Download The XIII th Cell Wall Meeting

Abstract Book
Nantes, France
7-12 July 2013
Committees
Sponsors
Local Organizing Committes (INRA, Nantes)
Marie-Christine Ralet
Adelin Barbacci
Fanny Buffetto
Anne-Laure Chateigner-Boutin
Fabienne Guillon
Jane Métayer
Isabelle Mauboucher
Scientific Organizing Committee
Debra Mohnen
The University of Georgia,
USA
Roula Abdel Massih
University of Balamand,
Lebanon
Daniel Cosgrove
Penn State University, USA
Stephen Fry
The University of Edinburgh, UK
Scientific Sessions Committees
1. Plant Cell Wall Architecture : Structure, Interactions
& Cross-Links of Cell Wall Components
Maija Tenkanen, Lennart Salmen, Vincent Bulone
2. Dynamics of Plant Cell Wall Components :
from Biosynthesis to Remodelling
Intracelullar synthesis & trafficking,
Synthesis & assembly at the plasma membrane, Remodelling in muro,
Transcriptional and post-transcriptional control
Elisabeth Jamet, Staffan Persson, Tony Bacic
3. Evolution & Diversity of Plant Cell Walls
Zoë Popper, David Domozych
4. Functions of Plant Cell Walls in planta
Growth, morphogenesis & development, Signaling & defense,
Response to environment
Giulia De Lorenzo, Sìlvia Coimbra, Herman Höfte
5. Advanced Understanding of Cell Wall Structure,
Biosynthesis & Function
Bioinformatic & omic approaches,
approaches
Computational & biophysical
Anja Geitmann, Arezki Boudaoud, Joachim Selbig
6. Uses of Plant Cell Walls & Derived Products
Food, feed, chemicals & fuel, Renewable biomedical & smart materials
Bernard Cathala, Simon Mc Queen Mason, Henk Schols
Abstracts from oral presentations listed by session and order of talks
Session 1 : Plant Cell Wall Architecture; Structure, Interactions & Cross-Links of CW
Components
Session 2 : Dynamics of plant CW Components: from Biosynthesis to Remodelling : Intracellular
synthesis & trafficking, Synthesis & assembly at the plasma membrane, Remodelling in muro,
Transcriptional and post-transcriptional control
Session 3 : Evolution & Diversity of plant CW
Session 4 : Functions of Plant CW in planta : Growth, morphogenesis & development, Signaling &
defense, Response to environment
Session 5 : Advanced Understanding of CW Structure, Biosynthesis & Function : Bioinformatic &
omic approaches, Computational & biophysical approaches
Session 6 : Uses of plant CW and derived products : Food, feed, chemicals & fuel, Renewable
biomedical & smart materials
Session 1 : Plant Cell Wall Architecture; Structure, Interactions & Cross-Links of
CW Components
O1-01
CesA dimers are the fundamental building blocks of a plant cellulose synthase complex
Olek A.T. (a), Rayon C. (b), Makowski L. (c), Ciesielski P. (d), Paul L.N. (e), Kim H.R. (f), Ghosh S. (f), Kiharaf D. (g),
Crowley M. (d), Himmel M.E. (d), Bolin J.T. (f), Carpita N.C. (aef)
(a) Department of Botany & Plant Pathology, (e) Bindley Bioscience Center, (f) Department of Biological Sciences, (g)
Department of Computer Science, Purdue University, West Lafayette, IN, USA; (b) EA 3900-BIOPI, Université de Picardie
Jules Verne, Amiens, France; (c) Department of Electrical and Computer Engineering, Northeastern University, Boston, MA,
USA; (d) Biomolecular Science Group, National Renewable Energy Laboratory, Golden, CO, USA.
Cellulose is a para-crystalline array of three dozen (1→4)-β-D-glucan polysaccharides that forms the fundamental
scaffold of plant cell walls. Recombinant catalytic domains of rice CesA8 cellulose synthase reversibly form
dimers dependent on protein concentration and the presence of thiol reducing agents. Small-angle x-ray scattering
(SAXS) predicts an elongated two-domain monomer, with the smaller domain functioning to couple two
monomers into a dimer. Homology modeling of the catalytic domain truncated to remove the plant-conserved
region (P-CR) and the class-specific region (CSR) gives close alignment with the Rhodobacter sphaeroides
cellulose synthase catalytic domain. Binding stoichiometry studies suggest a single site for UDP-glucose binding
and a second binding site for a phosphorylated glucosyl moiety. Structure modeling against 18 additional targets
gives closest similarity to an E. coli chondroitin polymerase when the P-CR and CSR are included and a
molecular shape consistent with the findings from SAXS studies. However, combinatorial modeling against other
targets with optimal structural similarity to the P-CR and CSR conserves the structural homology of the fourcomponent active site. Independent sites of glycosyl transfer on each monomer and production of two (1→4)-βD-glucan chains per dimer account for a size consistent with known synthase complex dimensions. We offer this
new perspective for construction rosette complex to stimulate new approaches to engineer the complex for
enhancing efficiency of biofuel production or for synthesis of new materials and nanoproducts.
This work was supported by the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research
Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Award Number DE-SC0000997. Use
of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.
O1-02
Cellulose ultrastructure is influenced by the GPI-anchored protein COBL4
McNair G.R. (a), Samuels L. (b), Mansfield S.D. (a)
(a) Department of Wood Science, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada; (b) Department of
Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
At the plasma membrane, cellulose is synthesised by cellulose synthase complexes (CSCs) to produce elementary
fibres that then associate to form cellulose microfibrils in the apoplast. Although it is well accepted that cellulose
synthases (CesA) are intricately involved, the exact underlying molecular mechanisms controlling the ordered
deposition of cellulose still unclear. However, it is apparent that several associated proteins localised in the
apoplastic space contribute to normal cellulose biosynthesis. For example, AtCOBRA-LIKE4 (AtCOBL4), a GPIanchored protein concentrated at the outer leaflet of the plasma membrane appears to be involved in normal
cellulose deposition. Null mutants of cobl4 and the rice homolog display normal plant growth, but reduced
cellulose content and thinner secondary cell walls. In an attempt to better understand AtCOBL4 function we have
chemically and ultrastructurally examined the properties of secondary cell walls associated with secondary cell
wall specific over-expression of AtCOBL4 in hybrid poplar. Transgenic lines were shown to be altered in cell wall
polysaccharide content, with glucose abundance being elevated up to 15% in some transgenic lines relative to
wild-type controls. The increase in glucose levels was positively correlated with AtCOBL4 transcript abundance,
and shown to manifest in significant increases in α-cellulose. The ultrastructural properties of the cellulose
deposited showed altered cell wall crystallinity with no significant change in microfibril angle. Furthermore, the
degree of polymerisation (DP) of cellulose was shown to be 10-40% longer than that of wild-type poplar.
Interestingly, morphological characteristics, such as xylem fibre length, were shorter in the AtCOBL4 overexpression lines relative to wild type. These findings clearly suggest AtCOBL4 and its poplar orthologs function
to organise newly synthesised cellulose at the plasma membrane-cell wall interface, and play an important role in
controlling the quantity and quality of cellulose synthesised.
O1-03
Structure and biochemistry of (1,3)-β-glucan
Naumann M. (a), Dalüge N. (a), Eggert D. (b), Reimer R. (b), Voigt C.A. (a)
(a) Phytopathology and Biochemistry, Biocenter Klein Flottbek, University of Hamburg, 22609 Hamburg, Germany; (b)
Electron Microscopy and Microtechnology, Heinrich Pette Institute, 20251 Hamburg, Germany.
In plants, the cell wall polymer (1,3)-β-glucan, known as callose, is essential for developmental processes and
pollen formation. Additionally, callose is generally deposited in response to abiotic and biotic stress, which
includes deposition at wounding sites and sites of pathogen attack. Regarding the latter, we could recently proof
that callose can have a decisive role in preventing fungal penetration. The overexpression of the callose synthase
GSL5, which is responsible for stress-induced callose formation in Arabidopsis thaliana, resulted in an elevated
early callose deposition at sites of attempted powdery mildew infection and complete penetration resistance [1, 2].
In this study, we wanted to know whether GSL5 overexpression not only affected the amount of deposited callose
but also its structure. By using the non-invasive method of direct stochastic optical reconstruction microscopy
(dSTORM), we were able to receive a structural resolution of < 50 nm for aniline blue-stained callose polymer
fibres in pathogen-induced deposits. Only callose deposits of the resistant GSL5 overexpression lines revealed
distinct structural differences in the orientation of callose fibres. In the dense central core of the callose deposit,
callose fibres formed a reticular, almost amorphic structure, whereas the surrounding field was characterized by a
radial orientation of callose fibres, which was enclosed by ring of circular oriented callose fibres. Callose deposits
of wild-type plants revealed an almost amorphic structure. For the biochemical characterization of callose
biosynthesis, we expressed the hydrophilic, cytosolic loop of the callose synthase GSL5 in Escherichia coli and
purified the synthesized 75 kDa peptide. In an activity assay, we were able to proof that the purified cytosolic loop
is sufficient for callose biosynthesis. Neither the transmembrane regions of the full length enzyme nor additional
factors as previously proposed for active complex formation were required for enzymatic activity. Moreover, the
cellulose precursor cellubiose, a (1,4)-β-glucose dimer, facilitated a higher callose synthase activity than the
callose precursor laminaribiose, a (1,3)-β-glucose dimer. This indicates a putative interaction of callose formation
with cellulose biosynthesis.
[1] D. Ellinger & M. Naumann et al. (2013) Plant Physiol., 161, 1433-1444; [2] M. Naumann et al. (2013) Plant Signal.
Behavior, 8, in press.
O1-04
Cellulose and an AGP play distinct roles in mediating the adherence of seed coat mucilage
Griffiths J.S., Voiniciuc C., Haughn G.W.
Dept. of Botany, University of British Columbia, 6270 University Blvd., Vancouver, BC, V6T 1Z4, Canada.
Cell-cell adhesion is a vital function of cell walls that is mediated by interactions between cell wall polymers. We
are exploiting the Arabidopsis seed coat mucilage system to examine cell wall polymer interactions using genetics
and biochemistry. Upon hydration, seeds release large amounts of pectinaceous mucilage that separates into two
distinct populations: an adherent layer that remains attached to the seed and a non-adherent layer that can be
removed by gentle shaking. The major component of adherent mucilage is rhamnogalacutonan I (RG I), but other
components including cellulose and xyloglucans are also present. Cytological analyses of adherent mucilage with
dyes that label cellulose and pectin indicate that it has a distinct structure consisting of strongly dark-staining rays
of cellulose and pectin that extend above the centre of each epidermal cell. CELLULOSE SYNATHASE 5 and
SALT-OVERLY SENSITIVE 5, which encode a cellulose synthase and a fasciclin like arabino-galactan protein,
respectively, have been shown to be required for mucilage adhesion through unknown mechanisms. It has been
suggested that SOS5 mediates adhesion through an influence on cellulose biosynthesis 1. We therefore investigated
the relationship between SOS5 and CESA5. Mutant cesa5 seeds show a general reduction in cellulose content,
less mucilage cellulose and shorter rays. In contrast, mutant sos5 seeds have a wild-type cellulose content but a
complete loss of ray-like structure in the mucilage. The double mutant, cesa5 sos5, shows a more severe loss of
mucilage adherence, and a complete loss of cellulose staining, indicating these two genes function independently
to mediate mucilage adhesion. Double mutant analysis combining cesa5 and sos5 with mutations in loci required
for normal mucilage pectin (mum2, fly1) indicate that sos5, but not cesa5, is required to suppress pectin-mediated
adhesion defects. These data suggests that cellulose and SOS5 contribute to cell wall adhesion through distinct
mechanisms. The implications of these data on polysaccharide interactions in the cell wall will be discussed. [ 1] S.
Harpaz-Saad et al.,(2011) Plant J., 68, 941–953.
O1-05
AGP31, an arabinogalactan protein, as a network-forming protein in A. thaliana cell walls
Hijazi M., Roujol D., del Rocio Cisneros Castillo L., Saland E., Jamet E., Albenne C.
LRSV, UPS/CNRS, 31326 Castanet-Tolosan, France.
AGP31 (arabinogalactan protein 31) is a remarkable plant cell wall protein displaying a multidomain organization
unique in Arabidopsis thaliana: it comprises, from N- to C-terminus, a predicted signal peptide, a short AGP
domain of 7 amino acids, a His-stretch, a Pro/Hyp-rich domain and a PAC (PRP-AGP containing Cys) domain.
AGP31 was found as a major protein of the wall proteome of growing etiolated hypocotyls [1]. It displays
different O-glycosylation patterns with arabinogalactans on the AGP domain and Hyp-O-Gal/Ara-rich motifs on
the Pro/Hyp-rich domain [2]. In vitro interactions assays were carried out on nitrocellulose membranes containing
polysaccharides probed with purified native AGP31 or recombinant PAC-V5-6xHis. After validation of the
arrays, we demonstrated that AGP31 interacts through its PAC domain with galactan, which are branches of
rhamnogalacturonan I (RGI). Besides, AGP31 was also found to bind methylesterified polygalacturonic acid
(m.e.PGA), probably through its His-stretch. Finally, we showed that AGP31 was able to interact with itself in
vitro through its PAC domain. These results permitted to propose a model of interactions of AGP31 with itself
and other cell wall components via non-covalent bounds. AGP31 is assumed to participate in complex
supramolecular scaffolds, which could contribute to the strengthening of cell walls of quickly growing organs like
etiolated hypocotyls.
[1] Irshad et al., 2008, BMC Plant Biol., 8, 94; [2] Hijazi et al., 2012, J. Biol. Chem., 287, 9623-9632.
O1-06
Wall
glycoproteins and membrane glycolipids
rhamnogalacturonan-II-borate cross-linking
act
as
‘chaperones’
promoting
Chormova D., Voxeur A., Fry S.C.
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, UK.
RG-II is a complex pectic domain, ubiquitous in vascular plant primary cell walls. [1] Its ability to dimerise via
tetrahedral borate bridges controls the wall’s growth, thickness and porosity. Simply mixing pure RG-II with boric
acid (H3BO3) results in slow dimerisation, but the process is strongly promoted by certain non-biological metals,
especially Pb2+. Nothing was known of how living cells rapidly dimerise their RG-II. Indeed, it was not clear at
what stage in the ‘career’ of an RG-II domain it dimerises in vivo. We have developed a gel electrophoretic assay
for quantifying monomeric and dimeric RG-II, and used this to explore borate cross-linking.
In Rosa cell cultures grown in boron-free medium, almost all the RG-II was monomeric. Adding H 3BO3 to such
cells failed to dimerise the existing RG-II within 24 h, but new RG-II made after H 3BO3 addition was largely
dimeric. Also, when monomeric [3H]RG-II was added to H3BO3-fed cells, it remained soluble and monomeric in
the medium. These observations indicate that dimerisation occurs mainly intraprotoplasmically, not in the wall.
We tested plant extracts for hypothetical enzymes that might ‘mimic’ Pb 2+, promoting RG-II bridging by H 3BO3
in vitro, but found no such activity. However, RG-II was rapidly dimerised by H 3BO3 in the presence of certain
wall glycoproteins and membrane glycolipids, which we propose act as molecular ‘chaperones’ of the RG-II and
H3BO3 respectively, manoeuvring them so as to favour cross-linking. The nature and specificity of the chaperones
will be discussed. Ascorbate (but not dehydroascorbic acid) inhibited the borate-bridging of RG-II in vitro, and,
along with the chaperones, may be another factor controlling the cross-linking of this pectic domain in vivo.
[1] M.A. O’Neill et al. (2004) Annu. Rev. Plant Biol., 55, 109.
We thank the UK BBSRC for funding this work.
O1-07
Half of the xyloglucan in cell walls of tissue cultures is linked to pectin via a highly branched
arabinan
Mort A., Fu J., Umezu S., Patil P.
Department of Biochemistry and Molecular Biology, Oklahoma State University, USA.
In 1973 a model of the primary cell walls of dicots was proposed in which the xyloglucan and pectin were
covalently linked to each other via an arabinan or an arabinogalactan . More recent models show no such linkage.
However, several groups have reported evidence that xyloglucan and pectin are linked together somehow at least
to a certain extent . We have found that after extensive digestion of both cotton and Arabidopsis cell walls 25%
KOH/ 0.1% NaBH4 solubilizes xyloglucan (XG) along with rhamnogalacturonan (RG) and xylogalacturonan
(XGA). About half of the XG does not adsorb to an anion exchange column whereas the other half does and coelutes with the RG and XGA at quite a high ionic strength. If the complex is incubated with avicel, most of the
XG and half of the RG adsorbs to it. Incubation of the complex with arabinosidase and then endoarabinanase
disrupts the linkage between the XG and RG/XGA. The majority of the XG is no longer adsorbed on the anion
exchange column. Incubation with the purified endoarabinanase alone does not disrupt the linkage between the
XG and RG/XGA. Thus the linkage must contain a branched arabinan.
[1] Keegstra, K., et al. (1973) Plant Physiol., 51,188-196; [2] Thompson, J.E. and S.C. Fry (2000) Planta,. 211, 275-286; [3]
Baydoun, E., et al. (2005) American Chemical Society; [4] Popper, Z.A. and S.C. Fry (2005) Ann. Bot. 96, 91-99; [5]
Popper, Z.A. and S.C. Fry (2008) Planta,. 227, 781-794.
O1-08
Distinct domains of glucuronoxylan may create interfaces for interaction with cellulose and lignin
Busse-Wicher M. (a), Bromley J.R. (a), Mortimer J.C. (a), Tryfona T. (a), Nikolovski N. (a), Brown D. (b), Dupree P. (a)
(a) Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK; (b) Shell Global
Solutions, Thornton, Chester, CH1 3SH, UK.
Despite the fact that cellulose, xylan and lignin are major components of angiosperm secondary cell walls, we
know very little of how these molecules may be interacting. We recently discovered that there are different
patterns of [methyl]glucuronic acid ([Me]GlcA) substitution of xylan in hardwood species. The major domain of
xylan is characterized by [Me]GlcA substitution on even-numbered xyloses with relatively long intervals between
substitutions (on average, on every 8-10 xylose). These kinds of substitutions span across the majority of the
xylan chain (hence the name Major Domain). The minor domain of xylan is more tightly substituted (every 5, 6 or
7 xylose) and covers about 20-30% of xylan.
Hypothetically, the even [Me]GlcA spacing pattern of the major domain may be significant if the xylan forms a
flat two-fold screw ribbon structure when interacting with the cellulose microfibrils in the cell wall. In our
proposed model, the orientation of all the [Me]GlcA side chains would be on one side of the xylan backbone
ribbon. Such a ribbon structure could create a potential surface for interaction of the xylan and cellulose chains,
and a charged surface for interaction with other cell wall components, such as lignin. To answer if and how the
major and minor domains are functionally different, we are currently investigating their role in binding to
cellulose and lignin using the gux mutants that lack each of these distinct xylan domains.
Bromley et al. (2013) Plant J., in press.
O1-09
The impact of arabinoxylan structure on the properties of cell walls in wheat grain endosperm
Ying R. (a), Rondeau-Mouro C. (a), Barron C. (b), Guillon F. (a), Saulnier L. (a)
(a) UR1268 BIA, INRA, F-44316 Nantes, France; (b) UMR 1208 IATE, INRA-CIRAD-UMII-Supagro, F-34000 Montpellier,
France.
Arabinoxylans (AX) and (1→3)(1→4)-β-D-glucans (BG) are the main components of the cell walls in the
endosperm of wheat grain [1]. The relative occurrence of these two polysaccharides and the fine structure of the
AX are highly variable within the endosperm, but little is known about the impact of these variations on the
properties of the cell walls.
Films of AX and BG were used as simplified models of the cell wall to study the impact of polymer structure on
the hydration and mechanical properties of the cell walls [2]. Effective moisture diffusivities (Deff) were
influenced by the water content, and the structure of polysaccharides. Higher Deff were obtained for films made
with highly substituted AX compared to values obtained for films made with BG or lowly substituted AX. Proton
dipolar second moments M2 and water T2 relaxation times measured by TD-NMR, indicated that the highly
branched AX films exhibited a higher nano-porosity, favoring water motions within films. In addition, traction
tests showed that BG films have much higher extensibility than AX films, and strength and extensibility of AX
films was impacted by arabinose to xylose ratio. The water content and the polymer structure within films
strongly influence interactions between polysaccharides and the nanostructure of films. These results bring new
lightning on the possible impact of polysaccharide structure on cell wall properties in wheat grain endosperm. A
higher effective diffusion of water is observed with highly substituted AX films compared to BG or lowly
substituted AX films, which is in line with the structure of polymers in the different cell types and possible cell
wall hydration requirement in the endosperm. For example, substitution of AX is higher in the cell walls of the
transfer cells that are supposed to play an active role in solute uptake for grain filling. In conclusion, the changes
in cell wall polymers structure observed during grain development and within grain tissue could possibly
modulate the hydration properties of the cell walls.
[1] Saulnier et al. (2012) J. Cereal Sci., 56(1), 91-108; [2] Ying R et al. (2013). Carbohydr. Polym., 96(1), 31-38.
O1-10
Direct visualization of polysaccharide interactions in plant primary cell walls by AFM
Zhang T., Cosgrove D.J.
Center for Lignocellulose Structure and Formation and Department of Biology, Penn State University, University Park PA
16802, USA.
Cell wall properties, including strength and extensibility, emerge from the nano-scale interactions of matrix
polymers with cellulose microfibrils, but the details of these interactions have not been settled. To visualize the
spatial arrangement of cell wall components at the nm scale, we used atomic force microscopy (AFM) to map
variations in the surface height, modulus and tip adhesion across the surface of never-dried primary cell walls.
The position of individual cellulose microfibrils (~3 nm wide) and matrix substances were visible at the inner wall
surface (closest to the plasma membrane) without the need for extraction, which can cause significant
rearrangements of these delicate structures. Dehydration induced significant surface alterations. Addition of
calcium rigidified a pectic network on the wall surface, coagulating the soft and ‘invisible’ layer into strands now
visible by the AFM probe. Microfibril bundling and cross-lamellar layering were two common and prominent
features of primary cell walls of parenchyma and epidermis of onion scales, cucumber hypocotyls and maize
coleoptiles. Montages of AFM images showed that individual microfibrils emerged into and out of short regions
of microfibril bundles, resulting in an extensive reticulated pattern. We suggest that the short regions of
microfibril bundling are key determinants of wall mechanics and potential sites of wall loosening by expansins
and endoglucanases. Modulus mapping by AFM showed significant heterogeneity in wall stiffness at the nm
scale; matrix polymers close to the surface of microfibrils had a higher modulus than polymers at some distance.
AFM images before and after wall strain showed that deformation was non-uniform and complex. Our results
point out the spatial heterogeneity of primary walls at the nm scale and the utility of AFM to assess polymer
stiffness and motions during large-scale wall deformations.
O1-11
Monitoring large-scale ordering of cellulose in intact plant cell walls using sum frequency
generation (SFG) spectroscopy
Park Y.B. (a), Lee C.M. (b), Zhang T. (a), Koo B.-W. (c), Park S. (c), Cosgrove D.J. (a), Kim S.H. (b)
Departments of Biology (a) and Chemical Engineering (b) and Materials Research Institute (b), Pennsylvania State
University, University Park, PA 16802, USA; (c) Department of Forest Biomaterials, North Carolina State University,
Raleigh, NC 27695, USA.
Sum-frequency-generation (SFG) vibration spectroscopy can selectively detect crystalline cellulose without
spectral interference from non-cellulosic components. Here we show that the cellulose SFG spectrum is sensitive
to cellulose microfibril alignment and packing within the cell wall. In primary walls of Arabidopsis, cellulose
exhibited a characteristic SFG peak at 2904 cm -1, whereas in secondary walls it had peaks at 2944 cm -1 and 3320
cm-1. Removal of matrix polysaccharides from primary walls resulted in an SFG spectrum resembling that of
secondary wall cellulose. A similar change was found in the xyloglucan-deficit mutant xxt1/xxt2, where the SFG
spectrum of primary walls resembled that of secondary walls. This observation was extended with atomic force
microscopy of cell wall surfaces, which showed highly dispersed microfibrils in wild-type but well aligned
microfibrils in the mutant. The result implies that xyloglucan influences microfibril alignment at the nm scale,
possibly via effects on cellulose crystallinity and microfibril stiffness. SFG intensity at 2944 cm -1 correlated well
with crystalline cellulose contents of various regions of the Arabidopsis inflorescence while changes in the
2944/3320 cm-1 ratio suggested subtle changes in cellulose ordering as tissues mature. SFG analysis of two
cellulose synthase mutants (irx1/cesa8, irx3/cesa7) indicated a reduction in cellulose content without evidence of
altered cellulose structure. Our results show that SFG spectroscopy is sensitive to the ordering of cellulose
microfibrils in plant cell walls at critical length scale that is important for cell wall architecture.
O1-12
Determining the structure of cellulose and hemicellulose in the primary plant cell wall using small
angle neutron scattering [SANS]
Huang S.-C. (ab), Park Y.B. (ac), Cosgrove D.J. (ac), Maranas J.K. (ab)
(a) Center for Lignocellulose Structure and Formation, (b) Department of Chemical Engineering, (c) Department of Biology,
Penn State University, University Park, PA 16802, USA.
Hemicellulose influences in primary cell walls, either through a tethered network with cellulose, or, as recently
suggested, as an adhesive between adjacent microfibrils. The distribution and structure of hemicellulose is
unknown. Further, measurement of its structure is challenging using scattering methods because the atomic
compositions of hemicellulose and cellulose are similar. We provide the missing structural information using
small angle neutron scattering in combination with contrast matching. Measurements are made with depectinated
primary petiole walls of wild-type and xyloglucan-deficient [xxt1xxt2] Arabidopsis Thaliana, with cellulose and
hemicellulose highlighted in different samples. The cellulose microfibril diameter of ~3nm and microfibril
spacings [20-40 nm in WT] are consistent with available data. Interfibril spacing in the mutant is wider [10-70
nm], which we interpret as the formation of loose bundles lacking preferential orientation relative to one another.
Hemicellulose in wild type forms extended coils and short [49 nm] coatings on microfibrils. One third of the
xyloglucan is in this configuration, coating 16% of the cellulose surface, in agreement with NMR [<10%]. Some
of us have recently suggested that the biomechanical strength of is due to <1.5% of the total cellulose in close
physical contact with xyloglucan. We suggest that these “hotspots” arise from contact between two coated
microfibrils. Negligible coating is observed in the mutant, consistent with its reduced mechanical strength and
absence of hotspots.
[1] Y. B. Park and D. J. Cosgrove, Plant Phys., 158, 465–75 (2012); [2] Y. B. Park and D. J. Cosgrove, Plant Phys., 158,
1933–43 (2012).
O1-13
Orientation analysis of cellulose in complex plant tissues by X-ray diffraction
Rüggeberg M. (ab), Saxe F. (c), Metzger T.H. (c), Sundberg B. (d), Fratzl P. (c), Burgert I. (ab)
(a) Swiss Federal Institute of Technology Zurich (ETH Zurich), Institute for Building Materials, Schafmattstrasse 6, CH8093 Zurich, Switzerland; (b) Swiss Federal Laboratories for Materials Science and Technology (EMPA), Applied Wood
Materials, Überlandstrasse 129, CH-8600 Dubendorf, Switzerland; (c) Max-Planck-Institute of Colloids and Interfaces,
Department of Biomaterials, Am Mühlenberg 1, D-14476 Potsdam, Germany; (d) Umea Plant Science Centre, Department of
Forest Genetics and Plant Physiology, Swedish University of Agricultural Science SLU, SE-90183 Umea, Sweden.
The orientation distribution of cellulose microfibrils in the plant cell wall is a crucial parameter for anisotropic
plant growth and mechanical behaviour. The direct visualisation of the cellulose microfibrils in the different
layers of the plant cell wall has been proven to be very challenging. X-ray diffraction is one of the most common
methods for analysing orientation of cellulose in single cells and plant tissues, but the interpretation of the
diffraction data is complex. We present a simulation procedure, which reveals the entire orientation distribution of
the cellulose microfibrils in the multi-layered cell wall. Measurements on aspen wood and Arabidopsis
demonstrate the versatility of this method and show that simplifying assumptions on geometry and distribution
parameters can lead to errors in the calculated microfibril orientation patterns for complex plant tissues. The
simulation routine is a valuable tool for better understanding the impact of genetic modifications on the structure
of plant cell walls.
O1-14
Modification of sugar beet pectin by pectin acetylesterases and structural elucidation of the
modified pectins by novel LC-MS approaches
Remoroza C. (a), Wagenknecht M. (b), Schols H.A. (a), Moerschbacher B. (b), Gruppen H. (a)
(a) Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD, The Netherlands; (b) Westfälische
Wilhelms-Universität Münster, Hindenburgplatz 55, D-48143, Münster, Germany.
Sugar beet pulp (SBP) can be regarded as a potential raw material for the pectin industry because it contains high
proportions of pectic substances. The commercial application of SBP in food so far is being limited because of its
poor gelling property, due to the presence of acetyl groups on the homogalacturonan part of SBP.
The aim of the study was to enzymatically fingerprint methylesterified and acetylated sugar beet pectins using
pectin lyase (PL) and polygalacturonase (endo-PGII) and to analyze the generated oligomers by hydrophilic
interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) and
evaporative light scattering detection (ELSD). To characterise different SBPs in a more quantitative manner, new
parameters for the description of the methyl ester and acetyl distribution over the pectin backbone were
established.
The novel enzymatic fingerprinting approach was not only used to establish the distribution of methyl and acetyl
substituents over the pectin, but was also used to reveal the mode of action of novel Bacillus pectin
acetylesterases.
O1-15
MALDI mass spectrometry imaging (MSI) as a new and promising method to investigate
polysaccharides in wheat grains
Veličković D., Ropartz D., Durand S., Saulnier L., Guillon F., Rogniaux H.
INRA, UR1268 Biopolymers Interactions Assemblies F-44316 NANTES, France.
Matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry emerged recently as a new imaging
method with outstanding features. It provides a means of resolving both the spatial distribution of many kinds of
molecules and their molecular structure in tissue sections. After preparing tissue section and coating with an
appropriate MALDI matrix, the instrument captures a series of mass spectra, each of which represents the mass
profile of a specific region in the sample [1]. Sensitivity, high information content and rapidity of tissue analysis,
in addition to the fact that no previous knowledge is required on the monitored molecules, may give MSI
tremendous advantage over other imaging techniques (e.g. microscopy with immune-labelling).
In this work, MSI was used to monitor the distribution and chemical variability of arabinoxylans (AX) and βglucans (BG) in cross sections of different development stages of Triticum aestivum L. These two polysaccharides
are the main cell wall components of wheat endosperm and their structure have a significant effect on wheat grain
development as well as on the end-uses and functional properties of the grain [2]. To the authors’ best knowledge,
this study is the first attempt to image cell wall polysaccharides in intact tissue by MS. The presentation will
highlight some unique results obtained on developing wheat grains.
[1] R. Goodwin. (2012) J. Proteomics., 75, 4893-4911; [2] S. Philippe et al. (2006) Planta., 224, 449-461.
Session 2 : Dynamics of plant CW Components : from Biosynthesis to Remodelling
Intracellular synthesis & trafficking, Synthesis & assembly at the plasma membrane,
Remodelling in muro, Transcriptional and post-transcriptional control
O2-01
Nucleotide sugar transporters are involved in the biosynthesis of rhamnose- and arabinosecontaining cell wall polysaccharides.
Temple H. (a), Moreno I. (a), Doñas D. (a), Blanco M.F. (a), Greve M. (a), Moreno A. (a), Dardelle F. (c), Mortimer J. (b),
Rautengarten C. (d), Mollet J.-C. (c), Dupree P. (b), Scheller H.V. (de), Orellana A. (a)
(a) Centro de Biotecnología Vegetal, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile; (b)
Department of Biochemistry, University of Cambridge, Cambridge, U.K. (c) Laboratoire de Glycobiologie et Matrice
Extracellulaire Végétale UPRES-EA 4358, Université de Rouen, 76821 Mont Saint-Aignan, Cedex, France; (d) Joint
BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA; (e) Department of Plant & Microbial
Biology, University of California, Berkeley, CA, USA.
Golgi-localized nucleotide sugar transporters (NSTs) are responsible for taking nucleotide sugars into the lumen
of the organelle, place where several glycosyltransferases involved in cell wall polysaccharide biosynthesis are
located. Given the function of these proteins, we hypothesized that changes in the activity of NSTs should lead to
changes in cell wall composition. Here we show that AtUTr8 is a UDP-rhamnose transporter, expressed during
seed development, in a pattern that resembles the expression of MUM4, an enzyme involved in the synthesis of
UDP-rhamnose. Mutants in AtUTr8 exhibited an altered pattern in mucilage staining and sugar composition
analyses revealed a decrease in rhamnose and galacturonic acid. Since mucilage is mainly composed by
rhamnogalacturonan-I our results suggest that AtUTr8 plays an important role in the biosynthesis of this
polysaccharide in Arabidopsis seeds. In addition, we have identified AtUTr10, a NST expressed in pollen.
Mutants in this NST show a decrease in the content of arabinose in the cell wall from pollen tubes.
Immunocytochemical analyses of this structure showed differences in the reactivity to LM13 and LM2, antibodies
that recognize arabinan and arabinogalactan proteins, suggesting that AtUTr10 is involved in the incorporation of
UDP-arabinose in Arabidopsis thaliana. All these results support the importance of NSTs in the biosynthesis of
cell wall polysaccharides in the Golgi apparatus.
Supported by: FONDAP-CRG 5090007, ICM-P10-062-F, PFB-16.
O2-02
Characterization of a Arabidopsis UMP / UDP-Gal, UDP-Rha and UDP-Ara antiporter family
Ebert B. (a), Rautengarten C. (a), Temple H. (b), Orellana A. (b), Heazlewood J. (a), Scheller H.V. (ac)
(a) Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA; (b) Centro de Biotecnología
Vegetal, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile; (c) Department of Plant & Microbial
Biology, University of California, Berkeley, CA, USA.
The matrix of plant cell walls consists of polysaccharides, which are assembled by glycosyltransferases in the
Golgi apparatus. The precursors for polysaccharides are activated sugars that are generally synthesized in the
cytosol. To transfer nucleotide sugars into the Golgi lumen a family of nucleotide sugar transporters (NSTs) has
evolved. We investigated NST activities by expressing the Arabidopsis proteins in yeast and reconstituting them
into liposomes. The activities were determined with radiolabeled nucleotide sugars and with LC-MS/MS analysis
of nucleotide sugar uptake. Here we present evidence that the Golgi-localized NST UDP-GalT2[1] is not only
capable of transporting UDP-Gal, as previously reported, but also UDP-Rha and to a lesser extent UDP-Ara in
vitro. Transport activity was strictly dependent on counter-transport of UMP. The same transport activities could
be observed for two homologous NSTs. While similar, the three transporters differ in the relative preference for
the three substrates. Mutant cell wall monosaccharide analysis confirmed the biological function of UDP-GalT2 in
plants. galt2-1 and galt2-2 mutants have significantly reduced levels of galactose in Arabidopsis leaves whereas
overexpression of GalT2 results in an increase of up to 50%.
[1] H. Bakker et al. (2005) Glycobiology, 15(2), 193-201.
O2-03
Genetic analysis of de novo and salvage pathways of nucleotide sugars for plant cell walls
Geserick C., Reboul R., Endres S., Tenhaken R.
University of Salzburg, Dept. Cell Biology, Plant Physiology, Salzburg, Austria.
Cell wall polymers are synthesized by glycosyltransferases using nucleotide sugars as glycosyl donors. We are
studying two de novo pathways for the synthesis of UDP-glucuronic acid, involving UDP-glucose dehydrogenase
(UGD) [1] and myo-inositol oxygenase (MIOX) [2] as key enzymes. This nucleotide sugar is the common
precursor for apiose, arabinose, galacturonic acid and xylose in cell wall polymers. Analysis of knockout mutants
indicates a dominant role of UGDs for precursor biosynthesis of cell wall polymers. A reduction in MIOX activity
is compensated by the UGD pathway and therefore causes no changes in the cell wall of miox knockdown
mutants. However miox mutants are more resistant to nematode infections. We have recently also focused on
salvage pathways for UDP-sugars. Some of the enzymes are essential for pollen development. The analysis of
mutants surprisingly reveals a much more important role of nucleotide sugar recycling as previously thought [3].
[1] R. Reboul et al. (2011) J. Biol. Chem., 286, 39982-92; [2] S. Endres and R. Tenhaken (2011) Planta, 234, 157-169; [3] C.
Geserick and R. Tenhaken(2013) Plant J. doi: 10.1111/tpj.12116.
O2-04
Investigation of the mechanism of (1,3;1,4)-β-D-glucan biosynthesis and assembly in Poaceae spp
Doblin M.S. (a), Wilson S.M. (a), Ho Y.Y. (a), Ilc T. (ab), Bain M. (a), Zhou J. (a), Zeng W. (a), Beahan C.T. (a), van der
Meene A. (a), Livingstone M. (a), Bacic A. (ac)
(a) ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Australia; (b) current address:
Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire des Plantes, Universite de Strasbourg,
France; (c) Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia.
Human societies are fundamentally reliant on plant cell wall polysaccharides in the form of timber, paper, textiles
such as cotton and linen, food, dietary fibre, biocomposites and increasingly biofuels. However, the process by
which the cell wall is assembled and regulated is still poorly understood. The overall aim of our work is to
determine the genes, proteins, biochemical pathway and molecular mechanism of synthesis and assembly of noncellulosic polysaccharides in the Poaceae, with a major focus on the predominant arabinoxylans and (1,3;1,4)-βD-glucans. We recently proposed that (1,3;1,4)-β-D-glucan is biosynthesised in a two-phase process at two
different sub-cellular sites: (1,4)-β-D-oligoglucosides are made in the Golgi apparatus, then subsequently
transported via a glycoprotein or glycolipid intermediate to the plasma membrane where they are assembled into a
polysaccharide chain by another enzyme with either glucosyltransferase or endotransglucosylase activity [1-5].
We have been testing this hypothesis with multi-disciplinary approaches using various ‘omic platforms,
biochemistry, cell and molecular biology techniques. We present our latest findings and discuss these in relation
to the proposed model.
[1] M.S. Doblin et al. (2009) Proc. Nat. Acad. Sci. USA, 106, 5996-6001; [2] S.M. Wilson et al. (2006) Planta, 224, 655-667;
[3] G.B. Fincher (2009) Plant Physiol., 149, 27-37; [4] M.S. Doblin et al. (2010) Funct. Plant Biol., 37, 357-381; [5] R.A.
Burton et al. (2010) Nature (Chemical Biol.) 6, 724-732.
O2-05
Sub-cellular-location of the Cellulose Synthase-Like CslF and CslH genes in members of the
Poaceae
Wilson S.M. (a), Doblin M.S. (a), van de Meene A.M.L. (a), Bacic A. (ab)
(a) ARC Centre of Excellence in Plant Cell Walls, School of Botany, (b) Bio21 Molecular Science and Biotechnology
Institute; The University of Melbourne, VIC 3010, Australia.
(1,3; 1,4)-β-D-glucan is a relatively abundant polysaccharide of grass species and is of particular interest because
of its benefits to human digestive health. The Cellulose Synthase-Like CslF and CslH gene families are known to
be involved in (1,3; 1,4)-β-D-glucan biosynthesis [1, 2] but the mechanism of synthesis is still little understood. It
has long been accepted that the non-cellulosic polysaccharides including (1,3;1,4)-β-D-glucan are synthesized at
the Golgi and our Transmission Electron Microscope (TEM) observations using antibodies to cell wall
polysaccharides show this to be true for xyloglucan, arabino-(1-4)-β-D-xylan, and hetero-(1-4)-β-D-mannan.
However, no gold labeling is ever seen over Golgi stacks when we apply the (1,3; 1,4)-β-D-glucan antibody to
thin sections of various grass species leading us to question the sub-cellular location of (1,3;1,4)-β-D-glucan
synthesis. Peptide antibodies have been generated to recognize the CSLF6 or CSLH1 proteins of cereals. These
antibodies have been applied to high pressure-frozen and freeze-substituted tissues. Despite both CSLF6 and
CSLH genes being involved in (1,3; 1,4)-β-D-glucan biosynthesis, the two antibodies do not co-locate at the subcellular level and therefore appear to play different roles in the synthesis of this polysaccharide. We present a
revised model of (1,3; 1,4)-β-D-glucan synthesis [2] in light of these data [3].
[1] Burton et al. (2006) Science 311, 1940-1942; [2] Doblin et al. (2009) PNAS, 106(14), 5996-6001; [3] Burton et al. (2010)
Nature Chemical Biology, 6(10), 724-732.
O2-06
Pectin as a domain of cell wall polysaccharides and proteoglycans and biological function of pectin
biosynthetic GAUT1 and the GAUT gene family
Mohnen D. (abd), Atmodjo M.A. (ab), Tan L. (abd), Amanda D.-C. (a), Petrascu I. (a), Mohanty S.S. (ad), Hao Z. (acd),
Biswal A.K. (ad), Amos R. (ab)
(a) Complex Carbohydrate Research Center, (b) Department of Biochemistry and Molecular Biology, (c) Plant Biology
Department, (d) BioEnergy Science Center; University of Georgia, Athens, GA, USA 30602-4712.
Pectin is described as a class of cell wall polysaccharides that contains galacturonic acid linked at the 1- and the 4position, and many plant cell wall models depict a matrix of separate cellulose, hemicellulose and pectic
polysaccharides with ~10% diverse structural and enzymatic proteins. However, the structure and putative subunit
composition of the pectin biosynthetic GAUT1:GAUT7 complex (1) and the identification of the cell wall
proteoglycan arabinoxylan-pectin-arabinogalacton protein 1 (APAP1) (2) lead us to propose that pectins are
synthesized as domains of wall polysaccharides and/or proteoglycans (3) and that, at least in some cell types,
pectin and hemicellulose matrix polysaccharides are present in the same proteoglycan structure.
Homogalacturonan (HG) accounts for ~65% of pectin and thus down-regulation of HG synthesis would be
expected to modify plant growth and development. Our recent characterization of a GAUT1 T-DNA insertion
mutant line, the only line recoverable to-date, confirms the critical biological function of GAUT1 for normal plant
growth and development. Homozygous gaut1-/- seedlings are recovered at only low frequencies (~5%) and have a
severely stunted phenotype. Models for cell wall structure and HG and pectin synthesis, as well as results
suggesting a role for GAUT1 in vegetative and reproductive stages of Arabidopsis growth and development will
be presented. The possible functions of the other GAUTs in matrix polysaccharide and proteoglycan synthesis
will also be discussed.
[1] Atmodjo et al. (2011) PNAS, 108, 20225-20230; [2] Tan et al. (2013) Plant Cell, 25, 270-277; [3] Atmodjo et al. (2013)
Annu.Rev. Plant Biol., 64, 28.1-28.33.
Supported by USDA AFRI 2010-65115-20396, NSF-MCB 0646109, and BioEnergy Science Center grant DE-AC05-00OR22725. The
BioEnergy Science Center is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and
Environmental Research in the Department of Energy’s Office of Science.
O2-07
The wall polysaccharide O-acetylation machinery
Schultink A. (a), Xiong G. (a), Gille S. (a), Liu L. (b), Cheng K. (a), Naylor D. (b), Pauly M. (ab)
(a) Energy Biosciences Institute, University of California, Berkeley, USA; (b) Department of Plant Biology, Michigan State
University, USA.
Most plant cell wall polysaccharides are O-acetylated including the various hemicelluloses and the pectic
polysaccharides. O-acetyl substituents aid in making the polymer soluble in aqueous solutions but inhibit
enzymatic degradation through steric hindrance. While the biological function of polysaccharide O-acetylation in
planta is not well described, acetate as a byproduct of the processing of lignocellulosics can be a major inhibitor
of microbial fermentation.
The molecular mechanism of polymer O-acetylation in plants is not known limiting our ability to tailor plants
with defined O-acetylation levels. Recently, putative specific wall polysaccharide O-acetyltransferases have been
identified for the hemicelluloses xyloglucan and xylan. These genes are part of an extensive gene family
(trichome-birefringece like, TBL) present in plants. In addition, another, smaller gene family (reduced wall
acetylation, RWA) has been identified as affecting the O-acetylation status of multiple polysaccharides present in
a wall giving rise to the hypothesis that RWA might represent a Golgi-membrane localized acetyl-precursor
transporter.
In an attempt to identify additional genes/proteins involved in polysaccharide O-acetylation we screened a
chemically mutagenized Arabidopsis population for mutants with altered xyloglucan structures (axy-mutants) by
oligosaccharide mass profiling utilizing a xyloglucan specific endoglucanase. One of the mutants, axy9, exhibited
a 15% reduction in xyloglucan O-acetylation. The corresponding axy9 knock-out allel showed an even further
reduction of xyloglucan O-acetylation by 58%. Interestingly, the O-acetylation status of other wall polymers are
also affected (xylan – 28% decrease; mannan – 8% decrease). AXY9 is annotated as a protein of unknown
function, but our data suggests that it is important novel factor in the polysaccharide O-acetylation machinery
present in plants. The data and the resulting model concerning a potential complex of AXY9/RWA/TBL will be
discussed.
O2-08
Homogalacturonan biosynthesis and methylesterification in Arabidopsis depend on a family of
plant-specific Golgi-associated proteins
Kim S.-J. (a), Held M.A. (b), Zemelis S. (a), Wilkerson C.G. (ac), Brandizzi F. (ad)
(a) Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI 48824, USA; (b) Department of
Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA; (c) Department of Biochemistry and Molecular
Biology, Michigan State University, East Lansing, MI 48824, USA; (d) MSU-DOE Plant Research Lab, Michigan State
University, East Lansing, MI 48824, USA.
