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Page 1 Maybe have the WoW logo and the dates and location of the conference and all that sort of thing. Page 2 I made a map showing Michael Smith Laboratories (red arrow) and Beaty Biodiversity Museum (orange arrow). I don’t think that the full image should be cropped any more, because some people will be staying at Gage or Totem Park Residences, and they’ll need to know how to walk over from there. I am including a cropped image that we can put as an inset, though. I’ve also included the banner from the Biodiversity Museum website so that people will know to look for the whale skeleton to be sure that they’re in the right place. We could write something like “You can’t miss the reception venue: it’s under a giant Blue Whale skeleton.” (Do we know how to party, or what?) Addresses and Wayfinding: Michael Smith Laboratories 2185 East Mall Vancouver, BC V6T 1Z4 Beaty Biodiversity Museum – Djavad Mowafaghian Atrium 2212 Main Mall Vancouver, BC V6T 1Z4 Page 3 Here’s the schedule. Abstracts and the specific order of presenters can go on a later page. June 1, 2011 16:00-18:30 18:30-20:30 Registration, Michael Smith Building lobby Reception, Beaty Biodiversity Museum June 2, 2011 8:45-9:00 9:00-10:00 10:00-10:30 10:30-12:10 12:10-13:30 13:30-14:30 14:30-15:10 15:10-15:30 15:30-16:30 16:30-18:30 Opening remarks Keynote speaker, Simon Turner, University of Manchester Coffee break Oral presentations Lunch Invited speaker, Debra Mohnen, CCRC, University of Georgia Oral presentations Coffee break Oral presentations Poster session and barbecue June 3, 2011 9:00-10:00 10:00-10:30 10:30-12:10 12:10-13:30 13:30-14:30 Invited speaker, Taku Demura, RIKEN and NAIST Coffee break Oral presentations Lunch Invited speaker, Clint Chapple, Purdue University 14:30-15:10 15:10-15:30 15:30-16:10 16:10-16:25 16:25-17:45 Oral presentations Coffee break Oral presentations Break (judges will meet to decide on award winners) Awards (best student poster and oral presentation) Page 4 Full abstracts and titles and such! Invited speakers Simon Turner, University of Manchester: Analysis of carbohydrate biosynthesis in the Arabidopsis secondary cell wall Debra Mohnen, CCRC, University of Georgia: Pectin biosynthesis: a surprisingly complex story with protein complexes, protein processing and multiple GAUT proteins Taku Demura, RIKEN and NAIST: Key transcription factors regulating secondary cell wall formation Clint Chapple, Purdue University: Is dwarfism an obligatory phenotype in lignin down-regulated plants? Oral presentations June 2, 2011 session 1 (10:30-12:10) Molecular phenotyping of cell wall expansion in Arabidopsis inflorescence stems Hardy Hall, Rodger Beatson, Thomas Berleth, and Brian Ellis Michael Smith Laboratories, University of British Columbia, Vancouver, Canada Plant cell walls are sophisticated, dynamic structures that play a vital role in coordinating the directional growth of plant tissues. The rapid elongation of the inflorescence stem in the model plant Arabidopsis is accompanied by radical changes in cell wall structure and chemistry, but the study of the underlying mechanisms has been hampered by difficulties in sampling discrete developmental states along the developing stem. I have developed a novel sampling approach that allows me to sample stem tissues representing specific and distinct developmental phases (elongation rate increase, maximum growth rate, and growth cessation) from individual plants, by use of time-lapse imagery and computational analysis of growth kinematic profiles. This high-resolution growth phenotyping enables the harvest of pooled, developmentally-matched samples that I have used for immunohistochemical analysis of growth-associated cell wall epitopes in stem cross-sections, and for transcriptome profiling of both growthcharacterized stem segments and laser capture micro-dissected cell types within those segments. This study has produced a comprehensive view of the cellular events accompanying the transition from early differentiation through maximum anisotropic cell expansion to growth cessation and secondary cell wall maturation, in a single organ. The resulting transcript profiles have identified dozens of genes, both known and novel, whose expression is coupled to these growth transitions, and knock-out mutants for many of these are currently being examined for growth/developmental phenotypes. Carbohydrate partitioning to wood development and cell wall biosynthesis in hybrid aspen Melissa Roach, L. Gerber, A. Gorzsas, M. Mahboubi, T. Niittyla, and B. Sundberg Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Umeå, Sweden Cell wall biosynthesis is a major sink for carbon during wood development. Our group has investigated some key carbohydrate active enzymes that control or modify carbon mobilization into metabolic pathways. We have analyzed the individual effects of RNAi downregulation of the main wood expressed sucrose synthases (Susy; PttSus1A, PttSus1B) and fructokinases (FRK; PttFRK2A, PttFRK2B) in hybrid aspen (Populus tremula x tremuloides). Both constructs resulted in decreased bulk wood density, but had no drastic effects on overall growth and development of the trees. Chemical analyses of RNAi-FRK and RNAi-SUS wood by FT-IR, Py-GC/MS, 2D-NMR and mechanical strength analysis indicated remarkably similar cell wall chemotypes. For RNAi-SUS trees in-depth chemical and mechanical analyses revealed increased porosity and a dramatically altered wall ultrastructure. RNAi-SUS also showed altered soluble sugar levels, but no differences in UDP-Glc or hexose phosphate levels. RNAi-FRK showed alterations in the levels of soluble sugars, as well as decreased hexose phosphates and UDP-Glc content, but no effect on the quantities of other carbohydrates, particularly those involved in primary metabolism. Though these transgenics produce remarkably similar cell wall chemotypes, the data suggest they act in overlapping pathways to allocate carbon to secondary cell wall biosynthesis. Further understanding the mechanisms of carbon partitioning to cell wall polymer biosynthesis is important to efficiently optimize wood biomass production in the future. Homogalacturonans in the pit membrane annulus may influence xylem vulnerability to embolism Lenka Plavcová and U.G. Hacke Renewable Resources, University of Alberta, Edmonton, Alberta, Canada Pits are small openings in the cell wall that connect adjacent cells. In xylem, bordered pits have several important functions. Inter-conduit pits allow water movement between the xylem conduits; nevertheless, they still provide a significant resistance to the flow. Pits also act as safety valves that prevent air from entering functional conduits. Hence, pits affect both xylem efficiency and safety. Another type of pit occurs between ray parenchyma cells and xylem vessels, thus providing boundary between the living and dead components of the xylem. The function of pits is intimately linked with their structural and chemical properties. Pit membranes develop from the primary cell wall and middle lamella. However, not much is known about their actual chemical composition. Using immunogold labeling, we studied the distribution of selected polysaccharides in the pit membranes of four angiosperm tree species. We focused primarily on pectic homogalacturonans (HGs) as they are believed to influence porosity and permeability of pit membranes. In all four species, HGs were surprisingly not detected in the inter-conduit pit membrane except for the membrane margin called the annulus. In contrast, the entire membrane of ray pits showed strong labeling. The immunolabeling pattern is interpreted in conjunction with hydraulic measurements. Our results suggest that the pit