Anne Lacey Samuels

Prospective Graduate Students / Postdocs

This faculty member is currently not looking for graduate students or Postdoctoral Fellows. Please do not contact the faculty member with any such requests.


Research Classification

Research Interests

plant cell biology
plant cell walls

Relevant Thesis-Based Degree Programs

Affiliations to Research Centres, Institutes & Clusters


Research Methodology

cryo fixation; electron microscopy
live cell imaging

Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Monolignol export by sink-driven diffusion in lignifying plant biomass (2022)

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. The question of how lignin precursors (monolignols) move from inside the cells, where they are synthesized, across the cell membrane to the cell walls, where they are polymerized by laccase and peroxidase enzymes, has remained unanswered. Computer modelling indicates that monolignols could passively diffuse through lipid bilayer membranes, however, this has not been studied experimentally. This study tests the hypothesis that monolignol diffusion occurs when monolignols in the cell wall are consumed by lignin polymerization, due to the activity of oxidative enzymes like laccases and peroxidases. Liposomes containing monolignol-polymerizing laccase enzymes were developed as a model system to test monolignol diffusion across synthetic lipid bilayers. Not only did diffusion across the membrane occur, but lignin-like polymers formed within the liposomes. In parallel, two-photon microscopy of lignifying Arabidopsis root xylem vessels was used to visualize lignin polymer in the cell wall and soluble phenolics inside cells. Loss of function laccase mutants had monolignols accumulation in the vacuoles of neighbouring cells as detoxified monolignol glucosides, consistent with a diffusion-based transport (Chapter 2). The interaction of monolignol production and consumption was studied by using plants with upregulation in monolignol synthesis and overexpression of laccases. Monolignol over-producing lines were severely dwarfed, while the addition of laccase in the cell walls restored wild type growth and metabolic status. This suggests that the balance of monolignol production and consumption is finely controlled and has wide reaching transcriptional and developmental consequences when perturbed (Chapter 3). When lignin monomers were over-produced or left unpolymerized, increased monolignol glucosides stored in the vacuole were observed, and this was correlated with increased transcription of ATP binding cassette (ABC) transporters ABCC6 and ABCC7. The double mutant of abcc6abcc7 was generated by CRISPR-Cas9 gene editing, but there was no apparent change in phenolic transport into vacuoles. It may be that additional redundancy with other ABCC transporters is obscuring detectable phenotypes (Chapter 4). This thesis illustrates a new paradigm for monolignol export across biological membranes by sink-driven diffusion, and the challenges of investigating monolignol transporters and lignin-modification induced dwarfism.

View record

Developmental cell biology of specialized metabolite production and storage in cannabis glandular trichomes (2021)

Glandular trichomes on the flowers of Cannabis sativa L. (cannabis) produce and store a wealth of cannabinoids and terpenes, the metabolites consumed for both medicinal and recreational purposes around the world. Substantial progress has been made towards elucidating the enzymes involved in metabolite biosynthesis, as well as the diversity of metabolites produced. In contrast, we know very little about the cellular processes that facilitate the sheer abundance of metabolite biosynthesis and storage within the glandular trichomes. To fill this gap in knowledge, I used several microscopical approaches to assess the structure and composition of glandular trichomes throughout their development. A combination of fluorescence microscopy and scanning electron microscopy provided evidence that stalked trichomes develop from sessile-like trichomes during floral development, whereas bona fide sessile trichomes can be distinguished from a stalked trichome based on disc cell number, intrinsic fluorescence, and metabolite organization within their storage cavity. Evaluation of the glandular trichome ultrastructure with cryofixation sample preparation for transmission electron microscopy (TEM) led me to propose a new model of cannabinoid trafficking and secretion. An abundance of membrane contact sites near the apical plasma membrane suggests lipophilic metabolites may traffic from plastids to plasma membrane by partitioning into membrane bilayers and subsequent lipid exchange at membrane contact sites. The cell wall of the cannabis trichome was characterized using glycomic analysis followed by immunolocalization of cell wall-directed antibodies, which revealed dynamic changes in xyloglucan, pectin, and glycoproteins in the glandular trichome cell wall. Together, this thesis provides insights into the cell biology of cannabis glandular trichomes, in particular, how they produce and store abundant specialized metabolites.