Pectins are important cell wall polysaccharides involved in sustaining plants’ life. Pectins undergo
methylesterification, which regulates their cellular roles. Pectin methylesterification occurs in the Golgi, but no
genuine methyltransferase is known. So far, only indirect evidence supports a role of QUA-like proteins and of an
unrelated plant-specific protein, CGR3, in pectin methylesterification. The existence of different types of putative
methyltransferase domains in CGR3 and QUA2 led to the hypothesis that CGR3 belongs to a separate class of
proteins involved in homogalacturonan methylesterification. Here we report on the characterization of a close
homolog of CGR3, named CGR2, and of a cgr2-1 cgr3-1 double knockout. We localized CGR2 in the Golgi with
its enzyme active site in the lumen, as expected for a pectin methyltransferase. We also established that a cgr2-1
cgr3-1 knockout has defects in growth as well as homogalacturonan methylesterification, as deduced by immunofluorescence microscopy and cell wall analyses. Finally, through biochemical assays of microsomes of gain- and
loss-of-function mutants and in vitro methyltransferase activity assays with purified proteins, we demonstrate that
CGR2 and CGR3 share overlapping functions as methyltransferases. Therefore, our data support that CGRs
constitute a family of plant-specific proteins with key roles in development and homogalacturonan
methylesterification.
Ph. D, Iowa state (2010) ; Postdoc - 2010-, under Dr. Federica Brandizzi.
O2-09
Phosphorylation as a key mechanism in (1,3)-β-glucan biosynthesis
Naumann M., Manisseri C., Voigt C.A.
Phytopathology and Biochemistry, Biocenter Klein Flottbek, University of Hamburg, 22609 Hamburg, Germany.
The cell wall polymer (1,3)-β-glucan, known as callose, is involved developmental processes of plants. In
addition, callose deposition ubiquitously occurs after both, abiotic and biotic. At wounding sites, callose
deposition is fast response to seal directly affected cells. After pathogen attack, callose is deposited at sites of
penetration.
In our studies, we examined the role of phosphorylation in regulating callose biosynthesis. As a target, we chose
the callose synthase GSL5 (=PMR4) from Arabidopsis thaliana. This enzyme is responsible for stress-induced
callose formation [1]. The constitutive expression of GSL5 resulted in complete penetration resistance to powdery
mildew. Regarding alterations of the cell wall, the most prominent change in the mutant was detected in
hemicelluloses composition; a 30% increase in glucose. After powdery mildew infection, the main difference to
wild-type was the strongly elevated callose deposition a sites of attempted fungal penetration. Previous studies
indicated that a serine of the intracellular loop of GSL5 can be phosphorylated [2]. We mutated this serine (S) to i)
an alanine (A) to prevent phosphorylation and ii) aspartate (D) to a mimic phosphorylation. As a result, the
constitutive expression of the GSL5 containing the S->A mutation did not show any changes in callose deposition
compared to wild-type and lost its resistance phenotype. In contrast, the S->D showed the same complete
penetration resistance to powdery mildew as the mutant constitutively expressing the native GSL5 based on
elevated early callose deposition. Moreover, the S->D mutant revealed aberrant, additional callose deposition in
unchallenged leaves. We observed this additional callose formation neither in wild-type nor the other GSL5
mutants.
[1] Ellinger D, Naumann M, Falter C, Zwikowics C, Jamrow T, Manisseri C, et al. Elevated early callose deposition results in
complete
penetration
resistance
to
powdery
mildew
in
Arabidopsis.
Plant
Physiol
2013.
http://dx.doi.org/10.1104/pp.112.211011
O2-10
Control of cellulose deposition: COBRA vs. MONGOOSE
Sorek N., Szemenyei H., Somerville C.
Energy Biosciences Institute, UC Berkeley, USA.
The cellulose microfibrils of the cell wall are crystalline structures formed from the organized interaction of
glucan chains. Cellulose synthase complexes (CSCs) synthesize these individual glucan chains at the plasma
membrane, but it remains unclear how the organization into the microfibril is established. The expression of
COBRA (COB) is highly correlated with that of the primary cellulose synthase (CesA) genes, and the mutant has
been shown to exhibit altered cellulose morphology. In an effort to understand the function of COB, we analyzed
localization of a fluorescently tagged COB that complemented the cob-6 mutant. We observed a similar
localization pattern to that of the CSCs, and treatment with isoxaben caused depletion of the protein from the
plasma membrane, suggesting an association with the CSCs. Additionally, binding assays reveal that COB can
bind to cellulose as well as individual glucan chains. Furthermore, we performed a suppressor screen of the cob-6
mutant in an attempt to find new genes involved in cellulose genes, and to gain more insight into the function of
COB. We named the screen MONGOOSE, and have successfully identified 10 suppressors. We have identified
several candidate genes and are currently characterizing these mutants and their implications on cellulose
deposition.
O2-11
Internalization of cellulose synthase complexes is mediated via clathrin-based endocytosis
Sánchez-Rodríguez C. (a), Wallace I. (b), Lindeboom J. (c), Ehrhardt D. (c), Persson S. (a)
(a) Max-Planck-Institute for Molecular Plant Biology, Am Muehlenberg 1, 14476 Potsdam, Germany; (b) Energy
Biosciences Institute, 130 Calvin Hall, MC 5230, Berkeley, California 94720-3102, USA; (c) Department of Plant Biology,
Carnegie Institution for Science, 260 Panama St, Stanford, CA 94305, USA.
Plant cell walls are essential for cell differentiation, and for environmental adaptation of the plant. During rapid
cell growth, new cell wall components are continuously recycled through a fine-tuned endo-exocytosis
mechanism. The main endocytic process in plants is mediated by clathrin coats, also referred to as CME. Several
studies suggest a role of CME during cell plate formation and cell wall deposition, but no direct evidences for this
have been presented. Using bioinformatic and proteomic approaches, we have identified a protein complex which
acts as a novel adaptor structure for CME in plants, and that drives CME. One of these proteins, named TPLATEFcHo-Like (TFL), is encoded by a gene with an expression pattern similar to the cellulose synthase ( CesA) genes.
In addition, TFL knock-down plants are highly sensitive to cellulose synthesis inhibitors, supporting a role of TFL
on cellulose synthesis in plants. Biochemical analyses of the cell walls in mutants affecting different aspects of
clathrin-mediated endocytosis clearly show a reduction in cellulose. Moreover, both chemical and genetically
impairment in CME alters the accumulation of CesA in brefeldin (BFA) bodies. In addition, we have confirmed
that several CME early adaptors, including TFL, interact with the primary wall CesAs in yeast. These data support
that the CesA complexes are internalized via CME, and provide for a first direct role for CME in cell wall
synthesis.
O2-12
Regulation of cellulose synthesis through clathrin-mediated endocytosis
Bashline L., Li S., Gu Y.
Center for Lignocellulose Structure and Formation, Department of Biochemistry and Molecular Biology, Pennsylvania State
University, University Park, PA 16802, USA.
In an attempt to identify proteins that may be involved in cellulose biosynthesis, a yeast two-hybrid (Y2H) screen
was performed in which the central domain of Cellulose Synthase 6 (CESA6) was used as bait to screen an
Arabidopsis cDNA library for potential interaction partners of CESA6. The Y2H screen identified μ2, a homolog
of a Clathrin Mediated Endocytosis (CME) adaptin, as a putative interaction partner of CESA6. Arabidopsis μ2 is
homologous (49% identity) to the medium subunit 2 (μ2) of the mammalian AP2 complex. In mammals, the AP2
complex acts as the central hub of Clathrin mediated endocytosis (CME) by docking to the plasma membrane
while concomitantly recruiting cargo proteins, clathrin triskelions, and accessory proteins to the sites of
endocytosis. We confirmed that μ2 interacts with multiple CESA proteins through the μ Homology Domain
(MHD) of μ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent
with its role in mediating endocytosis of cargos at the plasma membrane, μ2-YFP localized to transient foci at the
plasma membrane and loss of μ2 resulted in defects in bulk endocytosis. More specifically, loss of μ2 led to
increased accumulation of YFP-CESA6 particles at the plasma membrane. Our results suggest that CESA
represents a new class of CME cargo proteins, and that plant cells might regulate cellulose synthesis by
controlling the abundance of active CESA complexes at the plasma membrane through CME.
O2-13
Phosphoinositides regulate cell wall deposition in Arabidopsis leaves
Krishnamoorthy P. (a), Sánchez-Rodríguez C. (a), Heilmann I. (b), Persson S. (a)
(a) Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany; (b) Martin-Luther-Universität HalleWittenberg, Institut für Biochemie und Biotechnologie, Halle, Germany.
Plant cell-wall assembly involves a complex pathway were different macromolecules are deposited into the cell
wall to form an intricate matrix. Cellulose is one of the most abundant components of plant cell walls. It forms a
part of the load-bearing network that both controls and maintains cell shape, while allowing regulated cell
expansion that is essential during growth. The cellulose microfibrils are deposited by cellulose synthase (CESA)
complexes, which are secreted to the plasma membrane by Golgi derived vesicles. The recycling of the CESAs is
likely executed by a tight interplay of exocytosis and endocytosis, which in turn is controlled by various factors.
The PI(4,5)P2 is one class of membrane lipids which has emerged as a key component in regulating membrane
trafficking [1]. These bi-phosphates are one of the major by-products of the plant phosphoinositide (PI) pathway,
catalyzed by specific PIP-Kinases (PIPKs) by phosphorylation at the 5 th position of the inositol ring. Our results
show that a double pipk mutant, affecting PIP5K1 and PIP5K2, cause a six-fold increase in epidermal cell wall
thickness in leaves, and almost a three-fold increase in the cellulose levels. Various other cell wall components
were also altered in these double mutants. Fluorescently tagged versions of these kinases localize to distinct but
dynamic foci at the plasma membrane, which strongly suggests a role for them in endocytosis. We will investigate
the exact mechanisms of how these phosphoinositides are involved in regulating membrane trafficking events by
using various molecular biology techniques and advanced imaging tools.
[1] Thole, J.M. and Nielsen, E. (2008) Curr Opin Plant Biol. 11(6): p. 620-31.
O2-14
Root hair cell wall organization is sensitive to the expression of VTI13, a SNARE required for
trans-Golgi network function in Arabidopsis
Larson E.R. (a), Domozych D.S. (b), Tierney M.L. (c)
(a) Cellular, Molecular, and Biomedical Science Program, University of Vermont, Burlington, VT 05405, USA; (b)
Department of Biology, Skidmore College, Saratoga Springs, NY, USA; (c) Department of Plant Biology and Cellular,
Molecular, and Biomedical Science Program, University of Vermont, Burlington, VT, USA.
PRP3 encodes a structural cell wall protein that is localized to the root hair cell wall and is required for normal
root hair growth. To examine if changes in cell wall structure result in an alteration of gene expression that
contributes to the prp3 root hair phenotype, we used microarray analysis to identify gene expression changes in
prp3 roots compared to those of wild type seedlings. These studies identified several genes whose protein
products are predicted to function in vesicle trafficking pathways, including VTI13 (At3g95100), a SNARE
whose gene expression is upregulated in the prp3 mutant. Genetic analysis demonstrated that VTI13 is required
for root hair growth under a variety of media conditions. Using transgenic plants expressing a GFP-VTI13 fusion,
we showed that VTI13 localizes to both the vacuole membrane and the trans-Golgi network (TGN). We also
demonstrated that VTI13 function is required for the transport of SYP41 to the TGN, implicating a role for VTI13
in the organization of specific TGN compartments. Further, a prp3 vti13 double mutant was found to suppress the
branched root hair phenotypes observed in the vti13 and prp3 parental lines, suggesting that the misregulation of
VTI13 expression in prp3 contributes to its root hair phenotype. Based on these observations, we propose that
VTI13 functions in endocytic trafficking pathways important for cell wall metabolism in growing root hairs.
O2-15
Expansin interactions with plant cell walls
Cosgrove D., Georgelis N., Yennawar N., Tabuchi A., Wagner E., Bruenig L.
Department of Biology, Penn State University, University Park, PA 16802 USA.
Currently we recognize three classes of expansins with distinctive roles.
(1) Alpha-expansins are key mediators of ‘acid growth’, which is important for cell enlargement, abscission and
other events requiring wall loosening.
(2) Beta-expansins are prominent in grasses and one subclass of beta-expansins accumulate to high levels in grass
pollen where they function to loosen walls of the stigma and style to facilitate pollen tube invasion. The betaexpansins from maize pollen solubilize arabinoxylans and homogalacturonans from the middle lamella 1, causing
cell separation, detachment of the cuticle and release of a hydrophilic layer that resembles an intact middle
lamella. Application of maize pollen beta-expansin to diverse tissues from grasses profoundly weakens cell-cell
adhesion and lowers tensile strength by >80%, but has no effect on a variety of tissues from dicots.
(3) Bacterial expansins are found in a small but phylogenetically diverse group of bacteria that colonize plant
surfaces or vasculature. By combining crystallography of expansin-ligand complexes with site-directed
mutagenesis, binding assays and activity studies, we have a detailed understanding of the interactions of the
protein with cell wall glycans2. Expansin binding to cellulose is of key importance for wall loosening activity, is
entropically driven, and determined by three aromatic residues on the conserved surface of domain 2. Binding of
domain 1 to the wall is too weak to measure, but key acidic residues are essential for wall loosening. These results
show that different expansins have diverse biological roles, biochemical targets and biophysical effects on the cell
wall, but they are united in the similarity of their structure and the lack of detectable lytic activity.
[1] Tabuchi et al. (2011) Plant J 68: 546-559; [2] Georgelis et al. (2012) Proc Natl Acad Sci 109:14830-5.
O2-16
Activation tag screening reveals the functions of polygalacturonases in cell wall expansion and
plant development
Xiao C., Anderson C.T.
Department of Biology and Center for Lignocellulose Structure and Formation, The Pennsylvania State University,
University Park, PA 16802 USA.
Plant growth and development require the expansion of the walls that encase plant cells, and mutations that
abolish wall expansion are thus likely to result in early lethality. To circumvent this problem, we screened
approximately one million Arabidopsis thaliana activation tag seedlings containing randomly inserted enhancer
elements for increased dark-grown hypocotyl elongation, which occurs mainly via cell expansion. We identified
637 lines displaying long hypocotyls and sequenced the activation tag insertion point for 27 of them. Two
insertions were located upstream of genes encoding putative polygalacturonases, proteins that hydrolyze the
galacturonic acid backbones of pectins, potentially loosening the pectin networks that can constrain wall
expansion. We named these genes POLYGALACTURONASE INVOLVED IN EXPANSION 1 (PGX1) and 2 and
determined that both genes are expressed in multiple tissues throughout plant development. Constitutive
overexpression of each gene recapitulated the long hypocotyl phenotype seen in the activation tag lines,
confirming their roles in cell expansion. Despite the potential for redundancy among the 68 polygalacturonase
genes in Arabidopsis, we found that knockout mutants lacking PGX1 expression displayed shorter hypocotyls and
cells than wild type controls, and that this phenotype was rescued by expression of PGX1 in the mutants. A
PGX1-GFP fusion localized to cell walls and intracellular compartments, and purified PGX1 cleaved
polygalacturonic acid in vitro, confirming its biochemical activity. Interestingly, we also discovered that PGX1
activation tag plants displayed larger rosette leaves and a higher percentage of flowers with extra petals than wild
type controls, suggesting that this gene functions in multiple developmental contexts to promote cell expansion.
O2-17
New molecular players of the glyco-network control the polarized growth at a single plant cell
level
Velasquez S.M., Ricardi M.M., Salgado Salter J., Gloazzo Dorsz J., Estevez J.M.
Instituto de Fisiología, Biología Molecular y Neurociencias (IFIByNE-CONICET), Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires, Argentina.
Root hairs are single cells that develop by tip growth, a process shared with pollen tubes and fungal hyphae. Root
hairs cell walls are composed of polysaccharides and hydroxyproline-rich glycoproteins that include extensins
(EXTs) with several Ser-(Hyp)4 repeats and Tyr-crosslinking motifs. It was shown that O-glycosylation on EXTs
is essential for root hair cell growth. Here we demonstrate that prolyl-4-hydroxylase 5 (P4H5) present in a
multimeric P4H complex with P4H2/13 is pivotal for determining Hyp-O-arabinosylation. In addition, SGT1 is
responsible for serine-O-galactosylation of EXTs. Overall, both O-glycosylation types are required for correct
EXT function. Finally, we provide evidence that O-glycosylation modulates EXTs self assembling. Our results
highlight that correct protein O-glycosylation affects EXT-network development and in turn modulates single
plant cell growth.
[1] S.M. Velasquez, J. Salgado Salter, J. Gloazzo Dorosz, B.L. Petersen & J.M. Estevez. 2012. Recent advances on the posttranslational modifications of EXTs and their roles in plant cell walls. Frontiers in Plant Science 3(93). doi:
10.3389/fpls.2012.00093; [2] S.M. Velasquez, et al. & J. M. Estevez. 2011. Essential role of O-glycosylated plant cell wall
extensins for polarized root hair growth. Science 332, 1401-1403.
O2-18
Functional identification
of a hydroxyproline-O-galactosyltransferase
arabinogalactan-protein biosynthesis in Arabidopsis
specific
for
Basu D., Liang Y., Liu X., Himmeldirk K., Faik A., Kieliszewski M., Held M., Showalter A.M.
Molecular and Cellular Biology Program, Department of Environmental and Plant Biology (D.B., Y.L., X.L., A.F., A.M.S.),
and Department of Chemistry and Biochemistry (K.H., M.K., M.H.), Ohio University, Athens, Ohio 45701–2979, USA.
Although plants contain substantial amounts of arabinogalactan-proteins (AGPs), the enzymes responsible for
AGP glycosylation are largely unknown. Bioinformatics indicated AGP galactosyltransferases (GALTs) are
members of the Carbohydrate-Active enZyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in Nand O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme
activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to
hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [14C]Gal from
UDP-[14C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous
Hyp residues, such as [Ala-Hyp] repetitive units exemplified by chemically synthesized [AO] 7 and HFdeglycosylated d[AO]51. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [ 14C]Gal
to [AO]7, and the resulting product co-eluted with [AO] 7 by reverse phase HPLC. Acid hydrolysis of the [ 14C]Gal[AO]7 product released [14C]radiolabel as Gal only. Base hydrolysis of the [ 14C]Gal-[AO]7 product released a
[14C]radiolabeled fragment that co-eluted with a Hyp-Gal standard after HPAEC fractionation. AtGALT2 is
specific for AGPs as substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2
uses only UDP-Gal as the substrate donor and requires Mg 2+ or Mn2+ for high activity. Additional support that
AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knockout mutants, which demonstrated
lower GALT activities and reductions in β-Yariv-precipitated AGPs compared to wild type plants. Confocal
microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated AtGALT2 is likely
localized in the endomembrane system consistent with its function.
[1] D. Basu et al. (2013) J. Biol. Chem., First Published on February 19, 2013, doi: 10.1074/jbc.M112.432609.
O2-19
Cellular and genetic dissection of FASCICLIN LIKE ARABINOGALACTAN PROTEIN4
Xue H., Acet T., Seifert G.J.
University of Natural Resources and Life Science, Vienna; Department of Applied Genetics and Cell Biology, Muthgasse 18,
1190 Vienna, Austria.
The Arabidopsis thalianaSOS5 locus encodes the fasciclin-like putative arabinogalactan-protein 4 (FLA4). FLA4
is required for normal growth and salt tolerance in roots and is involved in cellulose deposition in various tissues
[1]. Previous analyses are consistent with a role of FLA4 in cell wall performance and integrity signalling. To
closer examine this possibility we used the sos5 mutant background to identify non-additive genetic interactors in
a forward genetic screen and tested the involvement of selected signalling pathways using double mutants and
pharmacological analysis. We identified a suppressor locus offla4 (slof9) that might act downstream cell wall
integrity signalling is involved in the homeostasis of reactive oxygen species (ROS). Chemical and genetic
evidence suggest an interaction of FLA4 with abscisic acid signalling. Moreover, we investigate localization and
structure with a fluorescently tagged functional FLA4 fusion.
[1] Shi, H., Kim, Y., Guo, Y., Stevenson, B., and Zhu, J. K. (2003) Plant Cell15, 19-32.
This work is supported by the FWF (The Research Fund). Grants: P21782-B12, I1182-B22, H.X. is supported by the Chinese Scholarship
Council.
O2-20
Transcriptional regulation of xylan biosynthesis
Jensen J.K., Fode B., Johnson N., Wilkerson C.G.
Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA.
A large number of biosynthetic enzymes are involved in plant cell wall formation. Identifying transcription factors
regulating these genes will likely enhance our understanding about how these genes coordinately function to
produce these structures, and may lead to the identification of more genes involved in cell wall formation.
Manipulation of cell wall specific transcription factors presents an opportunity for altering cell wall formation in
ways that may not be achievable by manipulating cell wall biosynthetic enzymes one by one.
To discover transcription factors regulating xylan biosynthetic genes we analyzed gene expression in the Psyllium
mucilaginous layer, which is a tissue that contains large quantities of xylan. We reasoned that such a tissue would
have xylan-specific transcription factors expressed at high levels. Using this strategy, we identified the
Arabidopsis homeodomain proteins KNAT7 and BLH6 as candidate transcription factors regulating xylan
biosynthesis. We have demonstrated, through knockout mutants and protoplast trans-activation assays, that BLH6
functions redundantly with its closest homolog, BLH7, and that both KNAT7 and BLH6/7 are necessary for
activation of the xylan biosynthetic gene IRREGULAR XYLEM 10 (IRX10). Moreover, chromatin
immunoprecipitation experiments reveal direct association of KNAT7 with the IRX10 promoter in vivo and
investigations of KNAT7 knockout mutant plants show altered xylan formation in inflorescent stems. A number
of other genes, such as At4g08160, a glycosyl hydrolase family 10 protein, are also down regulated in the KNAT7
knockout mutant. We are currently conducting experiments to define the KNAT7-BLH6/7 regulon and we are
investigating the genes in the regulon for possible roles in xylan biosynthesis.
O2-21
Differences and similarities of xylan synthesis in dicots and grasses
Lovegrove A. (a), Wilkinson M.D. (a), Freeman J. (a), Pellny T.K. (a), Tosi P. (a), Wan Y. (a), Gritsch C. (a), Saulnier L. (b),
Tryfona T. (c), Dupree P. (c), Karp A. (d), Shewry P.R. (a), Mitchell R.A.C. (a)
(a) Plant Biology and Crop Science and (d) Agroecology Depts., Rothamsted Research, UK; (b) INRA-BIA, Nantes, France;
(c) Biochemistry Dept., University of Cambridge, UK.
The well-characterised structural differences between xylan in secondary cell walls of dicots and xylan in cell
walls of grasses can now be related in detail to transcriptomes of tissues synthesising these, together with
experimental evidence on gene function from multiple systems, to build a picture of the genes responsible. Here,
we contrast the RNA-Seq transcriptomes of developing starchy endosperm of wheat and developing stems of
willow to illustrate commonalities and differences in expression of candidate xylan synthetic genes. Differences in
transcript abundance seem to explain the different substitutions [methyl-glucuronic acid versus arabinofuranose
(Araf)] and presence/absence of backbone acetylation and of feruloylated Araf. We have shown by RNAi
suppression that IRX9 and IRX10 homologues are responsible for arabinoxylan (AX) backbone synthesis in
wheat endosperm. Further analyses of AX from the transgenic wheat lines also give clues as to the different roles
for these which may be common to all higher plants. We also speculate on possible explanations for the apparent
much greater chain length of xylan in wheat endosperm compared to dicot secondary cell walls and absence of the
characteristic reducing-end pentasaccharide.
O2-22
Non-cell autonomous post-mortem lignification of xylem vessels
Pesquet E.
Umeå Plant Science Centre (UPSC), Department of Plant Physiology, Umeå University, 901 87 Umeå, Sweden.
Lignification and programmed cell death (PCD) are of fundamental importance to the production of functional
tracheary elements (TEs) – cellular corpses which require PCD to hollow out their content and lignification to
reinforce their walls to transport sap. Live cell imaging of TE formation showed that lignification occurred after
TE PCD1. The interplay between PCD and lignification during TE formation was studied at the cellular level
using in vitro xylogenic cultures, at the genomic level using differential subtractive libraries and at the whole
plant level by analyzing the cell wall biochemistry of 51 Arabidopsis KO mutants. Pharmacological modulation of
TE lignin monomer biosynthesis resulted in dead unlignified TEs, which could partially lignify post-mortem when
supplied externally with lignin monomers. Pharmacological modulation of TE PCD in the differentiating TEs
blocked both cell death and lignification without affecting xylan/cellulose secondary wall deposition. Differential
libraries constructed from TE cell cultures inhibited or not to perform PCD allowed to identify 693 differentially
expressed genes involved in PCD-triggered lignification. Among these genes were identified known cell wall
biosynthesis, lignin monomer biosynthesis and PCD related genes. Interestingly, cinnamoyl-CoA reductase
(CCR) and cinnamyl alcohol deshydrogenase (CAD), two lignin monomer synthesis genes, were expressed
beyond normal TE lifespan. In situ localization using IS-RT-PCR of CAD and CCR revealed that both genes were
expressed in cells analogous to xylem parenchyma. Cell wall composition analysis of 51 Arabidopsis mutants in
genes differentially responding to TE PCD inhibition were characterized by reverse genetic approaches and
exhibited changes in lignin composition in whole plants although gene expressions were restricted to xylem
parenchyma. Altogether, our results suggest that lignin is mostly made through a post-mortem and cooperative
process in xylem vessels2.
[1] E. Pesquet et al. (2010) Curr. Biol., 20, 744-749; [2] E. Pesquet et al. (2013) Plant Cell, in press.
O2-23
Lignin deposition in xylem relies on non-lignifying neighbouring cells
Smith R. (c), Mansfield S.D. (b), Ellis B. (c), Samuels L. (a), Schuetz M. (a)
(a) Department of Botany, University of British Columbia, Vancouver, BC, Canada; (b) Department of Wood Science,
University of British Columbia, Vancouver, BC, Canada; (c) Michael Smith Laboratories, University of British Columbia,
Vancouver, BC, Canada.
The survival of land plants is dependent upon a functional network of xylem tissue, some which is composed of
lignified secondary cell walls. It is widely accepted that lignifying cells contribute to the lignification of their own
cell walls, but whether this contribution is pre- or post-mortem is still under debate. Autoradiography was used to
demonstrate that tracheary elements are living during lignification, but that lignification continues post-mortem.
The “good neighbour” hypothesis suggests that xylary parenchyma cells adjacent to lignifying cells may be
producing and exporting monolignols to tracheary elements that are actively lignifying. To test this hypothesis,
genes encoding lignin biosynthetic enzymes in Arabidopsis thaliana were targeted for silencing in cells producing
thickened secondary cell walls, but not their neighbours. Employing transgenic plants expressing 35S::miRNA
suppression vectors, all cell types displayed drastic reductions in lignification. However, when the expression of
the same miRNA was driven only in cells with secondary cell walls, lignification of tracheary elements in roots
and stems, and in stem xylary fibres, was unaffected, whereas interfascicular fibres had severely reduced
lignification. These findings imply that “good neighbour” cells may indeed be operating within vascular bundles,
but not in extraxylary fibres.
O2-24
PtxtXyn10A regulates cellulose microfibril angle in aspen wood
Derba-Maceluch M. (a), Takahashi-Schmidt J. (a), Awano T. (ab), Ratke C. (a), Lucenius J. (c), Serimaa R. (c), Kallas Å. (d),
Ezcurra I. (d), Berthold F. (e), Kosik O. (f), Dupree P. (f), Mellerowicz E. (a)
(a) Department of Forest Genetics and Plant Physiology, SLU, Umeå Plant Science Centre, Umeå, Sweden; (b) Division of
Forest and Biomaterials Science, Kyoto University, Kyoto, Japan; (c) Department of Physics, University of Helsinki,
Finland; (d) School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden; (e) INNVENTIA AB, Stockholm,
Sweden; (f) Department of Biochemistry, University of Cambridge, UK.
The mechanism and function of post-synthetic modifications of xylan in cell walls are poorly understood. We
report here cloning, expression and reverse genetic studies of the hybrid aspen (Populus tremula L. x tremuloides
Michx.) PtxtXyn10A gene, which belongs to the xylanase family GH10, and is highly upregulated during
secondary wall biosynthesis. We found that PtxtXyn10A is proteolytically processed before accumulation in
wood cell wall as a 68 kDa peptide. A 50% reduction of PtxtXyn10A expression in hybrid aspen induced a
decrease in trans-xylanase activity without change in xylan chain length, suggesting that PtxtXyn10A acts as a
trans-xylanase rather than xylan hydrolase in developing wood. Interestingly, transgenic plants had a reduced
cellulose-microfibril angle in wood fibers, as well as longer internodes and larger leaves, and had a reduced
tendency to form tension wood compared to the wild-type plants. We propose that PtxtXyn10A functions in
determining the orientation of cellulose microfibrils in secondary walls, thereby affecting primary and secondary
xylem mechanical properties, and plant development.
Session 3 : Evolution & Diversity of plant CW
O3-01
The algal origins of hemicellulose biosynthesis
Mikkelsen M.D. (a), Fangel J.U. (a), Johansen I.E. (a), Domozych D.S. (b), Ulvskov P. (a), Harholt J. (a), Willats W.G.T. (a)
(a) Department of Plant Biology and Biochemistry, University of Copenhagen, Faculty of Life Sciences, Bülowsvej 17-1870
Frederiksberg, Denmark; (b) Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College, Saratoga
Springs, New York, 12866, USA.
Plant cell walls are sophisticated fibre composite structures that have evolved to fulfill a wide range of biological
roles that are central to plant life. The evolution of some cell wall components can be traced to prokaryotes whilst
others appear to have first emerged with the green algal ancestors of land plants. The fact that the cell walls of
charophycean green algae (CGA) are remarkably similar to those of land plants implies that the ability to produce
particular cell wall components may have been an aspect of pre-adaptation in the ancestral CGA, explaining why
they alone gave rise to the land plant lineage. However, our current understanding of the genetic mechanisms
underlying the early evolution of plant cell walls generally remains limited.
We have established a multi-disciplinary strategy that integrates plant cell wall metaglycomic analysis with
existing genomic and transcriptomic data sets. This approach enables us to map the phylogenetic distribution of
cell wall polysaccharides across the plant kingdom, and to relate this to the evolution of the glycosyl transferase
(GT) encoding genes involved in their biosynthesis. Selected GTs are cloned, expressed and biochemical
activities determined through heterologous expression.
Of particular interest are GTs involved in mannan and xyloglucan biosynthesis, which can be traced to the
chlorophyte and charophyte algae respectively. Specifically, we propose that the sole cellulose synthase like gene
present in certain chlorophytes is an ancestral mannan synthase, and that xylosyltransferases participating in
xyloglucan biosynthesis are present in the CGA.
O3-02
Novel hemicellulose-remodeling transglycanases from charophytes: towards the evolution of the
land-plant cell wall
Franková L., O'Rourke C., Fry S.C.
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, UK.
The plant primary wall matrix constitutes a flexible and metabolically active extraprotoplasmic compartment.
It is equipped with >20 enzyme activities which cleave or cleave and regenerate glycosidic bonds. ‘Cutting-andpasting’ transglycosylases (TGs) remodel wall polymers, thereby having critical impact on many physiological
processes[1]. Unlike xyloglucan endotransglucosylase, widely studied in land plants, little is known about
charophyte wall-modifying enzymes, information that would promote our appreciation of the ‘primordial’ plant
cell wall. We are exploring wall composition and wall-remodelling processes in charophytes - the closest extant
relatives of land plants. Our recently devised glass fibre-blotting assay allowed us to discover several homo- and
hetero-TG activities (e.g. trans-β-xylanase and mannan : xylan transglycanase) in diverse enzyme extracts from
pteridophytes and charophytes. Chara and Coleochaete TGs were further purified, characterised and screened for
their substrate specificities. These activities were also proved by in-situ and in-vivo approaches. Chemical
fractionation of hemicelluloses followed by specific digestive analysis revealed the presence of mannans and
xylans but a lack of conventional xyloglucan and 1,3-1,4-β-glucan (MLG) in Chara and Coleochaete. These algae,
however, possess xyloglucan- and MLG-remodelling TG activities besides 4 other so far unreported activities
which indicates that mechanisms modifying wall structure were probably highly conserved in ancient
charophytes. An overview of transglycanase and transglycosidase action, newly developed in-vitro screens,
discovery and characterisation of novel TG activities accompanied by florescence microscopy in-situ observations
will be presented, shedding new light on algal evolution.
[10] L. Franková and S.C. Fry (2011) Plant J., 67, 662-681.
Supported by the Leverhulme Foundation.
O3-03
Cell-wall deposition during asymmetric cell division in the brown algal fucoid zygote
Le Moigne M.-A. (a), Torode T. (b), Jam M. (a), Knox J.P. (b), Hervé C. (a)
(a) UMR1739 Marine Plants and Biomolecules, Station Biologique de Roscoff, Place Georges Teissier, 29688 Roscoff,
France; (b) Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K.
Multicellularity is one of the most successful innovations in the evolution of living organisms that allows cellular
diversification and sub-functionalization. Asymmetric cell division, by creating two daughter cells that differ in
their fate, is tied to the generation and maintenance of such cell diversity. Zygotes and embryos of the brown alga
Fucus have long served as classical models for understanding cell polarization and asymmetric cell division [1].
Previous studies have demonstrated an essential role of the cell-wall in polar axis fixation [2] and cell-fate
determination [3], but the precise integration of the polysaccharide components involved remains elusive. Brown
algal cell walls share some components with plants (cellulose) and animals (sulfated fucans), but they also contain
some unique polysaccharides that might have plaid a major role for the acquisition of complex multicellularity in
brown algae (alginates) [4]. Our recent results show some cell-wall components regulate the normal cell cycle
progression in the fucoid zygotes and that their disruption leads to a symmetric first cell division. In collaboration
with J. Paul Knox, we have now developed ten new monoclonal antibodies, which recognize various substructures
of fucans and alginates. These novel cell-wall probes allow the detailed study of cell-wall deposition during early
embryogenesis in Fucus, in normal or altered conditions. The roles of the cell-wall components will be discussed
in relation to: cell adhesion, cell expansion and cell division.
[1] S.R. Bisgrove et al. (2008) In: Plant Cell Monographs, Verma D, Hong Z (eds), pp 323-341; [2] D.L. Kropf et al. (1988)
Science, 239, 187-190; [3] F. Berger et al. (1994) Science, 263, 1421-1423; [4] G. Michel et al. (2010) New Phytol., 188, 8297.
O3-04
Building the pollen wall - Interplay of biosynthetic pathways, ABC transporters, and
sporopollenin assembly
Quilichini T.D., Samuels A.L., Douglas C.J.
Department of Botany, University of British Columbia, Vancouver BC Canada.
The pollen wall, containing a durable biopolymer called sporopollenin, protects pollen from terrestrial stresses.
Sporopollenin is an outer wall polymer composed of fatty acids and oxygenated aromatic compounds, which is
synthesized in the tapetum and is likely to include polyketide(s) produced by a highly conserved biochemical
pathway requiring ACOS5, PKSA/B, and TKPR1 enzymes localized in tapetum cells. ABCG26, an Arabidopsis
ATP-binding cassette transport protein, is thought to function in sporopollenin export from tapetum cells, but its
substrate remains unknown. In the abcg26 mutant, sporopollenin precursors are predicted to accumulate in
tapetum cells due to defective export. Through the analysis of anthers by two-photon microscopy, tapetum lipids
and autofluorescent metabolites were visualized. abcg26 tapetum cells contain autofluorescent vacuoles with
bright puncta not observed in wild type. TEM supports these findings, with enlarged, debris-filled vacuoles in
abcg26 tapetum cells. The nature of these putative ABCG26-trafficked sporopollenin components was
investigated by examining double mutants affecting both the transporter and enzymes required for sporopollenin
biosynthesis and/or deposition. This analysis revealed the disappearance of tapetum inclusions in double mutants
of abcg26 and acos5, pks-a/pks-b, and tkpr1. These data suggest that the proposed aliphatic polyketide
synthesized by the sequential action of ACOS5, PKSA/B, and TKPR1 is transported from tapetum cells by
ABCG26. We also observed novel autofluorescent and unexpected extracellular bodies that accumulate around
tapetum cells in acos5, pks-a/pks-b, and tkpr1, but not in abcg26 anthers. We will discuss how the elucidation of
the chemical nature of these bodies further informs our understanding of pollen wall trafficking and assembly in
Arabidopsis.
O3-05
Natural variation in the polysaccharide components of Arabidopsis seed coat epidermal cells
North H. (a), Ralet M.-C. (b), Saez-Aguayo S. (a), Tran J. (a), Poulain D. (b), Berger A. (a), Crépeau M.-J. (b), Sallé C. (a),
Loudet O. (a), Ropartz D. (b), Marion-Poll A. (a)
(a) Institut Jean-Pierre Bourgin, UMR1318, INRA-AgroParisTech, Versailles, France; (b) INRA, UR1268 Biopolymères
Interaction Assemblages, 44316 Nantes, France.
During Arabidopsis seed differentiation the epidermal cells of the testa produce a variety of cell wall structures.
Fully differentiated cells are characterised by reinforced proximal and radial cell walls and a central columella of
secondary cell wall material, a distal primary cell wall, and mucilage polysaccharides surrounding the columella.
The latter are released on imbibition to form a hydrogel that surrounds the seed. We have previously shown that
Arabidopsis mucilage is formed of a mixture of polysaccharides in two structurally distinct layers, both of which
are mainly composed of the pectin rhamnogalacturonan I [1]. A simple visual screen for the absence of mucilage
release has allowed the identification of natural Arabidopsis variants affected in two novel loci that influence
polysaccharide properties; the MUM2 beta–galactosidase and the PMEI6 pectin methylesterase [2, 3, 4]. Most
Arabidopsis accessions release mucilage when imbibed and we have examined the extent of natural variability in
soluble mucilage characteristics. Mucilage composition varied little between accessions; in contrast the amount
and physicochemical properties of mucilage vary widely. Analyses have been carried out to link these variations
with particular habitats and potential mucilage functions.
[1] A. Macquet et al. (2007a) Plant Cell Physiol., 48, 984-999; [2] G. Dean et al. (2007) Plant Cell, 19, 4007-4021; [3] A.
Macquet et al. (2007b) Plant Cell, 19, 3990-4006; [4] S. Saez Aguayo et al. (2013) Plant Cell, 25, 308-323.
O3-06
Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls
against desiccation
Moore J.P. (a), Nguema-Ona E. (ab), Vicré-Gibouin M. (b), Sørensen I. (cd), Willats W.G.T. (c), Driouich A. (b), Farrant J.
(e)
(a) Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, South Africa; (b)
Glyco-MEV Laboratoire, Université de Rouen, Mont Saint Aignan, France; (c) Department of Plant Biology and
Biotechnology, University of Copenhagen, Denmark; (d) Department of Plant Biology, Cornell University, NY, USA; (e)
Department of Molecular and Cell Biology, University of Cape Town, South Africa.
A variety of desiccation-tolerant resurrection plants from Southern Africa were surveyed using high-throughput
cell wall profiling tools [1,2]. Hydrated and desiccated leaf (frond) material, were analysed using monosaccharide
analysis, CoMPP and FT-IR spectroscopy coupled with chemometrics [3]. Arabinan-rich pectin and an abundance
of arabinogalactan proteins were found associated with the resurrection fern Mohria caffrorum [4] and the woody
angiosperm Myrothamnus flabellifolia [5]. In contrast, Craterostigma plantagineum up-regulated wall proteins
and osmoprotectants [3]. The hemicellulose-rich walls of the grass-like Xerophyta spp. and the resurrection grass
Eragrostis nindensis possess highly arabinosylated xylans and arabinogalactan proteins for protection [3]. These
data support a general evolutionary mechanism of ‘plasticising’ the cell walls of resurrection plants to desiccation
and implicate arabinose-rich polymers (pectin-arabinans, arabinogalactan-proteins and arabino-xylans) as the
major contributors in ensuring flexibility is maintained and rehydration is facilitated in these remarkable plants.
[1] J.P. Moore et al. (2009) Trends Plant Sci., 14(2), 110-117; [2] E. Nguema-Ona et al. (2012) Carbohydrate Polymers., 88,
939-949.; [3] J.P. Moore et al. (2013) Planta., 237, 739-754.; [4] J.M. Farrant et al. (2009) Plant J., 57, 65-79.; [5] J.P.
Moore et al. (2006) Plant Physiol., 141, 651-662.
Session 4 : Functions of Plant CW in planta
Growth, morphogenesis & development, Signaling & defense, Response to environment
O4-01
The IC proteins; new players of the cellulose synthase complex
Endler A., Kesten C., Persson S.
Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
Cellulose is produced at the plasma membrane by rosette-like cellulose synthase complexes (CSCs) that move
along tracks of cortical microtubules (MTs) while synthesizing cellulose into the apoplastic space. So far only
very few components of the CSCs are described, i.e. the cellulose synthase (CESA) proteins, which are believed
to be the catalytic subunits of the complex, and CSI1/POM2; a proteins that mediates the guidance of the CESAs
along cortical MTs. Using a spilt-ubiquiting system, we identified a new protein family that appears to interact
directly with the CESAs, named Interactors of CESAs (ICs). GFP fusion proteins of IC1 and IC2 co-migrate with
tdT:CESA6 in the plasma membrane and IC1 bind to MTs both in vitro and in vivo. However, live cell imaging
and genetic analysis suggest a distinct function of CSI1/POM2 and ICs during cellulose production. Instead the
proteins seem to be important for the behavior of CSCs in response to certain stresses. We believe that IC proteins
represent a new class of proteins regulating cellulose production in higher plants.
O4-02
Dissecting the molecular mechanism underlying intimate relationship between cellulose
microfibrils and cortical microtubules
Lei L., Li S., Gu Y.
Center for Lignocellulose Structure and Formation, Department of Biochemistry and Molecular Biology, Pennsylvania State
University, University Park, PA 16802, USA.
A central question in plant cell development is how cell wall determines directional cell expansion and therefore
the final shape of the cell. As the major load-bearing component of the cell wall, cellulose microfibrils are laid
down transversely to the axis of elongation, thus forming a spring-like structure reinforcing the cell laterally and
favoring longitudinal expansion in most growing cells. Although mounting evidence suggest that cortical
microtubules determines the deposition of cellulose microfibrils, the precise molecular mechanisms linking
microtubules to cellulose organization remain unclear until the recent discovery of a linker protein1. Cellulose
synthase interactive protein 1 (CSI1), initially identified through a yeast two-hybrid screen for CESA interactive
proteins, is associated to both primary CSCs and microtubules. Arabidopsis encodes two CSI1-like proteins,
namely CSI2 and CSI3. Here we investigated roles of CSI1 and CSI1-like proteins in regulation of cellulose
biosynthesis, especially their role in the co-alignment between CSCs and microtubules and the regulation of the
activity of CSCs. Our results suggest that CSI1 is a key player required for guidance of primary CSCs along
microtubules during cellulose synthesis. CSI1 has a major role in guidance of CSCs along microtubules whereas
CSI3 is dispensable for co-alignment of CSCs and microtubules. Both CSI1 and CSI3 influence the velocity of
CSC possibly through accessory proteins yet to be identified. We will also discuss the potential mechanism by
which CSI proteins associate with CSCs and microtubules.
[1] Li et al. (2012) Proc. Natl. Acad. Sci., 34, 12866-12871.
O4-03
Spatiotemporal secretion of PEROXIDASE36 responsible for seed coat mucilage extrusion in
Arabidopsis thaliana
Kunieda T. (ab), Shimada T. (a), Kondo M. (c), Nishimura M. (c), Nishitani K. (b), Hara-Nishimura I. (a)
(a) Grad. Sch. of Sci., Kyoto Univ; Japan; (b) Grad. Sch. of Life Sci., Tohoku Univ; Japan; (c) Cell Biol., NIBB, Japan.
The epidermal cells of the Arabidopsis seed coat, which correspond to the second layer of the outer integument
(oi2), accumulate large quantities of a pectic polysaccharide called mucilage within the apoplastic space beneath
the outer periclinal cell wall. Immediately after seed imbibition, the mucilage is extruded and completely envelops
the seed in a gel-like capsule. Previously, we reported that NAC transcription factors, NAC REGULATED SEED
MORPHOLOGY1 (NARS1) and NARS2, are involved in seed coat development.[1] The nars1 nars2 double
mutant failed to extrude the mucilage from seed coat, suggesting that NARS1 and NARS2 regulate expression of
mucilage-related factors. We found that a class III peroxidase family protein, PEROXIDASE36 (PER36), was
significantly down-regulated in the nars1 nars2 double mutant. Expression of PER36 occurred only in oi2 cells for
a few days around the torpedo stage. A PER36-GFP fusion protein was secreted into the outer cell wall in a
polarized manner. per36 mutants were defective in mucilage extrusion after seed imbibition due to the failure of
outer cell wall rupture, although the mutants exhibited normal monosaccharide composition of the mucilage. This
abnormal phenotype of per36 was rescued by pectin solubilization, which promoted cell wall loosening. These
results suggest that PER36 functions as a mucilage extrusion factor through regulation of the degradation of the
outer cell wall. Taken together, this work indicates that polarized secretion of PER36 in a developmental stagedependent manner plays a role in cell wall modification of oi2 cells [2].
[1] T. Kunieda et al. (2008) Plant Cell, 20, 2631-2642 ; [2] T. Kunieda et al. (2013) Plant Cell, in press.
O4-04
Identification of laccases involved in lignin formation in Brachypodium distachyon
Wang Y. (a), Dalmais M. (b), Antelme S. (a), Cézard L. (a), Léger F. (a), Bouchabké-Coussa O. (a), Soulhat C. (a),
Bendahmane A. (b), Jouanin L. (a), Lapierre C. (a), Sibout R. (a)
(1) IJPB, Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon, Route de StCyr (RD10), 78026 Versailles France ; (2) URGV, Unité de Recherche en Génomique Végétale, Université d'Evry Val
d'Essonne, INRA, 2 rue Gaston Crémieux CP 5708, 91057 EVRY cedex, France.