membrane annulus affects xylem vulnerability to embolism. We hypothesize that the degree of HG esterification and HG-calcium interactions within the annulus control pit membrane stretching and deflection, which in turn affect the membrane permeability to air. Seed mucilage synthesis: screening for novel pectin mutants using an EMS population Aleksandar Vasilevski1, Abdul Ahad2, George Haughn2, and Björn Usadel1 1 MPI für Molekulare Pflanzenphysiologie, Berlin, Germany, 2 Department of Botany, University of British Columbia, Vancouver, Canada A significant portion of the carbon fixed by plant photosynthesis is incorporated into cell wall carbohydrates. However, cell wall synthesis and its regulation are still not fully understood. This might be partly due to the fact that screening for cell wall mutants might be hampered by the complexity of the cell wall and therefore screens have only produced a few dozen mutants. As the easily extractable Arabidopsis seed coat mucilage is nearly entirely composed of pectins, it provides an ideal system to study pectin synthesis by screening mutant populations. Thus we screened an Arabidopsis EMS-mutagenized population of ca. 1700 lines for changes in the polysaccharide composition of the extractable mucilage using a HAPEC based method. We found 16 candidate lines with a confirmed change in their mucilage composition or amount including two mutants showing a strong reduction in rhamnose and galacturonic acid mutants with an increase in rhamnose, glucose and galacturonic acid. The mutants were divided into 4 groups, according to their phenotype and the mutants from the same group have been subjected to complementation tests. We will present the mutant collection and a preliminary biochemical and histochemical analysis for some of interesting candidate lines. CESA-6 like cellulose synthases are involved in secondary cell wall biosynthesis and mucilage adherence properties in Arabidopsis seed epidermal cells Jonathan S. Griffiths, V. Mendu, S. Persson, J. Stork, C. Voiniciuc, A.B. Downie, S. DeBolt, and G. Haughn Department of Botany, University of British Columbia, Vancouver, Canada The seed coat epidermis is a unique cell type designed to protect the embryo from the environment until germination. The differentiation of Arabidopsis epidermal seed coat cells involves secondary cell wall biosynthetic processes. Two such events are the production of a donut-shaped apoplastic mucilage pocket, and a highly reinforced secondary wall that fills the cell creating a volcano shaped columella. Cellulose is thought to play a major role in secondary wall development and in mucilage production. Which cellulose synthase genes are involved in these processes remains unclear. Here we investigated the relationship of the CESA-6 like cellulose synthases: CESA2, CESA5, and CESA9. We identified their role in the developing seed coat by examining cell wall composition, epidermal cell morphology, and mucilage hydration properties using reverse genetics. We found significant changes in cell wall composition, a loss of cell shape uniformity, reduced secondary wall thickening, and a loss of mucilage adherence in cesa mutant seeds. CESA2 and CESA9 are involved in secondary wall cellulose biosynthesis, while CESA5 plays a role in both mucilage biosynthesis and in secondary wall cellulose biosynthesis. We have identified specialized roles for different CESA subunits that are developmentally regulated in one cell type, and demonstrate the importance of cellulose in Arabidopsis epidermal seed coat cells. June 2, 2011 session 2 (14:30-16:30) Cortical microtubules influence cellulose synthase movement and the proportion of crystalline cellulose in rapidly elongating cells Miki Fujita, Shawn D Mansfield, and Geoffrey O Wasteneys Department of Botany, University of British Columbia, Vancouver, Canada The direction of plant cell expansion is governed by the mechanical properties of the wall. Cellulose microfibrils align parallel to each other and at right angles to the growth axis to restrict the expansion predominantly in one direction against isotropic turgor pressure. Previous studies using the mor1-1 (microtubule organization 1) mutant, which undergoes radial swelling upon altered cortical microtubule organization and dynamics, have demonstrated that well-ordered cellulose microfibrils are not sufficient to drive unidirectional cell expansion. These puzzling results led us to hypothesize that cortical microtubules play an additional role in controlling the proportion of crystalline cellulose in cell wall. Using X-ray diffraction analysis we determined that the reduced microtubule polymer mass in mor1-1 at high temperature is associated with both a relatively high degree of crystallinity and reduced growth anisotropy. By comparing the cell wall crystallinity in other microtubule-related mutants, results suggest that maintaining a critical microtubule polymer mass is required for modulating cell wall crystallinity during cell expansion. To understand the relationship between microtubule polymer mass and cellulose crystallinity we performed 2D coincidence analysis of fluorescently tagged microtubules and cellulose synthase complexes (CSCs). We determined that more CSCs track in microtubule-free domains as a result of reduced microtubule polymer mass in mor1-1 and that the average velocity of CSCs was increased. Taken together, our X-ray diffraction and live cell imaging strategies indicate that microtubules domains at the plasma membrane influences CSC activity and the proportion of crystalline cellulose in the cell wall. Specific Arabidopsis arogenate dehydratase isoforms are involved in biosynthesis of phenylalanine as a precursor for lignin Oliver R.A. Corea, C. Ki, C.L. Cardenas, L.B. Davin, and N.G. Lewis Institute of Biological Chemistry, Washington State University, Pullman, Washington, USA The strength and rigidity of plant cell walls is due to the deposition of lignin during secondary development. Lignin is a product of the phenylpropanoid pathway, which is fed by phenylalanine from the chloroplast-localized shikimate/chorismate pathway. Although the phenylpropanoid pathway has been extensively studied, upstream factors controlling carbon flux into it are not currently well understood. In this regard, we were interested characterizing arogenate dehydratases (ADTs), which catalyze the final step of phenylalanine biosynthesis in plants. To examine the role of ADTs in Arabidopsis, we obtained SALK T-DNA insertion lines for five of six ADT isoforms, with these being crossed together to form double and triple knockouts in all combinations. Analysis of these knockouts identified candidate ADTs that are involved in phenylalanine biosynthesis for lignin production. One double knockout and two triple knockouts had substantially weakened stems compared to WT; therefore these lines (and the single knockouts from which they derived) were selected for analysis. Histochemical staining of stem cross sections with phloroglucinol-HCl and Mäule reagent suggested G-lignin was virtually absent in the interfascicular (if) region of double and triple knockouts, while one single also displayed a moderate decrease in the if. These results were confirmed by thioacidolysis, which showed the double and triple knockouts ranged from ~5070% reduction in G+S lignin, compared to WT. Single knockouts had either modest (<20%) reduction or no change compared to WT. This is the first time a chloroplast-localized enzyme has been demonstrated to differentially alter carbon allocation into lignin. Expression of an oomycete chitin synthase gene in Arabidopsis and production of chitin in transformed plants Gea Guerriero1, Vaibhav Srivastava1, Qi Zhou1, Sophia Ekengren2, Frank