View record

Mapping xylan biosynthesis in plant Golgi and teaching biology using example answers (2019)

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

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

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

Functional genomic analysis of novel secondary cell wall genes in poplar (Populus trichocarpa) (2018)

Secondary cell walls (SCWs) contain a significant amount of fixed carbon that can be harnessed for the production of renewable energy. However, efficient conversion of wood-based biomass for use as an alternative fuel source is constrained by lignin, a phenolic polymer that is recalcitrant to enzymatic degradation. Many aspects of SCW biosynthesis remain enigmatic, including how genes of broad functional classes affect lignin content and composition. A genetic association mapping (AM) study in poplar (Populus trichocarpa) previously identified novel genes genetically associated with lignin trait variation. To test the hypothesis that these genes influence SCW biosynthesis, I screened 27 lignin-associated genes using in silico analyses and transfer-DNA (T-DNA) mutant phenotyping of Arabidopsis (Arabidopsis thaliana) homologs and identified two genes for in-depth functional characterization. First, Coiled-coil Protein of Unknown Function (CPU) was identified to be highly expressed in xylem and co-expressed with well-known SCW-related genes including SND1, a key transcriptional regulator of SCW biosynthesis in fibres. While AM predicted CPU to be significantly associated with total lignin content variation, this was not found in transgenic poplar over-expressing poplar CPU. Instead, transgenic poplars exhibited altered fibre length compared to wild-type. In Arabidopsis, a genetic interaction for CPU and Cellulose-Microtubule Uncoupling (CMU) was identified as the cpucmu1cmu2 triple mutant had decreased SCW thickening in fibre cells compared to wild-type, suggesting CPU to also influence microtubules during SCW deposition. Secondly, P. trichocarpa Nitrate Peptide Family 6.1 (PtNPF6.1), a member of the nitrate1/peptide transporter superfamily was characterized. PtNPF6.1 is expressed in the vascular tissue, as detected from transgenic PtNPF6.1pro:GUS lines. Transgenic poplar suppressed in PtNPF6.1 had elevated levels of total nitrogen corresponding to elevated levels of free glutamic acid and aspartic acid compared to wild-type. Under luxuriant nitrogen conditions, PtNPF6.1-suppressed lines produced wood with less syringyl lignin compared to wild-type. The findings suggest PtNPF6.1 may help regulate the long-distance transport of nitrogen. Altogether, previously unsuspected classes of genes identified through AM has broadened our understanding of genes that impact the cellular and physiological processes that contribute to wood formation which may enable further optimization of woody plants for a diversity of applications including bioethanol production

View record

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.

View record

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.

View record

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.

View record

Good neighbours: The role of non-lignified cells in arabidopsis lignification (2014)

Lignin is a critical structural component of plants, providing vascular integrity and mechanical strength. Lignin precursors, monolignols, must be exported to the extracellular matrix where random oxidative coupling produces a complex lignin polymer. The objectives of this study were twofold: to determine the timing of lignification, with respect to programmed cell death during Arabidopsis thaliana primary xylem development, and to determine which cells are contributing to the lignification of tracheary elements and fibres. This thesis demonstrates that lignin deposition is not exclusively a post-mortem event, but also occurs prior to programmed cell death. Radiolabelled monolignols were not detected in the cytoplasm or vacuoles of tracheary elements or neighbours. To experimentally define which cells in lignifying tissues contribute to lignification in intact plants, a microRNA against CINNAMOYL CoA-REDUCTASE1, driven by the promoter from CELLULOSE SYNTHASE 7 (proCESA7:miRNA CCR1), was used to silence monolignol biosynthesis in cells developing secondary cell walls. When monolignol biosynthesis was knocked down specifically in the cells with thickened secondary cell walls, but not in the neighbouring cells, lignin was still deposited in the xylem secondary cell walls. This indicates that “good neighbour” cells are sufficient to produce lignin in the vascular bundles. Surprisingly, this was not the case in the interfascicular fibres, where a dramatic reduction in cell wall lignification demonstrates that these extra-xylary fibers undergo cell autonomous lignification. When a fibre-specific promoter (proAtPEROXIDASE64) was used to drive the miRNA, autonomous extraxylary fibre lignification was again observed, as was non-cell autonomous lignification between xylary fibres and neighbouring tracheary elements. These effects may have reflected compensatory mechanisms in response to lignin downregulation, so to demonstrate that discrete cell populations, such as xylem parenchyma, do contribute to lignification, genes encoding enzymes catalyzing the synthesis of novel monolignol conjugates were introduced into wild-type Arabidopsis using cell population-specific promoters. The detection of novel monolignol conjugates in the cell wall by chemical analysis and fluorescence microscopy supported the contribution of tracheary elements and fibres to lignification and also revealed that xylary parenchyma cells are producing monolignol substrates and acting as “good neighbours” to tracheary elements and xylary fibres during lignification.