Lignin is a phenolic polymer exclusively made by plants. It contributes to the hydrophobic nature and integrity of
plant cell walls in xylem and sclerenchyma tissues. The monomeric subunits of the polymer are produced in the
cytosol, exported into the cell wall, oxidized and polymerized in the polysaccharide matrix. LACCASES were
recently shown to be involved in monolignol polymerization in Arabidopsis thaliana (1). Interestingly,
Arabidopsis mutants with substantial decrease of laccase activity produced higher saccharification yields. Up to
now, the biological functions of most laccase genes remain unknown in grasses. In this study, we report the
identification of Brachypodium genes and the production of mutants affected in laccase activity. We identified
series of mutants for BdLAC5, BdLAC6 and BdLAC8 by TILLING. The corresponding genes are highly
expressed in lignified tissues of wild-type plants. Furthermore, these genes are co-expressed with specific genes
involved in secondary cell wall formation. Chemical analysis supported by stained stem cross sections allowed the
detection of significant changes in lignin content and composition of mutants when compared to wild-type plants.
Genetic complementation experiments both in Arabidopsis and Brachypodium mutants showed partial rescue of
mutant phenotypes. The consequences of laccase disruption on cell wall composition and plant development will
be discussed.
[1] Berthet et al. (2011) Plant cell, 23, 1124-1137.
O4-05
Periplasmic AGP-Ca2+ is an essential component of the calcium oscillator that regulates plant
growth and development
Lamport D.T.A. (a), Várnai P. (b), Seal C.E. (c)
(a) University of Sussex, UK; (b) Imperial College, University of London, UK; (c) Royal Botanic Gardens Kew, Wakehurst
Place, West Sussex, UK.
Classical ArabinoGalactan glycoProteins (AGPs) that cover the plasma membrane (1) are involved in many
aspects of plant biology (2). But how? AGPs are highly glycosylated by numerous polysaccharides strung along
an extended polypeptide thread like beads on a necklace (3). These beads are acidic ArabinoGalactans O-linked to
hydroxyproline (Hyp-AGs) that although apparently heterogeneous and complex consist of highly conserved
repetitive 15-sugar arabinogalactan subunits with paired glucuronic carboxyls (4). NMR data and molecular
dynamics simulations (movie) identify these carboxyls as potential intramolecular Ca 2+-binding sites (5). Rapid
ultrafiltration assays showed that AGPs bind Ca 2+ tightly and stoichiometrically (Kd ~6.5 µM) at pH 5 but with
50% released at ~pH 3.2 consistent with the pKa of glucuronic acid. Indeed, plant cells can secrete acid (HCl) to
yield pHs even as low as pH 0.5 (6). Thus periplasmic AGPs, each containing ~30 Ca 2+-binding subunits,
represent a Ca2+ capacitor discharged when stretch-activated plasma membrane H +-ATPases generate a low
periplasmic pH. Hence AGPs are a substantial source of cytosolic Ca 2+ entry via Ca2+ channels that may also be
stretch-activated (7). We propose that these Ca2+ waves prime the ‘Calcium Oscillator’, a signal generator
essential to the global Ca2+ signalling pathway of green plants. This explains how AGPs are involved in such a
wide range of processes and resolves the paradox of peripheral glycoproteins that nevertheless play a central role
in the regulation of plant growth and development (8,9).
[1] D.T.A. Lamport et al. (2006) New Phytologist 169, 479-492; [2] M. Ellis et al. (2010) Plant Physiology 153, 403-419;
[3] Z.D. Zhao et al. (2002) Plant J.31, 431-444; [4] L. Tan et al. (2010) Journal of Biological Chemistry 285, 24575-24583;
[5] D.T.A. Lamport, P. Varnai (2013) New Phytologist 197, 58-64; [6] M.D. Lazzaro, W.W. Thomson, (1995) Physiologia
Plantarum 94, 291-297; [7] B.G. Pickard, M. Fujiki (2005) Funct. Plant Biol. 32, 863-879; [8] M.C. Cannon et al. (2008)
PNAS 105, 2226-2231; [9] D.T.A. Lamport, University of Cambridge (1963): http://gradworks.umi.com/3504823.pdf.
O4-06
AGP6 and AGP11 biological mode of action in Arabidopsis pollen and pollen tube growth
Costa M. (ab), Nobre S. (ab), Amorim M.I. (ab), Masiero S. (c), Pereira L.G. (ab), Coimbra S. (ab)
(a) Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Portugal; (b) BioFIG,Center for
Biodiversity, Functional and Integrative Genomics, Portugal; (c) Dipartimento di Bioscienze, Universitá degli Studi di
Milano, Italy.
Arabinogalactan proteins (AGPs) are cell wall proteoglycans that were shown to be important for pollen
development. An Arabidopsis double null mutant for two pollen-specific AGPs (agp6 agp11) showed reduced
pollen tube growth and compromised response to germination cues in vivo. So, in order to understand the mode of
action of these AGPs, an Affymetrix ATH1 genome array in the agp6 agp11 double null mutant pollen tube was
performed. The lack of two specific AGPs induced a meaningful shift of the gene expression profile. Calciumand signaling-related genes were found to be altered, which gives support to the known roles of such genes in
pollen tube growth. The presumed involvement of AGPs in signaling cascades was also reinforced. Cysteine-rich
proteins have been proposed to play a role in recognition and fertilization, and it was thus quite relevant that such
genes were found to be differentially expressed. The putative involvement of AGPs in signaling cascades through
calmodulin and protein degradation via ubiquitin was found. Also, stress related genes were found to be affected,
which supports the recognized similarities between signaling pathways in both defense and pollen tube growth
(Costa et al., 2013). Yeast two-hybrid experiments gave further support to these signaling pathways and revealed
putative AGP6 and AGP11 interactors implicated in the process of recycling by endocytosis of cell membrane
components, through clathrin-mediated endosomes and multivesicular bodies. A model for AGP6 and AGP11
biological mode of action in pollen tube growth is presented.
[1] Costa, M., Nobre, S., Becker, J., Masiero, S., Amorim, M.I., Pereira, L.G. and Coimbra, S. 2013. On hand, putative
ligands for arabinogalactan proteins in Arabidopsis pollen development. BMC Plant Biology, 13:7.
O4-07
Disruption of PME activity alters hormone homeostasis and triggers compensatory mechanisms
controlling adventitious rooting
Guénin S. (ab), Mongelard G. (b), Demailly H. (b), Novak O. (c), Lee K. (d), Bouton S. (a), Amakorova P. (c), Strnad M. (c),
Knox J.P. (d), Mouille G. (e), Bellini C. (f), Gutierrez L. (b), Pelloux J. (a)
(a) EA3900-BIOPI and (b) CRRBM, UPJV, 33 Rue St Leu, F-80039 Amiens, France; (c) Laboratory of Growth Regulators &
Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, CZ-783 71 Olomouc, Czech
Republic; (d) School of Molecular and Cellular Biology, University of Leeds, LS2 9JT Leeds, United Kingdom; (e) IJPB,
UMR1318 INRA-AgroParisTech, Bâtiment 2, Route de St Cyr (RD 10), F-78026 Versailles, France ; (f) Umeå Plant Science
Centre, Swedish University of Agricultural Sciences, 90183 Umeå, Sweden.
We recently showed that the decreased PME activity in Arabidopsis pme3 mutant, in which the PECTIN
METHYLESTERASE 3 gene was knocked-out, led to increase the degree of methylesterification (DM) of pectins
as well as the adventitious root formation in hypocotyl [1]. In order to gain insights into the relationship between
DM of pectins and adventitious rooting, we analyzed the Arabidopsis pme36 knock-out line. In wild-type, PME36
is expressed during seed maturation and in seedling hypocotyl up to 72-hours after germination. As expected,
pme36 displayed a decrease in PME activity during seed maturation and a resulting increase in the DM of pectins
in the mature seed. Unexpectedly, 48-hours after germination, PME activity in dark-grown hypocotyl of pme36
was higher than in the wild-type. This led to a decrease in the DM of pectins and in the number of adventitious
roots in the mutant. While confirming the positive correlation between DM of pectins and adventitious rooting,
these results highlight the existence of a mechanism overcompensating the absence of PME36 in pme36
hypocotyl. We found that this compensatory mechanism involved transcriptional regulations, other PME genes
being overexpressed in pme36 hypocotyl. In addition, looking at hormone contents and assessing the expression
of genes involved in the hormone signaling pathways shown to control adventitious rooting [2], we found a strong
alteration of hormone homeostasis in pme36 hypocotyl. Altogether these results suggest that adventitious rooting
is controlled by a regulatory network involving crosstalk between hormone signaling and PME activity, which is
modulated through a compensatory mechanism triggered by variations in DM of pectins.
[1] S. Guénin et al. (2011) New Phytol., 192, 114-126; [2] L. Gutierrez et al. (2012) Plant Cell, 24, 2515-2527.
O4-08
Versatile roles of PME and pectin in plant development
Hongo S. (a), Yokoyama R. (a), Sato K. (a), Kunieda T. (ab), Shimada T. (b), Kondo M. (c), Nishimura M. (c), HaraNishimura I. (b), Negi J. (d), Moriwaki K. (d), Konishi M. (e), Nakano T. (d), Kusumi K. (d), Hashimoto-Sugimoto M. (d),
Schroeder J.I. (f), Yanagisawa S. (e), Iba K. (c), Nishitani K. (a)
(a)Tohoku University, Japan; (b) Kyoto University, Japan; (c) NIBB, Japan; (d) Kyushu University, Japan; (e) University of
Tokyo, Japan. (f) University of California, San Diego, USA.
Secondary cell walls, which contain lignin, have traditionally been considered essential for the mechanical
strength of the shoot of land plants, whereas pectin, which is a characteristic component of the primary wall, is not
considered to be involved in the mechanical support of the plant. Contradicting this conventional knowledge, lossof-function mutant alleles of Arabidopsis PME35, which encodes a pectin methylesterase, showed a pendant stem
phenotype and an increased deformation rate of the stem, indicating that the mechanical strength of the stem was
impaired by the mutation. PME35 was expressed specifically in the basal part of the inflorescence stem.
Biochemical and immunohistochemical analysis using JIM5, JIM7 and LM20 monoclonal antibodies showed that
the activity of pectin methylesterase was significantly reduced in the primary cell wall of the cortex and
interfascicular fibers in the basal part of the mutant stem, but lignified cell walls in the interfascicular and xylary
fibers were not affected in the mutant. This indicates that the PME35-mediated demethylesterification of the
primary cell wall directly regulates the mechanical strength of supporting tissue [1]. We also report roles of pectin
demethylesterification in guard-cell maturation [2] and mucilage extrusion from seed coat [3] in Arabidopsis.
[1] S. Hongo et al. (2012) Plant Cell 24, 2624-2634 ; [2] J. Negi et al. (2013) Curr. Biol. in press; [3] T. Kunieda et al. (2013)
Plant Cell in press.
O4-09
The Arabidopsis LRR-RLK LRI is a novel regulator of cell wall integrity sensing
Boutrot F. (a), McKenna J. (b), van der Does D. (a), Segonzac C. (a), Miedes E. (c), Molina A. (c), Hamann T. (bd), Zipfel C.
(a)
(a) The Sainsbury Laboratory, Norwich, UK; (b) Imperial College London, London, UK; (c) Centro de Biotecnología y
Genómica de Plantas (CBGP, UPM-INIA) Universidad Politécnica de Madrid (UPM), Madrid, Spain; (d) Norwegian
University of Science and Technology, Trondheim, Norway.
Early recognition of danger by plants is essential for their ability to adapt to changes in the environment. Upon
microbial attack, plants can recognize pathogen-associated molecular patterns or damage-associated molecular
patterns (plant-derived molecules released during pathogen infection) through surface-localized patternrecognition receptors (PRRs), resulting in pattern-triggered immunity (PTI) [1]. Evidence is emerging that plant
cell wall integrity (CWI) signalling is an active mechanism as in yeast, and that plant cell wall damage (CWD)
results in responses similar to PTI responses [2,3]. The components involved in CWI signalling are however still
largely unknown. Using the model plant Arabidopsis thaliana, we identified a novel leucine-rich repeat receptorlike kinase (LRR-RLK) similar in structure to known PRRs, required for responses to the cellulose biosynthesis
inhibitor isoxaben (ISX), a chemical widely used to induce CWD in a controlled manner [3,4]. We tentatively
named this LRR-RLK LRI for LRR-RLK REQUIRED FOR ISOXABEN RESPONSE. Mutant lri plants are
impaired in ISX-induced accumulation of reactive oxygen species (ROS), jasmonic acid production, lignin
deposition, and seedling growth inhibition. In addition, mutants in two key regulatory LRR-RLKs in PTI
signalling, BAK1 and BKK1, displayed impaired ISX-induced responses as well, suggesting that CWI sensing
relies on a mechanism similar to PTI. Importantly, treatment with CW extracts from ISX-treated wild-type plants
resulted in an LRI-dependent ROS burst, while extracts from mock-treated plants did not elicit a response.
Together, these data suggest that LRI is an important regulator of CWD perception in plants.
[1] T. Boller and G. Felix (2009) Annu. Rev. Plant Biol., 60, 379-406; [2] L. Denness et al. (2011) Plant Phys., 156, 13641374; [3] S. Wolf et al. (2012) Annu. Rev. Plant Biol., 63, 381-407; [4] T. Desprex et al. (2002) Plant Phys., 128, 482-490.
O4-10
New aspects of cell wall integrity signalling revealed through post-synthetic modifications
Pogorelko G. (a), Lionetti V. (b), Young Z. (a), Bellincampi D. (b), Zabotina O. (a)
(a) Iowa State University, USA; (b) ”Sapienza” University of Rome, Italy.
The existence of cell wall integrity (CWI) signaling in plants has been demonstrated, but little is known about the
actual signaling pathways involved. CWI is maintained through a highly dynamic balance between cell wall
biosynthesis involving a broad spectrum of synthetic enzymes localized in Golgi and cell wall post-synthetic
modifications involving a similarly broad spectrum of hydrolytic enzymes localized in plant apoplast or secreted
by plant-invading organisms. Hydrolytic enzymes are the key components involved in cell wall remodeling, the
main process involved in cell wall adjustments during plant development and response to environmental cues.
Therefore, employment of such enzymes with well characterized specificities, overexpression of which in plant
apoplast causes particular cell wall component structural modifications or their cross-linking, represents a
promising approach to studying plants’ responses to these modifications and involvement of different cell wall
components in these responses. We have created a set of homozygous Arabidopsis and Brachypodium transgenic
lines expressing different specific microbial glycosyl hydrolases or esterases and characterized their cell walls.
These transgenic plants represent a toolset that provides a new approach to study CWI signaling.
Expression of two different A. nidulans acetylesterases and subsequent reduction of cell wall acetylation initiated
defense-related responses leading to higher resistance to necrotrophic pathogens in both Arabidopsis and
Brachypodium plants. Moreover, de-acetylation of either xylan or pectins resulted in up-regulation of different
defense-related genes, suggesting that plants respond differently to modification of different polysaccharides.
These plants also showed up-regulation of three acetyltransferases involved in acetyl group transfer onto
polysaccharides during biosynthesis. Arabidopsis plants expressing A. nidulans α-fucosidase have reduced fucose
content in their cell walls and show up-regulation of several fucosyltransferases (FUT) differentially expressed in
different organs. While FUT4 was higher expressed in roots, expression of FUT3 and FUT12 was higher in stems,
and FUT6 and FUT11 were up-regulated in leaves of these transgenic plants. De-fucosylation differently affected
growth of roots and hypocotyls in the dark. The functions of FUT3, FUT11 and FUT12 are unknown, and further
analyses will assist in their functional characterization.
O4-11
A receptor protein links cell wall surveillance with brassinosteroid signalling
Wolf S. (a), Kolbeck A. (a), Greiner S. (a), Höfte H. (b)
(a) Centre for Organismal Studies (COS) Heidelberg, University of Heidelberg, Germany; (b) Institut Jean-Pierre Bourgin,
INRA-AgroParisTech, Saclay Plant Science, Versailles, France.
Modification of the pectic cell wall polysaccharide homogalacturonan is thought to play a key role in
development. Recently, we have shown that interference with pectin modification triggers activation of the
brassinosteroid (BR) signalling pathway, which in turn orchestrates a compensatory response involving cell wall
remodelling [1]. In the absence of BR-mediated feedback signalling, altered pectin modification severely
compromises cellular integrity, ultimately resulting in cell rupture, and thus demonstrating the relevance of cell
wall surveillance and signalling. Through the use of a forward genetic screen we have identified CNU2, a
receptor-like protein essential for the cell wall-induced activation of the BR pathway. Through genetic,
biochemical, and transcriptomic approaches, we demonstrate that CNU2 is not itself a component of the BR
pathway, but instead conditionally activates hormone signalling when triggered by cell wall-related cues.
Furthermore, our data suggest that CNU2 directly interacts with components of the BR receptor complex,
providing a mechanism for signalling integration.
[1] S. Wolf et al. (2012) Curr. Biol., 22 (18), 1732-7.
O4-12
The Rab GTPase SMG1 and its effector PMR4 are involved in signaling cascade and the
regulation of CWI in plants
Ellinger D., Glöckner A., Naumann M., Manisseri C., Voigt C.A.
Phytopathology and Biochemistry, Biocenter Klein Flottbek, University of Hamburg, Germany.
In recent years, accumulating evidence indicated that a cell wall integrity (CWI) signaling mechanism (may)
exist(s) that maintains functional integrity of the cell wall. It is supposed by the available data that plant CWI has
to be regarded as a distinct maintenance mechanism, as it is in yeast [1, 2]. While in S. cerevisiae CWI is well
characterized, our understanding of the mechanism in plants is rather limited although the evidences suggest that
similar signaling cascades may be involved and particular protein activities may be conserved between plant and
yeast. It is thought that in plants as in yeast downstream signalling and cell wall repair, e.g. by callose depositions,
can be induced by GTPases through activation of gene transcription and by posttranslational regulation of glucan
synthase. Based on microarray data, we identified the small G-protein SMG1 from Arabidopsis as being up
regulated on transcriptional level after induction of CWI by inoculation with the powdery mildew Golovinomyces
cichoracearum. To study the function of SMG1, we generated transgenic Arabidopsis lines that overexpress the
GTPase. Apart from early and more massive callose production also non-cellulosic cell wall monosaccharide
composition is changed in cell walls of this transgenic line upon treatment with the G. cichoracearum. The
detected strong callose deposits prevented fungal ingress into epidermal cells and lead to a total resistant
phenotype. In addition, we found that SMG1 interacts with stress-induced callose synthase PMR4 (= GSL5) [3, 4,
5] depending on the bound nucleotide and live cell imaging showed the presence of SMG1 at the plasma
membrane and in colocalization with callose deposits as site of attempted fungal penetration. With our findings
that constitutive expression of the small G-Protein SMG1 from the Rab-family induces callose depositions we did
a first step towards elucidating signaling cascades and the regulation of CWI in plants.
[1] Hamann (2012) Front. Plant. Sci., 3, 77; [2] Wolf et al. (2012) Annu. Rev. Plant. Biol., 63, 381; [3] Jacobs et al. (2003)
Plant Cell, 15, 2503; [4] Nishimura et al. (2003) Science, 301, 969; [5] Ellinger et al. (2013) Plant Physiol, 161, 1433.
O4-13
The triple mitogen-activated protein kinases ANPs regulate immunity triggered by
oligogalacturonides and orchestrate signaling and ROS formation in mitochondria, plastids
and nucleus
Savatin D.V., Marti L., Gigli-Bisceglia N., Cervone F., De Lorenzo G.
Dipartimento di Biologia e Biotecnologie "Charles Darwin", Sapienza, Università di Roma, Italy.
During pathogen infection or after injury, homogalacturonan (HGA), a major component of pectin in the plant cell
wall, is broken down into oligogalacturonide fragments (OGs) that act as damage-associated molecular patterns
(DAMPs) and activate the plant immune response. An extensive overlap exists between early responses triggered
by OGs and those induced by bacterial pathogen-associated molecular patterns (PAMPs). In plants, like in
animals, signal transduction pathways leading to immunity include Mitogen-Activated Protein Kinase (MAPK)
phosphorylation cascades. A MAPK cascade consists of a core module of three kinases that act in sequence: a
MAP kinase kinase kinase (MAPKKK) that activates, via phosphorylation, a MAP kinase kinase (MAPKK),
which in turn activates a MAP kinase (MAPK). We investigated the role of the Arabidopsis ANP
(ARABIDOPSIS NPK1-RELATED PROTEIN KINASE) gene family, which encodes the MAPKKKs ANP1-3,
in immunity triggered by OGs and by a representative PAMP, i.e. elf 18, a peptide derived from the bacterial
Elongation Factor Thermo Unstable (EF-Tu). We found that ANPs, besides playing a crucial role development,
are required for OG- and elf18-triggered defense responses and protection against the necrotrophic fungus
Botrytis cinerea. In physiological conditions, ANPs localize in mitochondria and in the cytoplasm, but, after
elicitor perception, localize also into plastids and nucleus, revealing a dynamics that is unique in plant cell
biology. Sites of elicitor-induced ROS accumulation and ANP localization coincide, and ANPs are required both
for ROS generation and ROS signaling. Our findings point to ANPs as key transduction elements that coordinate
PAMP- and DAMP-triggered immunity and central hubs in the orchestration of ROS accumulation and signaling.
O4-14
SignWALLing: Signals derived from Arabidopsis cell wall activate specific resistance to pathogens
Miedes E. (a), Sopeña S. (a), Jordá L. (a), Bacete L. (a), Riviére M.P. (a), Sánchez-Vallet A. (a), Sánchez-Rodríguez C. (a),
Escudero V. (a), Ranocha P. (b), Denance N. (b), Bartel X. (c), Boutrot F. (e), Marco Y. (c), Goffner D. (b), Zipfel C. (e),
Hahn M.G. (d), Molina A. (a)
(a) Centro de Biotecnología y Genómica de Plantas (UPM-INIA). Universidad Politécnica Madrid. Madrid, SPAIN; (b)
Unité Mixte de Recherche Centre National de la Recherche Scientifique Univ Toulouse III. Castanet Tolosan, FRANCE; (c)
Centre National de la Recherche Scientifique Instiut National de la Recherche Agronomique. Castanet Tolosan, FRANCE;
(d) Complex Carbohydrate Research Center. Athens, GA, USA; (e) The Sainsbury Laboratory, Norwich, UK.
The cell wall is a dynamic structure that regulates both constitutive and inducible plant defence responses. The
activation of plant innate immune system can be triggered by damaged-associated molecular patterns (DAMPs),
that are molecules released from plant cell walls upon pathogen infection or wounding. To further characterize the
function of cell wall on the regulation of immune responses, we performed a biased resistance screening of
putative/well-characterized primary/secondary Arabidopsis thaliana cell wall mutants (cwm). In this screening we
dentified 20 mutants with altered susceptibility/resistance to at least one of the following pathogens:
Plectosphaerella cucumerina, Ralstonia solanacearum, Hyaloperonospora arabidopsidis and a powdery mildew
fungus. We found that cell wall extracts from some of the selected cwm plants conferred enhanced resistance to
particular pathogens when they were applied to wild-type plants. This resistance was associated to the activation
of early immune responses, further suggesting the presence of DAMPs in the cwm wall extracts. Using glycomic
profiling we determined the carbohydrate structures of the wall fractions from selected cwm plants. Our data
support a specific interconnection between cell wall structure/composition and resistance to pathogens.
O4-15
Cell wall acetylation is important for surface permeability and resistance to Botrytis cinerea
Nafisi M. (a), Stranne M. (a), Fimognari L. (a), Ren Y. (b), Martens H.J. (a), Atwell S. (c), Manabe Y. (b), Kliebenstein D.
(c), Scheller H.V. (b), Sakuragi Y. (a)
(a) University of Copenhagen, Faculty of Sciences, Department of Plant and Environmental Sciences; VKR Research Centre
“Pro-Active Plants”, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; (b) DOE Joint Bioenergy Institute,
California, USA; (c) Department of Plant Sciences, University of California Davis, California USA.
Many cell wall polymers are O-acetylated, which contribute to physical properties of the polymers. However the
exact biological role of acetylation in vivo is unclear (1). We have previously identified an acetylation mutant of
Arabidopsis thaliana, reduced wall acetylation 2 (rwa2), with reduced acetylation of pectin and hemicelluloses
(2). Mutant plants showed enhanced resistance to the necrotrophic fungal pathogen Botrytis cinerea. Recently, a
conditional phenotype was observed, where the reduction in cell wall acetylation had a profound effect on plant
surface structures as rwa2 had collapsed trichomes, a more permeable cuticle and increased cell wall thickness.
Phenotypic analysis of the pectin- and xyloglucan-specific acetylation mutants (tbl10, tbl32, tbl38, tbl40, axy4-3,
axy4-4) revealed a wild-type level of susceptibility to B. cinerea and surface permeability. Notably, the tbl29/esk1
mutant, defective in xylan-specific acetylation, is not reported to have increased surface permeability (3,4). These
results indicate that the increased permeability observed for the rwa2 mutant is unique and is caused by
combinatorial effects due to reduced acetylation in more than one class of cell wall polymers. Transcriptome
analysis by mRNA sequencing revealed alterations between rwa2 and wild type in a number of genes, many of
which are involved in abscisic acid signalling. These results highlight a link between cell wall acetylation, surface
structural integrity, and plant hormone signalling.
[1] S. Gille and M. Pauly (2012) Front. Plant Sci, 3,12 ; [2] Y. Manabe et al., (2011) Plant phys, 155, 1068-1078 ; [3] G.
Xiong et al., (2013) Mol Plant, Jan 23 ; [4] V. Lefebvre et al., (2011) PLoS ONE, 6, e16645.
O4-16
The role of DEFECTIVE KERNEL1 (DEK1) in mechano-sensitive growth of plant cells
Johnson K. (a), Amanda D. (a), Ingram G. (b), Bacic A. (ac)
(a) School of Botany, University of Melbourne, Australia; (b) Reproduction et Développement des Plantes, ENS Lyon,
France; (c) Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia.
Plant cell walls provide a crucial structural function for the growing plant by harnessing the turgor pressure of
cells in a highly regulated manner; allowing controlled turgor-driven tissue growth whilst maintaining turgordependent structural support. Cells are thought to respond to the mechanical pressure by altering the
thickness/cross-linking of the cell wall [1] and by varying cell-wall extensibility which can be modified by
regulating either the production of cellulose, matrix polysaccharides that associate with cellulose or wallmodifying enzymes that influence the relationship between the two [2]. How this process is regulated across a
field of growing cells is still largely unknown but requires signals from the expanding cell to be transduced to the
cytoplasm to regulate production of new polysaccharides and wall-modifying enzymes [3].
We have preliminary evidence to suggest that an essential plant gene, DEFECTIVE KERNEL1 (DEK1), acts as a
mechano-sensor at the plasma membrane to perceive and transduce mechanical signals to promote cell wall remodelling and growth [4]. DEK1 is a large modular protein composed of a sizable integral membrane region
(predicted to contain up to 24 transmembrane spans and an extracellular loop), a cytoplasmic region adjacent to
the membrane, and a domain sharing strong homology to mammalian CALPAINS, a class of intracellular cysteine
proteases. Over-expression of the CALPAIN domain of DEK1 results in plants with thicker walls with increased
cellulose composition and up-regulation in the expression of cell wall-modifying enzymes. Data will be presented
that show the DEK1 CALPAIN domain regulates both cell growth (division and expansion) and cell wall
modification.
[1] Seifert GJ, Blaukopf C. (2010). Plant Physiol 153(2): 467-478; [2] Cosgrove DJ. (2005). Nat Rev Mol Cell Biol 6(11):
850-861; [3] Keegstra K. (2010). Plant Physiol 154(2):483-6; [4] Johnson KL et al. (2008). Plant Cell 20(10), 2619-30.
O4-17
The molecular basis of cell wall stiffness at the shoot apical meristem: a specific role of the CSLD
gene family
Wightman R. (a), Milani P. (c), Mortimer J. (b), Dupree P. (b), Hamant O. (c), Meyerowitz E. (ad)
(a) Sainsbury Laboratory, University of Cambridge, Bateman Street, Cambridge. CB2 1LR. UK; (b) Department of
Biochemistry, University of Cambridge, Downing Site, Cambridge. CB2 1QW. UK; (c) Laboratoire de Reproduction et
Développement des Plantes, UCB Lyon 1, 46 Allée D’Italie, 69364 Lyon Cedex 07, France ;(d) Division of Biology,
California Institute of Technology, Pasadena, California. USA.
In plants, the shoot apical meristem contains the population of stem cells that give rise to the aerial organs.
Initiation of organ primordia is known to require dynamic changes in the cell wall matrix including cell wall
loosening and pectin modifications but little is known about the cell wall structure of the cells that make up and
maintain meristem form and function. We have found members of the CSLD gene family to have important roles
within the meristem. Specific mutant combinations exhibit meristem termination after making between one and
four flowers. Prior to termination, the meristem is small and misshapen and the boundary region that divides
organ and meristem fails to form properly. Amplification of stresses within the meristem results in mechanical
failure of the cell walls in internal tissue layers and suggests csld walls are weaker. This has been confirmed by
direct measurements of wall stiffness using atomic force microscopy. We are currently investigating the nature of
the polymer that the CSLD proteins produce in the meristem and propose a model in which different load-bearing
components in the cell wall are recruited as cells acquire new fates.
Session 5 : Advanced Understanding of CW Structure, Biosynthesis & Function
Bioinformatic & omic approaches, Computational & biophysical approaches
O5-01
Another brick in the [cell] wall
Barbacci A. (a), Lahaye M. (a), Videcoq P. (a), Magnenet V. (b)
(a) INRA, UR 1268 BIA, F-44316, Nantes, France; (b) ICube, Université de Strasbourg, UMR CNRS 7357, 72 Route du Rhin
C.S. 315, 67411 Illkirch Cedex, France.
Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is
possible if (i) a tensile stress is developed in the cell wall due to coupling effect between turgor pressure and the
modulation of its mechanical properties by enzymatic and physicochemical reactions and if (ii) new cell wall
“bricks” are synthesized and assembled to the existing wall. In other words, expansive growth is the result of
coupling effects between mechanical, thermal and chemical energy. To better understand the process of growth,
models must describe the interplay between physical or mechanical variable with biological processes. In this
presentation a general unified and theoretical framework is proposed, based on irreversible thermodynamics [1,2],
which is adapted to model growth in function of energy forms and their coupling.
This communication presents the theoretical paradigm and then applied it to model growth of the internodal cell
of C. corallina modulated by changes in pressure and temperature. The results of the model describe faithfully the
growth of the cell in term of both length increment and cell pectate biosynthesis and addition to the expanding
wall. These results [3] are in full agreement with the experimental evidences [4]. The model proposed is able to
take into account the role of turgor pressure, which is not only the trigger of growth but is also implied in cell wall
construction regulation loop [5]. The creation of such biosynthesis dependent growth model can be viewed as the
first step towards a more integrative biophysical modelling of growth.
[1] Callen H (1985) Thermodynamics and an introduction to thermostatistics. 2nd edition. New York: Wiley. [2] Cunat C
(2001) Mechanics of Time-Dependent Materials 5: 39–65; [3] Barbacci A et al. Plos One (2013) submitted. [4] Proseus TE,
Boyer JS (2008) Plant, cell & environment 31: 1147–55; [5] Kroeger JH et al. (2011) PloS one 6: e18549.
O5-02
Regulation of plant cell growth through cell wall mechanics
Chebli Y. (a), Kroeger J. (b), Zerzour R. (a), Bou Daher F. (a), Geitmann A. (a)
(a) Institut de recherche en biologie végétale, Département de sciences biologiques, Université de Montréal, Québec,
Canada; (b) Raymor Industries, Boisbriand, Québec, Canada.
Plant cell morphogenesis is regulated through modulation of the mechanical properties of the cell wall. Both
spatial control (shape formation) and temporal control (long term and short term kinetics) depend on how the
material properties of the wall are regulated. In pollen tubes, the growth process occurs unidirectionally and the
resulting shape is axially symmetric. Moreover, the growth process can easily be reproduced in vitro, and it occurs
very rapidly on time scales that allow monitoring of drug- or enzyme-induced effects within minutes. These
features make the pollen tube an ideal system for cell biological studies and biomechanical modeling. The pollen
tube cell wall is characterized by a precisely defined spatial distribution of its components - a profile that is easily
disturbed upon external influence as we demonstrated by exposing the cells to simulated microgravity and
hypergravity conditions [1]. The pollen tube also displays an oscillatory growth kinetics that lends itself to the
analysis of the causal relationship between cellular features such as exocytosis, cell wall expansion, and ion
fluxes. In a series of models we have shown that these oscillations are governed by the mechanical properties of
the apical cell wall and we demonstrated how changes in turgor pressure influence the oscillation parameters such
as frequency and amplitude [2]. Here we will illustrate how an interdisciplinary approach to plant cell biology
allows developing new research questions that help us to understand how plant development is regulated at
cellular level.
[1] Y. Chebli et al. (2013) PLoS One, 8, e58246 ; [2] J.H. Kroeger & A. Geitmann (2012) Current Opinion in Plant Biology,
15, 618–624.
O5-03
Analysis of mechanical properties of cell walls by MEMS-based Cellular Force Microscopy
Draeger C. (a), Vogler H. (a), Weber A. (d), Eichenberger C. (a), Knox J.P. (b), Felekis D. (c), Smith R.S. (d), Nelson B.J.
(c), Grossniklaus U. (a), Ringli C. (a)
(a) Institute of Plant Biology & Zürich-Basel Plant Science Center, University of Zurich, Zollikerstr. 107, 8008 Zürich,
Switzerland ; (b) Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK ; (c) Institute of Robotics and Intelligent
Systems, ETH Zürich, Tannenstr. 3, 8092 Zürich, Switzerland ; (d) Institute of Plant Sciences, University of Bern,
Altenbergrain 21, 3013 Bern, Switzerland.
Plant cell growth largely depends on the ability of cell wall to expand. The knowledge of the mechanical
properties of cell walls, which have a strong influence on this process, is still limited. Recently, micro-indentation
experiments using Cellular Force Microscopy were used to measure mechanical properties of pollen tube (PT) cell
walls of Lilly. Combined with a mathematical model based on the Finite Element Method, this allowed us to
calculate the turgor pressure in the PT and the elasticity (Young’s modulus) of cell walls (Routier-Kierzkowska et
al., 2012; Vogler et al., 2012). This experimental system was expanded to the much smaller PTs of A rabidopsis
thaliana. In addition to the wild type, the xyloglucan-deficient xxt1 xxt2 mutant was analyzed. The xxt1 xxt2
mutant displays only a moderate growth phenotype at the whole-plant level. In PTs, however, it displays a strong
aberrant growth phenotype in vitro and in vivo which is reflected by a reduced fertilization efficiency. The
analysis of the mechanical properties revealed that the cell wall of xxt1 xxt2 mutant PTs have a decreased stiffness
compared to wild-type PTs. Hence, the defect in growth properties is paralleled by changes in the mechanical
properties of the cell wall. Additional analyses indicate a possible compensatory mechanism to cope with XyGdeficiency by structural cell wall proteins, both in terms of PT growth as well as the mechanical properties of the
cell wall.
Routier-Kierzkowska, A.-L., Weber, A., Kochova, P., Felekis, D., Nelson, B.J., Kuhlemeier, C., and Smith, R.S. (2012);
Cellular Force Microscopy for in vivo measurements of plant tissue mechanics. Plant Physiol. 158, 1514-1522; Vogler, H. et
al. (2013). The pollen tube: a soft shell with a hard core. Plant J. 73: 617-627.
O5-04
Coarse-grained simulation of cell wall load-bearing network structure
Fan B. (a), Maranas J.K. (b)
(a) Department of Chemical Engineering, Penn State University, University Park, PA 16802, USA; (b) Department of
Material Science, Penn State University, University Park, PA 16802, USA.
We use coarse-grained molecular dynamics simulation to study components of the load-bearing structure in
primary cell walls: cellulose microfibrils and xyloglucan. The model for the cellulose Iβ microfibril is constructed
from atomistic simulation of a 40 glucose-unit-long microfibril by requiring consistency between the chain
configuration, intermolecular packing, and hydrogen bonding of the two levels of modeling. We simulated
microfibrils with 100 to 400 residues, comparable to the smallest naturally occurring microfibrils. (See the figure
below) Microfibrils longer than 100nm form bends along their longitudinal direction. Multiple bends are observed
in the microfibril containing 400 residues. These bends reform after applying force on the chain ends to straighten
it, and are energetically favorable compared to unbent microfibrils. The torsional potential and van der Waals
potential decreases and are responsible for stabilization of the bended structure. The model for xyloglucan is also
constructed using atomistic simulation as a target to evaluate model performance. As a starting point, we use
chains with five XXXG motifs. We use explicit solvation with the coarse-grained water bead encompassing two
water molecules. Development of these models facilitates study of xyloglucan chains in solution, and provides a
valuable tool to study properties of the primary wall load-bearing network structure.
O5-05
Modeling mesoscale structure and mechanics of the secondary wood cell wall
Aigner N. (ab), Colombo J. (a), Burgert I. (b), Del Gado E. (a)
(a) ETH Zurich, IfB, Microstructure and Rheology of Building Materials, Suisse; (b) ETH Zurich, IfB, Wood Materials
Science, Suisse.
Controlling structure-property relationships of the secondary wood cell wall on the nanometer to micron length
scale is crucial for the mechanical response of the material but still far from being achieved [1]. We propose a
numerical model that allows us to investigate structural and mechanical features on this crucial scale and consists
of aligned flexible rod-like inclusions (cellulose) embedded in a soft gel (hemicellulose and lignin). We translate
structural information from AFM measurements [2] and data on chemical composition into a particle based
model, with specifically designed bonded and non-bonded interactions. Using a molecular dynamics integration
scheme we are able to investigate the influence of effective interactions on the structural organization and the
mechanical response to deformation of the model cell wall.
In the present work we discuss the range of parameters that the model can explore and the choice for the effective
interactions of our model. We characterize the structure by means of the structure factor (which can be directly
related to available SAXS measurements) and pore size distribution calculations in order to link the model length
scales to those of the real material. Furthermore, we apply tensile, compressive and shear deformation to our
model. From this we obtain the stiffness of the nano-composite as a function of effective interactions and
composition. We compare these results with recent experimental data on cell wall stiffness as obtained by e.g.
Orso and coworkers [3]. This allows us not only to draw conclusions about the magnitude of effective interactions
but also to get new insight into their importance with respect to mechanical properties. By linking supramolecular
interactions and organization to global cell wall properties we intend to eventually shed some new light on the
structure-property relationships of the secondary cell wall.
[1] Keckes J, Burgert I, Frühmann K, Müller M, Kölln K, Hamilton M, Burghammer M, Roth V S, Stanzl-Tschegg S, Fratzl P
(2003) Nature Mater 2, 810 - 813; doi: 10.1038/nmat1019; [2] Zimmermann T, Thommen V, Reimann P, Hug H J (2006) J.
Struct. Biol. 156 (2), 363 - 369; doi: 10.1016/j.jsb.2006.06.007; [3] Orso S, Wegst U G K, Arzt E (2006) J. Mater. Sci. 41,
5122 - 5126; doi: 10.1007/s10853-006-0072-1.
O5-06
Structural comparison of the cytosolic domain of cellulose synthases in bacteria and plants
Haigler C.H. (a), Davis J.K. (a), Yingling Y. (b), Zimmer J. (c)
(a) North Carolina State University, Depts. of Crop Science and Plant Biology, Raleigh, NC 27695 USA; (b) North Carolina
State University, Dept. of Materials Science and Engineering, Raleigh, NC 27695 USA; University of Virginia, Center for
Membrane Biology, Dept. of Molecular Physiology and Biological Physics, Charlottesville, VA 22908 USA.
Understanding the mechanistic operation of plant cellulose synthase is a prerequisite for biochemical engineering
of the enzyme to produce superior biomaterials and biomass feedstocks. Structure-function relationships in a
prokaryotic cellulose synthase were recently revealed by the crystal structure of an active BcsA from
Rhodobacter sphaeroides, including a glucan chain emanating from the catalytic site (Morgan et al., 2012, Nature
493: 181-187). All cellulose synthases share some highly conserved motifs, but such conserved regions are often
found widely spaced amid much more variable sequences. For this reason the BcsA structure provides only
limited insight into the function of the plant cellulose synthase proteins (CESAs). To gain insight into the
structure of plant CESA in the absence of a crystal structure, we carried out a structural co-alignment between the
glycosyltransferase domains of BcsA and a computationally-predicted, atomistic, tertiary structure of the cytosolic
domain of a secondary wall CESA from cotton (GhCESA1; Sethaphong et al., 2013, doi:
10.1073/pnas.1301027110). After editing both sequences to include only homologous regions, the structural coalignment showed a 3.9Å root-mean-square deviation (RMSD) between the backbone atoms of the two structures.
This similarity confirmed the bioinformatics methods used to generate the predicted GhCESA1 structure because
the BcsA crystal structure was not used in modeling GhCESA1. Here we will describe the overall commonalities
and differences in the bacterial and plant proteins and highlight previously unidentified motifs in plant CESA that
now have a predicted function based on the structural co-alignment with BcsA. Several point mutations in CESAs
of Arabidopsis thaliana are co-localized with newly predicted functional motifs in plant CESA, providing insight
into the likely mechanism underlying the cellulose-deficient phenotypes. This work was supported by the U.S. Department of Energy, Office of
Science and Office of Basic Energy Sciences (award no. DE–SC0001090 to the Center for LignoCellulose Structure and Formation, an Energy Frontier Research Center).
O5-07
New insights into xyloglucan biosynthesis: functional organization of a multi-enzyme complex
Chou Y.H., Morris R., Tietze A.A., Pogorelko G., Steadham J., Zabotina O.
Iowa State University, Ames, Iowa, USA.
Significant progress has been made in revealing different glycosyl transferases (GTs) involved in the synthesis of
various plant polysaccharides. However, detailed molecular mechanisms of polysaccharide biosynthesis and its
regulation at biochemical levels are unknown. Apart from cellulose, xyloglucan is the best structurally
characterized cell wall polysaccharide, and significant progress has been made in identifying and characterizing
the enzymes involved in its synthesis in Arabidopsis. We, therefore, chose xyloglucan for our studies directed to
understand the functional organization of GTs involved in the synthesis, their regulation, and their catalytic
activity.
Employing various techniques from cell and molecular biology, biochemistry, structural biology, and
computational modeling, we study, both in vivo and in vitro, the protein-protein interactions among seven
enzymes, CSLC4, XXT1, XXT2, XXT5, XLT2, MUR3 and FUT1, to elucidate their organization in the Golgi
during xyloglucan biosynthesis. In addition, we investigate protein-protein interaction residues, catalytic activity,
and substrate binding of three xylosyltransferases (XXTs). Obtained results demonstrate all seven enzymes form
hetero- and homo-complexes among each other, suggesting that they are organized into a multiprotein complex.
Using in vitro pull down assays, we demonstrated that GTs interact via their domains localized in the Golgi
lumen. Moreover, while the formation of three homo-complexes of CSLC4, XXT2 and FUT1 involves disulfide
bonding, most of the hetero-complexes do not. Next, mutagenesis studies will be performed to reveal the residues
involved in these interactions. To further understand the catalytic mechanism of XXTs, the mutagenesis analysis
was performed to elucidate the function of DXD motifs. Our results confirmed the importance of two distinct
DXD motifs for XXT2 enzymatic activity and one DXD motif for XXT5 functioning in vivo. The latter results are
the most intriguing since the catalytic activity of XXT5 has not been demonstrated before. However, our
computational simulations using the YASARA software suggested that XXT5 has all potentially required
structural features and organization for carrying D-xylosyltransferase activity.
[1] Chou et al. (2012) Plant Physiol., 159: 1355-1366 ; [2 ]Zabotina et al. (2012) Plant Physiol., 159: 1367-1384.
This research project is supported by National Science Foundation under Grant Number# 1121163
O5-08
The role of genes from the cellulose synthase superfamily in xylan biosynthesis
Burton R.A., Fincher G.B.
ARC Centre of Excellence in Plant Cell Walls, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064,
Australia.
Heteroxylans are common constituents of plant cell walls and generally contain a backbone of (1,4)-β-xylan
substituted mainly with single α-L-arabinofuranosyl and/or α-D-glucuronopyranosyl residues. The identification
of genes involved in the synthesis of the (1,4)-β-xylan backbone of heteroxylans has so far proved to be elusive,
although members of the GT8, GT43, GT47 and GT61 families of enzymes have been implicated in various
aspects of heteroxylan biosynthesis.
Here we have used deep transcript profiling in selected tissues from barley ( Hordeum vulgare) and Plantago
ovata to identify genes that are highly expressed where cell walls are rich in heteroxylans. The highest transcript
abundance values were observed for members of the cellulose synthase gene superfamily and these genes, which
encode family GT2 glycosyl transferase enzymes, became candidate genes for the synthesis of the (1,4)-β-xylan
backbone of the heteroxylans. Subsequent over-expression of the candidate genes in transgenic barley, driven by
an endosperm-specific promoter, led to increases of arabinoxylan content of about 50% on a per grain basis and
an elongated grain phenotype. Immunocytochemical examination using the LM11 antibodies confirmed the
increases in heteroxylan content in walls of the starchy endosperm in the transgenic grain. These effects persisted
into the T4 generation. Transient expression of the candidate genes in heterologous systems provided further
evidence for their participation in (1,4)-β-xylan biosynthesis.
O5-09
The plant Golgi apparatus – insights from quantitative proteomics
Nikolovski N. (ab), Rubtsov D. (a), Shliaha P. (ab), Sadowski P. (ab), Lilley K.S. (ab), Dupree P. (a)
(a) Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK; (b) Cambridge Centre for
Proteomics, Cambridge Systems Biology Centre, University of Cambridge, Cambridge, CB2 1QR, UK.