Meulewaeter3, Marc De Block3, Cortwa Hooijmaijers1, Gustav Sundqvist1, and Vincent Bulone1 1 Division of Glycoscience, School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden 2 Department of Botany, Stockholm University, 3Bayer CropScience, Ghent, Belgium There is a great potential to develop new carbohydrate-based materials that combine environmental friendliness and biocompatibility with high performance and increased functionality through the engineering of plant cell walls. For instance, the paper, materials, pharmaceutical and textile industries are keen to develop chitin/cellulose and chitosan/cellulose blends. Chitin and chitosan are rich in free NH 2 groups that can be readily modified, as opposed to cellulose which contains much less reactive OH groups. Furthermore, the chemical modification of the OH groups in cellulose leads to the loss of the cellulose structure and its mechanical properties. Thus, the introduction of chitin/chitosan in plant cell walls significantly increases the possibility of modifying cellulose-based materials without affecting their mechanical properties. For example, the introduction of NH 2 groups in cotton cell walls would significantly improve the dyeing properties of cellulose for textile applications. We have engineered Arabidopsis thaliana through stable transformation with a previously characterized oomycete chitin synthase gene. Chitin was detected in trichomes and leaves of transgenic plants, by using specific labeling and various analytical techniques. This work shows the potential of redirecting plant metabolism towards the biosynthesis of exogenous cell wall polysaccharides for the production of new carbohydrate composites with increased functionality and modified mechanical properties. Interestingly, the transgenic lines were more resistant to infection by Pseudomonas syringae than wild-type plants and showed up-regulation of genes involved in pathogen response (microarray and QPCR). Thus, in addition to the formation of new composites the concept may be exploitable for the generation of pathogen-resistant crops. A perplexing biopolymer: how autofluorescence is uncovering clues to sporopollenin composition and assembly Teagen D. Quilichini, A. Lacey Samuels, and Carl J. Douglas Department of Botany, University of British Columbia, Vancouver, Canada The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Analyses of male sterile mutants defective in pollen wall formation, primarily in the model plant Arabidopsis, have revealed genes required for sporopollenin biosynthesis and/or deposition, including MS2, ACOS5 and PKS-A/PKS-B, TKP1 and ABCG26. Based on genetic and biochemical analysis of these genes and the corresponding enzymes, a model was proposed by Grienenberger et al. (2010), which suggests that synthesis of an aliphatic polyketide sporopollenin monomer occurs in the tapetum, followed by its export and delivery to developing pollen grains by putative transport proteins, such as ABCG26 and lipid transfer proteins. However, phenylpropanoid-derived phenolic compounds are also thought to be sporopollenin substituents synthesized in the tapetum. Through the analysis of mutants affecting sporopollenin production and deposition by live-cell two-photon microscopy, the occurrence of autofluorescent phenolic compounds in these mutants can be visualized. Mutants affecting ACOS5, PKS-A/PKS-B, and TKP1, thought to affect the lipidic polyketide pathway in sporopollenin biosynthesis, exhibit autofluorescence at the periphery of tapetal cells inside locules. Conversely, the ABC transport protein mutant abcg26 exhibits autofluorescence in tapetum vacuoles. Light and transmission electron microscopy support the findings made by live-cell imaging, with enlarged, debris-filled vacuoles in the tapetum cells of abcg26 mutants. These results support the existence of a second pathway for the biosynthesis of a phenolic component of sporopollenin, which is exported from the tapetum by ABCG26 before copolymerization with lipidic polyketide components to generate the sporopollenin matrix. Monolignol export and plant cell wall lignification Mathias Schuetz 1,2, Rebecca Smith 1,2, Brian Ellis 1,2, and Lacey Samuels 1, 1 Department of Botany and 2Michael Smith Laboratories, University of British Columbia, Vancouver, Canada Plant vascular systems are comprised of xylem and phloem cell types, which are critical avenues for long distance transport throughout the plant. A prominent feature of xylem vessels is a thick secondary cell wall that provides mechanical strength. The polymerization of lignin monomers (monolignols) in secondary cell walls of xylem vessels and fibers in woody plants results in the high strength and durability of wood. A key step in lignin biosynthesis is the delivery of precursor monolignols to the site of polymerization, but the mechanisms of monolignol export from the place of synthesis in the cytosol to the subsequent deposition in the cell wall are unknown. Several global gene expression studies using tissues undergoing lignification have correlated expression of members of the ATP-binding cassette (ABC) transporter gene family with phenylpropanoid (lignin) biosynthesis. ABC transporters are characterized by their ability to transport a variety of molecules across plasma membranes, coupled to ATP hydrolysis. We are using ABC proteins fused to GFP under the control of native promoters to allow us to observe detailed spatio-temporal distribution profiles and subcellular localization in Arabidopsis inflorescence stems, which are rich in lignifying fibers and vascular bundles. In order to elucidate the functional roles of the candidate ABC genes, analysis of loss of function mutants from available T-DNA collections are being performed in parallel to microRNA mediated gene knockdown approaches. By designing synthetic microRNA constructs, we expect to silence multiple ABC transporter genes during discrete stages of xylem development in order to overcome gene redundancy among closely related ABC genes. The results of this ongoing study will be presented. June 3, 2011 session 1 (10:30-12:10) Requirement for matrix polysaccharides in G-type secondary wall development Michael K. Deyholos, M.J. Roach, A. Badhan, N. Hobson, M. DePauw, and T. Gorshkova Biological Sciences, University of Alberta, Edmonton, Alberta, Canada The gelatinous (G-type) secondary cell walls of tension wood and phloem fibers are rich crystalline cellulose, but are relatively deficient in xylans and other hemicelluloses commonly found in other types of secondary walls. Previous studies have shown that development of G-type walls in phloem (bast) fibers of flax (L. usitatissimum) involves a galactan rich matrix that is gradually replaced by crystaline cellulose. The maturation of the cell wall has been previously correlated with expression of a putative beta-galactosidase. Here, we demonstrate that development of a secondary wall rich in crystaline cellulose is in fact dependendent of the normal activity of beta-galactosidase. We founds that developing stems of transgenic (RNAi) flax with reduced beta-galactosidase activity had lower concentrations of free galactose, and significant reductions in the thickness of mature cellulose G-layers as compared to controls. The domain of the secondary wall that was labeled by the galactan-specific LM5 antibody was greatly expanded in the RNAi plants. These results demonstrate a specific requirement for hydrolysis of tissuespecific galactans during cellulosic cell wall formation. Furthermore, we show by mechanical testing that the high tensile strength of normal flax stems is dependent on galactosidase-mediated remodelling of the galactan-rich matrix. These observations demonstrate a novel role for matrix