View record

Plant lipid trafficking: the cell biology of cuticular lipid export and membrane contact sites of Arabidopsis thaliana (2013)


View record

The role and behavior of Arabidopsis thaliana lipid transfer proteins during cuticular wax deposition (2012)

The primary aerial surfaces of terrestrial plants are coated with a protective hydrophobic layercomprising insoluble and soluble lipids. The lipids are known collectively as cuticular wax. Togenerate the waxy cuticle during elongative growth, plants dedicate half of the fatty acidmetabolism of their epidermal cells. It is unknown how cuticular wax is exported from the plasma membrane into the cell wall, and eventually, to the cuticle at the cell surface. I hypothesized that lipid transfer proteins (LTPs) were responsible for plasma membrane to cell wall transport of cuticular lipids. Using an epidermis-specific microarray, I identified five candidate Arabidopsis LTPs. I discovered that mutations in gene At1g27950 result in a stem wax phenotype: reduced cuticular lipid nonacosane resulting in reduced total wax compared to wildtype. This gene encodes a glycosylphosphatidylinositol (GPI)-linked LTP and thus was namedLTPG. In contrast, to LTPG, no detectable wax phenotype was found in mutants for classical LTPs. Inphylogenetic analyses, these LTPs clustered into a weakly related group that I named LTPAs. Inan attempt to overcome genetic redundancy I made double and triple mutants from the candidateLTPAs. None of these mutants displayed detectable changes in wax compared with wildtype.Using live cell imaging, I showed that LTPG is localized to the epidermal cell plasma membraneand the cell wall and accumulates non-uniformly on the plant surface. I employed fluorescencerecovery after photobleaching to demonstrate that, in the plasma membrane, LTPG is relativelyimmobile and exhibits a complicated recovery, the latter appears linked to the flux of cuticular lipids through the plasma membrane. LTPG accumulates over the long cell walls of stemepidermal cells and this protein moves when observed over 1 min intervals. I created a GPIlinkedLTPA and demonstrated that it can rescue the ltpg-1 mutation.I demonstrate that LTPG is required for wax export by associating with the plant cell wall. This is the first experimental evidence linking the lipid transfer function of a plant LTP to a biological role, which in this case is lipid movement through the cell wall to the cuticle.

View record

Elucidation of Secondary Cell Wall Secretion Mechanisms of Arabidopsis Thaliana, Poplar (Populus Deltoides X P. Trichocarpa), and Pine (Pinus Contorta) (2009)

No abstract available.

Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Exploring Cannabis sativa's response to powdery mildew (Golovinomyces ambrosiae) infection (2022)

Powdery mildew infection, which is caused by the fungal pathogen Golovinomyces ambrosiae (Erysiphaceae), has been extensively characterized in Arabidopsis (Brassicaceae), and other species, but not in Cannabis sativa (Cannabaceae), a commercially important plant species. In this thesis, I explore the response of C. sativa to powdery mildew infection. I used microscopy to examine the phenotypic response and RNA-sequencing to examine the transcriptomic response to powdery mildew. I also examined differences between resistant (AGA10) and susceptible (Purple Kush) genotypes. Microscopy revealed that there are differences between the two genotypes in germination and hypersensitive response (programmed cell death). RNA-sequencing revealed differences in expression of genes encoding enzymes of the phenylpropanoid and monolignol biosynthetic pathways, phytoalexin production, and other plant-pathogen responses. Gene ontology (GO) functional analysis shows that the two genotypes also differ in expression of genes related to carbohydrate metabolic processing and other enzymatic processes. I conclude that a likely main factor behind the AGA10 resistant phenotype is increased reactive oxygen species and hypersensitive response, due to earlier or increased expression of genes such as oxidoreductases, peroxidases, phenylalanine ammonia lyase and trans-cinnamate 4-monooxygenase.