Polysaccharide synthesis in the Golgi apparatus is considered a multistep reaction that requires the cooperative
and orchestrated action of several enzymes, mostly glycosyltransferases (GTs). The organisation of such
biosynthetic machinery determines the properties of the polysaccharide made: its size, substitution degree and
pattern, translating into specific rheological behaviour, defining the polysaccharide function. The mechanism of
synthesis is poorly understood; not only is very little known about the specificity of the GTs, but even less about
their interaction or the identity of other types of proteins involved. Studies towards determining all the machinery
components and their abundances are therefore of high importance. In order to obtain such insight we explored
three different quantitative proteomic approaches. Firstly, the LOPIT technique led to the localisation of nearly
200 proteins in the Golgi apparatus, 79 of which have not been previously reported [1]. We found, unexpectedly,
a number of novel putative GT families which will extend the base for understanding polysaccharide synthesis.
Further analysis on multiple LOPIT experiments enabled an even greater list of Golgi residents, with confident
assignment of over 500 proteins. This list can be further extended to nearly 1000 proteins by the addition of
homologues, and thus possibly comprise the complete Golgi proteome and a finite list of GTs and other synthetic
components. Secondly, the label-free approach (MS e and IMS-MSe) allowed ranking of the Golgi proteins by their
abundances along three orders of magnitude, led to novel hypotheses on polysaccharide synthesis. Lastly, a
targeted absolute quantitation method (SRM) was employed for precise measurement of the abundances of the
xylan synthetic enzymes and their stoichiometry. These different proteomics techniques led to a fuller insight into
the Golgi proteome composition and organisation, specifically into the control of xylan structure and function.
[1] N. Nikolovski et al. (2012) Plant Physiol., 160, 1037-1051.
O5-10
Membrane trafficking dynamics during cell wall development, polar expansion and cell adhesion
in the model unicellular green alga, Penium margaritaceum
Domozych D.S., Ochs J.
Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College, Saratoga Springs, New York, 12866,
USA.
The unicellular Charophycean Green Alga, Penium margaritaceum, grows by a distinct polar expansion
mechanism focused at a narrow band at the cell’s isthmus and encompasses coordinated activation and interaction
of the cell’s membrane trafficking networks, cytoskeleton and wall assembly mechanism. In this study, we
performed a comprehensive analysis of the subcellular systems involved in production of the cell wall and
extracellular polymeric substance (EPS). At the expansion band, cellulose synthesis is accompanied by
secretion/insertion of high-esterified homogalacturonan (HG) and rhamnogalacturonan I (RGI) into the cellulose
network. At the edges of this band, HG fibrils are secreted through the wall where pectin methylesterase deesterifies the HG and calcium cross-linking occurs to yield the latticed outer wall layer. The secretory network is
symmetrically compartmentalized and is centered around an elaborate Golgi apparatus consisting of 100-150
stationary Golgi bodies that are positioned in internal valleys of cytoplasm. Pectin-containing vesicles emerge
from the trans face cisternae or trans Golgi Network (TGN) while large EPS vesicles arise from the medial-trans
loci of the Golgi stack. All vesicles move to the peripheral cytoplasm where they become part of the cortical
streaming network that consists of an extensive network of cortical actin bundles. Pectin-carrying vesicles are
directed to specific secretion sites including the cytokinetic apparatus that entails both a cell plate and an ingrowing peripheral furrow. Disruption of the actin cytoskeleton by Latrunculin B or cytochalasin E inhibits cell
plate formation but not furrowing and results in viable filamentous division products. EPS-vesicle secretion target
zones are highly transient and occur at surface sites that come into contact with a substrate. FM4-64 and FM1-43
labeling coupled with incubation in wortmannin reveals a novel endocytic network consisting of cortical vesicles
that transport to, and fuse with, a peripheral vacuolar network positioned between the Golgi and cortical
cytoplasm.
O5-11
Quantitative proteomics reveals that plant plasma membrane microdomains are involved in
molecular transport, stress responses and callose biosynthesis
Srivastava V., Malm E., Sundqvist G., Bulone V.
Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106
91 Stockholm, Sweden.
The plasma membrane (PM) is considered as one of the most interactive and dynamic membrane structures of the
cell. As being the interface between the cell and the extracellular environment, it is involved in many biological
processes such as metabolite and ion transport, endocytosis, defense against pathogens, cell differentiation and
proliferation, gaseous exchanges, etc. The PM contains microdomains enriched in sphingolipids and sterols,
which are resistant to certain concentrations of detergents. The aim of this work was to determine the main
functions of such microdomains in poplar through a comprehensive quantitative proteomic analysis using gelbased and solution (iTRAQ) approaches. Compared to PM, a total of 102 proteins related to cell wall
biosynthesis, stress responses and signaling processes, and molecular and ion transport were found to be
significantly enriched in DRM. The majority of the DRM-enriched proteins were predicted to contain up to 16
transmembrane domains and/or membrane-anchoring acylation sites. In addition, the number of transmembrane
domains and their length were higher in DRM proteins compared to the total PM proteins. An important
proportion of the most enriched proteins in DRM corresponded to (1 3)-β-glucan synthases and related proteins,
indicating that the isolated microdomains are the site for callose biosynthesis and regulation. The proteins
identified most likely reflect the biological specialization of the isolated microdomains in cell surface specific
responses that trigger callose formation, e.g. stress responses, as well as a potential role in plasmodesmata
formation and structure. The data will be presented and discussed in relation with these key biological processes
in plant cells.
O5-12
Candidate Substrate-Product Pairs (CSPP): structural characterization of plant metabolites and
its relevance for lignin pathway engineering
Morreel K., Vanholme R., Saeys Y., Dima O., Vanholme B., Boerjan W.
Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Bioinformatics, Ghent University,
Technologiepark 927, B-9052 Gent, Belgium.
Lignin is a major obstacle in the use of lignocellulosic biomass for chemical pulping or for conversion to second
generation biofuels and biomaterials. These industries would greatly benefit from processing lignocellulosic
feedstock with either less lignin or with a type of lignin that is easier to degrade 1. Evidently, altering lignin content
and composition in crop species requires fundamental knowledge about the lignin biosynthetic pathway.
Metabolomics is an important tool to study pathway architecture. Nowadays, thousands of metabolites are
profiled using gas and/or liquid chromatography-mass spectrometry and their abundances statistically analyzed.
Disappointedly, most of the differentially accumulating metabolites cannot be identified, a situation that is even
much more experienced in the plant kingdom where secondary metabolites fully outnumber those in primary
metabolism. Efforts in constructing mass spectral databases have been initiated, but chances that they finally
might cover all metabolites hardly exist. Complementary high-throughput approaches are therefore necessary.
Here, we describe a new algorithm employing biochemical information to enhance the structural characterization
of as many compounds as possible in liquid chromatography-mass spectrometry-based metabolomics.
[1] R. Vanholme et al. (2012) New. Phytol., 196(4), 978-1000.
Session 6 : Uses of plant CW and derived products
Food, feed, chemicals & fuel, Renewable biomedical & smart materials
O6-01
Dry separation of ground maize stems provides fractions with distinct enzymatic degradation
patterns
Guillon F. (a), Barron C. (b), Looten R. (a), Bonnin E. (a), Lapierre C. (c), Barrière Y. (d), Saulnier L. (a), Rouau X. (b),
Devaux M.-F. (a)
(a) INRA, UR 1268 BIA, 44316 Nantes, France ; (b) INRA, UMR 1208 IATE, F-34398 Montpellier, France (c) UMR 1318
INRA-AgroParisTech, IJPB, F-78026 Versailles, France, (d) INRA, UR GAPF FR-86600 Lusignan, France.
The enzymatic saccharification of lignocelluloses depends on their physical state and chemical composition,
which is related to tissue properties. The objective of the present work was to study the enzymatic degradation
pattern of particles isolated through a dry separation process from maize plant.
Whole plants without ears were ground and fractionated by turbo-separation. The fine- and medium-size particles
were submitted to electrostatic sorting. The IMATORE reactor [1] allowed us to monitor the compositional
changes and size evolution of the particles during saccharification. In all fractions, glucose and xylose were the
major sugars. However, minor sugars displayed distinct distributions, fine particles being richer in arabinose and
uronic acids. While the large particles were found to be lignin-richer, the fine ones exhibited the lowest lignin
amount and a higher ratio of syringyl over guaiacyl units. The enzymatic degradation patterns differed between
fractions. Not unexpectedly, large and fine particles were respectively the less and the more degraded, as revealed
by their compositional and size changes. Interestingly, positive-deviated medium size particles had a particular
behaviour with most of the sugar released during the first 30 min without any noticeable change in particle size.
The negative-deviated medium size particles behaved like the large ones.
The present work confirms that plant heterogeneity should be considered in the biorefinery chain. Next steps will
be (i) to find the relationships of particles degradation pattern to stem histology and (ii) to improve the dry
separation process in order to increase the saccharification yield.
[1] M.F. Devaux, et al. (2006) Journal of Food Engineering, 77, 1096-1107.
Acknowledgements: this work was supported by the INRA AIC Histochem and ApSaLi grants. The technical assistance of Frédéric Legée
and Laurent Cézard for lignin analyses is gratefully acknowledged.
O6-02
Increasing C6 cell wall sugar content by engineering the accumulation of a low recalcitrance
polysaccharide in plants
Vega-Sánchez M.E. (a), Loqué D. (a), Lao J. (a), Sharma V. (a), Catena M. (a), Heazlewood J.L. (a), Scheller H.V. (a),
Ronald P.C. (ab)
(a) Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, USA; (b) University of California at Davis, USA.
The production of biofuels from lignocellulose remains a challenging process due primarily to the inherent
resistance of plant cell walls to deconstruction (commonly referred to as recalcitrance). In addition, the presence
of significant amounts of 5 carbon (C5) sugars in plant biomass that are not readily fermented by microorganisms
is another obstacle to efficient conversion to fuels. For these reasons, reduced cell wall recalcitrance and increased
C6 monosaccharide content are desirable traits in potential biofuel crops, as long as these biomass modifications
do not significantly alter normal growth and development. Mixed-linkage glucan (MLG), a cell wall
polysaccharide comprised of glucose monomers linked by both β-1,3 and β-1,4 bonds, is a good example of a low
recalcitrance and C6-containing polymer. As opposed to cellulose, MLG is non-linear and cannot aggregate into
microfibrils due to the presence of the β-1,3 bonds, which makes it more soluble in aqueous environments. We
have previously shown that Nicotiana benthamiana plants transiently over-expressing the rice mixed-linkage
glucan synthase CslF6 using a 35S promoter accumulate large amounts of MLG (over 10% of dry weight).
However, previous data in barley and our own results in N. benthamiana have shown that constitutive production
of MLG in plants severely compromises growth and development. In order to bypass these negative effects, we
have successfully developed a strategy to engineer Arabidopsis plants to accumulate significant amounts of MLG
in the cell wall without growth defects. This approach offers a new engineering alternative to enhance cell wall
properties of lignocellulosic biofuel crops.
O6-03
Cell-wall compositional analysis of the energy crop Miscanthus as a means to optimise its
application as a feedstock for bioenergy and biorefining
Costa R., Winters A., Bosch M.
Institute of Biological Environmental and Rural Sciences, Aberystwyth University, United Kingdom.
Dedicated lignocellulosic energy crops from the genus Miscanthus constitute high potential candidate feedstocks
for biofuel production. However, there are gaps in our understanding concerning the relationship between
cell-wall structural components and the accessibility of its energy rich polysaccharides to exogenously applied
hydrolytic enzymes. Plant cell-wall recalcitrance to saccharification therefore remains a key bottleneck to the
cost-effective production of second-generation biofuels and industrial biomaterials. At IBERS, Aberystwyth
University, we have an extensive Miscanthus germplasm collection, of which 25 lines were chosen for an in-depth
analysis of lignocellulosic biomass following a multidimensional approach which considers: different
developmental stages, stem vs. leaf compositional variability and various genotypes selected to give a diverse
range in cell-wall composition. A combination of biochemical and cell biological approaches are being applied,
which includes: the analysis of cell-wall polysaccharides using an ELISA-based method for glycome profiling[1],
acetyl bromide lignin determination[2] and a bioassay using Clostridium phytofermentans for the determination of
biomass digestibility[3]. The results from these studies will be correlated with saccharification data, with the aim of
improving the cost-effectiveness of Miscanthus biorefining. Nonetheless, preliminary results have already
provided support to the pertinence of this multidimensional approach to acquiring a more detailed understanding
of cell-wall features in the context of lignocellulosic feedstocks. Ultimately this will lead to more effective
biorefining treatments and help guide the breeding and engineering of high-quality lignocellulosic energy crops.
[1] S. Pattathil et al. (2012) Methods in Molecular Biology (Clifton, N.J.), 908, 61-72; [2] R. Fukushima & R. Hatfield (2001)
J. Agric. Food Chem., 49, 3133-3139; [3] S. Lee et al. (2012) Biotechnol, Biofuels., 5:5.
O6-04
Altering plant lignins for improved processing
Ralph J. (ab), Lu F. (ab), Kim H. (ab), Tobimatsu Y. (b), Zhu Y. (a), Padmakshan D. (a), Karlen S. (a), Wilkerson C. (c),
Withers S. (c), Sedbrook J. (d), Cass C. (d), Grabber J.H. (e), Park J.Y. (f), Mansfield S. (f)
(a) Great Lakes Bioenergy Research Center, the Wisconsin Energy Institute, University of Wisconsin, Madison, Wisconsin
53726, USA; (b) Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA;(c) Great Lakes
Bioenergy Research Center, Michigan State University, East Lansing, Michigan, MI 48824, USA; (d) Department of
Biological Sciences, Illinois State University, Normal, IL 61790, USA; (e) U.S. Dairy Forage Research Center, USDA-ARS,
Madison, WI 53706, USA; (f) Department of Wood Science, University of British Columbia, Vancouver, BC V6T 1Z4,
Canada.
Lignin remains one of the most significant barriers to the efficient utilization of lignocellulosic substrates. Up- and
down-regulating genes for enzymes in the monolignol biosynthetic pathway can produce at times striking
alterations in lignin composition and structure that may positively or negatively impact a given processes. A few
approaches hold considerable promise for reducing processing severity and energy demands. We are gaining
insight into what features are required by ideal lignin monomers and are beginning explorations into possible
lignin monomer replacements. And now that monomer substitution in the lignification process is well
authenticated in various transgenic plants, we are beginning to actually design lignins to improve the ease with
which they can be removed from the cell wall. Here we highlight recent progress in the use of promising
monolignol conjugates for in planta lignification to produce plant materials that are better optimized for
processing, a strategy highlighted in recent papers and reviews [1-4].
[1] J.H. Grabber et al. (2008) Biomacromolecules, 9, 2510-2516; [2] J. Ralph. (2010) Phytochemistry Reviews, 9, 65-83; [3]
R. Vanholme et al. (2012) New Phytologist, 196, 978-1000 ;[4] Y. Tobimatsu et al. (2012) ChemSusChem, 2012, 5, 676-686.
O6-05
Modifications of cell wall properties by production of recombinant resilin composites in transgenic
plants
Preis I., Lapidot S., Abramson M., Shoseyov O.
Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Israel, Rehovot, Israel.
Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions. The elastic properties
of resilin are achieved by formation of di-tyrosine bridges within resilin monomers, generated by peroxidase
enzymes, in the insect cuticles[1]. This highly resilience and elastic protein was optimized for expression in
tobacco and was directed to the cell wall by a signal peptide. Cross-linking of resilin to the cell wall was enabled
by utilizing the presence of natural plant cell wall peroxidases and formation of di-tyrosine bridges between
resilin monomers and cell wall phenol gropes. Transgenic stems had shown a reduction in young`s modulus and
an increase in the ability to strain before deformation. Moreover, changes in the formation of the plant cell wall,
due to the cross-linking of resilin, improved sugars release from plant material. Tobacco is a model plant in this
research. Transformation of resilin to Eucalyptus tress is currently in progress. The future implications of this
discovery range from second generation biofuel, to elastic fibers from transgenic forest trees for elastic paper,
elastic wood products, wind resilient trees and replacement of syntactic materials with biodegradable ones.
[1] Qin, G., Lapidot, S., Numata, K., Hu, X., Meirovitch, S., Dekel, M., Podoler, I., Shoseyov, O., and Kaplan, D.L. (2009).
Expression, Cross-Linking, and Characterization of Recombinant Chitin Binding Resilin. Biomacromolecules 10, 3227-3234.
O6-06
Effect of cell wall diferuloylation on agronomic fitness and silage quality in maize
Barros-Rios J. (ac), Santiago R. (a), Jung H.-J.G. (b), Malvar R.A. (a)
(a) CSIC-Misión Biológica de Galicia, Department of Maize Breeding and Genetics. Apartado 28, C.P.: 36080, Pontevedra,
Spain; (b) USDA-Agricultural Research Service, Plant Science Research Unit, 411 Borlaug Hall, 1991 Upper Buford Circle,
St Paul, MN 55108, USA; (c) Present address: Department of Agronomy and Plant Genetics, University of Minnesota, 411
Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, USA.
Covalent cross-linking of grass cell wall components through diferulates (DFAs) has a marked impact on cell wall
properties. However, results on genetic selection for DFA concentration have not been reported for any grass
species. We report here the results of direct selection for ester-linked DFA concentration in maize stalk pith
tissues and the associated changes in corn borer resistance, agronomic performance, and cell wall polysaccharide
biodegradability. After three cycles of divergent selection, maize populations selected for higher total DFA
(DFAT) content (CHs) had 31% higher DFAT concentrations than populations selected for lower DFAT content
(CLs), suggesting that DFA deposition in maize pith tissues is a highly heritable trait that can be modified trough
conventional breeding. DFAT concentration was negatively correlated with stem damage by corn borers, and stem
tunneling was 29% greater in C3L than in C3H (31.7 cm vs. 22.6 cm). Results on other agronomic traits indicate
that CHs were slightly earlier in flowering, and showed higher plant height, and grain yield than CLs. Regarding
the cell wall structure, CHs had lower total cell wall concentration than CLs, suggesting that increased crosslinking of feruloylated arabinoxylans results in repacking of the matrix and in firmer and thinner cell walls. As
expected, a more DFA ester cross-coupled arabinoxylan network had a negative impact on in vitro ruminal cell
wall polysaccharide degradability. Interestingly, 8–8-coupled DFAs, previously associated with cell wall strength,
were the best predictors of cell wall biodegradability. These results provide the first direct experimental evidence
of the important role of polysaccharide-polysaccharide cross-linking through ester-linked DFAs in crop protection
and animal feed technology. We highlight that grass stem cell wall fortification through diferulates could improve
agronomic fitness in varieties intended for grain production but may limit accessibility to fermenting microbes
when use as whole plant silage to be fed to cattle or as feedstock in biofuels production.
O6-07
Wood and cereal hemicelluloses as future bio-based oxygen barrier materials
Mikkonen K.S., Heikkinen S.L., Tenkanen M.
Department of Food and Environmental Sciences, University of Helsinki, Finland.
Packages need to assure the safety and quality of foods. Proper packaging contributes to retarding undesirable
changes in foods by preventing the migration of gases and moisture in addition to providing support against
mechanical forces. Recyclability or compostability are other important requirements for future packaging
materials. Oxygen gas plays a crucial role in many reactions that affect the shelf life of foods. Presently,
aluminium foil and synthetic polymers are mainly applied as oxygen barriers in food packaging. Xylans and
mannans, the two main groups of hemicellulose, are abundant plant cell wall heteropolysaccharides that could be
recovered from various side streams of agriculture and forestry industry. They have property to form films, which
is foreseen as one highly interesting application for these potential future biorefinery products [1]. Xylan- and
mannan-based films and coatings have shown low oxygen permeability and, in some cases, relatively low water
vapour permeability and high tensile strength. We have studied extensively the role of chemical structure of
xylans and mannans on the mechanical and barrier properties of films. The use of plasticizers, cross-linking
agents, and addition of wood-derived nanocellulosic reinforcements for improved film properties will also be
highlighted.
[1] Mikkonen and Tenkanen (2012) Trends Food Sci. 28, 90-102.
O6-08
New ultra-thin nanostructured films based of lignin and cellulose with variable optical properties
Hambardzumyan A. (abc), Foulon L. (ab), Chabbert B. (ab), Aguié-Béghin V. (ab)
(a) INRA, UMR614 Fractionnement des AgroRessources et Environnement, Reims, France ; (b) University of Reims
Champagne Ardenne, UMR614 Fractionnement des AgroRessources et Environnement, Reims, France c University of
Erevan, Laboratory of Chemistry and Physics, Erevan, Armenia.
Within the framework of research on the valorisation of by-products from 2 nd generation biorefineries, we have
designed novel nanocomposite materials made of lignin and cellulose nanocrystals. These materials were prepared
without chemical modification or functionalization of the biopolymers either by spin-coating to carry out
successive deposits of each polymer solutions to build a multilayer assembly [1] or by evaporation starting from a
mixture of these polymers [2]. Both nanocomposites were in the form of thin films with a thickness in the range of
several nm to several μm, and transparent on glass or quartz slides. These films exhibit variable optical properties
according to the process used. The modulation of the interactions between the lignin and the surface of the
cellulose nanocrystal makes it possible to obtain either anti-reflective films or anti-UV films with low wettability
and good resistance to water. The combination of the optical properties of films made from cellulose nanocrystals
and the spectroscopic properties of lignins are of particular interest for surface coverings (optical glass, filters,
wood protection, etc.). Antibacterial and mechanical properties can be added to these properties.
[1] A. Hambardzumyan et al. (2011) CR Biol., 334, 839-850 ; [2] A. Hambardzumyan et al. (2012) Biomacromolecules, 13,
4081-4088.
O6-09
Development of flexible lignin nanotubes for the smart delivery of therapeutic agents
Vermerris W. (ab), Ten E. (ab) , Ling C. (bc), Srinastava A.(bcd), Dempere A. (ef)
(a) Department of Microbiology and Cell Science, USA;(b) Genetics Institute, USA;(c) Powell Gene Therapy Center,USA;
(d) Department of Molecular Genetics and Microbiology, USA; (e) Department of Materials Science and Engineering,USA;
(f) Major Analytical Instrumentation Center, University of Florida, Gainesville FL 32610, USA.
Development of high-value co-products has the potential to increase the economic sustainability of biorefineries
that convert lignocellulosic biomass to fuels and bulk chemicals. We synthesized flexible lignin nanotubes in a
sacrificial membrane template using lignin from the waste stream of a biorefinery. A base layer of lignin is
crosslinked to the inner walls of an activated alumina membrane and layers of dehydrogenation polymer are
added to the base layer via a peroxidase-catalyzed reaction. The lignin nanotubes are released by dissolving the
membrane in phosphoric acid. By using different phenolic monomers, we were able to synthesize nanotubes with
a wall thickness of 15-45 nm, or nanowires with a diameter of 200 nm [1]. Optical properties could also be varied.
Electron microscopy and nano-indentation studies revealed that morphology and mechanical properties of the
nanotubes depend on the biomass species (hardwood, softwood, grass) and lignin isolation procedure (NaOH,
sulfuric acid, thioglycolic acid). Based on cytotoxicity experiments with human cell cultures (HeLa cells), lignin
nanotubes have lower cytotoxicity than multiwalled carbon nanotubes with the fullerene structure. Furthermore,
lignin nanotubes enable the uptake of DNA, as shown by the expression of green fluorescent protein (GFP)
following transfection of HeLa cells with plasmid DNA. The ability to select physico-chemical properties and the
ease with which lignin nanotubes can be functionalized make them very attractive candidates for the delivery of
DNA in gene therapy, as well as for the smart delivery of pharmacological agents.
[1] Caicedo et al. (2012). Nanotechnol, 23, 10560.
Funding provided by USDA-BRDI project 2011-10006-303508.
Abstracts from poster presentations listed by session and order of posters
Session 1 : Plant Cell Wall Architecture; Structure, Interactions & Cross-Links of CW
Components
Session 2 : Dynamics of plant CW Components: from Biosynthesis to Remodelling : Intracellular
synthesis & trafficking, Synthesis & assembly at the plasma membrane, Remodelling in muro,
Transcriptional and post-transcriptional control
Session 3 : Evolution & Diversity of plant CW
Session 4 : Functions of Plant CW in planta : Growth, morphogenesis & development, Signaling &
defense, Response to environment
Session 5 : Advanced Understanding of CW Structure, Biosynthesis & Function : Bioinformatic &
omic approaches, Computational & biophysical approaches
Session 6 : Uses of plant CW and derived products : Food, feed, chemicals & fuel, Renewable
biomedical & smart materials
Session 1 : Plant Cell Wall Architecture; Structure, Interactions & Cross-Links of
CW Components
P1-01
Isolation and Structural Characterisation of Rhamnogalacturonan in Ginseng Roots
Sun L. (ab), Ralet M.-C. (b), Zhou Y. (a)
(a) School of Life Sciences, Northeast Normal University, Changchun 130024, PR China; (b) INRA, UR1268 Biopolymères
Interactions Assemblages, 44300 Nantes, France.
Panax ginseng C. A. Meyer (ginseng) has been used as a traditional medicine in Asia for thousand years. As one
of the active components in ginseng, polysaccharides have many pharmaceutical activities. Ginseng
polysaccharides are composed of neutral glucans and pectin. In this study, ginseng pectin was first de-esterified
by alkali, and then hydrolyzed by endo-polygalacturonase. The hydrolysate was fractionated by a combination of
anion-exchange and size exclusion chromatography, and four pectic domains (1A, 1B, 2A and 2B) were obtained.
IR and NMR spectra indicated that there were nearly no methyl-ester and acetyl groups in these fractions. They
were all composed of GalA, Rha, Ara and Gal as the main components. Fractions 1A and 2A were homogeneous
and their molecular weights were 8.3×104 and 1.2×105 Da, respectively. The ratios of Rha/GalA were 0.96 and
0.65, for 1A and 2A, respectively, typical of RG-I domains. Both fractions could be hydrolyzed by
rhamnogalacturonan hydrolase, which gives further evidence that they are RG-I type pectin. With its Rha/GalA
ratio close to 1, fraction 1A is the most purified RG-I domain from ginseng up to now. Fractions 1B and 2B
exhibited molecular weights of 6.4×103 and 6.5×103 Da, respectively. Based on a two-step mild acid hydrolysis
coupled to the analysis of the resulting fragments by mass spectrometry, they were indentified to be RG-II type
pectin. Fragments corresponding to RG-II backbone and side chains A, B and E were distinguished. This is the
first time RG-II structures are identified in ginseng roots.
[1] X. Zhang et al. (2009) Carbohydr. Polym., 77, 544–552; [2] L. Yu et al. (2010) Carbohydr. Polym., 79, 811–817; [3] M.
Séveno et al. (2009) Planta, 230, 947–957.
P1-02
Revisiting the cell wall of the wheat endosperm
Chateigner-Boutin A.-L., Bouchet B., Larre C., Guillon F.
INRA, UR1268 Biopolymères, Interactions, Assemblages, F-44316 Nantes, France.
The wheat grain is an important source of food, animal feed and industrial raw material. The grain starchy
endosperm is a storage tissue that corresponds approximately to the wheat flour. Cell walls only account for about
3% of the endosperm weight but they are prominent for wheat end-use quality (milling, bread-making) and as
dietary fibre, they have a major impact on nutritional quality. The cell wall polysaccharides in the wheat
endosperm consist in 70 % feruloylated arabinoxylans (AX) and 20% mixed-linked beta-glucans (MLG) which
are deposited in the walls during the grain development. Early works have shown that in addition to these major
polysaccharides, cellulose and mannans are also found in minor amounts in the dry grain endosperm. More
recently callose and xyloglucans were detected transiently in the developing endosperm. No pectins were ever
detected although pectins accumulate in the endosperm of the closely related Brachypodium distachyon.
By proteomic analysis of Golgi-enriched fractions obtained from the endosperm of developing wheat we
identified glycosyltransferases (GT) belonging to families implicated in the synthesis of xylans, MLG, mannans,
xyloglucans and pectins. We therefore decided to further analyse the cell wall composition in the endosperm.
Positive signals were obtained for mannans, xyloglucans and several pectin domains (homogalacturonans,
rhamnogalacturonans, galactans and arabinans) using specific antibodies and after removing the major
polysaccharides. Interestingly, some of the minor polysaccharides accumulate evenly in the whole endosperm
while others specifically in specialized cells. The transfer cells in the crease region appeared strongly labelled for
galactans, arabinans and xyloglucans. The endosperm cell wall is therefore more complex than once thought. We
are generating transgenic wheat to silence several genes potentially involved in the synthesis of these polymers to
evaluate their role in the developing grain.
P1-03
New light on the structure and function of the wheat arabinogalactan peptide
Wilkinson M. (a), Webster G. (a), Ward J. (a), Tosi P. (a), Knox J.P. (b), Lovegrove A. (a), Shewry P.R. (a)
(a) Plant Science & Crop Biology Department, Rothamsted Research, Harpenden, AL5 2JQ, UK; (b) Centre for Plant
Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
The Grain Softness Protein (GSP) and the arabinogalactan peptide (AGP) of wheat are encoded as a single
precursor protein by the gsp-1 gene, with AGP corresponding to the first 15 amino acid residues. Gsp-1 is located
at the Ha (Hardness) locus onchromosome 5D, that also encodes puroindolines a and b which are known to
account for approximately 70% of the variation in hardness among bread wheat cultivars. However, although GSP
was initially defined as contributing variation in grain texture, this has not been substantiated. AGP is estimated
to be present at a similar level in the wheat endosperm as water-extractable arabinoxylan(~0.3% dry weight) but
its location and function are unknown. AGP/GSP-RNAiwheat lines were produced in order to determine the role
and location of GSP and AGP in the wheat endosperm. Semi-quantitative RT-PCR showed a decreased
abundance of gsp-1 transcripts in the transgenic linescompared to the null controls while deceases in GSP and
AGP were shown by ELISA and Western blot analysis, respectively. Decreased abundance of AGP was also
demonstrated by NMR analysis of non-starch polysaccharide preparations. The availability of a new monoclonal
antibody specific for wheat AGP has also allowed its location within the endosperm cells to be determined. The
results presented here provide interesting new insights into the roles and locations of AGP and GSP in wheat.
P1-04
Epitope detection chromatography: a novel methodology to dissect the complexity of plant cell
walls
Cornuault V. (a), Manfield I. (b), Lee K.J.D. (c), Knox J.P. (a)
(a) Centre for Plant Sciences, (b) Astbury Centre for Structural Molecular Biology, (c) School of Molecular and Cellular
Biology, Faculty of Biological Sciences; University of Leeds, Leeds LS2 9JT, UK.
The structure and interactions of plant cell wall polysaccharides is poorly understood, especially with regards to
cell dynamics and biomechanical adaptations. Studying the composition of cell walls from different plant species,
cell types or at different stages of development is important to elucidate the biological functions of
polysaccharides. Understanding of the diversity of cell wall architectures has been facilitated by the use of in situ
analyses with large collections of monoclonal antibodies. Recent observations have indicated the widespread
occurrence of polysaccharide masking in which an abundant polysaccharide can block access to other
polysaccharides in cell walls suggesting associations or interactions between specific polysaccharides. Here we
present a new methodology, called Epitope Detection Chromatography (EDC), in which the existing panels of
monoclonal antibodies are used as detection tools for the analysis of cell wall polymers separated by
chromatography. This highly sensitive method allows analysis on a micro-scale (single organ, tissue or cell type)
and can reveal information on polysaccharide heterogeneity in addition to interconnections between epitopes and
polysaccharides. The method will also be useful in assessing changes to polysaccharide profiles in response to
mutations or environmental impacts. Progress in developing the EDC methodology and results from the analysis
of Arabidopsis cell walls will be presented.
P1-05
In situ analysis of cell wall polysaccharides in stems of Miscanthus species
Xue J. (a), Bosch M. (b), Knox J.P. (a)
(a) Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT, United Kingdom; (b) Institute of
Biological, Environmental & Rural Sciences (IBERS), Aberystwyth University, Aberystwyth, SY23 3EB, UK.
Plant cell walls are important cellular components underpinning growth and development and, in addition, can be
important sources of biomass and renewable energy. Miscanthus species are fast growing plants with high
biomass yield. Miscanthus x giganteus is the sterile hybrid between Miscanthussinensis and Miscanthus
sacchariflorus, with a faster and taller growth than its parents. A range of monoclonal antibodies have been used
in immunofluorescence in situ analyses to study the occurrence and interactions of non-cellulosic polysaccharides
in Miscanthus species. In this study, it was observed that heteroxylans and mixed-linkage-glucans (MLGs) were
abundant in the cell walls of stems of Miscanthus species and that their distributions varied in relation to the
vascular cells and interfascicular parenchyma. There were also clear differences in polysaccharide patterning
between the three species. Detection of pectic-homogalacturonan (HG) epitopes was restricted to the intercellular
spaces of parenchyma regions and, notably, the high methyl ester LM20 HG epitope was specifically abundant in
the pith parenchyma cell walls of M. x giganteus in comparison to its parents. It has been reported that some cell
wall polymer probes cannot access polymers, because they may be masked by other polymers. It was found in this
study that xyloglucan and pectic-galactan were masked by xylan and/or MLG in certain cell wall regions. The
heterogeneity of Miscanthus cell wall structures and molecular architectures found in this study are an important
basis for understanding Miscanthus cell wall properties and will also inform potential strategies for the efficient
deconstruction of Miscanthus biomass.
P1-06
Characterization of monoclonal antibodies against xylan epitopes
Avci U., Pattathil S., Clay R., Mazumder K., McCormick H., York W.S., Hahn M.G.
Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, GA, 30602 USA
A detailed understanding of plant cell wall function requires, among other aspects, knowledge of how cell wall
components are organized within the matrix. It is also vital to observe how wall composition, and the structures of
wall components and their interrelationships change with development and in response to different environmental
factors. Cell wall directed monoclonal antibodies of known specificity are powerful tools for addressing these
questions. We have recently generated a large and diverse set of 35 monoclonal antibodies directed against xylan
epitopes that are classified into several clades based on their binding patterns to a series of xylan preparations
from different plants. In this study, we define the specificities of 11 antibodies that bind to purified, unsubstituted
xylo-oligosaccharides of different lengths. Along with these xylan backbone-directed antibodies, we also show the
labeling patterns of the other xylan-directed antibodies to cell walls of diverse plants, including representative
monocots and dicots, and well-characterized xylan mutants. We further show base treatment of sections can alter
the labeling patterns of some, but not all antibodies, suggesting that base-labile substituents (e.g., acetylation,
methyl esterification) on xylans can affect observed xylan localization patterns using these antibodies. These
antibodies provide new well-characterized tools for studies of xylan deposition in diverse plants.
[Supported by a grant from the U.S. National Science Foundation Plant Genome Program (IOS-0923992)]
P1-07
Towards a New Screen for Detecting Changes in Plant Cell Wall Composition and Structure
Using Time-of-Flight Secondary Ion Mass Spectrometry
Tsai A.Y.-L. (a), Goacher R.E. (bc), Master E.R. (ac)
(a) Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada; (b) Department of Biochemistry,
Chemistry and Phyiscs, Niagara University, Niagara, NY, USA; (c) Department of Chemical Engineering and Applied
Chemistry, University of Toronto, Toronto, ON, Canada.
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface analysis technique recently adapted for
studies of woody biomass. Its inherent low material requirement, non-destructive analyses, minimal sample
modification, holistic data acquisition in addition to the high spectral and spatial resolution make it an appealing
alternative of lignocellulosic characterisation beyond the conventional spectroscopic and chromatographic
methods. ToF-SIMS spectra have been previously annotated for lignin and polysaccharides [1]; furthermore, the
monolignols have well defined signature peaks assigned [2]. Herein, we are proposing a ToF-SIMS based
Arabidopsis cell wall chemotyping platform that distinguishes plant variants based on cell wall compositions. The
interfascicular fibre region of wild type Arabidopsis thaliana and cell wall mutants irx3 and fah1 were used to
validate the applicability of wood-derived ToF-SIMS spectra annotations to describe the composition of
Arabidopsis cell walls. Principle component analyses readily distinguished ToF-SIMS spectra of irx3 and fah1
mutants from wild type Arabidopsis based on guaiacyl/syringyl lignin and polysaccharide content, respectively.
Further analysis of the cellulose deficient irx3 mutant also allowed for improved annotation of cellulosic peaks in
ToF-SIMS spectra. The potential to further improve ToF-SIMS spectra annotations for pectin and hemicellulose
are currently being assessed through the analysis of Arabidopsis hypocotyls and cell wall fractionation residues.
[1] R.E. Roacher et al. (2011) Anal. Chem., 83, 804-812; [2] K. Saito et al. (2005) Biomacromolecules, 6, 678-683.
P1-08
Effect of the mutation laurina on the monosaccharide composition of cell walls in Coffea Arabica
seeds from the fertilization to the harvest
Adler S. (a), Citerne S. (b), Tony P. (a), Fock-Bastide I. (a), Mouille G. (b), Noirot M. (c)
(a) Université de la Réunion, 97415 St Denis, Réunion, France ; (b) Institut Jean-Pierre Bourgin, UMR1318 INRAAgroParisTech Bâtiment 1 INRA Centre de Versailles-Grignon Route de St Cyr (RD 10), 78026 Versailles Cedex France ;
(c) IRD, UMR PVBMT, Pôle de Protection des Plantes, 97410, St Pierre, Réunion, France.
Coffea arabica ‘Laurina’ is a natural mutant of Coffea arabica ‘Bourbon’ and is commercially known as
‘Bourbon pointu’. The laurina mutation leads to pleiotropic morphologic effects as seed shape, tree habit,
internode length, leaf sizes and shoot apical meristem size. The mutation also increases dryness and cold
resistance. Especially, caffeine content in seeds is about 0.55% dry matter vs 1.1 % dmb in ‘Bourbon’). The aim
of this study was to highlight the role of the mutation on the evolution of monosaccharide composition of cell wall
in seeds over time.
Experiments were carried out on seeds of Bourbon and Bourbon pointu harvested every two weeks from the 8 th to
the 22th week after fertilization, the period corresponding to the fruit growth (8 th-18th weeks) and the beginning of
the fruit maturation (beyond the 18 th week). We will present the cell wall modifications occurring during these
various phases of the grain development.
P1-09
FTIR monitoring of cell wall compositional changes in maize stems at different developmental
stages
Chazal R. (a), Durand S. (a), Robert P. (a), Devaux M.-F. (a), Saulnier L. (a), Lapierre C. (b), Vigouroux J. (a), Cézard L. (b),
Leger F. (b), Guillon F. (a)
(a) INRA, UR1268 Biopolymères, Interactions, Assemblages, F-44316 Nantes, France; (b) AgroParisTech, Institut JeanPierre Bourgin, RD10, F-78000 Versailles, France.
Grass cell walls are major resources for the cellulose-to-ethanol conversion process. However, their enzymatic
saccharification is detrimentally affected by lignins and their cross-linking to hemicelluloses. Screening plant cell
walls more adapted to the process and monitoring their compositional changes during pretreatment and
saccharification steps call for a high-throughput technique of lignins and polysaccharide analysis. Such a
performance could be fulfilled by Fourier Transform Infrared (FT-IR) spectroscopy [1, 2]. In this work, FT-IR
spectroscopy was assessed as a tool to monitor the compositional changes occurring in the cell walls of the earbearing maize internode at three developmental stages. To this end, we first built a FT-IR spectral data base of
grass lignins and cell wall polysaccharides. While the FT-IR fingerprint of polysaccharides was in the 1200-800
cm-1 range, the 1800-1450 cm-1 and the 920-790 cm-1 ranges were selected for lignins and hydroxycinnamic acids.
This data base was then used to monitor the changes in cell wall composition of ground maize internodes at early
(9-leaf stage), medium (female flowering) and late (silage) developmental stages. At early stage, the lignin and pcoumaric acid FT-IR signals could not be detected. At flowering and silage stages, aromatic signals could be
observed, including the absorbance band at 833 cm -1 assignable to syringyl units and p-coumaric acid. Finally,
FT-IR micro-spectroscopy was applied to map the cell wall polymers in maize internode cross section (flowering
stage). In phloem, no lignin signals could be detected while these signals were prominent in the sclerenchyma.
The parenchyma cells displayed distinct fingerprints, with lignin signals in the cells far from the bundles and no
detectable lignin signals in the cells close to the bundles. In the 1200-800 cm -1 region, the cellulose signals were
found to be dominating in the case of sclerenchyma whereas xylan signals were prominent in parenchyma cells.
[1] Faix and Böttcher (1993) Holzforschung, 47, 45-49; [2] Kacurakova and Wilson (2001)Carbohydr. Polym., 44, 291-303.
P1-10
Lignin structural analysis by Raman spectroscopy: from purified molecules to plant cell walls
Barron C. (a), Lapierre C. (b), Guillon F. (c), Robert P. (c)
(a) INRA, UMR IATE 1208, F-34000 Montpellier, France ; (b) UMR 1318 INRA-AgroParisTech, Institut Jean-Pierre
Bourgin, RD10, F-78000 Versailles, France ; (c) INRA, UR1268 Biopolymères, Interactions, Assemblages, F-44316 Nantes,
France.
Enzymatic saccharification of lignocellulosic biomass is a bottleneck for its use in the green chemistry.
Differences in degradability were observed according to plant species but also according to cellular type within a
plant. Such variability was related to heterogeneity in composition. Lignin amount or lignin structure as well as its
interaction with polysaccharides were pointed out. Considering anatomical plant heterogeneity, lignin structure
must be analysed at different scale (from cell wall to tissue or organ). Spectral methods, in particular Raman
spectroscopy, are of particular interest due to (i) the high Raman scattering of phenolic compounds and (ii) the
ability to get spatially resolved information at micrometer scale. The objective of this study was first to create a
sample data set allowing spectral assignment of lignin structural features and secondly to evaluate the ability of
Raman spectroscopy to characterize lignin at tissue scale in planta.
Plant cell wall materials were prepared from different sources in order to cover a wide compositional range and
dioxane lignins were isolated from these cell walls. All these samples were analyzed by wet chemistry (sugar
analysis, lignin amount and composition, hydroxycinnamic acid content) and by Raman spectroscopy. Syringyl to
guaiacyl ratio could be evaluated in the 1270-1330 cm-1 spectral range. In cell wall material, while the lignin
amount was lower than 24%, spectra were dominated by lignin scattering in the 1200-1800 cm-1 spectral range.
Polysaccharides fingerprint was observed in the 800-1100 cm-1 region. Syringyl to guaiacyl ratio was still
detectable. Cell wall compositional changes of ear-bearing maize internodes were then analyzed at three
developmental stages. Lignification occurred between the 9-leaf stage and female flowering stage in parenchyma
cell walls. Difference in cell wall composition in different tissues (phloem, sclerenchyma) was also pointed out.
Acknowledgements: this work was supported by the INRA AIC Histochem grant.
P1-11
Exploring architectural cell wall heterogeneity: celery collenchyma as case study
Leroux O. (a,b), Sarkar P. (c), Allison G.G. (d), Auer M. (c), Knox J.P. (d), Popper Z.A. (a)
(a) Botany and Plant Science and the Ryan Institute for Environmental, Marine and Energy Research, School of Natural
Sciences, National University of Ireland, Galway, University Road, Galway, Ireland; (b) Pteridology, Department of
Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium; (c) Joint BioEnergy Institute, Physical
Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA; (d) Centre for Plant
Sciences, University of Leeds, Leeds LS2 9JT, UK.
The colonisation of land by plants ~470 MYA initiated a period of extensive morphological diversification in
concert with an equally impressive diversification in anatomy. Immobility has forced plants to acquire the ability
to adapt to environmental challenges, which in turn has impacted plant diversity. These evolutionary events and
mechanisms have contributed to the generation of complex body plans that require meticulously controlled
differentiation of tissues and cells throughout growth and development, and this specialisation is reflected in the
molecular design and dynamic nature of their cell walls. Architectural heterogeneity within a single cell wall may
involve compositional and/or structural differences and is controlled spatio-temporally in such a way that small
changes in wall architecture may have profound effects on the functional performance of the cell wall; thus
underpinning organ and tissue growth, differentiation and development. While analysis of fractionated cell walls
yields detailed structural information for individual polymers it results in destruction of the supramolecular
organisation essential for understanding cell wall complexity, heterogeneity and dynamics. However, a range of
methods for detailed in situ examination are available. Distinct lamellation of celery collenchyma cell walls has
been observed using transmission electron microscopy and we further investigated this heterogeneous feature
using immunocytochemistry, electron tomography, as well as Raman spectroscopy and chemometrics. Is
collenchyma cell wall architectural heterogeneity caused by structural and/or compositional variation?
P1-12
Cell wall composition changes with maturation of bamboo Phyllostachys bambusoides
Okahisa-Kobayashi Y. (ab), Mouille G. (a), Lapierre C. (a)
(a) UMR 1318 INRA-AgroParisTech, Institut Jean-Pierre Bourgin, F-78026 Versailles, France; (b) 8 Ichibancho, Chiyodaku, Tokyo 102-8472, Japan, Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellow for Research Abroad.
Bamboo grows widely in zones with a wet season ranging from the tropics to temperate areas. It grows faster than
any woody plant, and its uses have, since ancient times, ranged from basic tools to furniture and building
materials. Recently, some studies implied that bamboo has a potential as a bioenergy or fiber crop. However, there
is little information about bamboo cell walls especially on their growing stage. We investigated the structural
changes of cell wall polysaccharides and lignins of bamboo immature and mature culms.
Immature bamboo (0 year-old, 40 to 70cm-high) and mature bamboo (3 year-old, 10 m-high) internodes were
used. The determination of neutral sugars showed that only the amount of xylose increased with maturation. The
sugar linkage determination revealed that the ratio of 1,4-xyl to 1,4-glu increased with maturation and that t-Ara,
1,3,4-Xyl, t-Xyl, t-Gal, 1,5-Ara, 1,3-Glu were severely reduced as the plant ages. These results support the
occurrence of highly-substituted xylans in immature bamboo and of low-branched xylans in mature bamboo. Not
unexpectedly, the levels of lignins and of p-coumaric acid ester-linked to the cell walls increased with cell wall
maturation. Bamboo lignins were found to be composed of similar amounts of guaiacyl and syringyl units
together with trace amount of p-hydroxyphenyl units. As compared to other grass stems, ferulic esters were found
to occur in relatively low amount (in the 1-2 mg/g of extractive-free cell wall), which suggests lower ferulatemediated cross-linking possibilities between arabinoxylans and lignins. While arabinose units were essentially
acylated by ferulic acid in mature internode, acylation with p-coumaric acid occurred to a noticeable extent in
immature samples. These results further support the large structural changes of arabinoxylans as the plant ages.