polysaccharides in cellulose deposition. We also describe the annotation of the recently completed whole-genome sequence of flax, in the context of genes relevant for cell wall biosynthesis. Nucleotide sugar profiling reveals activation of the myo-inositol sugar pathway in carbon starved Arabidopsis plants Björn Usadel Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany Regulatory links between the availability of photosynthate and fluxes through the nucleotide sugar interconversion pathways that provide the precursors for plant cell wall biosynthesis are poorly understood. To facilitate the investigation of these regulatory interactions, we have established a novel MS-based method to quantify nucleotide sugar pool sizes in small samples. Using this method, we determined that myo inositol oxygenase mutant plants do not show any detectable changes in their nucleotide sugar levels during a diurnal cycle. However, as predicted from transcript measurements, this situation changes dramatically when plants experience acute carbon starvation by extending the dark period. Under these conditions the myo-inositol pathway is activated and accordingly miox mutant plants show a more than two-fold reduction in the UDP-glucuronic and UDP-galacturonic acid content compared to wild type plants. This suggests an intricate regulation of the nucleotide sugar interconversion and the myo inositol oxygenase pathway based on sugar availability. Good neighbours: the co-operative model during lignification Rebecca Smith1,2, Mathias Schuetz1,2, Brian Ellis1,2, and Lacey Samuels1 1 Department of Botany and 2Michael Smith Laboratories, University of British Columbia, Vancouver, Canada The survival of land plants is dependent upon the ability of the plant to transport water and minerals through cells called tracheary elements in the vascular system. During development, tracheary elements deposit thick secondary cell walls, which are strengthened with the polymer lignin to support and maintain the continuous network of xylem. The process of lignification has been widely studied not only because lignin plays a key role in maintaining plant structure, but also because the removal of lignin is required for the production of pulp and paper and for the conversion of plant biomass into biofuels. It is widely accepted that lignifying cells contribute to the lignification of their own cell walls, but there has also been some evidence that suggests xylary parenchyma cells adjacent to lignifying tracheary elements cells may be contributing to lignification. This is referred to as the co-operative model or the “good neighbour” hypothesis. I am using two complementary approaches to test this hypothesis. First, I am using monolignol immobilization (cryofixation) and localization (autoradiography) in Arabidopsis roots to determine whether neighbouring cells surrounding mature, dead tracheary elements are producing monolignols and exporting them to the tracheary elements. To complement the lignin localization experiments, I have built plant expression constructs that express artificial microRNAs, driven by a tracheary element-specific promoter. These miRNAs will target and silence the expression of specific lignin biosynthesis genes and should allow cell-specific manipulation of lignin precursors. From these results, I will be able to determine whether neighbouring cells are contributing to lignification. Plant PIRIN proteins are working on walls by regulating the late xylem maturation Sacha Escamez, B. Zhang, E. Pesquet, and H. Tuominen Plant Physiology, Umeå Plant Science Centre, Umeå, Sweden Maturation of xylem elements involves deposition of the secondary cell walls (SCWs) and programmed cell death (PCD). The SCWs consist mainly of carbohydrates, which can provide an attractive source of biofuels and other green chemicals. On the other hand, the SCWs also contain large amounts of lignin, which is known to have a negative impact on cellulose extractability. Hence, unraveling of the mechanisms underlying xylem maturation is expected to provide clues on factors affecting the woody biomass recalcitrance for further applications, such as saccharification. Recently, using an in vitro cell culture system in which cells from Zinnia elegans transdifferentiate into xylem tracheary elements (TEs) after a hormonal induction, it was possible to identify putative regulators of the late xylem maturation by differential expression analysis of TEs that were arrested in both PCD and lignification through the use of silver thiosulfate (STS). Differential gene expression analysis of Zinnia TEs revealed a gene called PIRIN as the most strongly suppressed gene after the STS treatment. Four PIRIN genes are present in the Arabidopsis thaliana genome and they are all expressed in the vascular tissues. Our results in Arabidopsis thaliana reveal the PIRIN2 protein as a completely novel regulator of lignin biosynthesis. PIRIN2 protein was also shown to interact with several papain-like cysteine proteases, which suggests that the PIRIN proteins might regulate the interplay between secondary cell wall lignification and cellular hydrolysis after xylem cell death. The Arabidopsis FLYING SAUCERS gene encodes a membrane protein required for connections to the cell wall Catalin Voiniciuc, G.H. Dean, J.S. Griffiths, and G.W. Haughn Department of Botany, University of British Columbia, Vancouver, Canada The genetic analysis of mutants defective in seed coat development facilitates the discovery of genes involved in cell wall biogenesis. The Arabidopsis thaliana seed coat epidermis is a dispensable cell layer that secretes large amounts of pectinaceous mucilage forming donut-shaped pockets between the primary cell wall and the plasma membrane. The epidermal cells then synthesize a volcano-shaped secondary wall, which protrudes through the center of the mucilage pocket where it connects to the primary wall. Hydration of mature seeds triggers the rapid expansion of pectins, which ruptures the outer tangential primary wall from the radial wall, and forms a mucilage halo around the seed. Although large fragments of tangential wall remain attached to the columella after mucilage extrusion, very little is known about what mediates this specific attachment. My research focuses on flying saucers (fly), a unique Arabidopsis thaliana mutant which is characterized by the presence of discs at the periphery of extruded mucilage. Preliminary evidence suggests that the fly discs are primary cell walls which have lifted off the columella, and are attached to mucilage which fails to expand properly upon hydration. Using positional cloning and sequence analysis, I found that FLY encodes a putative zinc finger transmembrane protein targeted for secretion. I hypothesize that FLY is a plasma membrane protein that anchors the primary cell wall. Functional characterization of the FLY protein should provide insight into the molecular machinery that mediates cell wall-plasma membrane attachment in plants. June 3, 2011 session 2 (14:30-16:10) Identification and analysis of seed coat epidermal-specific promoter in Arabidopsis thaliana and Brassica napus Elahe Esfandiari1, Zhaoqing Jin1, Ashraf M.A. Abdeen2, Jonathan Griffiths1, Tamara L. Western2 and George W. Haughn1 1Department of Botany, University of British Columbia, Vancouver, Canada 2 Department of Biology, McGill University, Montréal, Québec, Canada During differentiation of the Arabidopsis thaliana seed coat, dramatic changes occur including cytoplasmic rearrangement, proanthocyanidin biosynthesis, and production of secondary cell walls. The epidermal cells undergo an especially pronounced transformation highlighted by the synthesis and secretion of copious amounts of dispensable, pectinaceous