone was found to have higher stomatal conductance, transpiration rate, and leaf water potential, and lower growth rate in Experiments #2 and #1, respectively. Okanese also had lower accumulation of osmolytes such as raffinose, galactinol and proline, but higher overall levels of antioxidants such as catechin and dehydroascorbic acid. As such, it was proposed that osmotic adjustment as a mechanism for drought resistance in this clone is not as well developed in comparison to other clones investigated in this thesis, and that a possible alternative mechanism for the enhanced drought resistance displayed by Okanese may be due to differential allocation of resources towards root growth rather than osmotic adjustment.

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