Acknowledgement: the financial support of the Japan Society for the Promotion of Science is gratefully acknowledged.
P1-13
The Arabidopsis primary cell wall contains an unusual xylan, which is synthesised by a small set
of glycosyltransferases
Mortimer J.C. (a), Tryfona T. (a), Blanc N.F. (a), Ng A. (ab), Stott K. (a), Zhang Z. (a), Yu X. (a), Dupree P. (a)
(a) Biochemistry Dept, University of Cambridge, CB2 1QW, UK; (b) Current address: Chemistry Dept, Columbia University,
NY 10027, USA.
Some aspects of xylan synthesis appear to be conserved between dicots and grasses. In previous work, candidate
grass cell wall xylan biosynthetic genes were identified by their over-representation in grass EST collections
compared to dicots1, and some have been confirmed experimentally 2. Analysis of Arabidopsis microarray and
proteomic data revealed that possible orthologues of many candidate grass glucuronoarabinoxylan biosynthetic
enzymes were associated with tissues rich in primary cell wall (PCW).
Analysis by PACE, mass spectrometry and NMR of these tissues synthesising PCW has revealed a novel xylan
polysaccharide, with a structure different to the glucuronoxylan 3 found in dicot secondary cell walls (SCW).
Arabidopsis mutant plants lacking the candidate biosynthetic genes were isolated. Data describing the new xylan
structure will be presented, along with characterisation of the mutant plants.
[1] R.A. Mitchell et al. (2007) Plant Physiol. 34, 43-53; [2] N. Anders et al. (2012) Proc Natl Acad Sci USA 109, 989-93; [3]
J.C. Mortimer et al. (2010) Proc Natl Acad Sci USA 107, 17409-14.
P1-14
Compositional and structural characterization of cslf6 rice mutant
Smith-Moritz A.M. (a), Verhertbruggen Y. (a), Gonzalez Fernandez-Nino S. (a), Sharma V. (a), Scheller H.V. (a), Hao Z. (b),
Holman H.-Y. (b), Ronald P.C. (ac), Heazlewood J. (a), Vega-Sanchez M. (a)
(a) Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; (b) Berkeley Infrared
Strutural Biology Program, Lawrence Berkeley National Laboratory, Berkeley, CA 94720,USA; (c) University of California,
Davis, CA 94616, USA.
The CELLULOSE SYNTHASE-LIKE F6 (cslf6) gene has been recently shown to mediate the biosynthesis of
mixed-linkage glucan (MLG), a cell wall polysaccharide that is thought to be necessary for cell wall expansion in
the primary cell wall of young seedlings. A detailed analysis of a loss-of-function cslf6 rice mutant has been
recently conducted revealing surprising results. Though the mutant showed over 99% reduction of MLG content
in most tissues, the rice clsf6 knock out mutant showed only a slight decrease in growth compared to wild type,
demonstrating the flexibility of plant polysaccharide organization to compensate for changes within the cell wall.
The cell wall properties of both mutant and wild type were determined via biochemical and various spectroscopic
(Fourier Transform Mid-Infrared spectroscopy) analyses. We found that not only was the composition of the cell
wall dramatically altered, but the overall structure of major components of the cell wall were also affected.
Utilizing a synchrotron light source with FTIR spectroscopy, we were also able to characterization and compare
the cell wall of parenchyma cells of cslf6 mutants versus wild type coleoptiles.
P1-15
Understanding the role of O-acetylated matrix polysaccharides in woody plants
Prashant Mohan-Anupama P. (a), Derba-Maceluch M. (a), Chong S.-L. (b), Tenkanen M. (b), Madhavi Latha G. (c),
Jönsson L. (c), Mellerowicz E. (a)
(a) Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Sweden; (b)
Department of Food and Environmental Sciences, University of Helsinki, Finland; (c) Department of Chemistry, Umeå
University, Sweden.
The backbone or branches of hemicelluloses and pectins are often O-acetylated at specific positions to different
degree in different polysaccharides. Enzymatic and chemical deacetylation of lignocellulose increases sugar yields
in saccharification and improves fermentation, but on the other hand, artificial acetylation of wood makes it
resistant to biological attack. Is it then possible to reduce acetylation in woody species without compromising
plant biotic resistance and growth? Using transgenesis approach, we are trying to find out how modification of
acetylation in particular matrix polysaccharides affects plant cell wall architecture, and how it further impacts on
cell wall recalcitrance, and on plant development. To reduce wood acetylation, we have overexpressed fungal
acetyl xylan esterases in aspen and Arabidopsis. We have obtained a number of transgenic lines with reduced
xylan acetylation. These lines are being studied for alteration in plant growth, cell wall composition, resistance to
stresses, and saccharification. Reduction in xylan acetylation in aspen leads to a proportional increase in sugar
yield without pretreatment per g of dry weight. It also increased sugar yields after acid pretreatment but these
effects were not in a direct relation to the degree of deacetylation. We think that, modification in cell wall
acetylation could be useful approach to improve biomass recalcitrance without compromising plant growth and
stress resistance.
Acknowledgements: Eva-Miedes and Antonio Molina, Leonardo Gomez and Simon McQueen.Mason, Henrik Scheller et al., Gregory
Mouille and Herman Hoffte.
P1-16
Modifying plant cell walls by expression of cell wall degrading enzymes
Turumtay H. (a), Van de Wouwer D. (ab), Gheysen G. (a), Boerjan W. (ab), Vanholme B. (ab)
(a) Ghent University, Belgium; (b) Plant Systems Biology-VIB, Belgium.
To study the flexibility of the cell wall we express genes coding for cell wall degrading enzymes in the model
plant Arabidopsis thaliana. This approach provides a deeper insight into the role of different cell wall
polysaccharides in plant growth and development and circumvents the problems related with knock-down and
knock-out mutants to modify cell wall properties. The enzymes we currently use are derived from plant
pathogenic organisms (nematodes, fungi, and bacteria) and among them are cellulases, xylanases, pectate lyases,
polygalacturonases, galactosidases and the cell wall modifying proteins expansin and CBM. At this moment we
have a collection of over 40 different constructs in Arabidopsis and this unique collection is currently used for
gene stacking, where different lines are crossed to combine enzymes in one plant. Here, lignin mutants are
included to increase the accessibility of the cell wall for the different enzymes. For each of the lines, phenotypic
properties are determined focusing on plant growth and biomass production. Finally the cell wall composition and
saccharification yields of the different lines are determined.
P1-17
Structural characterisation of grass glucuronoarabinoxylan
Tryfona T., Sorieul M., Mortimer J., Rubtsov D., Dupree P.
School of Biological Sciences, Department of Biochemistry, Hopkins Building, The Downing site, Tennis Court Road,
Cambridge University, CB2 1QW Cambridge, UK.
Branched xylan is the main hemicellulosic polysaccharide in most energy crops. Glucuronoxylan is modified with
glucuronic acid or 4-O-methyl glucuronic acid and acetyl groups, and is found in willow and poplar wood. In
grasses, xylan carries additional substitutions, such as arabinosyl residues, forming glucuronoarabinoxylan
(GAX). The branching of xylan is thought to contribute to the recalcitrance of biomass to saccharification. It
inhibits the depolymerisation of xylan by enzymes. Moreover, the decorations can be covalently linked to lignin,
and they also modify the interaction of xylan with cellulose fibrils. Therefore, understanding the structure of GAX
and its synthesis is key to optimising biorefining and breeding for improved crops. Here we investigate the
structure of grass GAX using DASH (DNA sequencer-Assisted Saccharide High throughput analysis), HILIC
(Hydrophilic Interaction LIquid Chromatography) and high energy Matrix Assisted Laser Desorption/Ionisation
(MALDI)- Collision Induced Dissociation (CID) mass spectrometry. A number of different grasses are compared
in terms of their GAX structure and a model of grass GAX is proposed.
P1-18
Cell wall analysis of barley stems in modern cultivars, landraces and wild varieties
Voiniciuc C. (a), Usadel B. (ab), Günl M. (a)
(a) Institute of Bio- and Geosciences, IBG-2: Plant Sciences, Forschungszentrum Jülich, 52425 Julich, Germany; (b)
Institute of Biology 1, RWTH Aachen University, 52056 Aachen, Germany.
Barley (Hordeum vulgare) is the fourth most cultivated cereal crop worldwide. Barley production yields grains,
which are a valuable food commodity, and, in many countries, a surplus of straw. The vegetative tissues that make
up the straw are rich in cell wall polysaccharides that have a high economic potential, but their structure is
complex and is not well characterized. Moreover, the industrial application of this agricultural residue is limited
by the high recalcitrance of lignocellulosic biomass to degradation. The susceptibility of biomass to bioconversion
could be increased by identifying plants that have desirable cell wall changes such as reduced cellulose
crystallinity [1] and cell wall acetylation levels [2].
We have identified natural variation in the stem cell wall structure of eleven barley varieties that originate from
different parts of the world. The selected accessions comprise of three modern cultivars, three landraces or
traditional varieties, and five wild barleys (H. spontaneum and H. agriocrithon). We will present the matrix
polysaccharide composition, crystalline cellulose content, and wall acetylation levels of these lines.
[1] M. Pauly and K. Keegstra (2010) Curr. Opin. Plant Biol., 13, 305-312; [2] S. Gille and M. Pauly (2012) Front. Plant Sci.,
3, 12.
P1-19
The screening of cell wall polysaccharides composition and structure in plant collections
Le Gall S., Bittebière Y., Pouliquen C., Lahaye M., Rogniaux H.
INRA UR1268 BIA, BIBS platform, Nantes, France.
Cell wall characteristics define many plant biological functions as well as uses of agronomical crops. In depth
knowledge of their polysaccharides composition and structure with regard to genetic and environmental factors as
well as their physicochemical and mechanical properties would allow restraining crops qualitative variability and
exploring plant cell wall biodiversity. Recent works from the BIA research unit of INRA of Nantes on cereal
grains and fleshy fruits cell wall polysaccharides pinpointed compositional and structural variations of interest
with regard to food/feed values and fruit texture. These data allowed new insights of crop quality variability that
permit enriching genetic strategies for crop improvement. However, from a methodological point of view,
analytical approaches used in these studies hardly met the required throughput to handle large biological
collections (core collections, mutants, tilling populations, etc…). In this context, an instrumental biochemical
phenotyping laboratory was recently created within the BIBS (Biopolymers & Structural Biology) platform of the
BIA research unit of INRA of Nantes. It is dedicated to the screening of large plant collections by biochemical
analyses.
With regards to cell wall polysaccharides composition and structure, this biochemical phenotyping laboratory was
designed to overcome bottleneck points in sample preparations. Increased throughput of cell wall preparations is
achieved using Accelerating Solvent Extraction systems (ASE350, Dionex). A robotic platform (SwingXL,
Chemspeed technologies) equipped with several tools permits automated sample weighing, dispensing and
chemical derivatization/enzymatic digestion required to compositional and structural analyses. Automatized
prepared samples are then analysed on various chromatographic systems adapted to high throughput analyses
(GC, GC-MS, HPAEC) and other instruments from the BIBS platform.
This new instrumental set up will help screening cell wall polysaccharides variability among species of plants
(grain, seed, fruit) or marine (algae) origins with regards to genetic improvement and choice of plant raw material
for uses. Moreover, the expected throughput of the biochemical phenotyping laboratory should link with those of
“omics” methods and thus complete the set of available instrumental approaches for systems biology exploration.
P1-20
Application of polysaccharide analysis using carbohydrate gel electrophoresis (PACE) for
detecting novel mucilage mutants
Yang B. (a), Vasilevski A. (b), Usadel B. (ab)
(a) Institute for Biology 1, RWTH Aachen University Aachen, Germany; (b) Forschungszentrum Jülich, IBG2 Plant Sciences,
Jülich, Germany
Arabidopsis seed coat mucilage contains a layer consisting of rhamnogalacturonan I (RG I), which is easily
extractable, which can be used as a model system for pectin research [1]. Previous studies identified several genes
involved in the regulation of mucilage monosaccharide production and modification through staining and
monosaccharide analysis. However, there is no clear picture of the connection between RGI and the other pectic
components within the pectic network in muro. We plan to employ polysaccharide analysis using carbohydrate
gel electrophoresis (PACE) to screen mutants with alterations in mucilage oligosaccharide in an Arabidopsis EMS
population and will present first results.
PACE is a sensitive and robust method to separate and detect carbohydrates with reducing ends. Integrated with
specific hydrolases, it will be used to investigate mucilage oligosaccharide structures by comparing the enzymatic
fingerprinting of mutants and wild type. In addition, mass spectrometry (MS) could determine the identity of
oligosaccharides in specific bands [2], [3]. Using this method, we will screen for novel mutants with changes in
the mucilage oligosaccharide architecture.
[1] A. A. Arsovski et al. (2010) Plant Signal Behav., 5(7), 796-801; [2] F. Goubet et al. (2002) Anal. Biochem., 300, 53-68;
[3] C. J. Barton et al. (2006) Planta., 224, 163-174.
P1-21
Freeze fracture electron microscopy provides evidence for a cuticular layer on the surface of
leaves of the moss Physcomitrella patens
Ricci E.J. (a), Roberts A.W. (b), Haigler C.H. (c), Roberts E. (a)
(a) Rhode Island College, Providence, RI, USA; (b) University of Rhode Island, Kingston, RI, USA ; (c) North Carolina State
University, Raleigh, NC, USA.
The evolution of the plant cuticle, a hydrophobic layer coating the aerial surfaces of plants, was a significant
evolutionary adaptation that made possible the colonization of land. While the cuticle is ubiquitous in most groups
of vascular plants, it is less clear if it occurs on the leafy portions of moss gametophytes. While freeze fracturing
leaves of the moss Physcomitrella patens, we have observed that essentially all fractures pass through an
extracellular layer composed of lamellae or plates. These structures resemble the waxy cuticle identified in the
fern Adiantum and some other higher plants. It is not yet known if the structures observed in freeze fracture
replicas represents wax, cutin or a mixture of the two. To distinguish these possibilities, we are creating
Physcomitrella knockout mutants that are deficient in the homolog of the Solanum lycopersicum CD1 gene that
participates in cutin biosynthesis in higher plants.
P1-22
Diffusion of molecular probes in cell wall mimicking matrixes
Mutić J. (a), Nikolic J. (a), Crépeau M.-J. (b), Bonnin E. (b)
(a) University of Belgrade, Faculty of Chemistry, Studentski trg 12-16, 11000 Belgrade, Serbia; (b) INRA, UR 1268 Biopolymères-Interactions-Assemblages, BP 71627, 44316 Nantes, France.
The porosity of plant cell wall regulates the cellular exchange and the diffusion of macromolecules such as
enzymes during plant life. To better understand the enzyme action in the remodelling process in planta, one of the
possible approaches consists in using a model system mimicking cell wall network. For this, pectin gels or
pectin/cellulose binary systems were cast in cylindrical mould. To investigate the porosity of these assemblies,
pullulan was chosen as a molecular probe.
The diffusion of the pullulan was followed in the gels on the top of which the pullulan is deposited. The
cyclinders were sliced and pullulan present in each slice was degraded by a pullulanase. Progress of the pullulan
inside the gel was quantified by measurement of maltotriose concentration.
The total amount of pullulan deposited on the top of gel was found under maltotriose form after diffusion in the
gel and degradation by pullulanase, indicating that the developed method was relevant to quantify the pullulan
mobility in the gel.
With this method, we investigated the diffusion of pullulans with various gyration radia, and we varied the
cellulose concentrations in order to diagnose the role of polysaccharide concentration on matrix porosity. This
gave new insights on the role of the two polysaccharides in the porosity establishment.
The authors acknowledge support of the FP7 RegPot project FCUB ERA GA No. 256716. The EC does not share responsibility for the
content of the article.
P1-23
Size, conformation and interactions of polysaccharides
Morris G.A.
Department of Chemical & Biological Sciences, School of Applied Sciences,University of Huddersfield, Huddersfield,HD1
3DH, UK.
Polysaccharides and their derivatives have received a great deal of attention from, for example, the food, cosmetic
and pharmaceutical industries. Their size (weight-average molar mass, intrinsic viscosity, sedimentation
coefficient, radius of gyration and hydrodynamic (Stokes) radius); conformation (compact, flexible or rigid) and
interactions (bioactivity, self-association and synergistic interactions) are relevant to their commercial uses as
these properties play important roles in their function [1-5].
[1] Morris, G. A., García De La Torre, J., Ortega, A., Castile, J., Smith, A., and Harding, S. E. (2008). Food Hydrocolloids,
22, 1435-1442; [2] Morris, G. A., Ang, S., Hill, S. E., Lewis, S., Shafer, B., Nobbmann, U. and Harding, S. E. (2008).
Carbohydrate Polymers, 71, 101-108; [3] Inngjerdingen, K. T., Patel, T., Chen, X., Kenne, L., Allen, S., Morris, G. A.,
Harding, S. E., Matsumoto, T., Diallo, D., Yamada, H., Michaelsen, T. E., Inngjerdingen, M. and Paulsen, B. S. (2007).
Glycobiology, 17, 1299-1310; [4] Heinze, T., Nikolajski, M., Daus, S., Besong, T. M. D., Michaelis, N., Berlin, P., Morris,
G. A., Rowe, A. J. and Harding, S. E. (2011). Angewandte Chemie International Edition, 50, 8602 –8604; [5] Abdelhameed,
A. S., Ang, S., Morris, G. A., Smith, I., Lawson, C., Gahler, R., Wood, S. and Harding, S. E. (2010). Carbohydrate Polymers,
81, 145-148.
P1-24
Plant and fungal Pectin Methylesterase diffusion in pectin gels : a multiscale approach
Videcoq P. (a), Steenkeste K. (bcd), Bonnin E. (a), Garnier C. (a)
(a) INRA, UR 1268, Biopolymères Interactions Assemblages, BP 71627, 44316 Nantes, France ; (b) Univ Paris-Sud, Institut
des Sciences Moléculaires d’Orsay, UMR 8214, Orsay, F-91405, France ; (c) CNRS, Orsay, F-91405, France; (d) Univ
Paris-Sud, Centre de Photonique Biomédicale, Fédération LUMAT, FR 2764, Orsay, F-91405, France.
Pectins contribute to plant cell wall mechanical properties and are implied in cell-cell adhesion particularly by the
way of establishing pectin-calcium gels. The structure of pectin can be altered by plant or fungi pectin
methylesterases (PMEs). PMEs can affect pectin gelling properties by de-esterifying galacturonic acid. Indeed,
the increase of free galacturonic acid content in pectin enhances interactions between pectin and calcium ions and
then induces a gel reinforcement. PMEs de-esterify pectin in a specific pattern and lead to specific gelling
properties according to their origin. In plant cell wall, PMEs de-esterify pectins in the presence of calcium. In this
case, de-esterification and gelation occur simultaneously.
In this work, the diffusion of two PMEs with either fungal or plant origins and mode of action was characterized
with a multi-scale approach in different media consisting in pre-cast or not pectin gels. At microscopic scale,
Fluorescence Recovery After Photobleaching (FRAP) and Fluorescent Correlation Spectroscopy (FCS)
investigations showed similar diffusion behavior for both enzymes. On the opposite, at macroscopic scale, PMEs
diffuse in a different way: the fungal PME diffused faster and in a larger range than the plant PME. According to
local and macroscopic diffusion distances, plant and fungal PMEs exhibit different diffusion abilities linked to
their specific function in vivo: remodelling cell wall in the vicinity of the cell for plant PME and enhancing plant
colonisation for fungal PME [1].
[1] P.Videcoq et al. (2013) Soft Matter, DOI:10.1039/C3SM00058C.
P1-25
New insight into fine structure of red wine RGII
Buffetto F. (a), Ropartz D. (a), Zhang X.J. (b), Gilbert H.J. (b), Guillon F. (a), Ralet M.-C. (a)
(a) INRA, UR1268 Biopolymères Interactions Assemblages, 44300 Nantes, France; (b) Institute for Cell and Molecular
Biosciences Medical School, Newcastle University, Framlington Place, UK.
Rhamnogalacturonan II (RGII) is a pectic domain representing less than 5% of the dicotyledon primary cell wall.
It is a very complex structure containing 12 monosaccharides linked by more than 20 different linkages. The
homogalacturonan backbone is substituted by five side chains : chains C, D and E are neutral, chain B is slightly
acidic (one aceric acid unit) and chain A is highly acidic (3 uronic acid units). This original mega-oligosaccharide
is highly conserved over plant evolution however some structural variations have been pointed out [1,2]. Here we
report on the isolation of red wine RGII and characterisation of structural variations in side chain A. Side chains
were untied from the homogalacturonan backbone by mild acid hydrolysis. Fractionation of the hydrolysate by
anion-exchange chromatography allowed the purification of two main forms of chain A. Chain A fine structure
was studied by tandem mass spectrometry. Interestingly, variations in the methylesterification and
methyletherification forms were identified which could induce specific structural properties.
[1] N.J. Glushka et al. (2003) Carbohydr. Res. 338, 341-352 ; [2] M.A. Rodrigez-Carvajal et al. (2003) Carbohydr. Res. 338,
651-671.
P1-26
What is the conformation of MLG in the grass cell wall?
Kiemle S.N., Cosgrove D.J.
Department of Biology, Pennsylvania State University, University Park, PA, USA.
Mixed linkage glucan (MLG), or β-(1→3, 1→4) glucan, is an unbranched and unsubstituted homopolymer of βglucopyranosyl monomers linked through (1,3)- and (1,4)-linkages. While the structure of MLG is wellcharacterized, very little is known about how MLG is organized or assembled into a functional matrix of plant cell
walls. Several hypotheses have been proposed for the functional roles of MLG in plant walls such as coating
cellulose microfibrils, forming gel-like matrices, tethering cellulose microfibrils, and providing a source of
metabolizable glucose. We used enzyme digestion, chemical fractionation, biomechanical extension assays
including creep (long-term, irreversible extension) as well as atomic force microscopy to visualize structural
changes in grass walls. To test the MLG-cellulose tethering hypothesis, MLG was enzymatically digested with
endo-1,3(4)-β-glucanase in maize coleoptiles clamped in a constant force extensometer. The enzyme digestion
failed to induce wall creep, even after pretreatment with 60 mM NaOH to increase wall access by the enzyme. We
verified that characteristic (1,3)(1,4) β-glucan oligosaccharides were released by enzyme digestion. No xylose or
phenolics from arabinoxylans were released during enzymatic digestion that released 28% of the MLG, indicating
arabinoxylan is not linked to MLG. Chemical solubilization of the arabinoxylan with potassium hydroxide was
required to release the remaining MLG from the grass cell wall, indicating the possibility of two distinct MLG
domains, one that is easily accessable to enzymatic digestion (to supply glucose to the growing cell) and one that
is protected (serving a structural role). These results suggest that enzyme-accessible MLG does not tether
cellulose fibrils into a load-bearing network, that MLG does not bind covalently to arabinoxylan and that a portion
of MLG is inaccessible to soluble enzymes.
P1-27
Deciphering the structure of oligosaccharides by a new tandem mass spectrometry method based
on photo-activation in the VUV range
Ropartz D. (a), Giuliani A. (bc), Lemoine J. (d), Bittebière Y. (a), Ralet M.-C. (a), Rogniaux H. (a)
(a) INRA, UR1268 Biopolymers Interactions Assemblies F-44316 NANTES, France ; (b) Synchrotron SOLEIL, L’orme des
Merisiers, F-91190 Gif-sur-Yvette, France; (c) UAR 1008 CEPIA, INRA, F-44316 Nantes, France; (d) Institut des Sciences
Analytiques, UMR 5280, Université Lyon 1-CNRS, Université de Lyon F-69622 Villeurbanne cedex, France.
Polysaccharides or degraded polysaccharides from plant or marine origin are widely used in pharmaceutical,
cosmetics or food industries. The properties and end-uses of these molecules are tightly connected to their fine
structure, although - for many of them - they remain unresolved. Deciphering carbohydrates structures in complex
mixtures obviously challenges the field of analytical chemistry. Mass spectrometry, with its remarkable
sensitivity, rapidity and high information content, is a forefront method for that purpose, although classical
tandem mass spectrometry based on collision-activated dissociation (CAD) fails in many cases to achieve
definitive structural assignments of carbohydrates.
In this work, we explored an original instrumental setup using Vacuum Ultra-violet synchrotron radiation as
activation process for tandem mass spectrometry1. A complex mixture of oligogalacturonans, released by the
enzymatic degradation of highly methylated pectins and exhibiting many isomers differing by their methylation
pattern was investigated. In striking contrast with CAD, photon activation in the VUV yielded outstanding
information regarding these structures. In fact, the features that were observed on the fragmentation spectra are
remarkable and bring straightforward and highly valuable structural information. This enabled to reach the most
complete description by far of the products released from the enzymatic degradation of cell wall pectins, bringing
insights both on the structure of these polymers as well as on the enzyme specificity.
Milosavljevic et al.(2012) Journal of Synchrotron Radiation, 19, 174-178.
P1-28
Specific interaction of β-galactosyl Yariv reagent with β-1,3-galactan
Kotake T. (a), Kitazawa K. (a), Tryfona T. (b), Yoshimi Y. (a), Kaneko S. (c), Dupree P. (b), Tsumuraya Y. (a)
(a) Saitama University, Japan; (b) University of Cambridge, U.K.; (c) National Food Research Institute, Tsukuba, Japan.
Yariv phenylglycosides (Yariv reagents) are widely used as cytochemical reagents to perturb the molecular
functions of AGPs, as well as for the detection, quantification, purification, and staining of AGPs, but the target
structure in AGPs to which Yariv reagents bind has not been determined. In the present study, we identified the
structural element of AGPs required for the interaction with Yariv reagents by stepwise trimming of the
arabinogalactan moieties with specific glycoside hydrolases. Whereas the precipitation with Yariv reagents (Yariv
reactivity) of radish root AGP was not reduced after enzyme treatment to remove α-L-arabinofuranosyl and βglucuronosyl residues and β-1,6-galactan side chains, it was completely lost after degradation of the β-1,3galactan main chains. Among various oligosaccharides corresponding to partial structures of AGPs, β-1,3galactooligosaccharides (β-1,3-Galns) longer than β-1,3-Gal6 exhibited significant Yariv reactivity in the radial gel
diffusion assay. To the contrary, no Yariv reactivity was detected for β-1,6- Galns of any length. Yariv reactivity
of β-1,3-Gal9 was completely lost by a treatment with a specific enzyme endo-β-1,3-galactanase [1]. A pull-down
assay using oligosaccharides crosslinked to hydrazine beads detected interaction of β-1,3-Galns longer than β-1,3Gal5 with Yariv reagents. Based on these results, we conclude that Yariv reagents should be considered specific
binding reagents for β-1,3-galactan chains longer than five residues, and seven residues are sufficient for
crosslinking, leading to precipitation of the Yariv reagents.
[1] T. Kotake et al. (2011) J. Biol. Chem., 286, 27848-27854.
P1-29
Lignin-directed monoclonal antibodies
Cardenas C.L. (a), Avci U. (a), Davis R.H. (b), Dong R. (b), Ralph J. (cd), Kim H. (cd), Mansfield S. (e), Hahn M.G. (a)
(a) Complex Carbohydrate Research Center, Univ. of Georgia, 315 Riverbend Road, Athens, GA 30602, USA; (b)
Monoclonal Antibody Facility, Univ. of Georgia, Athens, GA 30602, USA; (c) Great Lakes Bioenergy Research Center, The
Wisconsin Energy Institute, Univ. of Wisconsin, Madison, WI 53726, USA; (d) Dept. of Biochemistry, Univ. of Wisconsin,
Madison, WI 53706, USA; (e) Dept. of Wood Science, Univ. of British Columbia, Vancouver, BC V6T 1Z4, Canada.
Lignin, the second most abundant biopolymer present on earth, is a major component of plant cell walls,
particularly secondary plant cell walls, and plays an important role in providing structural reinforcement for the
wall. Lignin, while crucial to the plant, is a prime factor limiting the utilization of plant biomass, particularly the
cell walls that make up the bulk of that biomass, in processes such as ruminant digestibility, production of pulp
and paper, and biomass feedstock conversion to other useful products. One of the biggest current research
impediments is the lack of lignin-specific tools that allow researchers to localize various molecular forms of lignin
in plant cells, tissues and organs, and to determine the timing of lignin deposition throughout cell wall
development, particularly with respect to cell wall polysaccharide synthesis. Monoclonal antibodies are a
powerful tool to address such questions. We have undertaken a comprehensive effort to generate lignin-directed
monoclonal antibodies and report here the first results of this effort. Solubilized lignin polymer preparations from
diverse plant sources were used to immunize mice, and hybridoma lines were generated from mice
immunologically responsive to these immunogens. We present the initial characterization of the antibodies
secreted by these hybridoma lines in terms of their specificity for diverse types of lignin. We also demonstrate the
utility of these antibodies for the localization of lignin in secondary wall-forming tissues, particularly vis-à-vis
cell wall polysaccharide synthesis.
[Supported by a grant from the U.S. Department of Energy (MSN138522)]
P1-30
Hemicellulose fine structure in apple as investigated by enzymatic profiling
Ray S., Vigouroux J., Quemener B., Bonnin E., Lahaye M.
INRA, UR 1286 Biopolymères – Interactions – Assemblages, 44 316 Nantes, France.
Texture changes during ripening impact fruit quality for their different uses. Changes in cell wall polysaccharides
i.e., pectins and hemicelluloses, structure and composition by the coordinated action of cell wall enzymes are
closely involved in this process [1]. However, the precise structural changes are still unclear and particularly that
occurring on xyloglucan (XyG), galactoglucomannan (GgM) and glucuronoxylan (GuX) hemicelluloses. To
detailed these hemicelluloses fine structure, they were extracted from partially depectinated cell walls of apple
parenchyma using DMSO doped by LiCl[2]. Unlike alkali solutions and as previously shown [3], this polar but
aprotic solvent solubilises hemicelluloses without removing the acetyl-ester groups. The extracts were
fractionated using a combination of ion exchange and size exclusion chromatographies into acetylated GuX, XyG
and GgM either as single major constituents or as mixtures. Oligosaccharides structure generated by β-glucanase,
β-mannanase and β-xylanase degradation of the hemicellulose fractions was assessed by MALDI-TOF mass
spectrometry. Partially acetylated xyloglucan-oligosaccharides of XXXG, XLXG, XXLG, and XXFG type were
detected, a series of acetylated xylo-oligosaccharides and a series of acetylated manno-oligosaccharides that
suggested the presence of mannans. Residual 4.0 M KOH-soluble hemicelluloses demonstrated a different
glucanase hydrolysis profile compared to LiCl DMSO soluble fractions. This study highlights the remarkable
structural diversity and complexity of XyG, GgM, GuX polysaccharides in apple cell wall. These hemicellulose
fractions will be used as substrates to investigate the enzymatic mechanisms of hemicelluloses structural changes
during fruit ripening process.
[1] F. Goulao et al. (2008) Trends in Food Sci. Techn., 19, 4-25; [2] L. Petruš et al. (1995) Carbohydr. Res., 268, 319-323;
[3] C. Assor et al. (2013) Carbohydr. Polym. (in press).
P1-31
Spatial structure of rhamnogalacturonans I
Mikshina P.V., Petrova A.A., Gorshkova T.A.
Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Kazan, Russia.
Plant cell wall polysaccharides, same as other polymers with non-template way of synthesis, have some variability
in many parameters of their structure (molecular weight, length and location of the side chains, presence and
distribution of modifying groups, etc.). The range of this variability is limited by "applicability for function", and
is supposed to be largely based on the parameters of spatial organization and the ability to form certain
supramolecular structures. This makes analysis of the polysaccharide spatial organization crucial to distinguish
between functionally divergent variations of the polymers, which are based on the same backbone. The extreme
example of variability of plant cell wall polysaccharide is rhamnogalacturonan I (RG I). Some versions of this
complex polysaccharide, each of which has some internal variability, are known to be organ-, tissue- or stagespecific. To understand the basis for functional divergence of different RGs I it is necessary to get the idea of their
spatial structure determinants. However, peculiarities of spatial organization of RGs I from various sources are
practically non-characterized. Using size exclusion chromatography, diffusion-ordered and traditional NMR
spectroscopy, dynamic light scattering and mass spectrometry, applied to the whole polymer and its fragments, we
have characterized the spatial structure of RG I from gelatinous cell wall, defined its physical and chemical
properties and hydrodynamic parameters. We established that the spatial structure of this polysaccharide is
determined by its ability to self-association by interaction of galactose chains. As a result, a specific compact
complex with a charged backbone at the periphery is formed. Such spatial structure of RG I corresponds to its
functional role in gelatinous cell wall, where this polysaccharide is captured by cellulose microfibrils and is a
cause agent of tension creation.
This work was partially supported by Russian Foundation for Basic Research (11-04-01602, 12-04-97077).
P1-32
Adsorption of cell wall polysaccharides to model cellulose substrates
Gu J., Catchmark J.
Department of Agricultural and Biological Engineering and Center for Lignocellulose Structure and Formation, Penn State
University, University Park PA 16802, USA.
In plant cell walls, the binding interactions among cellulose and non-cellulosic wall polysaccharides define the
primary structure of the cell wall network.In this study, the adsorption of four cell wall polysaccharides including
xyloglucan, xylan, arabinagalactan and pectin to model cellulose substrates have been characterized. Different
model cellulose substrates were used to represent natural cellulose with different structure (e.g. crystalline or
amorphous, different crystalline allomorphs). Highly crystalline cellulose nanowhiskers (CNWs) from both
Gluconacetobacterxylinus(cellulose Iα rich) and cotton (cellulose Iβ dominant), and amorphous cellulose derived
from CNWs (phosphoric acid swollen cellulose nanowhiskers, PASCNWs) have been prepared and characterized.
The amount of binding and the binding constant of cell wall polysaccharides to these model cellulose substrates
were determined by adsorption isotherms.The results were compared among different cellulose substrates and
different non-cellulosic polysaccharides. This work systematically studies the impact of cellulose structure on
binding interactions with other cell wall polysaccharides. With increased cellulose substrate surface area and
decreased porosity, the adsorption of hemicellulose/pectin was increased. The cellulose origin, allomorph and
degree of order may also impact the interactions.
P1-33
The effect of water-soluble polysaccharides on enzymatic digestibility and crystal orientation of
Gluconacetobacter xylinus cellulose
Fang L., Catchmark J.
Pennsylvania State University, Department of Agricultural and Biological Engineering, USA.
Hydrolysis of lignocellulosic biomass is complex as it is influenced by the structure and organization of many cell
wall components including cellulose, hemicelluloses and lignin. Material properties of cellulose such as
crystallinity, crystal organization and size, porosity, and the content and distribution of lignin and hemicellulose
could all contribute to digestibility. The aim of this study is to use Gluconacetobacter xylinus (G.xylinus) as a
model system to better understand how the interaction between cellulose and other biopolymers influences the
formation of cellulose and the resulting changes in its enzymatic hydrolysis. Xyloglucan, xylan, and glucomannan
produced from plant systems and two types of water-soluble exopolysaccharides (EPS) produced from G.xylinus
were added to the fermentation medium. Structure characterization of cellulose suggested that bacterial EPS is
more effective in crystal orientation modification. The incorporation of certain type of mannose-rich
polysaccharides produced from both plants and G.xylinus significantly improved the cellulose hydrolysis
efficiency without decreasing the mechanical properties of the network. Possible mechanisms were explored by
surface area and bundle morphology characterization.
P1-34
Cell walls and cotton fibre development
Hernandez-Gomez M.C. (a), Meulewaeter F. (b), Knox J.P. (a)
(a) Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK; (b) Bayer
CropScience NV-Innovation Center, Technologiepark 38, 9052 Gent, Belgium.
The developing cotton fibre is an excellent model to study cell wall biochemistry, structure and function as it is a
single cell with an unusual capacity for cell elongation and cell wall synthesis. Mature cotton fibres are comprised
mostly of cellulose, nonetheless the primary cell wall of developing fibres contains non-cellulosic polysaccharides
and glycoproteins that function in its growth and development and, ultimately, may determine quality traits such
as fibre length, fineness and mechanical properties. Using monoclonal antibodies, a range of developmental
dynamics of several non-cellulosic polysaccharide epitopes have been identified in developing cotton fibres. For
example, the esterified homogalacturonan (LM20) epitope is not detected after fibre elongation as a more rigid
cell wall is formed. Detection of the galactan (LM5) epitope is lost earlier during development compared to the
arabinan (LM6) epitope suggesting developmental changes in the RG-I structure at early stages of fibre
development. Moreover, two novel cellular structures were identified in the developing cotton fibre. The first
structure, named as polysaccharide-rich intercellular spaces (PRIS), contained sets of particles to which the LM15
(xyloglucan) and LM19 (non-esterified homogalacturonan) probes bound. PRIS were mainly found in the
developing fibre tissue during the elongation stage (from 9 to 17 days post anthesis) suggesting a possible role in
fibre cell adhesion. A second structure consisted of a distinctive pattern of paired cell wall bulges between
neighbouring cell walls, which contained xyloglucan and was present through the elongation stage into the early
maturation stage (22 days post anthesis).
P1-35
Long term exposure to microgravity triggers the downregulation of cell wall related genes and
corresponding modifications in cell wall architecture in Arabidopsis roots
Nakashima J., Sparks J.A., Tang Y., Blancaflor E.B.
Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.
Plants grown on earth are continuously exposed to gravity, which strongly dictates the manner in which they
grow. Because plants could potentially serve as renewable food resource and promote well-being of astronauts in
advanced life support environments for long term space exploration, it is critical to better understand how they
develop under microgravity. Through experiments utilizing the Biological Research in Canisters (BRIC) hardware
on board the STS-131 Space Shuttle Discovery in April 2010, we tested the impact of microgravity on seedling
development using Arabidopsis thaliana. Specifically we compared root development in etiolated wild-type and a
mutant (act2) that was disrupted in a root-expressed vegetative actin isoform. After 13 days of growth, seedlings
from space and ground controls were fixed in Glutaraldehyde, and embedded in LR White resin for light and
TEM studies. Independent sets of seedlings were fixed in RNA Later for transcriptomic analysis. We observed
that space-grown roots skewed vigorously toward one direction, and that appeared to be more pronounced in act2
mutants compared to wild-type. At the TEM level, there were clear structural deformities in cell walls of roots
particularly in act2 mutant grown in microgravity. A suite of monoclonal antibodies were employed to determine
specific components of the cell wall that were altered in microgravity. In one specific case, we found that spacegrown seedlings exhibited a decline in the labelling intensity of a fucosylated xyloglucan epitope. Consistent with
our microscopy studies, transcriptomic analysis revealed that 20% of genes down-regulated in space were genes
associated with plant cell wall function. Taken together our data indicate that microgravity affects root
development in part by modifying the expression of genes that are components of signaling networks that link the
cytoskeleton to structural components of the cell wall
Supported by NASA grant NNX10AF43G.
P1-36
Identification of the pI 4.6 extensin peroxidase gene in tomato
Dong W., Kieliszewski M., Held M.
Department of Chemistry and Biochemistry, Ohio University, Athens OH 45701, USA.
The insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall is
important for establishing proper cell wall architecture for mediating cell growth and plant defense responses. In
tomato (Lycopersicon esculentum), insolublization occurs by the formation of tyrosyl-crosslinks catalyzed
specifically by a pI 4.6 extensin peroxidase (EP) [1,2]. To date, neither the gene encoding EP nor the protein
itself have been identified. Here we have taken two approaches to identify the pI 4.6 tomato EP. The first involves
the identification of candidate genes by bioinformatics. A keyword search of the tomato genome database
(http://solgenomics.net/organism/Solanum_lycopersicum/genome) identified 111 peroxidase candidate genes.
Candidates were sorted by the presence of signal peptide and by the predicted pI of their encoding proteins. From
these candidates, eight genes were selected for further analysis. The second approach employed a biochemical
fractionation strategy aimed to directly purify the EP from tomato suspension cultures. Proteomic detection of the
fractionated EP identified a peroxidase that matched one of the candidate genes identified by bioinformatics. The
candidate EPs and several control peroxidases have been cloned as yellow fluorescent protein fusions. Confocal
microscopy of transiently expressed constructs in tobacco leaf epidermal cells has validated construct expression
and protein secretion. Stable expression of constructs in tobacco BY-2 cells is underway for protein purification
and in vitro characterization of the EP candidates. Recombinant proteins will be tested for peroxidase activity by
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays and for extensin crosslinking activity by
tomato P1 and P3 extensin crosslinking assays in vitro.
[1] Held et al. (2004) J. Biol. Chem. 279, 55474-82; [2] Schnabelrauch et al. (1996) Plant J. 9, 477-89.
P1-37
Structural characterisation of xyloglucans using electrospray ionisation mass spectrometry
Quemener B., Lahaye M.
INRA – Biopolymères, Interactions, Assemblages – Rue de la Géraudière BP 71627, F-44316 Nantes, France.
Xyloglucans of apple, tamarind and A. Thaliana were hydrolyzed by commercial endo ß-1-4 endoglucanase. The
released oligosaccharides (XGOS) were separated by HPAEC on CarboPac PA 200 analytical column in less 15
minutes using new unusual isocratic conditions. The main XGOS released from apple and tamarind xyloglucans
were purified for further structural characterization by electrospray ionization mass spectrometry using a
quadrupole time-of-flight (ESI-Q-TOF) analyser in negative ion mode. Commercial galacto-mannooligosaccharides (GMOS) were also studied to corroborate the fragmentation routes observed on XGOS. The
negative ion fragmentation spectra of the M-H] - species were characterized by glycosidic cleavage ions from the
C-series and by prominent double cleavage (D) ions corresponding to the side chains. Beside these important
diagnostic ions, cross-ring cleavage 0,2Ai ions, carrying linkage information, underwent consecutive loss of water
to produce 0,2Ai -18 fragment ions. Other 2,4A-type cross ring cleavage ions were also observed using negative ESIQ-TOF. CID spectra of sodium-cationised species acquired in the positive ion mode were more complex and less
informative. Interestingly, the fragmentation routes of the analyzed GMOS were similar to XGOS ones using
negative ion mode. All these characteristics provide sequence relevant informations so that isomeric structures
differing only in their substitution pattern can be differentiated by this approach.
P1-38
Specific inhibition of pectin remodelling enzymes
L’Enfant M. (a), Rayon C. (a), Domon J.-M. (a), Wadouachi A. (b), Kovensky J. (b), Pelloux J. (a), Pau-Roblot C. (a)
(a) Unité de Biologie des Plantes et Innovation, EA 3900 BIOPI, UFR des sciences, 33 rue Saint Leu, Université de Picardie
Jules Verne, 80039 Amiens ; (b) Laboratoire des Glucides, FRE 3517-CNRS ; UFR des sciences, 33 rue Saint Leu,
Université de Picardie Jules Verne, 80039 Amiens.
Pectins are major components of plant cell walls and constitute a valuable biomass for food and non-food
applications. Homogalacturonan (HG), the most abundant pectic polysaccharide, can be acetylated and/or methylesterified on specific carbons [1]. The degree of methyl-esterification (DM) and acetylation (DA) is controlled
within plant by specific enzymes such as pectin methylesterases (PME). Changes in DM can have dramatic
consequences on the rheological and chemical properties of the cell wall, modulating, for instance, the sensitivity
of HG to phytopathogens degrading enzymes such as polygalacturonases (PG). The action of PG induces cell wall
degradation and promotes colonization of host tissues by pathogens (bacteria and fungi) [2]. Substrates specificity
and mode of action of the enzymes (PME, and PG) are so far largely unknown in plants. To bring out new
potential applications of these polymers/oligomers (medicine, preventive treatment of crops against pathogens), a
better understanding of the relationships between their structure, modulated via the action of specific enzymes,
and their properties is required. After a production of PME and PG from plant (Arabidopsis thaliana) and
phytopathogens (Botrytis), an evaluation of their biological activities and their modes of action will be carried out.
Chemical modification of substrates is used to obtain substrate analogs which can be used as potential inhibitors.
The approach and results will be presented.
[1] Wolf et al. (2009). Mol. Plant, 2, 851-860; [2] Shah et al. (2009). Proteomics, 9, 3126-3135.
P1-39
Movements of radiocesium and radioiodine in forest trees
Hayashi T. (a), Yasukawa C. (a), Aoki S. (a), Nonaka M. (a), Itakura M. (a), Kaida R. (a), Sakata Y. (a), Uehara I. (a),
Obayashi H. (a), Baba K. (b)
(a) Tokyo University of Agriculture, Japan ; (b) Kyoto University, Japan.
Following the Fukushima nuclear power plant accident, radioactive compounds were dispersed over large forest
areas 50 km between the plant and Fukushima. Since the compounds attached to all surfaces of the forest trees,
particularly those on highly windward mountains that were more than 400 meters, they may have stagnated and
circulated between the mountains and the plant. Fallout radiocesium may have attached like via aerosol as metal
and its ions and incorporated into plant bodies from all plant surfaces, not only directly via the leaves but also via
the bark in the first year (2011). The alkali metal ions could also enter the xylem and fix in the heartwood, where
acidic residues such as the uronic acids of pectin and xylan could draw the ions via xyloglucan filters further into
the heartwood. Fallout radioiodine may also be incorporated as a gas into the leaves of poplar seedlings to bind
xyloglucan in the apoplastic spaces via the stoma because iodine was not incorporated into those of transgenic
poplars overexpressing xyloglucanase. Since the iodine disappeared with a short half-life of 8 days, it is evident
that the forests have become the largest sink of radiocesium, which has a half-life of 30 years and which is
recycled between the trees and the soil in their leaves, flowers, etc. as well as in other organisms.
P1-40
Maize glucuronoarabinoxylans taking part in elongation growth
Kozlova L.V., Mikshina P.V., Ageeva M.A., Ibragimova N.N., Gorshkova T.A.
Kazan Institute of Biochemistry and Biophysics, Russian Academy of Science.