mucilage. Thus, this cell type represents an excellent platform to study the biosynthesis and modification of cell wall components, particularly pectin. One tool required for molecular genetic analysis is a promoter that drives expression specific to this cell layer. To identify such a promoter, we analyzed Arabidopsis seed coat microarray data for genes specifically expressed in the seed coat. This led to the identification of 14 candidate genes. Based on RT-PCR results, 9 of these genes showed a seed-specific expression pattern. The transcriptional regulatory region of each of these candidate genes was fused to the GUS reporter gene. A histochemical GUS assay demonstrated that only one of the promoters, SEED COAT-SPECIFIC PROMOTER (SCSP) is able to express GUS specifically in the seed coat where expression was detected in the epidermal and palisade cell layers. qRT-PCR data using wild type seed coat RNA suggests that the promoter is particularly active at 7 days post anthesis. The SCSP was able to direct transcription of GUS in a similar pattern in the Brassica napus (Canola) seed coat. Thus, in addition to its application in studying the plant cell wall, this promoter will provide an experimental tool for expressing high-valued recombinant proteins as well as modifying seed coat traits in economically important crops. Investigations on protein-membrane and protein-protein interactions upstream of the phenylpropanoid pathway in Arabidopsis thaliana Jean-Etienne Bassard, J. Borch, H. Duan, F.Duval, P. Roepstorff, S. Sligar, D. Werck-Reichhart IBMP-CNRS, Strasbourg, France The phenylpropanoid pathway is a well-defined system that leads in particular to the biosynthesis of lignin. Metabolic intermediates are unstable, and have been assumed to be sequestered within supramolecular enzyme complexes. The upstream pathway involves two cytochromes P450 (CYP73A5 and CYP98A3 in A. thaliana) connected with soluble proteins (hydroxycinnamoyl ester transferase and 4-coumaroyl CoA ligases). This offers a unique possibility to elucidate how metabolon formation takes place at the molecular level. Such metabolons are fragile, difficult to isolate and to characterize. A key issue is to preserve stability and proper folding of the P450s which most likely anchor metabolons on membranes. Associations have been investigated using two complementary approaches. The first was protein fusions with fluorescent proteins expressed in vivo and analyzed by confocal microscopy strategies. In the second, P450 integrity was preserved by detergent assisted self-assembly into nanostructures of the heterologously expressed P450 and P450 reductase. In vitro interaction was investigated by Surface Plasmon Resonance and co-immunoprecipitation. This work revealed unexpected features of the target enzymes, including fast movement of the P450 enzymes with the plant endoplasmic reticulum and membrane binding of 4CL-1 and HCT, independent from the presence of the P450s. It indicated membrane relocalization of soluble enzymes in vivo in the presence of the P450s and demonstrated direct protein/protein interactions that were enhanced when all four partners were co-expressed. Furthermore, P450 homo- and hetero-oligomerization was revealed. Finally this work suggests that CYP98A3 plays an essential role in the formation of the lignin metabolon. The development of molecular resources for improving the fibre characteristics of industrial hemp (Cannabis sativa L.) Mihaela Voicu, J. Bernier, S. Koziel, C. Coffey, and J. Vidmar Alberta Innovates - Technology Future, Vegreville, Alberta, Canada Industrial hemp (Cannabis sativa L.) is regarded today as an alternative source of fibers in the pulp and paper, textiles and bio-composite industries. Our BioFibres team at Alberta Innovates – Technology Future is working at improving hemp characteristics with respect to fibre quantity and quality. In an effort to rapidly fulfill the need for newer highly productive hemp cultivars, we are using an integral approach that includes the use of mutagenized plants, screening of plants by reverse genetics and molecular marker development. An M1 mutagenized population of hemp was progressed to the M2 generation in the greenhouse. We observed several altered phenotypes among the individual plants from the M2 generation. To support the screening of the M2 plants we also isolated the full-length cDNAs of the genes involved in lignin biosynthesis by using the rolling circle amplification-RACE (rca-RACE) method. By using the above method we rapidly obtained the full-length cDNAs (full cds and most of the 5’ and 3’UTR) of the several genes: 4CL1 (4-coumarate-CoA ligase 1), 4CL2 (4-coumarate-CoA ligase 2), COMT1(Caffeic acid O-methyltransferase1), F5H (Ferulate 5-hydroxylase), PSKP (Phytosulfokine precursor), CCR1 (Cinnamoyl CoA reductase1), CCoAOMT (Caffeoyl-CoA 3-O-methyltransferase), transcription factor lim1 involved in lignin biosynthesis pathway, PCS3H (P-coumaroyl Shikimate 3’-hydroxylase), CAD (Cinnamyl-alcohol dehydrogenase), C4H (Cinnamate-4-hydroxylase ), and PAL (Phenylalanine Ammonia-lyase). A genomic library enriched in microsatellites (SSRs) was also constructed and sequenced: so far we were able to score 71 SSR markers with genomic DNA from 11 hemp varieties (61 yielded highly polymorphic bands). Identification of seed coat mucilage cohesiveness mutant in Arabidopsis thaliana Gabriel Levesque-Tremblay1, Shawn Mansfield2 and George W. Haughn1 1 Department of Botany, and 2Department of Forestry, University of British Columbia, Vancouver, Canada Pectin is an important and ubiquitous component of the cell wall. Understanding its structure, biogenesis and regulation is an important goal that could lead to the discovery of new applications in the pectin industry and help make possible, targeted modifications of the cell wall for manipulation of cell adhesion properties. In Arabidopsis, seed coat epidermal cells produce and secrete mucilage consisting mainly of pectin. Upon hydration, the pectinaceous mucilage swells to create a gel like capsule around the seed. We are using these epidermal cells as a model system to identify genes impacting pectin cohesion. Using both reverse and forward genetics approaches, we isolated mutant line with defects in mucilage cohesiveness. Pectin methylesterases (PMEs) are a large family of enzymes known to modify the property of the pectin molecule by removing the methyl group esterified to the galacturonic acid. The degree and pattern of pectin de-methylesterification modifies its properties, resulting in stiffening or loosening of the cell wall. Only a few genes in the PME family have been characterized and we have shown that at least 15 of 63 PMEs are highly expressed in Arabidopsis seed coat. Using immunolabelling with jim5, jim7 and 2f4 antibodies on thin section of resin-embedded seeds, we have shown that there is a high level of demethylesterified pectin present in the seed coat. Accordingly, we screened for seed coat phenotypes of Arabidopsis lines with loss-of-function mutations in each of the 66 genes encoding PMEs in Arabidopsis and isolated lines with seed coat phenotypes. Using a forward genetics approach we isolated three mutants with defects in mucilage cohesiveness and extrusion. Mucilage-modified mutants mum3 and mum5 produce mucilage that is much less cohesive than that of the wild type and remain unstained by the pectin stain ruthenium-red. Conversely the mutant wall-out mucilage extrusion is slower than that of WT and may be cause by an increase in pectin cohesiveness. We are using positional cloning (mapping) to identify the genes associated with these phenotypes. We hope to determine the role of each newly identified gene/enzyme in mucilage biosynthesis and provide