Anne Lacey Samuels

Professor

Research Interests

plant cell biology
plant cell walls

Relevant Degree Programs

 

Research Methodology

cryo fixation; electron microscopy
live cell imaging

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Master's students
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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.

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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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.

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

Theplantcuticlecoatstheprimaryaerialtissuesofalllandplantstoprovideprotectionagainstnon-­‐stomatalwaterloss.Thoughthebiosynthesisofcuticularlipidsisnowrelativelywellunderstood,themechanismsofcuticularlipidexportremainunclear.Theobjectiveofthisthesiswastocharacterizeseveraltransportersrequiredforcuticularlipidexportandtodeterminetherouteofcuticularlipidexportfromtheirsiteofsynthesisinsidethecelltotheirsiteofaccumulationonthecellsurface.BasedoninteractionstudiesbetweentwoATP-­‐bindingcassettetransporters(ABCG11andABCG12)andthechemicalphenotypesoftheirmutants,amodelfortheinfluenceofABCdimerizationonsubcellularlocalizationandsubstratespecificityofthesetransportersispresentedinthecontextofcuticularlipidexport(Chapter2).Analysisofseveralwell-­‐characterizedsecretorypathwaymutantsfurtherindicatesthatatleastsomecuticularlipidsreachtheseplasmamembranetransportersviavesiculartraffickingthroughtheGolgiapparatus(Chapter3).Furthermore,thesemutantstudiesrevealaformandfunctionrelationshipbetweenthestructureoftheendoplasmicreticulum(ER)anditsbiosyntheticcapacity,withrespecttolipidsynthesis(Chapter3).Finally,ER-­‐plasmamembranecontactsitesareinvestigatedasapossiblesecondrouteofcuticularlipidexport(Chapter4).Whilethefrequencyofcontactsitesisnotcorrelatedwithcuticularlipidexport,itremainspossiblethatlipidtraffickingmayoccuratthesesites.Isolationandproteomicanalysisofthesemembranesub-­‐fractionsrevealsapossibleroleforER-­‐plasmamembranecontactsitesinlipidremodelingorrecycling,ratherthancuticularlipidexport.Takentogether,theseresultsbuildacompletemodelofcuticularlipidexportfromthesiteofsynthesistothesiteoflipidaccumulation.

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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 (2010 - 2018)
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.

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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.

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Publications

 
 

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