Glucuronoarabinoxylan is the key hemicellulose in the primary plant cell walls of type II. Though it is known that
the ratio Ara/Xyl decreases during elongation, the mechanism of participation of hemicellulosic molecules in
elongation process is still unclear. On the way to understand the functional role of glucuronoarabinoxylans we
have compared features of the polymer in tissues of maize primary root before, during and after elongation. The
existence of biochemically distinct portions of glucuronoarabinoxylans and the peculiarities of their distribution
within cell wall were revealed by several approaches, including immunochemistry, treatment with specific
enzymes, analysis of isolated portions of the polymer by SEC, HPAEC and NMR-spectroscopy. The distinct
portions of glucuronoarabinoxylans have different degree of substitution and specific mode of interaction with
cellulose and mixed-linkage glucan. There are some evidences of the presence of high- and low-substituted
domains of glucuronoarabinoxylan within one and the same molecule. Proportion of various domains of
glucuronoarabinoxylans changes during elongation and is in correlation with cell growth rate.
Work was partially supported by Russian Foundation for Basic Research (project – 11-04-01016) and the President Program for State
Support of Leading Scientific Schools (825.2012.4).
P1-41
Interactions between cell wall hydration and mechanical behaviour in plant cell walls and
bacterial cellulose composites
Islam A., Thompson D.S.
University of Westminster, London, UK.
Manipulation of cell wall water content using high molecular weight osmotica demonstrates that cell wall
mechanical behaviour as determined by constant-load extensiometry is affected by the water content of the cell
wall in sunflower hypocotyls. Osmotic pressures of as little as 0.21 MPa, corresponding to water potentials that
would commonly be experienced by plants in vivo, significantly reduced long-term extensibility.
This effect also occurs in sheets of bacterial cellulose produced by Gluconacetobacter xylinum and composites of
bacterial cellulose incorporating plant cell wall polysaccharides. This has been used to investigate the role of
specific cell wall components on wall hydration and mechanical behaviour. Dehydration and rehydration and
mechanical behaviour under conditions of full hydration and after reduction of the water content using high
molecular weight polyethylene glycol (PEG 6000) were compared for cellulose composites with apple pectin,
polygalacturonic acid, rhamnogalacturonan II, xyloglucan, oat beta glucan and lichenan.
Composites with hemicelluloses (xyloglucan, oat beta glucan and laminarin) were similar to one another and were
substantially more extensible than bacterial cellulose alone or cellulose with pectins. Composites with pectin were
more extensible than cellulose alone but these materials were comparable in texture, whereas all of those
incorporating hemicelluloses were much more prone to thinning under stress or even manipulation and were
found to have substantially higher Poisson’s ratios. Consistent with this, composites with xyloglucan lost a much
greater proportion of their weight (72%) than cellulose (45%) or cellulose with apple pectin (44%) in PEG 6000
solutions with an osmotic pressure of 0.62 MPa. None completely rehydrated in control buffer alone after this
treatment, but did when subsequently treated with cucumber expansin.
P1-42
Modelling progression of fluorescent probes in bio-inspired lignocellulosic assemblies
Paës G. (ab), Burr S. (ab), Estephan M.-B. (abc), Molinari M. (c), Aguié-Béghin V. (ab), Chabbert B. (ab)
(a) INRA, UMR614 Fractionnement des AgroRessources et Environnement, Reims, France ; (b) University of Reims
Champagne Ardenne, UMR614 Fractionnement des AgroRessources et Environnement, Reims, France ; (c) University of
Reims Champagne Ardenne, Laboratoire de Recherche en Nanosciences LRN EA4682, Reims, France.
Lignocellulosic biomass is made of the plant cell walls from forest or cereal co-products and dedicated cultures. It
is considered as a renewable source of materials, biofuels and chemicals that substitute to fossil carbon. However,
chemical and structural complexity of plant cell walls make them be recalcitrant. Enzymes can improve their
transformation, but their potential is often restricted by factors that limit their accessibility. Progression of
enzymes in lignocellulosic biomass is thus a crucial parameter in biorefinery processes and it appears to be one of
the limiting factors in optimizing lignocellulose degradation.
In order to assay the importance of the chemical and structural features of the plant cell wall matrix on enzyme
mobility, we have designed and characterised bio-inspired model assemblies of secondary plant cell walls based
on arabinoxylan gels containing cellulose nano-crystals [1] with various concentration and water content. Then,
the mobility of fluorescent probes of various sizes has been measured by FRAP in these assemblies. Overall, the
dataset obtained has been used to model probe mobility and to rank the parameters varied in order of importance:
water content and probe size were shown to have the greatest effect, whereas polymers concentration had minor
effects. Although these assemblies are simplified templates of the plant cell walls, our strategy paves the way for
proposing new approaches for optimizing biomass saccharification, such as selecting enzymes with suitable
properties and finely tuning water content.
[1] G. Paës et al. (2012) Biomacromolecules, 13, 206-214.
P1-43
Hydrodynamics characterisation of plant cell wall polysaccharides and their assemblies - Proteinlike oligomerisation of carbohydrates
Morris G.A. (a), Harding S.E. (b)
(a) Department of Chemical & Biological Sciences, School of Applied Sciences,University of Huddersfield,
Huddersfield,HD1 3DH, UK; (b) National Centre for Macromolecular Hydrodynamics, School of Biosciences, University of
Nottingham, Sutton Bonington, LE12 5RD, UK.
Plant cell wall polysaccharides have been characterised using a variety of hydrodynamic techniques. Results
suggest that the polysaccharides selected are, in general, rigid or semi-rigid molecules with a large hydrated
volume, this is important in relation to their structure - function relationships. In addition the effect of chemical
modification was investigated for cellulose. Remarkably, for every aminocellulose studied, the sedimentation
coefficient distributions show between four and five discrete species with a stepwise increase in sedimentation
coefficient more typically seen in protein oligomerisation [1].
[1]. T. Heinze, M. Nikolajski, S. Daus, T. M. D. Besong, N. Michaelis, P. Berlin, G. A. Morris, A. J. Rowe & S. E. Harding.
Angew. Chem. Int. Edit., 50 (2011), 8602–8604.
P1-44
The biosynthesis and molecular weight distribution of hemicelluloses in cellulose-deficient maize
cells: an example of metabolic plasticity
de Castro M. (ab), Encina A. (a), Acebes J.L. (a), García-Angulo P. (a), Fry S.C. (b)
(a) Área de Fisiología Vegetal, Facultad de CC Biológicas y Ambientales, Universidad de León, E-2407, León, Spain; (b)
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences. The University of
Edinburgh, Edinburgh EH93JH, UK.
Habituation of maize cultured cells to DCB (an inhibitor of the cellulose biosynthesis) entails a reduction in their
cellulose content, partially compensated for by a modified arabinoxylan network. The aim of this work was to
investigate whether the metabolism and the molecular weight (Mr) distribution of hemicelluloses was modified in
cellulose-deficient cells. To this end, [3H]arabinose was added for various time intervals, at different stages of the
culture cycle, to non-habituated (Snh) and 1.5 µM DCB-habituated (Sh) cells. The radiolabelled soluble
extracellular polymers (SEPs) and hemicelluloses from several cell compartments (protoplasmic, and cell wall
bound extracted with 0.1 M or 6 M NaOH) were then isolated and characterised. DCB-habituated cells showed a
delayed biosynthesis of hemicelluloses, and a reduction in the proportion that were strongly wall-bound.
Moreover, the 3H-labelled hemicelluloses and SEPs from habituated cells were more size-homogeneous and had a
lower average Mr. Modifications observed could be related to a decrease in the possibilities for hydrogen-bonding
to cellulose and/or a reduced capacity to incorporate arabinoxylans into the cell wall by extraprotoplasmic
phenolic cross-linking.
P1-45
Water: the forgotten solvent
Huang S.-C. (a), Lin F. (b), Catchmark J. (b), Park Y.B. (c), Cosgrove D. (c), Maranas J.K. (a)
(a) Department of Chemical Engineering, (b) Department of Agricultural and Biological Engineering, (c) Department of
Biology; Penn State University, University Park, PA 16802, USA.
Water comprises 50-80 wt% of primary cell walls, yet is not frequently a target of investigation. This differs
within physical chemistry and related studies, where the properties of water are often investigated. Water forms
ordered layers on hydrophilic and hydrophobic surfaces, and these layers can be several orders of magnitude
slower than bulk water. The motion of solvated molecules, including polymers, proteins and small solvent are
slaved to the motion of the water solvent, which is slowed in contact with the solute. The thermal properties of
water change when confined within 40 nm, either not freezing or freezing below the normal melting point.
Thermal properties of water are altered in plant cell walls, which is not surprising given the 20-40 nm spacing
between cellulose microfibrils. We investigate the dynamics and thermal behavior of water on bacterial cellulose
microfibrils. In order to use quasi-elastic neutron scattering, we culture Acetobactor Xylinum with deuterated
glucose, producing deuterated cellulose as confirmed with FTIR. Because QENS primarily detects hydrogen,
this allows us to isolate the motion of water. Measuring at 20% water, where thermal measurements detect no
bulk water, find two dynamic classes of water, which we interpret as an outer hydration layer [76%, 35 times
slower than bulk water] and an inner hydration layer [24%, 100 times slower than bulk water]. As both
translation and rotation are slowed, this water is most likely ordered on the cellulose surface. To extend the
biological significance of these findings, we measured the dynamics in wild type and xxt1xxt2 Arabidopsis
petioles using a subtraction technique to isolate water mobility. At both 50-wt% and 20-wt% water, the mobility
of water is slowed in these walls, with the influence more apparent in wild type walls.
P1-46
Production and fine characterisation of new monoclonal antibodies against rhamnogalacturonan I
Buffetto F. (a), Cornuault V. (b), Gro Rydahl M. (c), Crépeau M.-J. (a), Tranquet O. (a), Willats W.G.T. (c), Knox J.P. (b),
Guillon F. (a), Ralet M.-C. (a)
(a) INRA, UR1268 Biopolymères Interactions Assemblages, 44300 Nantes, France ; (b) Centre for Plant Sciences, University
of Leeds, Leeds LS2 9JT, UK; (c) The Department of Molecular Biology, The University of Copenhagen, Ole Maaløes Vej 5,
2200 Copenhagen, Denmark.
The functional role of the complex pectic rhamnogalacturonane I (RGI) in the context of cell biology is still
unclear. RGI is mainly composed of a repeating disaccharide unit [,2)-α-L-rhamnosep-(1,4)-α-D-galacturonic
acidp-(1,]n (RU)n decorated primarily with arabinan and (arabino)-galactan side-chains. Monoclonal antibodies
(mAbs) are useful to probe pectin structural domains in situ. Several mAbs to the RGI backbone and to arabinan
and (arabino)-galactan side-chains have been developed but up to now, there is no mAb against
rhamnogalacturonan stretches bearing arabinose or galactose units. Here, we report on the production and fine
characterisation of new mAbs against RGI side chains connected to the rhamnogalacturonan backbone.
The study first focused on the generation of adequate antigens. A pool of low-branched RU oligosaccharides was
prepared and purified from potato pulp. mAbs were raised against low-branched RU oligosaccharides-ovalbumin
conjugates. Promising clones producing mAbs recognizing low-branched RU oligosaccharides but recognizing
neither unbranched RU oligosaccharides nor galactan oligosaccharides were recovered. The combination of
glycan microarray and sub-fractionation of the low-branched RU oligosaccharides pool by anion exchange
chromatography prior to competitive ELISA studies allowed fine mAbs characterisation. The use of these new
mAbs in immunocytochemistry is expected to give a better understanding of RGI role in planta.
P1-47
What’s new in the microscopy observation of the mature wheat ultrastructure
Gaillard C.
INRA UR1268 BIA Biopolymères, Interactions, Assemblages, Centre Angers-Nantes Rue de la Géraudière, 44316 Nantes
France
The microscopy tools including epi-fluorescence and laser scanning confocal microscopes, scanning and
transmission electron microscopes, atomic force microscopy, as well as dedicated sample preparation methods are
commonly used for a long time to study the structural organization of the wheat tissues. In particular, transmission
electron microscopy (TEM) allows observation of the wheat grain ultrastructure with a high spatial resolution.
However, such technique implies to prepare wheat samples with successive steps including chemical fixation,
dehydration, resin-inclusion and contrast-enhancement staining that may induce artefacts and reduce the image
resolution and valuable information.
To precise the distribution of the various layers of the wheat tissue, and ensure a good understanding of the
natural assemblies of biopolymers and mineral nutrients at the sub-cellular level, new microscopy-based
approaches for preparation of wheat grains avoiding any chemical fixation or resin inclusion steps, and for
observation with a high spatial resolution will be shown. For that, wheat grains are directly processed in their
native shape either to produce ultrathin slices for TEM analysis, or to get nanometer-sized smoothed surfaces for
AFM investigation, using a home-made device adapted for both AFM and ultramicrotome.
These methods those are particularly suitable for studying dried wheat grains, and can be adapted for hydrated
wheat grains, have revealed unique features in the wheat outer layers organisation due to both the capacity of
keeping the natural tissue contrast, free from any chemical artefacts, or the native mechanical stress between the
components, exempt from any alteration from resin infiltration. Moreover, taking advantage from the capacity of
keeping the wheat tissue in its native shape, the mechanical properties of the native wheat outer layers (intenal and
external pericarp, aleurone and sub-aleurone layers and starchy endosperm) have been characterised by AFM at
the nonometer scale.
P1-48
Molecular interactions and mechanical properties of bacterial cellulose composites
Gidley M., Mikkelsen D., Lopez-Sanchez P., Stokes J.R.
ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, The University of Queensland, St
Lucia, Queensland, Australia, 4072.
Fermentation of Gluconacetobacter xylinus in growth media containing cell wall polysaccharides can result in the
assembly of a range of cellulose-based composite materials depending on the chemistry and physical form of the
added polysaccharide. We report studies of composites formed in the presence of (1,3;1,4)-β-glucan and
arabinoxylan, characteristic primary wall components in cereals and grasses. In contrast to pectin and xyloglucan,
neither polymer family shows evidence for specific interactions with cellulose. In situ enzymatic dearabinosylation of arabinoxylan / cellulose composites results in self-association of arabinoxylan, rather than
cellulose / arabinoxylan association. A similar self-association phenomenon is found for galactose-depleted
xyloglucans in cellulose composites, suggesting a subtle balance between homotypic and heterotypic polymer
associations in plant cell walls.
We also report the effect of mechanical pressure on bacterial cellulose composites as an approach to assessing the
potential effect of turgor pressure. Compression of composites does not result in lateral expansion i.e. Poisson
ratio ~0, but does result in fibre coalescence in the absence of other polysaccharides, and is accompanied by a
mechanical collapse transition at pressures below those characteristic of turgor pressure. We further show that
incorporation of xyloglucan into composites results in strain hardening at high strain rates but not at low strain
rates, suggesting an in vivo role in resisting sudden mechanical insults. Xyloglucan composites are simultaneously
stronger at high compressive strain rates and weaker under slow strain rate tensile extension, the combination of
strength and extensibility that is ideal for the biological function of primary plant cell walls.
P1-49
In vivo and in vitro polymerization of lignin by plant laccases
Baratiny D. (a), Cottyn B. (a), Jouanin L. (a), Lapierre C. (a), Ducrot P.-H. (a), Demont-Caulet N. (ab)
(a) Institut Jean-Pierre Bourgin, UMR 1318 Inra/AgroParisTech, France; (b) UFR des Sciences du Vivant, Université Paris
Diderot-Paris 7, 75205 Paris Cedex 13, France.
Some plant cells are surrounded by a lignocellulosic cell wall constituted of polysaccharides (cellulose and
hemicelluloses mainly) and of lignin, a phenolic polymer accounting for 20% of the biomass. Lignins are heteropolymers constituted of three monolignols, p-coumaryl alcool, sinapyl alcool and coniferyl alcool. They are
oxidized in the cell wall by two types of enzymes peroxydases and laccases. These enzymes being part of
multigenic families (73 and 17 respectively identified in Arabidopsis thaliana) it is challenging to identify the one
specifically involved in lignin biosynthesis. Laccases (LAC) are multi-copper containing oxidases distributed in
plants, fungi, bacteria and insects that catalyses the oxidation of a wide range of different compounds. Well
characterized in fungi for their role in lignin-degradation, a little is known on their biological function in plants.
Three laccases found in Arabidopsis thaliana are involved in lignin polymerization in the stem (LAC4 and
LAC17) 1 or in flavonoids oxidation in the seed coat (LAC15) 2. In order to understand whether the biological
function of each enzyme is determined by its tissue specific expression or by its substrate specificity, in vivo and
in vitro analysis of these three laccases will be performed. Enzyme reactivity toward monolignols will also be
assayed using fungal laccases. These experiments would lead to the identification of factors that explain the
ability of these proteins to polymerize or degrade the lignin polymers.
[1] S.Berthet et al. (2011) Plant Cell., 23(3): 1124–1137; [2] L.Pourcel et al. (2005) Plant Cell., 17(11): 2966–2980.
P1-50
Structural study of O-acetyl-glucuronoxylan in Arabidopsis thaliana reveals spatial distribution of
acetyl substituents
Chong S.-L. (a), Virkki L. (a), Maaheimo H. (c), Derba-Maceluch M. (b), Koutaniemi S. (a), Tuomainen P. (a), Mellerowicz
E.J. (b), Tenkanen M. (a)
(a) Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, P.O. Box 27, 00014 University of
Helsinki, Finland; (b) Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University
of Agricultural Sciences, 901-83 Umeå, Sweden; (c) VTT Technical Research Centre, P.O. Box 65, 00014 Helsinki, Finland.
Glucuronoxylan (GX) isolation from Arabidopsis thaliana has usually been carried out by alkaline solutions.
Hence, structural studies of GX are lacking information about O-acetylation. In this work, native GX was isolated
from A. thaliana applying DMSO extraction and subjected to 2D heteronuclear single quantum coherence
(HSQC) nuclear magnetic resonance spectroscopy analysis. Degree of acetylation (DA) obtained for GX was
60%. Contents for O-3, O-2 and O-2,3 acetylated xylopyranosyl (Xylp) units were 29%, 15% and 6%,
respectively. O-2 linked (4-O-methyl)-α-D-glucopyranosyl uronic acid ((me)GlcpA) substituted Xylp residues
with and without O-3 acetylation contributed 10% and 3%, respectively. Direct endoxylanase (GH10) hydrolysis
of stem powder followed by mass spectrometry (MS) analysis revealed that mono- and di-acetylated xylobiose
were the most abundant neutral xylooligosaccharides produced. Based on the MS/MS fragmentation analysis, the
acetyl residues were found solely on the non-reducing end Xylp unit of xylobiose. The main peak observed in the
acidic xylooligosaccharides was di-acetylated (me)GlcpA-xylotetraose, MS/MS analysis of which suggested
alternated substitution of Xylp residues by acetyl residues. Analysis of xylan deficient mutants irx7, irx9-1, irx14
and irx10 revealed that mono-acetylation (O-3 and O-2) was reduced in irx7, irx9-1, irx14 resulting in DA of
31%, 32% and 42%, respectively. No change was observed in irx10. Endoxylanase-MS fingerprinting also
showed the clear reduction in xylan acetylation in irx7 and irx9-1, which also contain least xylan of the studied
mutants. The results obtained point out the relationships between the acetylation and other GX biosynthetic
processes.
Session 2 : Dynamics of plant CW Components : from Biosynthesis to Remodelling
Intracellular synthesis & trafficking, Synthesis & assembly at the plasma membrane,
Remodelling in muro, Transcriptional and post-transcriptional control
P2-01
Genetic and computational testing of the function of the region between cellulose synthase (CESA)
transmembrane helices (TMH) 5 and 6 in Arabidopsis thaliana
Slabaugh E. (ab), Sethaphong L. (ac), Mansouri K. (ab), Scavuzzo-Duggan T. (ad), Kubicki J. (ae), Roberts A.W. (ad),
Yingling Y.G. (ac), Haigler C.H. (ab)
(a) Center for Lignocellulose Structure and Formation, (b) Department of Crop Science, (c) Department of Materials
Science and Engineering; North Carolina State University, Raleigh, NC, USA; (d) Department of Biological Sciences,
University of Rhode Island, Kingston, RI, USA; (e) Department of Geosciences, The Penn State University, University Park,
PA, USA.
Recently, a putative structural model of cellulose synthase (CESA) transmembrane helices (TMH) 5-6 and the 30
amino acid intervening region was generated by the Yingling group using in silico homology modeling, molecular
dynamics simulations and quantum calculations. The model of this TMH5-6 region generated a bimodal folding
funnel consistent with two different structural conformations. We hypothesized that the region between TMH5-6
can move into and out of the membrane and may function in regulating cellulose biosynthesis. To further test this
in silico predicted structural model, specific amino acids in CESA1 and CESA7 of Arabidopsis thaliana were
targeted for site-directed mutagenesis based on structural and free energy predictions, as well as modeling of the
interaction between the TMH5-6 intervening region and other molecules. The altered gene constructs are being
transformed into the corresponding Arabidopsis cesa mutant lines, with alterations in primary or secondary wall
cellulose synthesis, in order to analyze potential phenotypic complementation and other cellulose-related
characteristics. Preliminary data show that the G949P mutation in CESA1 is deleterious while the F967G
mutation is tolerated. In CESA7, preliminary results suggest that the F913G mutation is also tolerated, along with
mutations that alter a central acidic motif (DDDD to NNNN). Analogous mutations are also being carried out in
Physcomitrella patens. This multi-disciplinary approach may lead to the discovery of new mutations that affect
cellulose biosynthesis and will help to uncover novel structure/function relationships in plant CESA proteins. In
the longer term, it will support the optimization of cellulose properties in plants targeted toward biofuel
production from lignocellulosic biomass.
This work was supported by the U.S. Department of Energy, Office of Science and Office of Basic Energy Sciences (award no. DE–
SC0001090 to the Center for LignoCellulose Structure and Formation, an Energy Frontier Research Center).
P2-02
An XET activating factor (XAF) in plant cells
Nguyen Phan C.T., Fry S.C.
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, UK.
XET, one of the two main activities of XTH proteins, is of interest because it is responsible for the modification
of xyloglucan tethers in the hemicellulose–cellulose microfibril network of the plant cell wall, providing a
molecular mechanism for controlled, turgor-driven wall expansion. In this study, we confirmed [1] the existence of
an ‘XET activating factor’ (XAF) in the cold-water-extractable polymers of cauliflower florets. The factor
remains water-soluble on boiling, unlike most proteins, and may thus be a polysaccharide or heavily glycosylated
glycoprotein. XAF solubilises Arabidopsis cell XTHs, increasing their XET activity on soluble xyloglucan up to
125-fold. XAF had effects similar to those of 15 mM Ca 2+ and 100 mM Na+ in this respect, although it was only
weakly ionic (at a highly effective concentration, its conductivity was equivalent to that of 8 mM NaCl). Gelpermeation chromatography on Sepharose CL-6B showed a peak of XAF activity with molecular weight ~10 4–
105. Acid hydrolysis (2 M TFA, 120°C and 6 M HCl, 110°C) of peak XAF-active fractions gave much Ara and
Gal, and smaller proportions of Glc, GalA, GlcA, Xyl, Man and Rha together with certain amino acids, including
Hyp and Ala, suggesting a predominance of arabinogalactan-protein. Some XAF-enriched fractions were Yariv
positive; however, cauliflower AGP purified by use of β-glucosyl Yariv reagent had little XAF activity.
Treatment of the fractions from CL-6B column with xyloglucan endoglucanase decreased their XAF activity and
yielded xyloglucan oligosaccharides, suggesting that xyloglucan is one component of XAF; however, Tropaeolum
xyloglucan had no XAF activity, indicating that XAF activity is not a feature of all xyloglucans. We are
conducting further experiments to determine the nature of XAF as well as its occurrence in growing plants. We
wish to characterise an endogenous modulator related to the mechanism of plant cell growth control.
[1] T. Takeda, S. C. Fry (2004) Planta, 219, 722-732.
P2-03
Transglycanase activity detected with pectic polysaccharides: a novel α-arabinan : xyloglucan
endotransglycosylase (AXE) activity ?
Holland C., Fry S.C.
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, UK.
Transglycanases are a class of cell-wall remodelling enzymes hypothesised to be involved in – among other
functions – cellular elongation and strengthening of the PCW via enzymic cleavage of tethering molecules.
Internal hydrostatic pressure separates microfibrils, expanding the cell, and new microfibrils and associated
polysaccharides are laid down on the innermost surface of the wall [1]. At present, four transglycanase activities
have been convincingly characterised but the potential existence of others is likely.
To detect novel activities we conducted fluorescent and radioactive assays using a variety of donor and acceptor
substrates in combination with enzyme extracts sourced from a range of plants representative of the majority of
the kingdom. Beansprout extracts repeatedly displayed significant retention of radioactivity and fluorescence
when incubated with α-arabinan : xyloglucan oligosaccharides (XGO) (donor : acceptor) indicating a potential
novel heterotransglycanase activity. Further analysis refuted the alternate theory that the activity detected was
representative of XET activity, as xyloglucan contamination was not present. Potential transglycanase activities
were also detected with the pectic polysaccharide β-(1→4)-galactan as donor polysaccharide and an XGO
acceptor. This result is to be further explored in future work.
[1] N. C. Carpita & D. M. Gibeaut (1993) The Plant Journal, 3, 1-30.
P2-04
Functional Analysis of Complexes with Mixed Primary and Secondary CESAs
Li S., Lei L., Gu Y.
The Center for LignoCellulose Structure and Formation, Department of Biochemistry and Molecular Biology, Pennsylvania
State University, University Park, PA 16801, USA.
In higher plants, cellulose is synthesized by cellulose synthase complexes, which may contain 36 heteromeric
CESA proteins. Among the total 10 CESA genes in Arabidopsis, recessive mutations at three of them cause the
collapse of mature xylem cells in inflorescence stems of Arabidopsis (cesa4, cesa7, cesa8). These genes are
considered as secondary cell wall CESAs. The others (the function CESA10 is still unknown) are thought to be
specialized for the primary cell wall. The split-ubiquitin membrane yeast two-hybrid system was used to assess
the interactions between 4 primary (CESA1, CESA2, CESA3, CESA6) and 3 secondary cell wall Arabidopsis
CESA proteins (CESA4, CESA7, CESA8). Our results showed that some primary CESAs could physically
interact with some secondary CESAs. Analysis of transgenic lines showed that CESA1 could partly rescue cesa8
null mutants, resulting in complementation of plant growth defect, collapsed xylem and cellulose content
deficiency. These results suggest that mixed primary and secondary CESA complexes are functional using
experimental set-ups.
[1] A Carroll et al. (2012) Plant Physiol., 160, 726-37; [2] S Li et al. (2013) Plant signal Behav., 8 (3).
P2-05
The Arabidopsis Cellulase KORRIGAN1 associates with the Cellulose Synthase Complex
Vain T. (abe), Crowell E.F. (ab), Timpano H. (ab), Biot E. (ab), Desprez T. (ab), Mansoori N.Z. (cd), Höfte H. (ab), Trindade
L. (d) , Gonneau M. (ab), Vernhettes S. (ab)
(a) INRA, UMR1318, IJPB, Saclay Plant Sciences, F-78000 Versailles, France ; (b) AgroParisTech, IJPB, RD10, F-78000
Versailles, France; (c) Graduate School Experimental Plant Sciences, Wageningen University, The Netherlands ; (d)
Wageningen UR Plant Breeding, Wageningen University and Research Centre, 6708 PD Wageningen, The Netherlands ; (e)
Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Umeå , Sweden.
Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the
cell wall. Cellulose is synthesized by a large protein complex, which comprises at least three different cellulose
synthases (CESAs) and also requires the presence of an endo-1,4-β-D-glucanase, KORRIGAN1 (KOR1).
Although an endo-glucanase is also required for cellulose synthesis in bacteria, its role is unknown in both
bacteria and plants . We studied the localization and dynamics of KOR1 fused to green fluorescent protein (GFP),
expressed with the endogenous KOR1 promoter in the kor1-1 Arabidopsis mutant. In the epidermis of etiolated
hypocotyls, GFP-KOR1 is present in the same cells and at the same time as CESAs . GFP-KOR1 and GFPCESA3 migrate with comparable velocities along linear trajectories at the cell surface. In addition, we find that
the cortical microtubule array is involved in the organization of GFP-KOR1 at the plasma membrane as has been
previously shown for CESAs. Using both in vitro and in planta protein interaction assays, we show that KOR1
interacts with CESAs. Although GFP-KOR1 localizes at the plasma membrane and to intracellular compartments
similar to the ones observed for primary cell wall CESAs, the intracellular accumulation pattern upon treatment
with the cellulose synthesis inhibitor CGA325’615 is different. Our data support a model in which KOR1 is an
integral part of the cellulose synthase machinery.
[1] C. Somerville (2006) Annu Rev Cell Dev Biol, 22, 53-78; [2] E.F. Crowell, et al. (2009) The Plant cell, 21, 1141-54.
P2-06
Identification of potential protein and/or lipid components involved in (1,3;1,4)-β-D-glucan
biosynthesis in Italian ryegrass (Lolium multiflorum) suspension-cultured cells (SCCs)
Ho Y.Y. (a), Ralton J. (b), McConville M. (b), Bacic A. (ab), Doblin M.S. (a)
(a) ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Parkville, VIC 3010,
Australia; (b) Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010,
Australia.
(1,3:1,4)-β-D-Glucan (referred to as mixed linkage glucan; MLG) is an important non-cellulosic polysaccharide of
the cell walls of grasses (Poaceae). MLGs also have important human health benefits being a major soluble
dietary fibre component. MLG consists of blocks of either two or three (1,4)- β-glucosyl (Glc) residues separated
by a single (1,3)-β-Glc, with ~10% being composed of longer cellodextrin units. The arrangement and proportion
of these blocks influences their solubility and functionality. The commelinoid monocot-specific Cellulose
Synthase-Like (CSL) F and CSLH multi-gene families were identified as encoding the putative catalytic
components of the MLG synthase enzyme. Given the paucity of knowledge of the mechanim(s) of assembly for
MLG (or any other plant polysaccharide), our objectives are to identify additional components involved in this
process. Lolium SCCs making large amounts of MLG are being used both as an enzyme source and to generate
transcript and protein databases (J.Z. Zhou, M.S. Doblin, K. Ford, A. Cassin, C. M. Chin, W. Zeng and A. Bacic,
unpublished data). We are using co-immunoprecipitation and blue native gel techniques to address whether
CSLF6 (the major MLG synthase isoform) acts in a protein complex to synthesize MLG. The absence of MLG in
the Golgi suggests a multi-stage mechanism of biosynthesis that may involve a glycolipid or glycoprotein primer.
We are using solvent partitioning and TLC to fractionate, and GC-MS to characterise, glycolipids in the major
membrane fractions of Lolium SCCs to identify potential intermediates involved in MLG biosynthesis. Proteomic
data from co-immunoprecipitation experiments and glycolipid profiles of Lolium SCCs will be presented.
P2-07
Role of rhamnogalacturonan-II in pollen germination and pollen tube elongation
Dumont M. (a), Lehner A. (a), Voxeur A. (ab), Mollet J.-C. (a), Lerouge P. (a)
(a) Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale, UPRES EA 4358, IRIB, Normandy University,
University of Rouen, 76821 Mont Saint-Aignan Cedex, France; (b) present address: Institute of Molecular Plant Sciences,
University of Edinburgh, Edinburgh EH9 3JH, United Kingdom.
Rhamnogalacturonan-II (RG-II) is the most complex pectic polysaccharide present in the primary cell wall of all
land plants. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom
suggesting that this polymer has fundamental functions in the primary cell wall organization. To date, very little is
known about the biosynthesis of RG-II. Recently, a bioinformatic study identified 24 new putative
glycosyltransferases possibly involved in the Arabidopsis RG-II biosynthesis [1]. Among them, two sialyl-like
transferases, At1g08660 and At3g48820, were selected and proposed to be involved in the transfer of Kdo ( 3deoxy-D-manno-octulosonic acid) and/or Dha (3-deoxy-D-lyxo-heptulosaric acid) on the homogalacturonan
backbone of RG-II [2] because sialic acid is absent in plants [3] and these three acidic monosaccharides share
common structural features. Mutation in At1g08660 was previously shown to induce defects in pollen
germination [4]. As a consequence, we focused our study on At3g48820 using Arabidopsis pollen tubes as
experimental model. Homozygous mutant line is not available for this gene. Analyses of two heterozygous lines
revealed a strong reduction in pollen tube germination both in vitro and in vivo. Moreover, pollen tubes exhibited
abnormal swollen tubes by comparison with the wild-type pollen. These data suggest that sialyl-like transferases
are important for the cell wall formation and stability during pollen germination and pollen tube growth.
[1] A. Voxeur et al., (2012) PLoS One, 7, e51129.; [2] M. Séveno et al., (2010) Glycobiology, 20, 617-628; [3] M. Séveno et
al., (2004) Nat. Biotechnol., 22, 1351-1352. [4] Y. Deng et al., (2010) J. Integr. Plant Biol., 52, 829-843.
P2-08
MiR397 overexpression, a strategy to engineer laccase activity and lignification in model plants
and in crop plants and trees
Le-Bris P. (a), Mazel J. (a), Berthet S. (a), Wang Y. (a), Meynard D. (b), Guiderdoni E. (b), Gendrot G. (c), Rogowsky P. (c),
Leplé J.-C. (d), Bouchabké-Coussa O. (a), Sibout R. (a), Lapierre C. (a), Jouanin L. (a)
(a) IJPB, UMR1318 INRA-AgroParisTech,, F-78026 Versailles, France; (b) CIRAD, F-34398 Montpellier, France; (c) RDP,
UMR5667 CNRS-INRA-ENSL-UCBL, ENS Lyon, F-69364 Lyon, France; (d) AGPF, INRA Orléans, F-45075 Orléans,
France.
We have recently established that several laccases are involved in the lignification of Arabidopsis stems [1]. To
substantially reduce their lignin level and therefore improve their enzymatic degradability, it was necessary to
down-regulate the two main laccases expressed in Arabidopsis stems. This was made possible by the production
of double knockout Arabidopsis mutants, a strategy not available for all plant species and particularly for
dedicated energy crops.
Laccases are the endogenous targets of the highly conserved microRNA miR397 in plants. This microRNA is not
expressed in normal growth conditions, but its expression is induced by various stresses (including copper
deficiency [2]).
In this work, the overexpression of miR397 was considered as a strategy to simultaneously silence the laccases
putatively involved in the lignification of various plants (Arabidopsis thaliana, Brachypodium distachyon, rice,
maize, poplar). Constructs for the expression of miR397 under the control of constitutive or lignin-specific
promoters were introduced in these target plants. We selected transgenic plants displaying a high miR397
expression in stems and obtained some homozygous lines. We evaluated the impact of this miR397
overexpression on the transcripts of laccases considered as lignin-specific and on stem lignification.
[1] Berthet et al. (2011) Plant Cell, 23, 1124-1137; [2] Sunkar and Zhu (2004) Plant Cell, 16, 2001-2019.
P2-09
The reaction of vitamin C with reactive oxygen species in the apoplast
Dewhirst R.A., Fry S.C.
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, UK.
Vitamin C (ascorbate and dehydroascorbic acid) is vital for plants and found throughout the plant cell including
the apoplast. The structure of ascorbate was determined eighty years ago; however, many of its degradation
pathways remain unclear. Numerous degradation products of ascorbate have been reported to occur in the
apoplast but many still remain unidentified 1. Ascorbate is well known as an antioxidant, and acts to quench
reactive oxygen species (ROS), such as hydrogen peroxide and ozone in the plant apoplast. The reaction of
ascorbate degradation products dehydroascorbic acid (DHA) and diketogulonic acid (DKG) with various ROS has
been investigated by use of radiolabelled ascorbate. The oxidation products were separated by high-voltage paper
electrophoresis. Differences were observed in the products formed from the various ROS, allowing a unique
fingerprint of oxidation products to be described for each ROS. Equally, different compounds were produced
depending on the starting substrate; for example cyclic oxalyl threonate was only observed in the reactions of
DHA and not DKG. A further role of ascorbate is as a metabolic precursor to compounds such as oxalate and
threonate, both of which are produced during the reaction of ascorbate with ROS. Oxalyl threonate and cyclic
oxalyl threonate have the potential to form oxalyl esters with cell wall components, which would theoretically
allow the formation of cross-links between cell wall polysaccharides. Experiments to test this hypothesis will be
described.
[1] H.T. Parsons et al. (2011) Biochem. J., 440, 375-383.
We thank the UK BBSRC and Vitacress Salads Ltd for funding.
P2-10
Role of IRX3 cysteine modifications in the function of the cellulose synthase complex (CSC)
Kumar M. (a), Atanassov I. (ab), Carr P. (a), Blacklock L. (a), Turner S. (a)
(a) University of Manchester; Faculty of Life Sciences; The Michael Smith Building; Oxford Road, Manchester M13 9PT,
UK; (b) Present Address: AgroBioInstitute; 8 Dragan Tzankov Blvd; 1164 Sofia; Bulgaria.
Ecological concerns have led to recent global drive to produce and use more biofuels. Cellulosic ethanol from
plant cell walls is an attractive alternative to fossil fuels. Secondary cell walls (SCW) are mainly composed of
cellulose, hemi-cellulose and lignin. There has been some progress regarding modification of lignin quantity and
quality to improve cell was digestibility. However, research into understanding how plants make cellulose and
efforts to increase cellulose content of plant cell walls is lagging behind. Cellulose is synthesized at the plasma
membrane by a very large membrane bound complex known as the cellulose synthase complex (CSC). For
secondary cell wall cellulose synthesis three different CESA proteins are essential (AtCESA4, 7 and 8, also
known as IRX5, 3 and 1 respectively) that were initially identified using genetics. In contrast to progress made in
identifying components of the CSC and live cell imaging of the CSC little progress has been made in biochemical
studies of the CSC. Consequently many questions about the number of glucan chains synthesized by a single CSC
and how CSC organisation contributes to the structure of the cellulose microfibril remain unclear. We have been
investigating why yields of the CSC during purification are so poor and whether protein modification contributes
to this poor yield. In this poster, we present our recent results about the importance of cysteine modifications of
IRX3 in the functioning of CSC. IRX3 has 26 cysteines spread across the length of the protein. We mutated each
of those cysteines to serines and then used complementation analysis to identify which of the cysteines are
important. We have some preliminary data about how the cysteines are modified. Role of the modifications in
cellular context is discussed.
P2-11
Fasciclin-like Arabinogalactan Proteins: Influence of FLA mis-expression on cell wall
biochemistry and biomechanics in flax and poplar
Roach M.J. (a), Hobson N. (b), Deyholos M.K. (b), Mansfield S.D. (a)
(a) Department of Wood Science, University of British Columbia, Vancouver, Canada;(b) Department of Biological
Sciences, University of Alberta, Edmonton, Canada.
In Arabidopsis, the fasciclin-like arabinogalactan proteins AtFLA11 and AtFLA12 have been shown to
significantly influence cell wall biochemical and biomechanical properties, such as tensile strength, elasticity,
cellulose microfibril angle, and cell wall composition 1. In tissues that deposit cellulose-rich (G-fibre) secondary
walls, such as tension wood in angiosperms (eg. Poplar spp.) and phloem fibres in flax, there is increased
expression of AtFLA12 orthologs2, as well as an increase in the number of AtFLA12 orthologs in these species in
general (8 FLA12 orthologs in flax, and 22 FLA12 orthologs in poplar).
Expression analyses in both flax and poplar were conducted to determine which AtFLA11/12 orthologs were
more highly expressed in tissues undergoing secondary wall development. RNAi constructs were then generated
to target down-regulation of multiple orthologs of AtFLA11/12 in both flax and poplar independently. The
resulting flax and poplar transgenics cell walls were analysed biochemically and assessed for biomechanical
characteristics.
[1] C. MacMillan et al. (2010) Plant J., 62, 689-703. [2] N. Hobson et al. (2010) Russ J Plant Physiol, 57, 321-327.
P2-12
VND-INTERACTING PROTEIN2 is controlled by ubiquitin-mediated proteolysis
Yamaguchi M. (ab), Matsuda K. (c), Kato K. (c), Demura T. (cd)
(a) IEST, Saitama University, Japan; (b) PRESTO JST, Japan; (c) NAIST, Japan; (d) BMEP, RIKEN, Japan.
Previously, an NAC domain protein, VND7, has been identified as a key regulator of the xylem vessel
differentiation (Kubo et al. 2005). In addition, we have isolated another NAC domain protein, VNI2, as an
interacting factor with VND7 (Yamaguchi et al. 2010). VNI2 negatively regulates the xylem vessel differentiation
by inhibiting VND7 activity, and protein stability of VNI2 is tightly regulated through a PEST motif located on its
C-terminus. Here, to understand how VNI2 function is controlled during the differentiation, we screened
interacting factors with VNI2 by using yeast two-hybrid system. When full length VNI2 was used as a bait, a
cDNA encoding a RING finger protein was isolated. The RING finger protein is known to be an ubiquitin E3
ligase by forming complex with other proteins. Interestingly, VNI2 lacking the C terminal region containing
PEST motif is unable to bind to the RING finger protein, suggesting that the RING finger protein could regulate
stability of the VNI2 protein via the PEST motif.
P2-13
Heat stress differentially affects XET activity in organs of durum wheat plantlets
Iurlaro A. (a), De Caroli M. (a), Piro G. (a), Dalessandro G. (a), Fry S.C. (b), Lenucci M.S. (a)
(a) Di.S.Te.B.A., Università del Salento, 73100 Lecce, Italy; (b) The Edinburgh Cell Wall Group, ICMB, The University of
Edinburgh, Daniel Rutherford Building, Kings Buildings, Edinburgh, EH9 3JH, UK.
The “dot-blot” assay for xyloglucan endotransglycosylase (XET) activity, using xyloglucan as donor and a
sulphorhodamine-labelled eptasaccharide (XXXG-SR) as acceptor substrates, [1] was applied on crude extracts
from leaves, germinated caryopsides and roots (4-5 cm long proximal portion and 2 cm long apical portion) of 5day old durum wheat (Triticum durum Desf.) seedlings incubated under control (25°C) or heat stress (42°C)
conditions for 2 and 4 hours. XET activity was not affected by heat stress in leaves and germinated caryopsides,
however a significant inhibition was observed in the extracts obtained from the proximal (>68%) and apical
(>42%) portions of root of stressed seedlings, regardless of incubation time. No statistically significant difference
was detected in total proteins concentration of the extracts obtained from both control and stressed organs.
The application of an in vivo real-time assay to assess the activity of XET, by incubating the seedlings roots into
an aqueous solution containing XXXG-SR as molecular probe followed by confocal microscopy observation and
fluorescence quantification [2], allowed the dissection of the effect of heat stress on enzyme activity in the few
first millimetres from the root tip, region where the XET action on endogenous donor substrates is most
prominent. The first millimetre from the root tip of durum wheat seedlings showed an increase in XETincorporated fluorescence of 60.3% and 69.5% after 2 and 4 hours of heat stress, respectively, compared with the
control. While, XET activity was significantly inhibited in the following 3 millimetres.
Taken together these results show that XET activity is differentially influenced by heat stress in different organs
of durum wheat seedlings and, within the root, depending on the gradient of cell differentiation.
[1] S.C. Fry (1997) Plant J., 11, 1141-1150; [2] K. Vissenberg et al., (2000) Plant Cell, 12, 1229-1237.
P2-14
Mixed-linkage glucan biosynthesis in Physcomitrella patens expressing a CSLH gene from rice
Kiemle S. (a), Haeger A. (b), Stein A. (c), Lai V. (b), Wilson S. (d), Burton R.A. (e), Liepman A. (c), Gretz M. (a), Roberts
A. (b)
(a) Michigan Technological University, USA; (b) University of Rhode Island, USA; (c) Eastern Michigan University, USA;
(d) University of Melbourne, Australia; (e) University of Adelaide, Australia.
CELLULOSE SYNTHASE-LIKE (CSL) genes are proposed to encode glycan synthases that polymerize the
backbones of noncellulosic cell wall polysaccharides. This hypothesis has been confirmed for CSLA, CSLC,
CSLF and CSLH genes. However, members of the CSLB, CSLE, CSLG, and CSLJ families have not been
functionally characterized. The complete genome sequence of the moss Physcomitrella patens lacks members of
these uncharacterized CSL families, as well as CSLF and CSLH genes. The utility of P. patens as a heterologous
expression system for analysis of CSL function was investigated. Wild type P. patens was transformed with an
expression vector carrying a CSLH gene from rice and expression of OsCSLH1 mRNA and protein was detected
in protonemal filaments of transgenic P. patens. The presence of (1,3;1,4)-β-D-glucan in the cell walls of these
transgenic lines was demonstrated by immunocytochemistry, MALDI-TOF mass spectrometry, and linkage
analysis of oligosaccharides released by endo-1,3(4)-β-glucanase digestion. Methods for enhancing accessibility
of P. patens cell wall polysaccharides to enzyme digestion and solvent extraction also are described. This study
shows the potential of P. patens as a host for functional studies of plant cell wall biosynthetic enzymes.
P2-15
Regulation of kiwifruit softening by differential cell wall modifications
Fullerton C. (abc), Schröder R. (a), Hallett I. (a), Schaffer R. (ac), Atkinson R. (a) , Perera C. (b), Smith B. (b)
(a) The New Zealand Institute For Plant & Food Research Limited (Plant & Food Research), 120 Mt. Albert Road, Mt.
Albert, Private Bag 92 169, Auckland, New Zealand; (b) Food Science Programmes, School of Chemical Sciences, (c) Joint
Graduate School of Plant and Food Science; University of Auckland, Private Bag 92 019, Auckland, New Zealand.