new insight into pectin biosynthesis and modification. Poster Presentations Albert Cairό – no abstract yet Cell wall secretion: the role of the TGN in the pectin secretory pathway in Arabidopsis Kimberley Carruthers1, Delphine Gendre2, Yoichiro Watanabe1, Heather McFarlane1, Gabriel LevesqueTremblay1, Rishi Bhalerao2, and Lacey Samuels1 1 Department of Botany, University of British Columbia, Vancouver, Canada, 2Umeå Plant Science Center, Umeå, Sweden The process of plant primary cell wall secretion lies predominantly in the endomembrane system. Pectin, a soluble wall polysaccharide component, is synthesized in the Golgi and secreted via the trans-Golgi network (TGN). For this study, seed coat cells are an ideal model as they secrete large quantities of pectin to the apical apoplast during development, forming pockets of a substance called mucilage. The product of the gene, ECHIDNA, has been localized to the TGN and the recently isolated Arabidopsis mutant, echidna, has a novel secretion phenotype. In the seed coat, echidna appears to accumulate mucilage intracellularly rather than in the extracellular pockets. This mislocalization of pectin is consistent with ECHIDNA having a role in secretion of primary wall components. Putatively, it is thought that pectin is either mistargeted to the vacuole or is contained within plasma membrane infolds suggesting ECHIDNA may also be involved in the recycling pathway. This ongoing study uses light microscopy, transmission election microscopy (TEM) and immunolabelling techniques to characterize the seed coat phenotype of echidna to elucidate how endomembrane trafficking at the TGN functions in secretion of soluble cell wall polysaccharides. The biological role of MPK20 in Arabidopsis Siyu Cheng, Albert Cairό, and Brian Ellis Michael Smith Laboratories, University of British Columbia, Vancouver, Canada Mitogen-activated protein kinase (MAPK) cascades form an important signal transduction system in eukaryotic organisms. In Arabidopsis thaliana, 20 MAPKs (MPKs) have been found which belong to two subtypes: the TEY subtype (MPK Groups A, B, and C) and the TDY subtype (MPK Group D). Very little is known about the biological roles of Group D MPKs, but previous studies have shown that the gene encoding one Group D MPK, AtMPK20, is co-expressed with the genes CESA1, 3, and 6, which suggests that MPK20 may play a role in primary cell wall formation or associated processes. Therefore, elucidating the biological functions of AtMPK20 may help us understand signalling associated with primary cell wall formation. According to the Membrane-protein Interaction Network Database 0.5 (http://www.associomics.org/Associomics/ Home.html), AtMPK20 doesn’t interact with any of the CesA proteins, and we have not detected any phosphorylation of recombinant CesA3 by recombinant MPK20 in vitro. Since CesA3 protein is known to be phosphorylated on multiple sites, it is possible that AtMPK20 can phosphorylate CesA proteins only after other phosphorylation events have occurred. To gain further insight into AtMPK20 function, I am characterizing mpk20 KO and MPK20 over-expression genotypes for growth and morphology differences from WT, as well as analyzing reporter gene expression patterns in MPK20pro:GUS lines. Heterologous Expression of Fungal Cellulases in Plants H. Klose, S. Wennekamp, M. Girfoglio, and Uli Commandeur Institut für Biologie VII, RWTH Aachen University, Aachen, Germany The hydrolysis of crystalline cellulose is one of the crucial steps in converting biomass to fermentable sugars. Currently cellulolytic enzymes are expensively produced by microbial fermentation. In planta expression of these enzymes could provide a more effective route to cellulosic biofuels. Enzymatic degradation of cellulose is a highly complex process which involves many different enzymatic activities. Endoglucanases break β-1,4-glycosidic bonds inside the polymeric chain, whereas exoglucanases cleave units from the ends of the exposed chains produced by endoglucanases. The resulting cellobiose units are finally cleaved by β-glucosidases into glucose. All enzymes were expressed transiently in Nicotiana tabacum and were targeted to the apoplast or retained in the ER of the plant cells. Functionality of the β-glucanases was tested with soluble substrates like Azo-CMC and 4-MUC. IMAC purified enzymes were used for activity studies on Ionic Liquid pretreated α cellulose exhibiting enhanced sugar release by using different combinations of recombinant β-glucanases. Our studies show the feasibility to express functional βglucanases from the mesophilic filamentous fungus Trichoderma reesei in tobacco plants. With purified enzyme preparations we were able to demonstrate synergistic effects on cellulose conversion. Discoordination of cellular expansion in twisting mutants and associated changes in primary cell wall extensibility Caitlin C.A. Donnelly, Yi Zhang, and Geoffrey O. Wasteneys Department of Botany, University of British Columbia, Vancouver, Canada The cortical microtubule array of the plant cell plays a role in directing cellulose synthesis during formation of the cell wall, allowing for controlled expansion of the cell. In cells that undergo rapid unidirectional elongation, such as root epidermal cells, cortical microtubules are organized into parallel arrays that are oriented perpendicular to the long axis of cellular expansion. If the organization of cortical microtubules is disrupted through a mutation or drug treatment, helical twisting of cell files around the long axis of an organ sometimes results. Twisting in the late elongation zone of the root is thought to indicate a discoordination of growth cessation between cells in adjacent tissue layers, and this loss of coordination is likely accompanied by local changes in the extensibility of the primary cell wall. This project focuses on a group of Arabidopsis thaliana mutants whose roots exhibit a twisting phenotype in response to treatment with a microtubule-targeted drug, propyzamide. The goal of the project is to gain a better understanding of the regulation of directional growth in plants, as well as of the changes in cell wall extensibility which accompany twisting. Cell-type specific antibody tagging of epitopes indicating growth cessation (i.e. demethylesterified homogalacturonan) will be used to verify whether there is a discoordination of growth cessation between different tissue layers in the root. Additionally, responses of twisting mutants to inhibitors of ethylene biosynthesis and signalling will be tested as a means of determining whether ethylene-induced growth cessation is involved in twisting. The transcriptional networks involved in xylem vessel formation Hitoshi Endo1, Masatoshi Yamaguchi1, Ko Kato1, Taku Demura1, 2 1 Nara Institute of Science and Technology, 2RIKEN Biomass Engineering Program Bio Science, NARA INSTITUTE of SCIENCE and TECHNOLOGY, Ikoma, Nara, Japan We established an in vitro system for vessel element transdifferentiation in Arabidopsis Col-0 suspension cells. Through a microarray analysis of the system we identified a number of genes with drastic changes in expression during the transdifferentiation. Moreover, we revealed that VASCULAR-RELATED NAC-DOMIN7 (VND7) encoding a NAC-domain transcription factor functions as a master regulator for vessel formation. However, the regulatory mechanism of VND7 expression is still largely unknown. Hence, in this study we aimed to identify transcription factors that regulate VND7 expression. So we first selected transcription factors up-regulated at the phase that most of the cell transdifferentiated into vessel element. Then we did particle bombardment-based transient assays to evaluate whether each candidates can induce VND7 