View record

Investigating early land plant cuticular waxes using physocomitrella patens as a model species (2022)

Cuticular wax research in Arabidopsis thaliana and other important model plant species has led to the elucidation of biochemical pathways for aliphatic wax synthesis in flowering plants, however, we still lack a critical understanding of how these important pathways developed in early land plants. Physcomitrella patens is a model moss species that is used in laboratory research and is believed to live in habitats similar to those found roughly 500 million years ago. Thus, the cuticle present in Physcomitrella can be used to explore the nature of the cuticles from early land plants. The goal of this thesis was to assess whether the proteins involved in production of wax components for the Physcomitrella cuticle are similar to those that produce the well-studied Arabidopsis cuticle. Six hypothesized cuticular wax genes were discovered in Physcomitrella based on sequence similarity to homologs known to be involved in wax biosynthesis in Arabidopsis. PpCERA, PpCERB, PpCERC, and PpCERD were discovered to have close sequence similarity to AtCER1 and AtCER3, while PpFAR1 and PpFAR2 displayed sequence similarity to AtCER4. Knock-out mutants were generated for each of the Physcomitrella candidate genes, except for Ppfar1, and the ensuing cuticular waxes were analyzed via gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectroscopy (GC-MS). A complementation experiment with Physcomitrella genes in knock-out mutants of the homologs in Arabidopsis was performed to determine if the moss genes could complement the wax-deficient phenotype. The Physcomitrella genes were also expressed in yeast to investigate their biological function, but these studies were discontinued due to technical difficulties. Physcomitrella mutants demonstrated that PpFAR2 participates in the production of primary alcohols and functions in the primary alcohol pathway. PpCERA, PpCERB, PpCERC, and PpCERD participate in the production aldehydes and alkanes. When PpCERC and PpCERD were expressed Arabidopsis alkane pathway mutants, Atcer1-4 and Atcer3-6, the wax deficient phenotype was complemented, and alkane pathway functionality was restored. Expression of PpCERC and PpCERD in Atcer4-3 failed to restore the primary alcohol pathway, suggesting that these enzymes may function in the alkane, not primary alcohol, pathway in Physcomitrella.

View record

Investigating Golgi retrograde dynamics through the xylan synthase complex in Arabidopsis thaliana (2022)

Xylan is a key hemicellulose in the plant secondary cell wall that contributes to structural integrity. As the third most abundant biopolymer in the world, xylan is an environmental cornerstone in woody species, thus it is important to understand the proteins that manufacture xylan. The xylan synthase complex (XSC), the enzymatic complex responsible for manufacture of the xylan backbone, is a group of Golgi resident proteins that maintains its position in the medial to trans-Golgi through unknown Golgi retrograde activity. To evaluate potential models of retrograde activity, two prevalent endomembrane transport systems were investigated: inter- Golgi tubule connections and coat protein 1 (COPI) vesicles. The effect of COPI vesicle inhibition was assessed by measuring secondary cell wall deposition and the growth phenotype of mutants of the small GTPase ARF1, which is required for COPI vesicle recruitment. COPI inhibition did not impact secondary cell wall formation. The drug brefeldin A (BFA) was also used to inhibit COPI vesicle formation, and the effect monitored with live cell imaging, but no change in the xylan biosynthetic enzyme was observed. Golgi resident protein dynamics were also investigated to determine the ability for proteins to move between Golgi stacks. Using a photoconvertible fluorophore, mEos, tagged to a component of the XSC, IRREGULAR XYLEM 9 (IRX9), sub-populations of the protein complex in individual Golgi could be tracked within a single cell. This tracking revealed the ability for resident proteins to move across Golgi stacks through an unknown trafficking method. This research reframes inter-Golgi trafficking models away from COPI and highlights the possibility that xylan biosynthetic enzymes can move between the scattered stacks of the plant Golgi.