The rate of softening in fruit has been shown to be mainly the result of differential enzyme activity, determining
how fast the fruit cell wall is deconstructed. In many fruit species this softening is controlled, at least in part, by
the ripening hormone ethylene. Kiwifruit (Actinidia spp.) is unusual because the fruit ripens to eating softness
seemingly independent of ethylene; however, application of ethylene can rapidly soften the fruit. Within
Actinidia there are closely related genotypes that show large differences in the rate of softening. We compared
three genotypes with different softening rates to investigate the biochemical, structural and molecular basis of
fruit softening with a view to identifying key softening regulators. Compositional analysis of cell walls during
softening showed differences in monosaccharide composition between genotypes at the same fruit firmness,
indicating that the rate of softening may not just be a matter of increased enzyme activity, but that there are
differences in the cell wall composition from early in development. Differences in the activities of key cell wall
enzymes, such as xyloglucan endotransglycosylase, suggest that the softening rate is influenced by the
modification of specific cell wall polysaccharides. Cell wall swelling, commonly observed in fruit cell walls
during softening, also occurred to a different extent amongst the three kiwifruit genotypes. Further work will
focus on the solubilisation and degradation of pectin and xyloglucan, using chemical, histological and molecular
techniques.
P2-16
Temporal deposition of β-1,4-galactan in the early stage of the development of Poplar gelatinous
layer
Yoshiura K., Awano T., Takabe K.
Laboratory of Tree Cell Biology, Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto
University Kyoto 606-8502, Japan.
Gelatinous layer (the G-layer) is formed in gelatinous fibre of tension wood and estimated to be involved in
tensional stress generation. The developed G-layer is known to have a high content of cellulose with high
crystallinity and a low content of hemicellulose and lignin. There is little information on the developing process of
the G-layer. We investigated immunolocalization of anti-β-1,4-galactan monoclonal antibody (LM5) in poplar
tension wood which was bent artificially at 30 degrees for 2 months and cut on the mid-July in Kyoto, Japan. In
the early stage of the development of gelatinous fibre, LM5 antibodies were localized in the entire area of the Glayer. In the later stage of development, however, labelling was restricted only in the outer part and the inner part
of the G-layer, which was same as a previous report on the mature G-layer [1]. We fractionated the developing
xylem into 2 parts (A: LM5 labelling was in the entire area of the G-layer, B: the labelling was restricted to the
outer and the inner part of the G-layer) and determined the neutral sugar composition of them by the alditolacetate method with gas chromatography. Fraction A contained 10.5% of galactose, whereas fraction B contained
2.8%. We measured the thickness of the G-layer along the radial direction from the cambium to the pith. The
thickness increased gradually toward the border between A and B, but in the gelatinous fibre next to the border in
B, the G-layer was thinner than that of the fibre next to the border in A (A:2.4μm, B:1.5μm). In B, the thickness
increased gradually toward the pith. In summary, β-1,4-galactan is deposited in the entire G-layer in the early
stage of the development. On the way of development, however, most of them would be removed, accompanying
with structural changes of the G-layer.
[1] Arend M (2008) Tree Physiology, 28, 1263-1267.
P2-17
Activity, localization and function of xylanases in differentiating poplar xylem
Tanaka R. (a), Awano T. (a), Takabe K. (a), Mellerowicz E.J. (b)
(a) Laboratory of Tree Cell Biology, Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto
University, Kyoto 606-8502, Japan ; (b) Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology,
SE-90183 Umeå, Sweden.
The mechanism and the function of xylan post-synthetic modifications in cell walls are poorly understood. On the
hypothesis that xylanases modify the xylan structure in differentiating xylem, we investigated xylanase activity
and Xyn10A, a poplar xylanase, in differentiating poplar xylem. PtxtXyn10A is a 101kDa peptide and has three
CBM22 domains, a GH10 catalytic domain and no signal peptide in silico. Polyclonal antibody 7597 was raised
against the C-terminus peptides of PtxtXyn10A. Proteins in differentiating poplar xylem were extracted under two
conditions: native conditions (extract A) and denaturing conditions (extract B). The antibody recognized
approximately 100, 63, and 56kDa proteins in extract B, however, only 56kDa one was detected in extract A.
Extract A has β-xylanase and trans-β-xylanase activity on xylohexaose. The activity didn’t decrease when the
antibody was added to the extract. Labeling by the antibody was localized in cell wall of xylem elements during
the secondary wall formation. This localization coincided with that of LM10 anti-xylan antibody labeling in
differentiating xylem. To undergo transglycosylation under these conditions, xylohexaose was successfully outcompeting an approximately 8900-fold molar excess of H 2O. Moreover, in Arabidopsis, more than 80% of xylan
molecules have a unique sequence of glycosyl residues at their reducing ends [1]. Therefore, it seems that particular
xylan molecules are hydrolyzed or transglycosylation is a major reaction. Xyn10A is likely to be secreted by
nonclassical pathway, binds to xylan, modifies xylan structure by transglycosylation and undergo proteolytic
cleavage to inactive form.
[1] Peña et al. (2007) The Plant Cell, 19, 549-563.
P2-18
Pectins demethylesterification and seed development
Turbant A. (a), Bouton S. (a), Fournet F. (a), Pageau K. (a), Marcello P. (b), Pelloux J. (a), Van Wuytswinkel O. (a)
(a) EA3900 BIOPI Biologie des Plantes et Innovation, UFR des Sciences, 33 rue Saint Leu, F-80039 Amiens France; (b)
Plateforme ICAP, UFR de Pharmacie, rue de Louvel, F-80039 Amiens France.
The cell wall surrounding plant cells is largely composed of polysaccharides, in particular cellulose microfibrils
and hemicellulose muddled in a pectic matrix [1]. Among pectins components, homogalacturonans (HGs) are the
most abundant. HGs are deposited in the cell wall as a highly methylesterified form, which can be the target of
cell wall enzymes such as pectin methylesterases (PMEs) [2]. PMEs activity controls HGs demethylesterification
leading to structural modifications involved in several developmental processes such as cell elongation or root
initiation [3]. The objective of this work is to study the influence of PMEs activity and of their specific inhibitors,
PMEIs, on seed development. Indeed, according to bio-computing data, PME and PMEI genes are specifically
expressed during Arabidopsis seed formation. Five PME and two PMEI genes expressed in the seed coat and
three PME genes expressed in the embryo were selected. Their expression specificity was confirmed by qRT-PCR
and promoter/reporter fusion constructs (GFP and GUS). In order to characterize their biological function,
mutants were obtained for each selected gene. Their phenotypic study seems to indicate a function of some seed
coat expressed PMEs in the structuration of the mucilage adherent layer.
[1] Cosgrove D.J. (2001) Plant Physiol., 125, 131-134; [2] Willats et al. (2006) Trends Food Sci. Tech., 17, 97104; [3] Pelloux et al. (2007) Trends Plant Sci., 12, 267-277.
P2-19
A role for pectin methylesterases (PMEs) in dark-grown hypocotyl elongation in Arabidopsis
Hocq L. (a), Sénéchal F. (a), Surcouf O. (b), Demailly H. (c), Mareck A. (b), Fournet F. (a), Assoumou Ndong Y. (a),
Mouille G. (e), Marcelo P. (d), Höfte H. (d), Peaucelle A. (d), Gutierrez L. (c), Lerouge P. (b), Lefebvre V. (a), Pelloux J. (a)
(a) EA3900-BIOPI, UPJV, 33 Rue St Leu, F-80039 Amiens, France; (b) EA4358-Glyco-MEV, IFRMP 23, Université de
Rouen, F-76821 Mont-Saint-Aignan, France; EA3900-BIOPI, UPJV, 33 Rue St Leu, F-80039 Amiens, France; (c) CRRBM,
UPJV, 33 Rue St Leu, F-80039 Amiens, France; (d) IJPB, UMR1318 INRA-AgroParisTech, Bâtiment 2, INRA Centre de
Versailles-Grignon, Route de St Cyr (RD 10), F-78026 Versailles, France; (e) ICAP, UPJV, 1-3 Rue des Louvels, F-80037
Amiens, France.
Understanding the role of pectin modifications in the control of cell expansion requires the use of a simple model
in which developmental and cell biology, genomics, biochemistry, and biophysics can be integrated at a cellular
level. The Arabidopsis dark-grown hypocotyl, which elongates in absence of cell divisions and is characterized by
two distinct growth phases, is therefore a powerful system to analyze the impact of the cell wall plasticity in cell
elongation. Transcriptome analysis of isolated hypocotyls showed that twenty-two genes involved in
homogalacturonan (HG) modifications (PMEs, PMEIs PGs, PLs) were differentially regulated during the growth
acceleration [1]. Here, we report the characterization of 2 PMEs, which are both expressed in dark-grown
hypocotyl during acceleration phase. Using isoelectric focusing, we showed the disappearance of PME activity
bands in both knock-out plants, allowing the identification of 2 PME isoforms. However, overall changes in total
PME activity were different when considering the two pme mutants. This is likely to be related to compensatory
mechanisms among the large PME gene family. The biochemical and phenotypical characterization of pme
mutants will be shown. Based on these results, a model for the roles of PMEs in the changes of HG structure
during hypocotyl elongation will be presented.
[1] Pelletier et al. (2010) New Phytol. 188, 726-739.
P2-20
Xylan deposition and lignification in differentiating xylem in Mallotus japonicus under tension
stress
Higaki A., Yoshinaga A., Takabe K.
Laboratory of Tree Cell Biology, Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto
University, Japan.
It has been hypothesized that xylan acts as a host structure for lignification [1]. We investigated the process of xylan
deposition and lignification in differentiating xylem of Mallotus japonicus under tension stress to give insight to
the relationship between lignin and xylan. A tree of M. japonicus was artificially inclined at 30-45˚. After about
three months, differentiating xylem forming tension wood was collected from the upper side of inclination.
Distribution of lignin was investigated by UV microscopy and TEM. Localization of xylan was observed by
immunolabeling with monoclonal antibodies (LM10 and LM11 [2]). In M. japonicus, very thin lignified layers (Llayers) were found in G-layers. Cell wall organization of tension wood fiber was S1+S2+G+n(L+G) (n=1 or 2),
where n means number of repetition. In gelatinous fibers, lignin was distributed in compound middle lamella
(CML), S1, S2, and L-layers, whereas no lignin was found in G-layers. Xylan labeling was found in lignified
CML, S1, S2 and L-layers. Lignification in CML and S1 and S2 layers proceeded during formation of G-layer.
Moreover, the labeling density of gold particles in S 1 and S2 layers increased during development of cell wall. The
thickness of S1 decreased until S2 layer formation, and that of S 2 layers decreased until early stage of G-layer.
Therefore increase in xylan labeling is probably due to the decrease in thickness of these layers rather than xylan
deposition during G-layer formation. This shrinkage of secondary wall layers may be caused by lignification or by
G-layer formation in relation to tension stress generation.
[1] D. Reis et al. (2004) Comptes Rendus Biologies 327, 785-790; [2] L. McCartney et al. (2005) J. Histochem. Cytochem.
53, 543-546.
P2-21
Xyloglucan β-glucosidase in Arabidopsis
Valdivia E.R., Sampedro J., Gianzo C., Revilla G., Zarra I.
Departamento de Fisiología Vegetal, Universidad de Santiago, Santiago de Compostela 15782, Spain.
The precise role of xyloglucan in the regulation of cell wall extension is still unclear. There is evidence that
xyloglucan chains can connect cellulose microfibrils and slow down their separation. However it is clear that
other cell wall components, such as pectins, can also regulate wall extension. Our group has been studying
Arabidopsis mutants deficient in different glycosidases that act on xyloglucan. The results have led us to conclude
that these enzymes can digest the non-reducing ends of polymeric xyloglucan and that their activity is necessary
for the correct elongation of siliques and sepals [1, 2].
We have identified an Arabidopsis mutant with an insertion in BGLC1 (At5g20950) that lacks extractable βglucosidase activity against xyloglucan oligosaccharides. In addition heterologous expression of BGLC1 in
tobacco resulted in high levels of β-glucosidase activity. The expression pattern of this gene, as shown by a
promoter-reporter construct, is similar to that of xyloglucan α-xylosidase and β-galactosidase. Expression levels
are high in cells undergoing elongation or cell wall remodelling, such as vascular bundles, stomata, trichomes,
anther filaments or abscission zones.
Lack of xyloglucan α-xylosidase or β-galactosidase results in the accumulation of partially digested xyloglucan
subunits within the polymer. These subunits did not accumulate in the xyloglucan of bglc1 plants, which has a
normal composition. Moreover bglc1 plants have normal siliques unlike plants deficient in other xyloglucan
glycosidases. We are currently exploring different explanations for this discrepancy.
[1] Sampedro et al. (2010) Plant Physiol., 154, 1105-1115; [2] Sampedro et al. (2012) Plant Physiol., 158, 1146-1157.
P2-22
Characterisation of Nucleotide Sugar Transporters in Grapevine (Vitis vinifera L.)
Utz D., Handford M.
Laboratorio de Biología Molecular Vegetal, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
In plants, the synthesis of most non-cellulosic polysaccharides occurs in the Golgi apparatus. Here, glycosylation
reactions are catalysed by glycosyltransferases. The substrates for the synthesis of these glycans are sugars that
are activated by the addition of a nucleotide, most of which are made in the cytosol. Nucleotide sugar transporters
(NSTs) are located in the membrane of the Golgi body and ER, and carry nucleotide sugars into the lumen of
these organelles [1]. Some NSTs in A. thaliana have been functionally characterised; the GONST1-5 family is
specific for GDP-sugars and localised in the Golgi [2]. In Vitis vinifera L., it has been determined that the noncellulosic polysaccharides contain sugars derived from GDP-sugars [3]. To determine the conservation of the
mechanism involved in the synthesis of non-cellulosic polysaccharides, the grapevine genome was analysed
bioinformatically for the presence of GONST orthologues. Two sequences with ≥ 78% identity at the amino acid
level were identified, both of which possess the molecular characteristics of those NSTs. We have called these
orthologues VvGONST-A and VvGONST-B. The cloning of both NSTs was performed successfully, and both are
expressed throughout berry development as determined by qRT-PCR. By transient transformation of tobacco
leaves with VvGONST-A::GFP and VvGONST-B::GFP, we determined that both are localised in the Golgi, as
demonstrated by their sensitivity to brefeldin A and their colocalisation with GmMan1 [4]. Finally, we are
determining their functionality in a nucleotide-sugar transport deficient mutant of GDP-sugars of S. cerevisiae and
experiments are underway to determine the transport specificity of VvGONST-A and VvGONST-B using
radiolabelled nucleotide sugars.
[1] F. Reyes et al. (2008) Curr. Opin. Plant Biol., 11, 244-251; [2] M. Handford et al. (2004) Mol. Gen. Genomics, 272, 397410 ; [3] K. Nunan et al. (1998) Plant Physiol., 118, 783-792 ; [4] B. Nelson et al. (2007) Plant J., 51, 1126-1136.
Funding: CONICYT 21090418 and 24120980.
P2-23
Relationship between microtubules and secondary cell wall in xylem vessels
Derbyshire P. (a), Ménard D. (b), Lloyd C. (a), Pesquet E. (b)
(a) John Innes Centre, Norwich Research Park, Colney Lane,NR4 7UH Norwich, UK.; (b) Umeå Plant Science Centre
(UPSC), Department of Plant Physiology, Umeå University, 901 87 Umeå, Sweden.
Xylem vessels are essential for transporting water and minerals transport as well as for mechanical resistance
against gravity. These key characteristics of xylem vessels directly depend on the development of specific
patterned secondary thickenings reinforcing the lateral cell walls. The patterns described by secondary wall
thickenings can be annular, spiral, reticulate or pitted. Disturbance of the overall pattern of the secondary cell wall
can be achieved by modifying microtubule stability. Two previously identified microtubule-associated proteins
(MAPs) – MAP70-5 and MIDD1 – were already shown to control xylem vessel secondary cell wall patterning in
xylogenic cell culture systems [1]. An inducible xylogenic system from Arabidopsis thaliana cell cultures was
used to better understand the microtubule dependant guiding mechanisms controlling xylem vessel secondary cell
wall deposition [2]. A systems biology approach, combining both transcriptomic and quantitative proteomic of
MAPs, was performed along in vitro xylem vessel ontogenesis. Using this approach, new insights on MAP
identity and levels of regulation were unravelled during vessel formation [3]. Altogether, pharmacological and the
genetic modulation of newly identified MAPs demonstrate that these cortical microtubules control the orientation,
the patterning, the symmetry and the deposition of the secondary wall of xylem vessels.
[1] E. Pesquet and C. Lloyd (2011) The Plant cytoskeleton; [2] E. Pesquet et al. (2010) Curr. Biol., 20, 744-749; [3] P.
Derbyshire et al. (2013) in preparation.
P2-24
Cell walls in developing wheat and rice grains
Palmer R. (a), Shewry P. (a), Tosi P. (a), Knox J.P. (b)
(a) Rothamsted Research, UK; (b) University of Leeds, UK.
Cell walls contribute about 2-3% of the mature wheat and rice endosperm, and, given the important role of these
cereals in the human diet, they represent an important source of dietary fibre. Whilst the cell wall polysaccharide
composition in wheat and rice grain is well characterized, little is known of the distribution of these
polysaccharides amongst the different grain tissues.
Through the use of immunocytochemistry, we aim to characterize the spatial distribution of the cell wall
polysaccharides in both wheat and rice grain, and how this may change during grain development.
Preliminary results show that the spatial regulation of cell wall polysaccharides changes dramatically from the
cellularisation stage (4 DAA) to maturity (~28 DAA). Immunolabelling with antibody specific for arabinoxylan
and mixed-linkage β-glucan show very little labelling in the 4 DAA starchy endosperm while uniform and strong
labelling can be observed in the same tissue at maturity. Conversely, 1-4 galactan antibodies strongly label the
newly cellularized endosperm but at maturity the labelling is confined to the aleurone and nucellar epidermis.
Clear differences can be observed between the cell wall of wheat at rice both in terms of their polysaccharide
composition, amount, and spatial distribution: in 12 DAA grains, for example, antibodies specific to mixedlinkage β-glucan, label all grain tissues in wheat while labelling is absent in the aleurone and nucellar epidermis of
rice.
P2-25
The Tale of GalT14 and its possible role in AGP glycan biosynthesis in Arabidopsis thaliana
Narciso J.O. (a), Doblin M.S. (a), Zeng W. (a), Lampugnani E. (b), Johnson K.L. (a) , Newbigin E. (b), Bacic A. (a,c)
(a) ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Parkville, VIC 3010,
Australia; (b) Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, VIC 3010,
Australia; (c) Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010,
Australia.
Arabinogalactan-proteins (AGPs), members of the hydroxyproline-rich superfamily, are proteoglycans that are
found in the plant cell wall and plasma membrane and in secretions. AGPs have been proposed to have a variety
of functions in planta. In Arabidopsis thaliana, these proteins are likely to be important in the initiation of female
gametogenesis, signal transduction pathways regulating plant growth and development, modulation of
phytohormone activity responsible for root growth and seed germination, pollen tube germination, cell division
and cell expansion. In general, AGPs are characterized by the presence of arabinose and galactose as the
predominant sugars in their carbohydrate moiety. The AGP polysaccharide belongs to the type II arabinogalactan
(AG), in which the backbone β–D-Gal is 3-, 6-, and 3,6-linked. How the carbohydrate decoration and the β-(1,3)Gal linkages of AGPs are synthesised is not well understood, and only a few glycosyltransferases (GTs) have
been associated with its synthesis. One GT family, GT31, has been predicted to form β-(1,3)-Gal linkages [1]. Our
research aim is to explore the functions of GT31 members GalT12 and GalT14 in the formation of β-(1,3)-Gal
linkages and determine whether they have roles in AGP biosynthesis. Through genotyping and phenotypic
analysis of a galt14 T-DNA insertion mutant, we have found this line has a number of phenotypic alterations in
comparison to wild-type. The characterization of the galt14 mutant will be described and the potential role of
GalT14 in AGP glycan synthesis discussed.
[1] Y. M. Qu et al., Plant Molecular Biology 2008, 68, 43-59.
P2-26
Pectin-related gene expression in flax (Linum usitatissimum): role of pectinesterases (PME) in bast
fiber development
Pinzon D., Galindo L., Deyholos M.K.
Department of Biological Sciences, University of Alberta, Edmonton, Canada.
L. usitatissimum is an annual eudicot for which two types are cultivated: linseed and fiber flax. The stem fibers of
linseed are not generally used commercially because they are of lower quality and yield than those obtained from
fiber flax. Moreover, the extraction of fibers by dew-retting is not possible in the climate of Canada. Flax fibers
elongate intrusively. Thus, the middle lamella between fibers and between fibers and surrounding tissues affects
fiber elongation and also fiber extractability. The degree and pattern of methylesterification of galacturonic acid
(GalA) residues in homogalacturonan (HG) influences the rigidity of the middle lamella and cell wall.
Pectinesterases (PME) mediate the de-esterification of GalA in muro, in a linear or random fashion, resulting in
wall rigidification, or wall loosening, respectively. Using transcript profiling assays (e.g. qRT-PCR, RNA-Seq),
we confirmed the expression patterns of 66 PMEs and 76 PMEIs in different tissues and developmental stages.
The degree of methylesterification of the fibers cell wall was also assessed using pectin specific antibodies. Based
on these expression data, and phylogenetic relationships with other PMEs in Arabidopsis and poplar, we selected
a subset of flax PMEs and pectate lyases (PLs) for further study, including heterologous expression and
biochemical characterization. Finally, we have developed a reverse genetics platform in linseed flax that allows
identification of mutations in genes of interest, including PMEs. We describe our progress towards obtaining lossof-function mutants in selected PME genes.
P2-27
Fucosylation of Arabidopsis arabinogalactan proteins
Tryfona T. (a), Lopez-Fernandez F. (a), Wagner T. (b), Keegstra K. (b), Dupree P. (a)
(a) School of Biological Sciences, Department of Biochemistry, Hopkins Building, The Downing site, Tennis Court Road,
Cambridge University, CB2 1QW Cambridge, UK; (b) Michigan State University, Department of Plant Biology and
Biotechnology and Molecular Biology, East Lansing, MI 48824, USA.
Plant type II AG polysaccharides are attached to arabinogalactan proteins (AGPs) usually at hydroxyproline
residues. Type II AGs are overwhelmingly diverse and heterogeneous but have some common structural
characteristics such as composing of a galactan backbone with β-1,6-linked galactan side chains modified with
arabinose or other less abundant sugars such as glucuronic acid or rhamnose. We have recently reported the
presence of fucose (Fuc) residues on Arabidopsis leaf AGPs [1] and we are interested in the structural analysis of
fucosylated AGPs and their function. In addition, we are interested in the discovery of glycosyl transferases (GTs)
involved in the biosynthesis of these molecules and in their function. Here we investigate the role of fucosylated
AG oligosaccharides in Arabidopsis fut4, fut6 and fut4/fut6 mutant plants. Under certain growth conditions the
fut4/fut6 mutant exhibited a shorter root phenotype. HPAEC-PAD monosaccharide analysis of Arabidopsis leaf
AGP extracts, revealed a significant reduction in Fuc content for fut4 mutant although the amount of Fuc was not
affected in fut6 mutant but was absent in fut4/fut6 mutant. In addition, Fuc was reduced in both fut4 and fut6
mutants in root AGP extracts and was absent in fut4/fut6 mutant. In all cases reduction of Fuc was accompanied
with reduction in xylose levels. Sequential digestion with AG specific enzymes and consequent analysis by
Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE), Matrix Assisted Laser
Desorption/Ionisation (MALDI)-Time of Flight (ToF)-Mass spectrometry (MS) and Hydrophilic Interactions
Liquid Chromatography (HILIC) revealed that FUT4 is solely responsible for the fucosylation of AGPs in leaves.
Both FUT4 and FUT6 enzymes however are required for the fucosylation of AGPs in roots. Structural
characterisation of root AGPs indicated the presence of an abundant fucosylated oligosaccharide. Detailed
structural characterisation with high energy MALDI-Collision Induced Dissociation (CID) revealed a unique
oligosaccharide structure.
[1] T. Tryfona et al. (2012) Plant Physiol., 160, 653-666.
P2-28
Cellulose Synthase Interactive 3 Coordinates with Cellulose Synthase Interactive 1 in the
regulation of Cellulose Biosynthesis
Lei L., Li S., Gu Y.
Center for Lignocellulose Structure and Formation, Department of Biochemistry and Molecular Biology, Pennsylvania State
University, University Park, PA 16802, USA.
The coordination between the orientation of cortical microtubules and the orientation of nascent cellulose
microfibrils is important for anisotropic plant cell growth. Cellulose synthase interactive 1 (CSI1) / POM-POM2
is a key scaffold for guidance of primary cellulose synthase complexes (CSCs) along cortical microtubules during
cellulose biosynthesis 1,2,3,4. Here, we characterize the function of CSI1-like proteins in Arabidopsis. csi1csi3
double mutants show enhanced cell expansion defect, which is consistent with additive reduction of CSC
velocities. Similar to CSI1, CSI3 associates with primary CSCs both in vitro and in vivo. However, pCSI1: GFPCSI3 cannot complement the anisotropic cell growth defect in csi1 mutants, suggesting that CSI3 is not
functionally equivalent of CSI1. Interestingly, the function of CSI3 is partially dependent on CSI1. We propose
that CSI3 is an important regulator in plant cellulose biosynthesis and plant anisotropic cell growth. The potential
mechanism how CSI1 proteins may influence the velocity of CSCs will be discussed.
[1] Li et al. (2012) Proc. Natl. Acad. Sci., 109, 185-190; [2] Bringmann et al. (2012) Plant Cell, 24, 163-177 ; [3] Lei et al.
(2012) Plant Signal. Behav., 7, 714-718; [4] Baskin et al. (2012) Cell Adhesion Migration, 6, 1-5.
P2-29
A GT47 family glycosyl transferase from Nicotiana pollen mediates the synthesis of (1,5)-α-Larabinan when expressed in Arabidopsis thaliana
Lampugnani E.R. (a), Moller I.E. (ab), Cassin A. (ab), Koh P.L. (a), Wilson S. (ab), Bacic A. (abc), Newbigin E. (a)
(a) School of Botany; (b) ARC Centre of Excellence in Plant Cell Walls, School of Botany; (c) Bio21 Molecular Science and
Biotechnology Institute, University of Melbourne, VIC 3010, Australia.
Tobacco pollen tubes are used to study plant cell wall synthesis and assembly because of their relatively simple
walls that are mainly composed of the polysaccharides (1,3)-β-D-glucan (callose) and (1,5)-α-L-arabinan, along
with lesser amounts of cellulose, homogalacturonan and xyloglucan. As part of an RNA-Seq analysis several
putative GT cDNAs were identified in Nicotiana alata pollen grains. One of these (NaARAT1) encodes a type II
membrane protein of 489 amino acids with high sequence similarity to AtARAD1, a presumed (1,5)-αarabinosyltransferase (AraT) from Arabidopsis belonging to CAZy glycosyltransferase family 47. Here we show
that the NaARAT1 promoter directs GUS expression in vegetative tissues of Arabidopsis as well as in pollen
grains, and that a fluorescently tagged version of NaARAT1, when transiently expressed in tobacco pollen tubes
and leaves, is targeted to the Golgi apparatus, a location that is consistent with a presumed role for enzymes
involved in arabinan synthesis. More importantly, Arabidopsis plants constitutively expressing NaARAT1 have
increased levels of arabinan in their cell walls, consistent with the identity of NaARAT1 as a (1,5)-α-L-arabinan
transferase.
P2-30
AtPME48 encodes a pectin methylesterase involved in Arabidopsis pollen grain germination
Lehner A. (a), Leroux C. (a), Guénin S. (b), Fournet F. (b), Kiefer-Meyer M.-C. (a), Pelloux J. (b), Driouich A. (a), Lerouge
P. (a), Mollet J.-C. (a)
(a) Laboratoire Glycobiologie et Matrice Extracellulaire Végétale, UPRES-EA 4358, PRIMACEN IBiSA, IRIB, Université de
Rouen, 76821 Mont Saint-Aignan Cedex, France; (b) Biologie des Plantes et Innovation, UPRES-EA 3900, Université de
Picardie-Jules Verne, 80039 Amiens, France.
Germination of pollen grain is a crucial step of plant reproduction. However, the molecular mechanisms involved
in pollen grain germination remain unclear. Thus, we investigated the role a pectin methylesterase implicated in
the remodeling of pectin, PME48, during the germination of Arabidopsis pollen grain. A combination of
functional genomics, gene expression, in vivo and in vitro pollen germination and immunolabeling were used on
wild-type and knock-out mutant for AtPME48. We showed that AtPME48 is specifically expressed in male
gametophyte and that its expression decreased rapidly upon imbibition and germination. Pollen grains from
knock-out mutant displayed a significant delay in imbibition and germination. Moreover, numerous pollen grains
showed two tips emerging from the same pollen grain. Immunolabeling experiments suggested that the content of
methylesterified pectic epitopes was higher in the mutant. Although pollen specific PME are traditionally
associated with pollen tube elongation, this study provides substantial evidence that pectin methylesterases are
also directly or indirectly implicated in the changes of the mechanical properties of the intine during maturation
and germination of Arabidopsis pollen grains.
P2-31
Plasma membranes of developing xylem of Norway spruce in monolignol transport studies
Väisänen E. (ab), Takahashi-Schmidt J. (abc), Blokhina O. (a), Fagerstedt K. (a), Kärkönen A. (b)
(a) Dept of Biosciences, Plant Biology, (b) Dept of Agricultural Sciences; Univ. of Helsinki, Finland; (c) Present address:
Swedish University of Agricultural Sciences, Umeå Plant Science Centre, Sweden.
The water transporting plant cells and cells of dead supporting tissues (fibers, stone cells) have a thick secondary
cell wall that is composed mostly of cellulose and lignin. Lignin is a strong, phenolic polymer that makes the cell
wall impermeable to water. Monolignols, the precursors of lignin, are synthesized in the cytoplasm via the
phenylpropanoid pathway. The precursors are then transported to the cell wall, where they are polymerized into
lignin. In Arabidopsis leaf tissue the transport of a major monolignol, coniferyl alcohol, has been observed to be
ABC-transporter-mediated [1]. In addition, an ABC-transporter that transports p-coumaryl alcohol, another
monolignol, has been identified in Arabidopsis [2]. However, it is not yet known whether the same transporters
are functioning in developing xylem and/or in other plant species.
We are investigating the monolignol transport mechanism in developing xylem of an economically important
gymnosperm species, Norway spruce (Picea abies), where coniferyl alcohol is the major constituent of lignin. We
have optimized a two-phase partitioning protocol [3] for plasma membrane (PM) vesicle enrichment for
developing xylem and phloem. The enrichment of PM can be shown by enzyme marker tests. We follow the
transport of [14C]coniferyl alcohol and its glucoside, [14C]coniferin, into the vesicles of the enriched PM
fraction. In contrast to the results by [1], our preliminary results do not show any ATP-dependent transport
activity for coniferyl alcohol in developing spruce xylem. However, ATP-dependent coniferin transport was
detected in xylem vesicles. This transport could not be observed with the PM of phloem. Our experiments do not
exclude the possibility that free coniferyl alcohol is transported by a non-ATP-dependent manner at the xylem
PM.
[1] Miao and Liu (2010) PNAS, 107, 22728-22733; [2] Alejandro et al. (2012) Curr.Biol., 22, 1207-1212; [3] Widell &
Larsson (1981) Physiol. Plant., 51, 368-374.
P2-32
Insights into the site of cleavage by GH12 and GH16 enzymes acting on poaceaen xyloglucan
Simmons T.J., Fry S.C.
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, University of Edinburgh, UK.
The hemicellulose xyloglucan appears ubiquitous in land plants but is absent elsewhere. It comprises a β-(1→4)D-glucan backbone with taxonomically dependent xylosylation patterns: XXXG prevails in dicots while XXGG
and XXGGG prevail in the Poaceae. 1 Xyloglucan endotransglucosylase activity (XET) is also ubiquitous and
thought to be crucial in plant physiology. 2 Here we devise a novel method to (a) demonstrate discrepancies
between XET and xyloglucan endoglucanase (XEG) sites of attack, and (b) investigate XET site of attack in vivo
and in vitro, by analysing the products of sequential XET–XEG treatments using reductively tritiated acceptor
substrates, e.g. [3H]XXLGol. Novel products (e.g. [3H]GGXXLGol), identified by β-D-glucosidase treatment,
were present when poaceaen, but not when tamarind, xyloglucan was used; this indicates that the two enzymes
cleaved the former but not the latter at different positions. Inferred recognition motifs show this is the product of
subunit length variation in poaceaen xyloglucan and are consistent with earlier work 3,4. To test whether this
distinction in site of attack represents a general GH16 : GH12 distinction, we subjected XET products to GH16
xyloglucan endohydrolase digestion, the results of which will be discussed. Finally, distinct qualitative differences
between products formed in different organs suggest structural dissimilarity between the xyloglucan present there,
with XXGGG prevailing in shoots and XXGG in roots.
[1] Y.S. Hsieh & P.J. Harris (2009) Mol. Plant, 2, 943–65; [2] J.K. Rose et al. (2002) Plant. Cell. Physiol., 43, 1421–35; [3]
Z. Jia et al. (2003) Carbohydr. Res., 338:1197–208; [4] M. Saura-Valls et al. (2008) J. Biol. Chem., 283, 21853–63.
We thank the BBSRC for funding.
P2-33
Functional characterization of a CELLULOSE SYNTHASE-LIKE B (CSLB) protein from
Populus trichocarpa
Díaz-Moreno S.M., Ekengren S., Mélida H., Vilaplana F., Srivastava V., Bulone V.
Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, SE10691 Stockholm, Sweden.
The cellulose synthase like (Csl) genes are distributed in 9 subgroups out of a total of 10 within the cellulose
synthase family of genes in higher plants. Based on reverse genetic approaches and heterologous expression, some
Csl genes have been shown to encode enzymes that are involved in the biosynthesis of the backbone of different
hemicelluloses. But the biochemical activity of most of these enzymes remains to be directly verified [1]. CSLB
are gathered in an unexplored group that occurs only in dicots but that is phylogenetically related to CSLH [2].
Members of the latter group are involved in the synthesis of mixed-linkage β-glucans in grasses [3]. Here, we
report the preliminary functional characterization of a member of the CSLB family from Populus trichocarpa
using different approaches. We have cloned one of the two PtCslB genes and expressed it in yeast and
Arabidopsis for biochemical and functional characterization. Interestingly, we have found differences in the cell
wall composition of the yeast expressing the protein that most likely reflect the catalytic activity of PtCSLB.
Moreover silencing of the orthologous gene in tobacco produced changes in the expression level of cell wall
related genes. A series of evidence that support the biochemical activity of PtCSLB will be presented.
[1] Carpita, N. C. (2011). Plant Physiol. 155, 171-184; [2] Yin, Y., J. Huang, et al. (2009). BMC Plant Biol. 9, 99; [3]
Fincher, G. B. (2009). Plant Physiol. 149, 27-37.
P2-34
New Transcription factors regulating lignified secondary cell wall formation in Eucalyptus
Wang H. (a), Soler M. (a), Yu H. (a), Plasencia A. (a), San Clemente H. (a), Ladouce N. (a), Hefer C. (b), Myburg A. (b),
Pinto Paiva J. (c), Grima-Pettenati J. (a)
(a) LRSV,UMR5546 Université Toulouse III /CNRS, 31326 Castanet Tolosan, France; (b) Forestry and Agricultural
Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa; (c) Instituto de Biologia Experimental e
Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.
Lignified secondary cell walls (SCW), the main component of wood, are the most abundant source of renewable
biomass on earth with enormous economic potential, notably for construction, pulp and bioenergy production.
Because transcriptional regulation is a major mechanism controlling SCW formation, our aim is to identify and
characterize master regulatory transcription factors dictating SCW deposition during wood formation in
Eucalyptus, the most planted hardwood genus worldwide. Taking advantage of recent release of E. grandis
genome, we have performed a genome-wide survey of several important gene families for wood formation
including MYB, Aux/IAA and ARF. We identified all members of these gene families and performed cophylogenetic analyses, which revealed woody plant preferential or highly expanded clades when compared to
Arabidopsis, rice, polar and grapevine. Using RNAseq and high-throughput RT-qPCR technique, we examined
the expression patterns of these genes in different tissues and organs during normal development and/or in
responses to environmental or hormonal cues. This enabled us to identify genes with preferential and/or specific
expression in wood cells undergoing SCW thickening. Among these genes we selected those exhibiting
differential expression between contrasting wood samples for functional characterization in planta using overexpression and dominant repression strategies. Preliminary results obtained with transgenic plants will be
presented and should help the identification of new factors underpinning the physiochemical propertied of wood
SCW.
P2-35
Transglycosylases in softening fruit
Schröder R. (a), Prakash R. (a), Johnston S.L. (a), Atkinson R. (a), Brummell D. (b), Hallett I. (a)
(a) The New Zealand Institute For Plant & Food Research Limited (Plant & Food Research), Auckland, New Zealand; (b)
The New Zealand Institute For Plant & Food Research Limited (Plant & Food Research), Palmerston North, New Zealand.
Transglycosylases in the plant cell wall are assumed to incorporate newly synthesised polysaccharides into the
existing network for restructuring during periods of growth. However, in senescing tissues such as softening fruit
where controlled disassembly of the cell wall is more prominent and cell wall synthesis is only limited, the need
for transglycosylases is not as evident. We characterised transglycosylase activities from softening fruits,
xyloglucan endotransglycosylase (XET) in kiwifruit [1], mannan endotransglycosylase in tomato [2], and xylan
endotransglycosylase in papaya [3] and showed that all three transglycosylases can also act as hydrolases. Thus, it
is possible that hydrolysis is the predominant action of these enzymes in planta. In softening kiwifruit, we found
xyloglucan oligosaccharides, which can act as substrates for XET, thereby reducing the molecular weight of
xyloglucan by polysaccharide-to-oligosaccharide transglycosylation rather than by hydrolytic activity. In
softening tomato, no molecular weight reduction of mannans is evident, suggesting polysaccharide-topolysaccharide transglycosylation may be predominant. However, in papaya, studies of recombinant protein in
vitro suggest the hydrolysis reaction appeared to be favoured, indicating the role of transglycosylases in fruit
softening may differ in different fruit. In peelable kiwifruit, we have found higher xyloglucan, mannan and xylan
transglycosylase activities in peel tissue than in the underlying flesh, suggesting a role for transglycosylation in
developing and maintaining tissue flexibility.
[1] R. Schröder et al. (1998) Planta, 204, 242-51; [2] R. Schröder et al. (2006) Planta, 224, 1091-1102; [3] S. L. Johnston et
al. (2013) Planta, 237, 173-187.
P2-36
Biosynthesis of pectic galactan: glycosyltransferases and UDP-galactose transporters.
Rautengarten C. (a), Ebert B. (a), Liwanag A.J.M. (a), Stonebloom S. (ab), Gondolf V.M. (ab), Stoppel R. (a), Temple H. (c),
Orellana A. (c), Loqué D. (a), Heazlewood J. (a), Scheller H.V. (ad)
(a) Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA; (b) Dept of Plant and
Environmental Biology, University of Copenhagen, Denmark; (c) Centro de Biotecnología Vegetal, Facultad de Ciencias
Biológicas, Universidad Andrés Bello, Santiago, Chile;(d) Department of Plant & Microbial Biology, University of
California, Berkeley, CA, USA.
β-1,4-galactan is an abundant sidechain of pectic rhamnogalacturonan I (RG-I). A β-1,4-galactan synthase,
GALS1, that elongates galactan chains has recently been described [1], but several additional components are
required for the efficient biosynthesis of β-1,4-galactan, including additional galactosyltransferases and UDPgalactose transporters. We have investigated the role of other members of the GT92 family of
galactosyltransferases in pectin biosynthesis and we have identified additional glycosyltransferases involved in
adding galactose to the RG-I backbone. The galactosyltransferases are often not limiting for pectin biosynthesis,
which requires efficient transport of UDP-galactose into the Golgi. We have identified a family of transporters
that are all capable of transporting UDP-galactose. Although the transporters have very similar activity in vitro,
one of the transporters was shown to preferentially affect β-1,4-galactan biosynthesis in vivo. Deposition of β-1,4galactan in secondary cell walls is a strategy to potentially improve bioenergy crops. By increasing the expression
of galactan synthase, UDP-galactose transporters and UDP-glucose epimerase in interfascicular fibers, either
alone or in concert, plants with substantially higher β-1,4-galactan accumulation and improved feedstocks
properties were generated.
[1] Liwanag et al. (2012) Plant Cell, 24, 5024–5036.
P2-37
Identification of lignin mutants by forward and reverse genetics in a flax EMS population
Chantreau M. (a), Grec S. (a), Chabbert B. (bc), Hawkins S. (a)
(a) Université Lille Nord de France, Lille 1 UMR INRA 1281, SADV, F-59650, Villeneuve d'Ascq cedex, France; (b) INRA,
UMR614 Fractionnement des AgroRessources et Environnement F-51100 Reims, France ; (c) Université de Reims
Champagne-Ardenne, UMR 614, Fractionnement des AgroRessources et Environnement F-51100 Reims, France.
Cellulose-rich, hypolignified fibers from the outer stem tissues of flax plants are economically important and are
used in both the textile and composite polymer industries. Furthermore, flax plants represent a novel model
system to investigate the molecular regulation of lignification since their stems also contain highly-lignified cell
walls in the xylem of inner-stem tissues 1. In order to increase our knowledge about this process we have created a
flax EMS mutant population (project PT-Flax). Tilling of DNA pools from 4,000 M2 families have enabled us to
identify a high number of flax cinnamyl alcohol dehydrogenase (cad) and coumarate 3-hydroxylase (c3h)
mutants. Interstingly, flax cad mutants show a typical brown midrib phenotype in xylem tissue. Analyses show
that we have created a highly mutagenized population that will constitute an extremely useful biological resource
to dissect lignification in this species. In parallel, a high-throughput cytological screening has also enabled us to
identify a number of mutants with lignified outer-stem fibers. FTIR spectroscopy and chemical analyses have
confirmed the increased lignification in these samples. To our knowledge, this is the first report of flax mutants
showing modifications in their fiber lignification pattern and represents an important step towards deciphering the
molecular regulation responsible for hypolignification in these atypical cells and will contribute to current
knowledge necessary for optimizing lignin-cellulose biomass in higher plants.
[1] Huis, R. et al. Natural hypolignification is associated with extensive oligolignol accumulation in flax stems. Plant
Physiol. 158, 1893–1915 (2012).
P2-38
Maize primary cell walls accumulate a lignin-like polymer in response to a lacking on cellulose
Largo-Gosens A. (a), Mélida H. (b), Pomar F. (c), de Castro M.B. (a), Alonso-Simón A. (a), García-Angulo P. (a), Acebes
J.L. (a), Álvarez J.M. (a), Encina A.E. (a)
(a) Área de Fisiol Veg, Univ de León, León, Spain; (b) Division of Glycosci, Royal Inst of Techn KTH, Stockholm, Sweden;
(c) Depart de Biol Animal, Biol Veg y Ecología, Univ de La Coruña, La Coruña, Spain.
Maize suspension-cultured cells with a reduced level of cellulose have been obtained by stepwise accomodation to
dichlobenil, a cellulose biosynthesis inhibitor [1]. Cellulose deficiency was accompanied by remarkable changes
in matrix polysaccharides and cell wall phenolics. At this respect, phenolic profile of cellulose-deficient cell walls
showed an increased content in ferulic acid, diferulates, coumaric acid [2], and the presence of a polymer that
resulted positive for phroglucinol-staining. In accordance with this, cellulose deficient cell walls showed a 600%fold increase in Klason-type lignin. Thioacydolisis/GC-MS analysis of cellulose-deficient cell walls indicated the
presence of a lignin-like polymer with S/G ratio of 1.45. At the biochemical level, dichlobenil-habituated maize
suspension-cultured cells showed a higher CAD activity and a putative higher rate of apoplastic H2O2
consumption. Gen-expression analysis of these cells indicated an overexpression of key-genes for lignin
biosynthesis (CAD, CCR and F5H). To get information on the relationship between cellulose deficiency, lignin
deposition, and stress responses a survey on stress signaling pathways was carried out. Our results would indicate
that a lack of cellulose provokes a defense response by overexpression of jasmonate-signalling pathway genes. All
these results point to a stress-response mechanism that invokes ectopic lignification in response to a cellulose
deficiency and reflects the structural plasticity of primary cell walls.
[1] Melida et al. (2009) Planta, 229:617–631;[2] Melida et al. (2010) Phytochem, 71: 1684-1689.
P2-39
Coniferin β-glucosidase localizes in lignifying and lignified cell walls
Tsuyama T., Takabe K.
Laboratory of Tree Cell Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
Lignin is one of major components of cell wall in vascular plant. Though biosynthesis and polymerization of
lignin precursors have been studied keenly, it remains to be elucidated how lignin precursors are transported to
cell wall, and what are transported as lignin precursors: monolignols or monolignol glucosides. Recently,
differentiating xylem of poplar showed ATP-dependent transport activity of coniferin, coniferyl alcohol glucoside
across tonoplast and endomembrane compartments. The localization of β-glucosidase (BGL) involved in cleavage
of coniferyl alcohol from coniferin may give us important information on lignification process.
We found the activity of coniferin BGL in both soluble and cell wall ionically bound fractions of poplar
differentiating xylem. Phylogenetic tree analysis revealed a putative protein of Populus trichocarpa has homology
with coniferin BGL, and named as PtrBGLC. Immunoprecipitation assay using anti-PtrBGLC antibody suggested
the antibody specifically recognizes coniferin BGL. Immunolabeling in poplar differentiating xylem indicated that
coniferin BGL localizes in the lignifying and lignified cell walls. These results support the hypothesis that
coniferin is transported to the cell wall in which BGL hydrolyzes coniferin to coniferyl alcohol for lignification of
differentiating xylem.
P2-40
Trehalose-6-Phosphate - part of the regulatory mechanism coordinating cellulose biosynthesis
with carbon metabolism?
Wormit A. (a), Trebus R. (a), Lunn J. (b), Usadel B. (a)
(a) Aachen University, Aachen, Germany; (b) Max-Planck Institute for Molecular Plant Physiology, Golm, Germany.