expression using VND7pro:Luciferase construct as a reporter. As a result, we found that several genes could up-regulate the VND7 expression including all VND family members. So now we are further analyzing these transcription factors to reveal their functions during vessel formation and trying to clarify the transcriptional relationship among these transcription factors. How modelling can elucidate microtubules' roles in root hair tip growth Ryan Eng1, Eric Cytrynbaum2, and Geoffrey Wasteneys1 1 Department of Botany and 2Department of Mathematics, University of British Columbia, Vancouver, Canada Tip growth is a highly polarized developmental process that can occur in cells of fungi as well as higher and lower plants. Root hairs of plants, particularly of Arabidopsis, are model cell types to study the mechanisms and processes of tip growth. One of these processes that aid in root hair tip growth is the regulation of microtubule organization and dynamics. Microtubules are dynamic polymers of the cytoskeleton that are thought to control the directionality of tip growth, although the exact relationship is unclear. Two Arabidopsis thaliana mutants, mor1-1 and ark1, have aberrant root hair polarity as a result of altered microtubule dynamics and organization. While MOR1 is known to promote growth and stability of microtubules, mor1-1 microtubules are observed to have reduced dynamics and exist as fragmented polymers as opposed to dynamic and lengthened polymers in wild-type conditions. As a result, mor1-1 root hairs have a wavy and bifurcated phenotype. Mutants of ARK1 have a similar root hair phenotype even though the putative role of ARK1 on microtubules is thought to be antagonistic to MOR1. Determining the exact role of ARK1 on controlling microtubule dynamics and organization is currently being performed using live cell imaging, in vitro assays, and biological mathematical modelling. Similar experiments are being done to further increase knowledge of MOR1 function and its potential interaction with ARK1. In doing so, the details on how specific microtubule dynamics and organization can lead to proper tip growth of root hairs will be elucidated. Discovery of the Collapsed Vessel gene by a reverse genetic approach in Arabidopsis thaliana Etienne Grienenberger and Carl J. Douglas Department of Botany, University of British Columbia, Vancouver, Canada The appearance of secondary cell walls was a major adaptation in land plant evolution, providing crucial structural, physiological and defensive innovations. Secondary wall formation is tightly regulated by developmental and environmental cues and recently, several transcription factors have been characterized defining a basic regulatory network that controls these processes. However, our understanding of the complexity of secondary wall formation and its regulation is far from complete. In order to identify new transcriptional regulators involved in secondary wall processes, we used a reverse genetic approach. Taking advantage of the latest genomic tools such as gene coregulation, conservation of genes amongst species and microarray based gene expression patterns, we selected 24 Arabidopsis gene candidates for their putative involvement in transcriptional regulation of secondary cell wall formation. Analysis of the corresponding mutants for cell wall and growth related phenotypes led to the identification of the Collapsed Vessel (COVE) gene. The cove mutant displays an irregular xylem phenotype with collapsed vessels, but lacks growth defects. COVE belongs to a small family of unknown genes in Arabidopsis and is highly conserved as a small family amongst the angiosperms. The protein lacks known domains and does not share similarity with other known proteins, but exhibits high structural conservation with all of its homologs and, interestingly, with bHLH transcription factors. RT-PCR experiments show the high expression of COVE in Arabidopsis stems while yeast two hybrid experiments reveal a transcriptional activation activity of the protein. All together these data suggest a role for COVE in secondary cell wall regulation. Investigation of a KNAT7-BLH-OFP transcription factor complex involved in regulation of secondary cell wall biosynthesis in Arabidopsis thaliana Yuanyuan Liu and Carl Douglas Department of Botany, University of British Columbia, Vancouver, Canada The plant secondary cell wall is a composite network of complex polymers (cellulose, lignin, and hemicellulose) that provides protective and structural properties to the cell wall. Based on previous research, the Arabidopsis KNOX gene KNAT7 has been shown to act as a transcription factor that regulates secondary wall formation in Arabidopsis inflorescence stems in coordination with Ovate Family Proteins (OFPs). Co-expression and yeast twohybrid analyses suggest that BEL1-LIKE HOMEODOMAIN (BLH) transcription factors could be part of a KNOXBLH-OVATE transcription factor complex regulating aspects of secondary cell wall formation, together with KNAT7 and OFP1/4. I investigated the interactions of BLH partners with KNAT7 and OFP proteins through yeast two-hybrid and in planta bimolecular fluorescence complementation analyses, and have identified a BLH protein BLH6 (At4g34610), from among six candidate BLH proteins as a BLH interacting partner of KNAT7. In addition, I demonstrated that OFP4 interacts with homeodomain of KNAT7 and BLH6 interacts with the KNAT7 MEINOX domain by yeast two-hybrid analyses. Furthermore, I investigated the function of BLH6 and an additional BLH protein, BLH5 (At2g27220), by characterizing the phenotypic effects of blh loss of function and BLH overexpression on stem anatomy. Phenotype analysis showed that blh5 knockout mutant and BLH5 overexpression mutant are indistinguishable from wild type. The blh6 knock out mutant displayed slightly thicker cell walls in interfascicular fibers. In addition, I employed protoplast transfection assay to demonstrate that BLH6 is a transcriptional repressor. This study provides new information regarding the existence of a BLH6-KNAT7-OFP complex and insights into the biological function of BLH6. PtCOBRA3 and PtCOBRA5, putative orthologs of AtCOBRA-like4 function in cellulose deposition during secondary cell wall development in poplar Grant R. McNair1, L. Samuels2, and S.D. Mansfield1 1 Department of Wood Science, Forestry, and 2Department of Botany, University of British Columbia, Canada Plant cell walls provide the structural support for maintaining orientated tree growth. The ordered process of secondary cell wall deposition within the confines of the primary cell wall provides the structural and load bearing capacity for plants. Lignin, hemicelluloses and cellulose are the major components of the secondary cell wall with cellulose contributing up to 45% of the cell wall. Reduced and or altered cellulose deposition may result in cell wall failure, irregular xylem, and reduced plant growth. In Arabidopsis, non-functional mutant alleles of a GPI-anchored protein, Atcobl4, present a normal plant growth phenotype, but have thinner secondary cell walls due to a reduction in cellulose content. Two putative orthologs of AtCOBL4 in poplar, PtCOBRA3 and PtCOBRA5, are highly expressed in the xylem of poplar. These finding suggest potential for PtCOB3 and PtCOB5 to function in secondary cell wall deposition, possibly in a redundant manner. To investigate this, RNAi-mediated gene suppression is being utilised to reduce the transcript abundance of PtCOB3, or PtCOB3 and PtCOB5 simultaneously. To complement this study, overexpression of AtCOBL4 in wood, using a promoter found in cells with developing secondary cell walls, will be undertaken to determine if increased transcript abundance alters the wood