View record

Identification and characterization of a cellulose synthase from the calcifying red alga Calliarthron tuberculosum (2021)

In land plants and algae, cellulose, a glucose polymer, is important for strengthening tissues and preventing breakage in the face of physical forces. Cellulose synthase enzymes (CESA) are responsible for producing cellulose. While our understanding of land plant CESAs has advanced for several species, it is unclear whether the systems surrounding cellulose synthesis are the same in all plant lineages. For example, no red algal CESAs have been functionally demonstrated. The objective of this thesis is to discover and characterize putative CESA encoding genes from the calcifying red alga Calliarthron tuberculosum and compare their function to those from the land plant eudicot Arabidopsis thaliana.Using a bioinformatics approach, I identified three candidate CESAs from Calliarthron tuberculosum’s transcriptome dataset (CtCESA1, CtCESA2, and CtCESA3). I explore the evolution of CESAs in gene tree analysis and find that while CtCESA1 was closely related to other red algal CESA sequences, CtCESA2 and 3 were more closely related to bacterial cellulose synthases (Ch 2). Using yeast and insect cell expression systems, I heterologously express, purify, and test the CtCESA1 protein in glucose tracer assays to look for polymer formation. CtCESA1 showed evidence of glucan synthase activity that was comparatively lower than plant (PttCESA8) and bacterial (BCSA/BCSB) cellulose synthases (Ch 3).Finally, I test for functional compatibilities between the land plant (A. thaliana) and red algal (C. tuberculosum) CESAs. A. thaliana encodes multiple non-redundant CESAs that function in primary cell wall and secondary cell wall regions as well as several other accessory proteins critical to cellulose synthesis. However, only some accessory proteins were recovered from C tuberculosum’s transcriptome in bioinformatics analyses (Ch 2). To ultimately test for functional differences, I introduced the CtCESA1 gene into A. thaliana cesa mutants deficient in cellulose production (Ch 4). The red algal CtCESA1 partially rescued the A. thaliana primary cell wall cesa6 mutant but not cesa3 or the secondary cell wall cesa7 mutant. This thesis collectively presents the first functional evidence of a red algal CESA and demonstrates a combination of both deeply conserved and largely distinct aspects of cellulose production between the red algal and land plant lineages.

View record

Setting the stage for lignin deposition: spatial distribution of enzymes directing lignification in Arabidopsis thaliana (2019)

Lignin is a critical phenolic polymer that reinforces secondary cell walls of plant cells. Lignin confers structural strength in fiber cells and enables water transportation in xylem vessel elements. Secreted enzymes, laccases (LACs) and peroxidases (PRXs), facilitate lignin polymerization by oxidizing lignin monomers called monolignols. In Arabidopsis thaliana, there are 17 LACs and 73 PRXs and the isoenzymes involved in lignification and their spatial distributions in plant tissues are poorly characterized. This study tested the hypotheses that LACs and PRXs occupy distinctive cell wall domains, and that the activity of these enzymes could enable deposition of lignin to different regions during development. Putative lignin-associated LACs and PRXs, AtLAC4, AtLAC10, AtLAC17, AtPRX42, AtPRX52, AtPRX71, and AtPRX72, were identified using publicly-available mutant and bioinformatics data. Enzymes were fluorescently tagged with an mCherry label and co-localized with lignin, visible as UV autofluorescence. AtLAC4, AtLAC17, and AtPRX72 localized to xylan-rich secondary cell walls of xylem vessel elements and fibers while AtLAC4, AtPRX64, AtPRX71, and AtLAC10 localized to the pectin-rich middle lamella in fibers. AtLAC10, AtPRX42, AtPRX52, and AtPRX71 localized to non-lignified tissues. Enzyme localization may be due to a combination of mobility in the cell wall and timing of expression. Expressing fluorescently-tagged AtLAC4, AtLAC17, and AtPRX64 under the promoter of the secondary cell wall-specific gene AtCESA7 resulted in similar cell wall localization patterns for all three enzymes in roots, indicating that changing the timing of expression was sufficient to alter localization. However, transgenes were transcriptionally silenced in stem tissue.Stem histochemistry demonstrated that both LAC and PRX oxidative activity was restricted to lignifying cells throughout development. Production of apoplastic reactive oxygen species, such as H₂O₂ for PRX activity, was also found exclusively in lignifying tissue and differed temporally between lignified cell types. Taken together, these results indicate that characteristic sets of oxidative enzymes localize to specific cell types and regions of the lignified cell wall. The location of oxidative enzymes and production of oxidative substrates, such as H₂O₂, could ultimately dictate lignin deposition spatially and temporally throughout stem growth.

