Download The appearance of secondary cell walls was a - Plant-o

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.