properties. Developmental roles of pectin methyl esterification in Arabidopsis thaliana cell walls Kerstin Mueller, S. Bartels, K. Weitbrecht, R. Thomann, G. Leubner, and A. Kermode Biological Sciences, Simon Fraser University, Burnaby, Canada Pectins are polygalacturonans that constitute a major component of the plant cell wall. The galacturonic acid residues of the cell wall pectins can be methyl-esterified, a modification that influences cell wall characteristics, and is mediated by pectin methyl esterases (PMEs). Endogenous PME inhibitors constitute one aspect of PME regulation. Notably these PME inhibitors can also participate in defense mechanisms against plant pathogens. Pectin deesterification leads to a decrease in cell wall pH, and de-esterified galacturonic acid residues can be cross-linked by Ca2+. PME activities thus regulate the biochemical and biophysical properties of the cell wall, including its extensibility, a property crucial for plant growth and development. PMEs are a large gene family with over 60 members in Arabidopsis, and knocking out a single or even a handful of PMEs does not typically cause a visible phenotype. We have therefore created Arabidopsis lines over-expressing an endogenous PME inhibitor, as a means of elucidating the developmental processes in which pectin methyl esterification plays a role. The over-expressing lines show strong developmental phenotypes such as twisted stems and siliques, lower seed production, altered seed germination and hypersensitivity to mechanic stress in seedling roots. Within germinating seeds, there are no differences in cell size or shape associated with PME-inhibitor over-expression, as determined by environmental scanning electron microscopy. Analyses of the size and shape of the cells comprising seedlings are planned, as are studies involving the FTIR spectroscopic analysis of the pectin methyl esters in the cell wall. Phosphorylation of VASCULAR-RELATED NAC-DOMAIN7; the master regulator for xylem vessel differentiation Yoshito Ogawa1, Masatoshi Yamaguchi1, Ko Kato1, Taku Demura1,2 1 Nara Institute of Science and Technology, 2RIKEN Biomass Engineering Program Nara Institute of Science and Technology, Ikoma, Nara, Japan The vascular system of plants consists of two types elongated cell files: xylem, through which water and dissolved minerals are passed, and phloem, in which amino acids and sucrose are transmitted. In Arabidopsis, several genes were reported to be involved in secondary cell wall formation or programmed cell death during xylem differentiation, and VASCULAR-RELATED NAC-DOMAIN7 (VND7) was identified as the master regulator for xylem vessel differentiation. Recently, we demonstrated that VND-INTERACTING2 (VNI2) has a function as a transcriptional repressor of VND7. However, the regulatory mechanisms by which VND7 function is regulated are still unknown. In this study, we are focusing on phosphorylation of VND7, which was discovered by heterologous expression of VND7 in BY-2 cells and was supposed to be closely associated with the regulation of VND7 function. To reveal the phosphorylated amino acid residues, we performed an in silico prediction, and then proceeded to alanine substitution of the candidate residues. As a result, we succeeded in the identification of a putative phosphorylation site of VND7. At present, we are examining the biological meaning of phosphorylaion of VND7. Identification and activity assessment of the Pectinesterases and Pectinesterase inhibitors in Linum usitatissimum David Pinzon and Michael K. Deyholos Biological Sciences, University of Alberta, Edmonton, Alberta, Canada Pectinesterases (PME) catalyze the demethylesterification of homogalacturonic acid units of pectins in the cell wall; their activity is regulated by pectinesterase inhibitors (PMEI). They have been implicated in the regulation of fibre length, cell separation, and susceptibility to pathogens, among others. Only 3 PMEs have been previously reported in flax, and their activity is not totally clear. The identification of the PME and PMEI families in flax is important to understand the different roles and regulations, and specifically, to determine their role in fibre development. The flax genome was sequenced by our research group and was searched to identify PMEs and PMEIs. 103 PME and 23 PMEI putative genes were found after blast and motif search. The type of PME, cleavage site, isoelectric point, and other sequence features were determined. Specific primers were designed to determine the expression of the genes in different tissues of flax by using TaqMan real time PCR. It was found that 79 PME and 21 PMEI are being expressed. The specific expression of each gene in 10 different tissues and its activity will be evaluated by protein purification. As well, mutants generated by EMS treatment will be screened for PME/PMEI mutations by a reverse genetics approach using next generation sequencing, and the phenotype will be assessed. Unraveling cellulose synthesis in poplars Haley Rupp and C.P. Joshi Forestry, Michigan Tech University, Houghton, Michigan, USA Genetic manipulation of cellulose biosynthesis in trees could provide important insights into the growth and development of trees. Recently cellulosic biofuels have become important as a part of global bioenergy agenda. Therefore, we are discovering novel ways of improving cellulosic biomass production for efficient biofuel production by understanding how trees synthesize cellulose. We are taking a multi-pronged approach. First, we have characterized expression of several cellulose synthesis related genes from poplar trees. Second, genetic manipulations of many of these genes in transgenic poplar trees have produced interesting phenotypes ranging from increase in the cellulose content and alterations of its properties to significant decrease in wood cellulose amounts. Finally, we have developed a virus induced gene silencing (VIGS) platform for rapid screening and identification of hitherto unknown genes involved in cell wall development with the hope to translate that knowledge to poplar trees. Our long-term goal is to unravel basic process of cellulose synthesis in trees in order to enable economically viable and ecologically sustainable utilization of bioenergy. Elucidating the function of Arabinogalactan protein during secondary cell wall biosynthesis Li Xi 1, Lacey Samuels2, and Shawn Mansfield1 1 Department of Wood Science, Forestry, and 2Department of Botany, University of British Columbia, Canada Arabinogalactan proteins (AGPs) appear to be important proteins in cell wall development that exist as a multigene family, consisting of 85 genes in Arabidopsis and 37 in poplar. The classical AGPs have been divided into three main types: fasciclin-like AGPs, lysine- rich AGPs and AG peptides. AGPs have been proposed to be integral to cell wall deposition, plant growth and development, and programmed cell death; however, the mechanisms of their roles are not yet clear. To date, the function of classical AGPs have been studied using both Arabidopsis mutant analysis with chemical reagents such as the AGP cross-linker β-glucosyl Yariv reagent (1,3,5-tris (4-β-Dglycopyranosyloxyphenylazo)-2,4,6-trihydroxy-benzene) and epitope localization with antibodies. Here we will discuss the expression pattern of AGP9, AGP14 and AGP18 from poplar using real time Q-PCR, immunofluorescence detection and promoter-Gus-fusion protein. Based on the gene expression results, PtAGP9 appears to be specific to the cambium and secondary xylem; while PtAGP14 is expressed in the cambial zone and phloem and PtAGP18 in the cambium. It appears that these three AGPs are active during cambium cell differentiation, xylem cell expansion and secondary cell wall formation.