View record

Understanding the patterned deposition of lignin in secondary cell walls (2017)

Lignin, one of the three main components of the secondary cell wall, is an important phenolic biopolymer that provides strength and rigidity to the cell walls of tracheary elements and fibers in vascular plants. Lignin is composed of phenolic alcohol monomers called monolignols, which are synthesized in the cytoplasm. These monolignols are exported to the apoplast where they polymerize by random radical coupling following oxidation by laccases and peroxidases. Two laccases found in Arabidopsis thaliana, LAC4 and LAC17, were localized to secondary cell wall, and required for lignification of protoxylem tracheary elements. The localization of LAC4 and LAC17 to spiral secondary cell walls could be due to either: 1) the diffuse secretion of laccases followed by remobilization to the secondary wall, or 2) a reorientation of post-Golgi vesicle trafficking to secondary cell wall specific plasma membrane domains. Localization studies with LAC4-RFP driven by a constitutive promoter found laccases localized to all regions of the primary cell wall prior to differentiation, then the localization shifted into the helical secondary cell wall bands during protoxylem tracheary elements differentiation. This change in localization suggests there is a change in vesicle traffic during secretion of secondary cell wall components (such as laccases). Furthermore, Fluorescence Recovery After Photobleaching (FRAP) was used to determine if laccase localization in secondary cell walls was due to constraint by the secondary cell walls or exclusion from the primary cell wall. Laccases were also found to be immobile in secondary cell wall domains, but mobile when expressed ectopically in primary cell wall domains. Further drug and mutant FRAP studies found laccases remain immobile in the absence of secondary cell wall: cellulose, xylan, lignin and xylan/lignin. These results suggest laccases are not only anchored to secondary cell wall specific components but may be anchored to multiple components of the secondary cell wall or an unknown component of the secondary cell wall.

View record

Laccase-dependent lignification of secondary cell walls of protoxylem tracheary elements in Arabidopsis thaliana (2014)

Lignin is a phenolic polymer that plays important roles in the structural integrity ofplants. Both peroxidases and laccases have been implicated in the polymerization of lignin, andmutant analyses have conclusively demonstrated a role for laccases in lignification ofArabidopsis thaliana stems. However, the oxidative enzymes that polymerize lignin inprotoxylem tracheary elements (TEs) have not been defined. Induction of the mastertranscription factor VASCULAR RELATED NAC-DOMAIN 7 (VND7) causes systemic transdifferentiationinto protoxylem TEs, providing an inducible-experimental model system to studyprotoxylem TE differentiation. The transcriptome of these lines has been well characterized, andtwo laccases, LAC4 and LAC17, are strongly expressed following induction of protoxylem TEdevelopment. To test if LAC4 and LAC17 are necessary for the lignification of protoxylem TEs,the inducible VND7 construct was transformed into the lac4-2/lac17 double mutant backgroundand fluorescently labeled monolignols were exogenously applied to differentiating protoxylemTEs. Labeled polymerized lignin was only detected in the wild-type protoxylem TEs, but not inlac4-2/lac17 protoxylem TEs. To test if laccases alone are sufficient to promote lignification, theconstitutive 35S promoter was used to drive either LAC4 or LAC17 in wild-type plants, resultingin strong ectopic lignification of primary cell walls upon application of fluorescently labeledmonolignols. Fluorescently tagged laccases were transformed into the inducible protoxylem TEssystem, where they specifically localize to the secondary, but not primary, cell walls ofprotoxylem tracheary elements. This research shows that LAC4 and LAC17 are necessary andsufficient for the lignification of secondary cell wall domains of protoxylem TEs and that theyare specifically localized to these domains.

View record



If this is your researcher profile you can log in to the Faculty & Staff portal to update your details and provide recruitment preferences.


Discover the amazing research that is being conducted at UBC!