Plant cells are surrounded by a wall composed of complex, interacting networks of polysaccharides associated
with highly glycosylated proteins and lignin (1). Plant cell walls are complex and highly dynamic structures,
responding and adapting to normal processes of growth and development as well as to biotic and abiotic stresses.
Cellulose is the main load-bearing component of cell walls and represents a major sink for photosynthates in plant
cells. Previous work suggests that a regulatory mechanism exists coordinating photosynthetic activity, sugar
metabolism and carbon sinks like lignin and cellulose biosynthesis (2, 3, 4, 5, 6). Trehalose-6-phosphate (T6P)
plays a central role in the regulation of carbon allocation and growth and development (7).
We show that inhibition of cellulose biosynthesis (by treating Arabidopsis thaliana seedlings with isoxaben)
causes transcriptional changes in three genes involved in trehalose metabolism. This is accompanied by an
increase in T6P levels starting at 8 hours of treatment. By using T-DNA insertion mutants we are analyzing the
effect of T6P levels on cell wall composition and nucleotide sugar contents. In addition overexpression lines are
currently generated to further characterize the role of T6P metabolism in the response to cellulose biosynthesis
inhibition and its effect on carbon metabolism.
[1] Burton et al. (2010) Nat. Chem. Biol., 6(10), 724-32; [2] Coleman et al. (2008) Plant Physiol., 148, 1229-1237; [3]
Rogers et al. (2005) J. Exp. Bot., 56, 1651-1663; [4] Peng et al. (2000) Planta, 211, 406-414; [5] Harrison et al. (1998) Plant
J., 13, 753-762; [6] Wormit et al. (2012) Plant Physiol., 159(1), 105-17; [7] Delatte et al. (2011) Plant Physiol., 157, 160174.
P2-41
Uncovering protein-protein interactions in glucuronoxylan biosynthesis
Bromley J.R. (abc), Lund C.H. (a), Stenbæk A. (a), Scheller H.V. (bcd), Sakuragi Y. (a)
(a) Department of Plant & Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark; (b) Feedstocks
Division, Joint BioEnergy Institute, Emeryville, CA, USA; (c) Physical Biosciences Division, Lawrence Berkeley National
Laboratory, Berkeley, CA, USA; (d) Department of Plant & Microbial Biology, University of California, Berkeley, CA, USA.
A growing body of evidence suggests that the complexity of plant cell wall carbohydrates is achieved through the
co-operative function of closely associated glycosyltransferases (GTs) [1]. To further probe protein-protein
interactions (PPIs) involved in glycan biosynthesis, we have modified a split Renilla luciferase complementation
assay (SLCA) for heterologous expression in Nicotiana benthamiana and have applied a split ubiquitin system
(SUS) in yeast to identify interactions between Golgi resident proteins.
In recent years, numerous GTs mediating the production of glucuronoxylan (GX) have been identified, including
those with backbone elongating and substituting activities. A multiprotein complex containing GT43, GT47 and
GT75 members has previously been demonstrated to have a role in glucuronoarabinoxylan synthesis in wheat [2]
however, little is known as to how GTs interact in GX synthesis in Arabidopsis. We have used both SLCA and a
yeast-based SUS to probe binary PPIs at the Golgi membrane amongst GT candidates previously demonstrated to
have a role in GX synthesis.
To further expand the GX protein interaction network, we have also produced cDNA libraries enriched for
inflorescence stem transcripts and have screened these by SUS against xylan-related GT candidates to identify
new members of the network.
[1] Oikawa et al. (2013) TiPS, 18, 49-58; [2] Zeng et al. (2010) Plant Phys, 154, 78-97.
P2-42
Pectin methylesterase and pectin remodelling differ in the fibre walls of two Gossypium species
with very different fibre properties
Liu Q., Talbot M., Llewellyn D.J.
CSIRO Plant Industry, P.O. Box 1600 Canberra ACT 2601, Australia.
Pectin, a major component of the primary cell walls of dicot plants, is synthesized in Golgi, secreted into the wall
as methylesters and subsequently de-esterified by pectin methylesterase (PME). Pectin remodelling by PMEs is
known to be important in regulating cell expansion in plants. We determined fibre PME transcript abundance,
total PME activity, pectin content and extent of de-methylesterification in fibre walls of two cotton species. There
was a higher transcript abundance of fibre-PMEs and a higher total PME enzyme activity in G. barbadense (Gb)
than in G. hirsutum (Gh) fibres, particularly during late elongation. Total pectin was high, but de-esterified pectin
was low during fibre elongation (5-12dpa) in both Gh and Gb. De-esterified pectin levels rose thereafter when
total PME activity increased and this occurred earlier in Gb fibres resulting in a lower degree of esterification in
Gb fibres between 17 and 22 dpa. Gb fibres are finer and longer than those of Gh, so differences in pectin
remodelling during the transition to wall thickening may be important in influencing final fibre diameter and
length, two key quality attributes of cotton fibres.
P2-43
Biochemical characterization of an Arabidopsis pectin methylesterase AtPME3 and a pectin
methylesterase inhibitor
Sénéchal F (a), L’Enfant M. (a), Domon J.-M. (a), Surcouf O. (b), Quéméner B. (c), Esquivel-Rodriguez J. (d), Mareck A.
(b), Guérineau F. (a), Hyung-Rae K. (d), Bonnin E. (c), Jamet E. (e), Mravec J. (f), Kihara D. (d), Ralet M.-C. (c), Lerouge P.
(b), Pelloux J. (a) , Rayon C. (a)
(a) EA 3900-BIOPI, Université de Picardie Jules Verne, 80039 Amiens, France; (b) UPRES-EA 4358, IFRMP 23, UFR des
Sciences et Techniques, 76821 Mont-Saint-Aignan, France; (c) INRA, UR 1268 BIA, BP 71627, 44316 Nantes, France; (d)
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; (e) UMR 5546 CNRS- Université P.
Sabatier, 31326 Castanet-Tolosan, France; (f) Department of Plant and Environmental Sciences, University of
Copenhagen,1871 Frederiksberg, Denmark.
Pectin methylesterase (PME) catalyses the de-methylesterification of pectin in plant cell walls during cell
elongation. While it is generally assumed that fungal PMEs have a random mode of action, plant PMEs are
thought to act in a processive manner to generate long stretches of non-methylesterified residues. However, it is
unlikely that all plant PMEs have a strictly similar mode of action. One A. thalianaPME gene (PME3) has been
over-expressed in an heterologous system and its protein product purified by affinity chromatography.
Homogalacturonans (HGs) from citrus pectin with varied degrees of methyl esterification (DM) have been
generated and characterized. Kinetic properties of PME3 have been determined. PME activity from the purified
PME3 was performed on the characterized HG substrates with respect to pH. PME3 shows a highest activity at a
basic pH but displays different affinities towards specific methyl esterification patterns on the pectin substrates.
Demethylesterified blocks assessed by measuring the degree of blockiness and absolute degree of blockiness will
be presented. PME activities are also regulated by endogenous pectin methylesterase inhibitors (PMEIs), which
control the DM of HG. PMEI7 inhibits PME3 enzyme activity. Docking analysis indicates that the inhibition of
PME3 occurs via the PMEI7 interaction with a PME ligand-binding cleft structure.
P2-44
Cell wall of gelatinous type: gene expression analysis to study development and function
Mokshina N.E. (a), Deyholos M.K. (b), Gorshkova T.A. (a)
(a) Kazan Institute of Biochemistry and Biophysics, KSC RAS, Kazan, Russia; (b) Department of Biological Sciences,
University of Alberta, Edmonton, Alberta, Canada.
Gelatinous type of cell wall is specifically developed in plant fibers and is quite important both for plant itself and
for establishing innovative technologies of plant biomass processing. Spatio-temporal characteristics of gelatinous
cell wall development are well characterized for several model systems, including flax stem. Combination of
possibility to isolate fibers with relatively simple and well-characterised composition of gelatinous cell wall,
which is devoid of lignin and xylan, together with full genome sequencing of flax permits to look for the tissuespecific profiles of gene expression to match them with the major peculiarities of gelatinous cell wall – cellulose
microfibril orientation, unusual composition of matrix polysaccharides and dynamic remodelling of the deposited
cell wall layers. Genes from several multigene families, which encode the proteins involved in cell wall formation
(including LusCESAs, LusCSLs, LusCTLs, LusBGAL), were checked for the expression in various flax plant
organs and tissues to find the ones specifically expressed during deposition of gelatinous cell wall. The obtained
data revealed the combination of several genes with tissue- and stage-specific expression with the ones that are
expressed during deposition of other cell wall types. Plants with modified gene expression were obtained to prove
the importance of certain genes with tissue-specific expression. Identification of genes, involved in gelatinous cell
wall formation and function may help to understand both the development of this type of the cell wall per se and
plant cell wall in general, helping to identify protein assemblages which are important for certain cell wall
characteristics.
This work was partially supported by the Russian Foundation for Basic Research (12-04-31418) and the President Program for State
Support of Leading Scientific Schools (825.2012.4).
P2-45
Focus on cell wall biogenesis in wheat grain using sub-cellular proteomic approach
Larre C., Chateigner-Boutin A.-L., Francin-Allami M., Alvarado C., Suliman M., Rogniaux H., Guillon F.
INRA, UR1268 Biopolymères, Interactions, Assemblages, F-44316 Nantes, France.
The wheat grain is an important source of food, animal feed and industrial raw material. Cell wall polysaccharides
are minor components of the grain but critical for plant fitness and valuable for human nutrition as dietary fiber.
The amount and chemical structure of cell wall polysaccharides varie according to tissues. Heteroxylans and
cellulose are the major polysaccharides in the outer layers of the grain while arabinoxylans (AX) and β(1-3)(1-4)
D glucans (MLG) are predominant in the endosperm. Polysaccharides except cellulose are synthesized in the
Golgi apparatus; the mechanisms underlying their synthesis have yet to be fully elucidated. To identify actors
involved in the wheat cell wall formation, a subcellular fractionation strategy was carried out to isolate
microsomal fractions from outer layers and endosperm harvested during active cell wall deposition. The proteins
extracted from these fractions were analyzed by immunochemistry and LC–MS/MS. Thousands of proteins were
identified and among them glycosyl transferase (GT) and glycosyl hydrolase (GH) families. GT families
implicated in xylan and MLG synthesis were found as well as GT families involved in the synthesis of less
abundant and less studied cell wall polysaccharides in wheat grain. Surprisingly, we also identified numerous GHs
some of which could be involved in cell wall remodeling. In particular, they were found abundant in the outer
layers. Altogether, these results provide new candidates potentially involved in cell wall biogenesis in wheat
grain.
P2-46
A cell wall peroxidase required for correct mucilage release in Arabidopsis seed coat
Francoz E. (a), Ranocha P. (a), Martinez Y. (b), Leru A. (b), Burlat V. (a), Dunand C. (a)
(a) LRSV, UMR5546 UPS/CNRS, 31326 Castanet-Tolosan, France; (b) FR AIB 3450 31326 Castanet-Tolosan, France.
Class III Peroxidases (Prx) predicted as secreted are largely detected in various cell wall proteomes [1,2]. Even
though their function is not clearly defined, their involvement in the development and remodeling of the plant cell
walls (loosening, crosslinking and lignification) is well reviewed [3]. However, it remains difficult to assign any
precise in vivo function to Prx. Thus, the in vivo spatiotemporal co-localization of a “cell-wall substrate” and
particular Prx is believed to confer the specificity of the reaction [3]. A preliminary in situ RNA Hybridization
(ISH) study of 22 of the 73 members of this multigenic family was realized to assess the expression pattern in
various A. thaliana embryogenic developmental stages. It already confirms and complements (micro-)
transcriptomic data [4] by refining gene expression pattern to specific cells. One candidate showed an interesting
expression profile within mucilage secretory cells (MSC), a well-described model of plant cell wall research [5].
During seed development, a burst of pectinaceous mucilage synthesis occurred in these specialized cells,
accompanied by the neosynthesis of a secondary wall inside the cell, the columella. During imbibition, the outer
tangential primary wall is broken at particular points, leading to mucilage extrusion. Our candidate may be
involved in this degradation by weakening the primary wall. A KO mutant shows delayed mucilage extrusion
phenotype associated with a peeling of the primary wall. SEM imaging revealed no visible structural differences
compared with wild-type seed coat. Further investigations are engaged to decipher the functional role of this Prx
in the remodeling of MSC wall.
[1] Y. Zhang et al. (2011) Phytochemistry., 72, 1109-1123; [2] M. Irshad et al. (2008) BMC Plant Biol., 8; [3] F. Passardi et
al. (2004) Trends Plant Sci., 9, 534-540; [4] BH. Le et al. (2010) PNAS., 107, 8063-8070; [5] GW. Haughn and TL. Western.
(2012) Front. Plant Sci., 3, 64-64.
P2-47
ICs and their Interactors – new Cellulose Synthesis related Proteins
Kesten C. (a), Endler A. (a), Mansoori N. (b), Trindade L. (b), Persson S. (a)
(a) Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany; (b) Laboratory of Plant Breeding,
Wageningen University and Research Centre, Wageningen, The Netherlands.
Cellulose is composed of hydrogen-bonded β-(1,4)-linked glucan chains, and is produced at the plasma membrane
by hexameric enzyme arrangements; the cellulose synthase complexes (CSCs). These complexes move along
tracks of cortical microtubules, and release newly synthesized cellulose into the apoplastic space. The only known
components of this complex are the cellulose synthase proteins (CESAs), which are believed to be the catalytic
subunits of the complex, and the recently identified CSI1/POM2, which guides the CSCs along the cortical
microtubules. We have discovered a new class of CESA interacting proteins, referred to as Interactors of CESAs
(IC). GFP-Fusions of IC1 co-migrated together with RFP:CESA6 in the plasma membrane. Through in vitro
pulldown-assays, we show a direct interaction of the protein with microtubules. Furthermore, the microtubule
interacting domain appears to change its three-dimensional structure during microtubule interactions, and more
detailed structural analyses are underway for this domain. To extend the knowledge regarding cellulose synthesis,
we performed split-ubiquitin screens and protein pull-down assays to identify additional components that may
contribute to cellulose synthesis. Interestingly, using GST-pulldown assays we identified some putative
interactors that may play roles in the regulation of the IC proteins. We conclude that a more detailed
characterization of the IC1, and its family members, will provide a deeper understanding for how plants regulate
cellulose production.
P2-48
Identification and analysis of the highly esterified mucilage mutant defective in seed mucilage and
embryo development
Levesque-Tremblay G. (a), Muller K. (b), Mansfield S. (a), Haughn G. (a)
(a) University of British Columbia Dept Bot, Vancouver, BC V6T 1Z4, Canada; (b) University of Simon Frazer, Dept Biol
Sci, Burnaby, BC V5A 1S6, Canada.
In Arabidopsis, seed coat epidermal cells secrete mucilage consisting primarily of pectin. Upon hydration, the
mucilage swells to create a gel-like capsule around the seed. This mucilage represents an important tool for
studying pectin biosynthesis inplanta. Pectin methyl esterases (PMEs) are enzymes known to modify the property
of pectin by removing the methlyl esterified group from galacturonic acid. We have shown that at least 7 of 66
PMEs are highly expressed in the Arabidopsis seed coat. We screened Arabidopsis T-DNA insertion lines for
loss-of-function mutants in each of the genes encoding putative PMEs in Arabidopsis and identified one line with
a seed phenotype. The highly esterified mucilage mutant (hem) has a reduced mucilage halo phenotype in water
and was later shown to be caused by a smaller sized embryo. HEM shows a specific increase of transcript at 7 day
post anthesis (DPA) in the seed coat which corresponds to the period of mucilage secretion. Using confocal
microscopy, the mucilage of hem seeds shows changes in labeling by different antibodies relative to wild type
mucilage. Moreover, hem mutant seeds are also morphologically distinct compared with WT as shown by
scanning electron microscopy. hem seeds show a strong defect in embryo development shown by a decrease in
cell size and oil content. The germination rate of hem mutants is also affected as shown by the delay in radicule
protrusion and testa rupture relative to wild type seeds. The hem mutant mucilage phenotype was partially
rescued upon a complementation test by crossing hem with wt. This suggests that the embryo development defect
leads to the shrinkage of the seed causing most of the mucilage phenotype. HEM-YFP proteins are found in the
cell wall of the external layer of the seed coat and in the embryo cells. A general decrease in PME activity occurs.
The study of hem underlines the importance of de-methyl esterification in seed development and mucilage
extrusion.
P2-49
Endomembrane Trafficking and Polysaccharide Deposition
Drakakaki G. (a), Park E. (a), Bulone V. (b), Diaz-Moreno S.M. (b), Worden N. (a)
(a) Department of Plant Sciences, University of California Davis, One Shields Avenue Davis, CA 95616, USA; (b) Royal
Institute of Technology (KTH), SE-100 44 Stockholm, Sweden.
A major challenge towards understanding endomembrane processes and their biological roles has been their
highly dynamic nature. We used chemical genomics and proteomics, to study components of the endomembrane
system that are present in trans-Golgi Network (TGN), a site of polysaccharide trafficking and recycling of
endosomal components.
We took an immunoisolation approach to separate TGN vesicles marked by the syntaxin SYP61, which is
localized at the TGN and analyzed their proteome. Interestingly, components of the cellulose biosynthesis
machinery were identified in the SYP61 vesicle proteome, suggesting that SYP61 vesicles are involved in the
trafficking of the CESA complexes. We are currently characterizing several components of the SYP61 vesicle
proteome of unassigned function and investigate their involvement in cell wall deposition [1].
Chemical genomics is a powerful approach to effectively study endomembrane trafficking. Towards this effort,
we have utilized a confocal microscopy based screen to discover small molecules [2] that affect the
endomembrane trafficking and polysaccharide deposition. Novel pharmacological inhibitors selected for their
unique effects on cell wall deposition, either during cell division or during cell elongation. Endosidin7 (ES7), a
small chemical inhibitor interferes specifically with vesicle trafficking to the cell plate during late cytokinesis.
This compound induces the formation characteristic cell plate “gaps’ labeled with the cell-plate specific markers,
and inhibits callose deposition in Arabidopsis. Using a genetic approach we identified potential ES7 targets that
are essential complexes during cell plate maturation and new molecular components involved in this specialized
vesicle trafficking pathway.
[1] Drakakaki et al. (2012) Cell research., 22 (2),413-424; [2] Drakakaki et al. PNAS., 108 (43),17850-17855.
P2-50
The Role of CSLD Proteins During Polarized Cell Wall Deposition in Arabidopsis
Gu F., Nielsen E.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, USA.
Plant cell expansion is governed by the deposition of new cell wall components, and cellulose microfibrils, the
major load bearing components in plant cell walls, are deposited along one or more entire faces of a cell during
diffuse growth. However, during tip growth, newly synthesized cell wall polysaccharides are deposited in a
restricted region. We have shown that cellulose-like polysaccharides are enriched at the tips of growing root hairs
and treatment with cellulose synthase inhibitor abolishes root hair growth. However, while two known essential
cellulose synthase (CESA) proteins failed to localize to the growing tips of root hairs, CSLD3 did localize to
apical plasma membranes in growing root hairs. ArabidopsisCSLD family has five members, of which three
(CSLD2, CSLD3, and CSLD5) are expressed in vegetative plant tissues. Here we report our progress on
understanding the functions of CSLD2 and CSLD5 during plant growth and development. As previously reported,
both csld2 and csld3 mutant plants show defects during root hair growth and development, but csld5 root hairs
remain largely unaffected in this mutant background. While only root hair morphology is affected in csld2 and
csld3 plants appear, csld5 plants display overall reduced stature. Here we show that csld5 phenotypes are
associated with cell division defects in diverse Arabidopsis organs and tissues, and these cell division defects are
more pronounced csld2csld5 double mutants. We also show that fluorescently-tagged CSLD2/3/5 proteins
localize to phragmoplasts in dividing cells. Taken together, these results indicate that, in addition to their roles in
cell wall deposition in tip-growing cells, CSLD proteins also function during phragmoplast formation in non-tip
growing cells undergoing cell division.
P2-51
Investigating cell wall biosynthesis in the secretory pathway using Free Flow Electrophoresis
Parsons H.T. (a), Verhertbruggen Y. (b), Joo M. (b), Batth T.S. (c), Petzold C.J. (b)
(a) Department of Plant and Environmental Sciences, Section for Plant Glycobiology, Faculty of Science, University of
Copenhagen, Denmark; (b) Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National
Laboratory, USA; (c) The Novo Nordisk Foundation Center for Protein Research, Copenhagen, Denmark.
Many cell wall biosynthetic processes are known to occur in the secretory pathway, particularly the ER and Golgi
apparatus, but the precise location and sequence of these reactions is unknown. We show here that linear
separation of much of the secretory pathway from early à late compartments is possible using the Free Flow
Electrophoresis (FFE) technique (1). A gradient of surface charge exists across much of the secretory pathway.
During FFE membrane vesicles are separated according to net surface charge, allowing separation of the
endomembrane system even at the sub-Golgi level. Separation has been demonstrated by tracking protein
migration in the endomembrane system using semi-quantitative mass spectrometry of charge-separated fractions.
This revealed sequential separation of enzymes in the N-glycosylation pathway, one of the few pathways for
which spatial arrangement of enzymes across the Golgi stack is known (2). These results are being verified by
quantitative mass spectrometry of selected marker proteins. An increase in polysaccharide complexity through the
endomembrane system has often been proposed but never before observed directly until now. Immunodot
blotting using monoclonal antibodies against specific polysaccharide epitopes showed an increase in
polysaccharide processing in later Golgi fractions, which was confirmed by immunogold localisation. Shotgun
proteomics has been performed on all fractions of separated secretory compartments. This will, once the extent of
membrane compartment separation has been described, provide an important resource for understanding plant cell
wall biosynthesis, protein modification and protein trafficking.
[1] Parsons et al. (2012) Isolation and Proteomic Characterization of the Arabidopsis Golgi Defines Functional and Novel
Components Involved in Plant Cell Wall Biosynthesis Plant Physiol. June; 159: 12-26; [2] Schoberer and Strasser (2011)
Sub-Compartmental Organization of Golgi-Resident N-Glycan Processing Enzymes in Plants. Mol Plant. March; 4(2): 220–
228.
P2-52
Reciprocal small-molecule probes localize polyionic structural carbohydrates in plant and fungal
cell walls
Mravec J. (ab), Kračun S.K. (a), Gro Rydahl M. (a), Pedersen H.L. (a), Vidal Melgosa S. (a), Miart F. (b), van Cutsem P.
(bc), Höfte H. (b), Willats W.G.T. (a)
(a) PLEN Department, Section for Plant Glycobiology, SCIENCE Faculty, University of Copenhagen, Denmark; (b) Institut
Jean-Pierre Bourgin, INRA Centre de Versailles-Grignon, France; (c) Unité de Recherche en Biologie cellulaire et
moléculaire Végétale, Université de Namur, Belgium.
Pectins and chitin are examples of abundant, evolutionary distinct structural polysaccharides which can undergo
post-biosynthetic enzymatic removal of alkyl groups from their monomeric subunits leaving polyionic molecules.
However, it is little know about spatiotemporal distribution and function of these modifications. Here we show
that the exact stereo chemical allocation of oppositely charged amino residues of de-acetylated form of chitinchitosan and carboxy groups of de-methylesterified homogalacturonan can be used to create small polar-bindingbased probes. Conjugates of the respective oligosaccharides with distinct fluorophores can serve as a one-step
detection substitute for currently used antibodies. We will present several specificity tests and examples of their
effectiveness for in situ and in vitro call wall analyses. These probes f enabled us or example to demonstrate the
involvement of the pectin methylesterase activity in maturation and sloughing-off the Arabidopsis root cap cells.
Hence, we identified previously unknown patterns of chitosan deposition in insect exoskeletons and fungal cell
walls.
P2-53
Inducible overexpression of the transcription factor SWN5 is sufficient to activate secondary cell
wall synthesis in Brachypodium
Valdivia E.R., Gianzo C., Revilla G., Zarra I., Sampedro J.
Departamento de Fisiología Vegetal, Universidad de Santiago, Santiago de Compostela 15782, Spain.
A subfamily of NAC transcription factors has been identified as master switches of secondary cell wall synthesis.
The grass model Brachypodium distachyon has eight SWN genes that belong to this subfamily. All of them were
able to induce ectopic cell wall thickenings in tobacco leaves. With the exceptions of SWN7 and SWN8 they also
caused cell death. Estradiol-inducible expression of SWN5 in Brachypodium seedlings resulted in accelerated
vascular development and ectopic secondary wall deposition in both roots and shoots. These results establish that
secondary cell wall synthesis in monocots is also controlled by NAC master switches [1].
Through microarray hybridization more than 1500 upregulated genes have been identified 6, 12 and 24 hours after
induction of SWN5 expression. Among these genes are three putative secondary cell wall cellulose synthases, as
well as several genes that are likely involved in xylan synthesis. Transcription factors from different families and
genes probably involved in cell death, can also be found among the strongly upregulated genes. We have obtained
promoter-reporter constructs for several of these targets, as well as different SWNs.
Transcription of XCP1, a cysteine protease expressed in developing tracheary elements, appears to be directly
activated by SWN5, but not SWN7 or SWN8. SWN5 binds two conserved SNBE motifs in the promoter. We are
currently characterizing the binding preferences of SWNs through yeast-one-hybrid assays to determine if cell
wall synthesis and cell death programmes can be activated independently.
[1] Valdivia et al. (2013) J. Exp. Bot., 64, 1333-1343.
P2-54
Characterization of suppressors of cell adhesion defective mutants
Verger S., Chabout S., Sormani R., Höfte H., Mouille G.
Institut Jean-Pierre Bourgin, INRA Centre de Versailles-Grignon, UMR 1318 INRA-AgroParisTech, route de Saint Cyr
(RD10), 78026 Versailles cedex, France.
Pectins, the most complex polysaccharides in plants, are essential components of the primary cell wall. They play
a crucial role in cell adhesion as well as mechanical strength, coordination of growth and development, and the
defence against microorganisms. Homogalacturonans (HG), an α-(1,4) Galacturonic acid chain, is the main
component of the pectins. Very little is known about the biosynthetic pathway and the functions of pectins.
Our group has identified and characterized two mutants, quasimodo1 and 2, displaying cell adhesion defects, a
high sensitivity to carbon/nitrogen imbalance, and a 50% decrease in HG content. QUASIMODO1/GAUT8 is a
galacturonosyltransferase (Bouton et al., 2002) and QUASIMODO2 is a putative pectin methyltransferase
(Mouille et al., 2007). These two enzymes seem to be partners in a complex involved in the synthesis of
methylesterified HG, but their exact role in HG biosynthesis remains to be determined. In addition, the link
between the decrease in HG content, the cell adhesion defect and the altered metabolism remained to be
understand. In order to gain more insight into the role of QUA1 and QUA2 in pectin synthesis and cell adhesion,
as well as the role of pectin in the metabolism and development of the plant, we decided to study the reversion of
the quasimodo phenotype using three different but complementary suppressing approaches:1) A genetic
suppressor screen, 2) a chemical genomic suppressor screen, and 3) we study the reversion of the quasimodo
phenotype through its growth on various conditions.
Detailed results on the genetic suppressors as well as the complementarity of the three approaches will be
presented.
Bouton, S., Leboeuf, E. Mouille, G., Leydecker, M.T., Talbotec, J., Granier, F., Lahaye, M., Höfte, H. and Truong, H.N.
(2002). Plant Cell, 14, 2577-2590. Mouille G., Ralet M.-C., Cavelier C., Eland C., Effroy D., Hématy, K., McCartney L.,
Truong H.N., Gaudon V., Thibault J.-F. , Marchant A., Höfte H. (2007). Plant J, 50, 605-614.
P2-55
Analysis of transgenic Arabidopsis thaliana with pinoresinol reductase gene derived from a soil
bacterium, Sphingobium sp. SYK-6
Tamura M. (a), Tsuji Y. (a), Kusunose T. (b), Okazawa A. (c), Kamimura N. (d), Hishiyama S. (e), Fukuhara Y. (d), Hara H.
(f), Sato K. (a), Muranaka T. (b), Katayama Y. (g), Fukuda M. (d), Masai E. (d), Kajita S. (a)
(a) Tokyo University of Agriculture and Technology, Tokyo, 184-8588 Japan;(b) Osaka University, 1-1 Yamadaoka, Suita,
Osaka 565-0871, Japan;(c) Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan; (d)
Nagaoka University of Technology, Niigata, 940-2188, Japan;(e) Forestry and Forest Products Research Institute, Ibaraki,
305-8687, Japan;(f) Okayama University of Science, Okayama, 700-0005, Japan;(g) Nihon University, Kanagawa, 2528510, Japan.
Resinol structure is found in plant secondary metabolites such as lignans or dilignol (lignin dimer). We try to
modify lignan composition and lignin structure via metabolic engineering of pinoresinol. A gene for pinoresinol
reductase (pinZ), which has been isolated from a soil bacterium, Sphigobium sp. SYK-6, the catalyzes the
conversion of pinoresinol, syringaresinol, and lariciresinol to lariciresinol, 5-5’ dimethoxylariciresinol, and
secoisolariciresinol, respectively. Catalytic activities of PinZ toward pinoresinol is significantly higher than those
of plant-derived enzymes with the same catalytic function. To transform Arabidopsis thaliana, we constructed
expression cassettes of pinZ with and without apoplast targeting signal sequence (hereafter designated as Sig-pinZ
and pinZ, respectively). Both of the constructs were introduced into A. thaliana and several independent lines for
each construct were generated. We extracted crude enzyme from roots and stems of the both transgenic and wildtype lines, and the resultant enzymes were reacted with pinoresinol and NADPH. Apparent PinZ activities could
be detected in the reaction mixtures prepared from the both trasgenic lines. Significant change in lignan
composition and no apparent differences in growth and development were observed in the pinZ lines. Analysis of
the Sig-pinZ lines is now in progress.
P2-56
Expression of xylanase in lignin mutants
Harholt J. (a), Vanholme B. (b), Turumtay H. (b), Lebris P. (c), Lapierre C. (c), Jouanin L. (c), Sibout R. (c), Boerjan W. (b),
Ulvskov P. (a), Jørgensen B. (a)
(a) University of Copenhagen, Denmark;(b) Ghent University, Plant Systems Biology-VIB, Belgium; (c) Institut National de
la Recherche Agronomique, France.
Constitutive expression of a thermostable xylanase (XynB) in Arabidopsis led to the accumulation of large
quantities of active enzyme in dried stems [1]. No visual phenotype could be observed and the only detectable
biochemical phenotype was a reduction in the degree of polymerisation of the xylan. After incubation of XynB
producing Arabidopsis stems, no difference was observed beyond the reduction in the degree of polymerisation of
the xylan seen in unincubated XynB producing stems. This leads to the conclusion that XynB did not have access
to the xylan present in the cell wall and the difference observed was due to depolymerisation during transport and
deposition of the xylan in the cell wall. To investigate the impact of lignin on the limited activity of XynB on
mature cell wall, crosses between different lignin mutants (comt1, 4cl-1, c4h-3, lac4-2 and ccoaomt1) and XynB
expressing plants were generated. In several of the double mutants clear phenotypic affects could be observed.
Additionally, changes in xylan quantity and/or degree of polymerisation of the xylan were detected. This indicates
that deposited lignin limits the access of XynB to xylan in mature cell walls. A finding that has implication on
enzyme usage in cell wall deconstruction.
[1] B. Borkhardt et al. (2010) Plant Biotechnol. J., 8, 363-374.
P2-57
VASCULAR-RELATED UNKNOWN PROTEIN 1 regulates secondary wall formation via
hormone regulatory pathways in Arabidopsis
Grienenberger E., Douglas C.J.
University of British Columbia, Vancouver, BC. Canada.
Despite a strict conservation of the vascular tissues in vascular plants (Tracheopytes), our understanding of the
genetic basis leading to differentiation of secondary cell wall containing cells in the xylem of Tracheopytes is still
far from complete. Using bioinformatic tools such co-expression analysis and phylogenetic conservation across
sequenced Tracheopytes genomes, we identified a number of the Arabidopsis genes of unknown function whose
expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULARRELATED UNKNOWN PROTEIN 1 (VUP1) gene, encoding predicted proteins of 20kD and 24kD (depending
on splice variant) with no annotated functional domains but is highly conserved amongst vascular plants, was
studied in depth. Consistent with a role in secondary wall formation, VUP1 expression determined by the GUS
reporter gene is associated with developing tissues while vup1 loss-of-function mutants exhibited vascular
defects, including collapsed morphology of xylem vessels cells. Ectopic expression of VUP1 induced a wide
range of pleiotropic developmental defects, including severe dwarfism and altered photomorphogenesis,
resembling brassinosteroid (BR)-deficient mutants, and overexpression of VUP homologs from multiple
Tracheopytes gave similar effects. Whole genome transcriptome analysis revealed that overexpression of VUP1
repressed the expression of BR and auxin-responsive genes. Additionally, protein deletions and site-directed
mutagenesis were used to identified critical domains and amino acids required for VUP1 function. Analysis of
VUP1 overexpression phenotypes in GA and BR signaling mutant backgrounds suggests that VUP1 functions as
downstream negative regulator in GA and BR signaling pathways, possible where they converge. Altogether, our
data suggest a role for VUP1 in regulating secondary wall formation during vascular development by locally
modulating hormone pathways.
P2-58
Investigation of a KNAT7-BLH-OFP transcription factor complex involved in regulation of
secondary cell wall biosynthesis in Arabidopsis thaliana
Liu Y. (a), Douglas C.J. (a)
(a) Department of Botany, 6270 University Boulevard, University of British Columbia, Vancouver, British Columbia V6T
1Z4, 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) [1]. Co-expression and yeast
two-hybrid analyses suggest that BEL1-LIKE HOMEODOMAIN (BLH) transcription factors could be part of a
KNOX-BLH-OVATE transcription factor complex regulating aspects of secondary cell wall formation, together
with KNAT7 and OFP1/4. I 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 by characterizing the phenotypic effects of blh loss of function
and BLH overexpression on stem anatomy. 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 BLH6KNAT7-OFP complex and insights into the biological function of BLH6.
[1] E. Li et al. (2011) Plant J., 67, 328-341.
Session 3 : Evolution & Diversity of plant CW
P3-01
Arabinogalactan proteins in seaweeds: taxonomic and seasonal variation
Raimundo S., Popper Z.A.
Botany and Plant Science and Ryan Institute for Environmental, Marine and Energy Research, School of Natural Sciences,
National University of Ireland Galway, Galway, Ireland.
Seaweed cell walls share some components, such as cellulose, with plants, but they also contain some unique
polysaccharides including carrageenans (from red seaweeds) and alginates (from brown seaweeds), which have
significant commercial value derived from their nutritional and pharmaceutical importance. This has motivated
research focusing on the structure, localization, metabolism and secretion of algal wall components. Three
lineages of seaweeds exist, green (Chlorophyta), red (Rhodophyta) and brown (Phaeophyta), which are only very
distantly related to each other and to land plants. Arabinogalactan proteins (AGPs) belong to a family of
hydroxyproline-rich glycoproteins present in the cell wall of flowering plants. Although recent advances have
been made their exact mechanism of action is not yet fully understood. However, the importance of AGPs is
highlighted by their implication in a wide range of physiological functions related to vegetative processes, sexual
reproduction, development, and signaling. AGPs are also interesting from an evolutionary perspective as they are
present in all land plants investigated, and have also been found in some microalgae and green seaweeds.
Preliminary evidence for the presence of AGPs in seaweeds was found using tissue prints immunolabelled with
monoclonal antibodies (mAbs), which recognize epitopes present in the AGPs of flowering plants. The present
study focused on the extraction and quantification of AGPs from a variety of seaweeds belonging to the three
lineages, and also from five species of brown seaweeds, Ascophyllum nodosum, Pelvetia canaliculata, Fucus
serratus, Fucus spiralis and Fucus vesiculosus, collected over the course of a year. Quantification was carried out
by colorimetric assay and radial gel diffusion with Yariv reagent. Here we give evidence for taxonomically- and
seasonally- derived variation of AGPs in seaweeds.
P3-02
Ceratopteris richardii (C-fern): a model for investigating adaptive modification of vascular plant
cell walls
Eeckhout S. (a), Leroux O. (ab), Willats W.G.T. (c), Popper Z.A. (b), Viane R.L.L. (a)
(a) Research Group Pteridology, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium;
(b) Botany and Plant Science and The Ryan Institute for Environmental, Marine and Energy Research, School of Natural
Sciences, National University of Ireland Galway, University Road, Galway, Ireland; (c) Department of Plant Biology and
Biochemistry, Faculty of Life Sciences, University of Copenhagen, Buelowsvej 17–1870 Frederiksberg, Denmark.
Plant cell walls are complex structures that underpin many aspects of cell and plant development, including cell
division, growth, cell-cell communication, and differentiation. They are dynamic and constantly remodelled to
allow growth and development. Evolutionary mechanisms have shaped the morphological diversity of plants,
resulting in complex body plans containing specialised tissues with distinct walls. To gain better insight into the
role of cell walls during evolution we need to investigate how, if at all, specific polymers are associated with the
observed diversity between different plant lineages, within a single plant, between cell-types and even within
individual walls. Our understanding of the diversity of plant cell wall components and their biosynthesis has been
revolutionised by the availability of sequenced plant genomes. The genomes of Physcomitrellapatens (bryophytes)
and Selaginella moellendorffi (lycophytes) were recently sequenced, and though full sequences of fern genomes
are still unavailable, Ceratopteris richardii (C-fern) has proven to be a valuable fern model system. It is a
homosporous fern with free-living haploid gametophytes and dominant diploid sporophytes in which vascular and
mechanical tissue is initiated. The aim of this study is to investigate if wall polymers associated with complex
tissues in its sporophyte are also present in its morphologically less complex gametophyte. We adopt a two level
antibody-based strategy; screening for specific cell wall components by probing glycan microarrays with
monoclonal antibodies (mAbs), complemented with detailed immunocytochemical analyses. Our analyses show
that mAbs developed against the wall components of flowering plants are able to recognise epitopes present in
both C-fern gametophytes and sporophytes, and reveal spatio-temporal diversity in their cell wall composition.
P3-03
WallEvo: a comprehensive online resource for plant cell wall evolution studies
Domozych D.S. (a), Popper Z.A. (b)
(a) Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College, Saratoga Springs, NY, 12866,
USA; (b) Botany and Plant Science and The Ryan Institute for Environmental, Marine and Energy Research, School of
Natural Sciences, The National University of Ireland Galway, Galway, Ireland.
The past decade has seen an enormous increase in research focused on the elucidation of cell wall evolution in
eukaryotic photosynthetic organisms and in particular of green land plants. Multiple technologies employed in
these studies derived from molecular genetics, biochemistry, cell biology and immunocytochemistry have
provided novel insight into cell wall origins and the adaptations that have evolved in various taxonomic lineages
and their inclusive cell/tissue/organ types. Currently no central web-based resource is available that serves to
highlight these discoveries and their importance for understanding plant processes. The WallEvo website will be
an on-line library for archiving research reports and disseminating news and research developments pertinent to
cell wall evolution. The site is an international effort that will provide the following: 1) relevant up-to-date lists of
research publications and associated pdfs; 2) information on evolution-based research activities and opportunities
available in various research laboratories globally; 3) links to websites of specific researchers/laboratories,
taxonomic groups, culture collections and other practical resources; d) a gallery of images and a library of
laboratory protocols related to cell wall evolution research and e) educational resources that can be clearly
understood by the general public, especially students. The International Cell Wall meeting will provide a conduit
for introducing the WallEvo website and identifying researchers interested in contributing to this effort. The
official launch of this website is planned for November 2013.
P3-04
Distribution of arabinogalactan proteins and pectins in cork oak female flower
Amorim M.I. (ab), Sousa C. (a), Costa M.L. (ab), Coimbra S. (ab)
(a) Department of Biology, Faculty of Science, University of Porto, Portugal; (b) Centre for Biodiversity, Functional &
Integrative Genomics – BioFIG, Porto, Portugal.
The Southern European evergreen cork oak (Quercus suber) is a monoecious tree species, with a long progamic
phase, that can provide a comprehensive system for comparative studies in development and reproduction in a
non-model plant. Cell surface proteoglycans such as arabinogalactan proteins (AGPs) play important roles in cell
wall growth and development. AGPs are a superfamily of highly glycosylated hydroxyproline-rich glycoproteins
cell-wall components found in the entire plant kingdom, in almost all plant organs and cell types from root to
flowers. At the subcellular level, AGPs can be found in the cell wall, in the apoplast or anchored to the plasma
membrane via a GPI anchor attached to the C-terminal domain of the AGP backbone. In reproductive tissues, the
expression of AGPs is associated with the sporophyte–gametophyte transition. Our own previous work has
shown a specific AGP expression pattern during plant gametogenesis in Arabidopsis thaliana and in Trithuria
submersa. A set of monoclonal antibodies (mAb) directed against the carbohydrate moiety of cell wall
polysaccharides were used for immunolocalisation of AGPs, such as: JIM8, JIM13, JIM16, MAC207 and LM2.
The labeling obtained with anti-AGP antibodies in Cork female flowers from different phenological stages
showed a dynamic distribution of those sugar epitopes in reproductive tissues. The distribution of AGPs in cork
oak female flower reveals that the expression of the identified AGPs is specific for some cell types, since the AGP
labeling obtained reveals a tissue specific expression pattern. Labeling with JIM5, JIM7 and LM7 for pectin
epitopes shows a very uniform labeling associated with almost all cell types and a strong presence in the pollen
tubes cell wall.
The authors are grateful for the financial support through FCT for the Project PTDC/AGR-GPL/118508/2010.
P3-05
Structural organization of plant cell walls is fairly conserved through evolution
Sarkar P., Correa J., Auer M.
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Biochemical composition of plant cell walls has gradually yet significantly changed throughout evolution [1, 2].
Our research objective is to determine whether these biochemical changes were also accompanied by
organizational changes in the cell wall architecture. We compared the three-dimensional (3D) architecture of cell
walls from different cell types of evolutionarily diverse plant groups, using electron tomography of cryoimmobilized tissue samples. A comprehensive qualitative and quantitative analysis of the macromolecularresolution cell wall structures revealed a high degree of similarity in the overall wall organization patterns,
indicating that an optimum basic structural organization might be essential for proper functioning of cell walls
universally, irrespective of the diversity of their biochemical compositions. We are further testing the idea of a
‘optimum cell wall structure’ by developing 3D computer-aided-design (CAD) models of the observed wall
structure and hypothetical alternative structures, and simulating the affects of mechanical stresses on these models
that cell walls generally encounter in living plants. The results of the simulation tests will reveal if our observed
wall organization has any mechanical advantage over other possible arrangements, and will possibly provide an
explanation for the unchanged wall architecture through evolution. The results of the electron tomography based
structural comparison and the mechanical stress simulations will be presented.
P3-06
Evolutionary transcriptomic study of cell wall related genes using RNA-seq and genome-wide
phylogenetic analysis
Hansen B. Ø., Ruprecht C., Persson S., Mutwil M.
Max Planck Institute for Molecular Plant Physiology, Golm , Am Mühlenberg 1. 14476 Potsdam, Germany.
Whole biological pathways, similarly to genes, can be duplicated and specialized to perform novel functions
(Ruprecht et al., unpublished data). Co-expression analysis utilizes the fact that genes with functional
relationships tend to be transcriptionally coordinated and can be used to uncover composition of biological
pathways (modules) that are often conserved across different species (Mutwil et al., 2011). We propose that the
duplication of these modules within species leads to increase the morphological complexity during evolution, but
exact composition and the event that created the duplicated modules is still unknown.
Recent advances within RNA sequencing (RNA-Seq) technology provide affordable means to determine gene
expression, with advantage of de novo detection of transcripts, high sensitivity and complete coverage of the
transcriptome. Based on publicly available RNA-seq data we have identified duplicated modules of genes within
Arabidopsis, which seem to be involved in a diverse range of cellular functions, such as protein synthesis, biotic
stress, and most prominently, cell wall biosynthesis. Genome-wide phylogenetic analyses can be used to
determine phylogenetic events and evolutionary periods that have created gene families. By combining
phylogenetic information with co-expression data, we show that primary cell wall, secondary cell wall, pollen and
root hair co-expression modules have been created in a very specific moment in plant evolution. Our method of
analyzing RNA-Seq data can detect duplication of whole biological pathways. Combining this with phylogenetic
analysis can reveal evolutionary periods that created the duplicated pathways. This, in future, will be used to link
morphological and molecular traits that made plants as they are today.
[1] Balakrishnan, C. N. et al. (2012). Genomics; [2] Giorgi, F. M. et al. (2013). Bioinformatics, 29(6), 717-724; [3] Persson,
S. et al. (2005). PNAS USA, 102(24), 8633-8638; [4] Stuart, J. M. et al. (2003). Science, 302(5643), 249-255.
P3-07
Xylans in Plantago Species
Burton R.A., Fincher G.B., Shirley N.J., Le-Lam-Thuy Phan J., Tucker M.R.
ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of Adelaide, Urrbrae, SA
5064, Australia.
The seed mucilage of members of the Plantago genus is predominantly composed of heteroxylan. Whilst the
biological function of the mucilage has been the subject of some debate, the heteroxylan is used as a dietary fibre
supplement and a gelling agent in food. We are now using these myxospermous seeds as a model for studying
xylan biosynthesis and structure. We have examined the histology, monosaccharide content, polysaccharide
structure and transcript patterns of mucilages from a variety of Plantago species. Differences are indicated by
variable xylose:arabinose ratios and oligosaccharide fingerprints, and these are being related to changes in global
transcript profiles. Using the natural variation in gene expression, combined with significant differences in
mucilage composition, we are defining enzyme families involved in xylan synthesis, backbone substitution and
polysaccharide turnover. This approach has been complemented by the construction of a gamma-irradiated
population of P. ovata that we are screening for mutants with altered mucilage production and composition.
P3-08
The DUF642 cell wall proteins: a new family of plant carbohydrate binding proteins ?
Roujol D. (a), Vázquez-Lobo A. (b), Hoffmann L. (a), Albenne C. (a), Gamboa de Buen A. (b), Jamet E. (a)
(a) LRSV, UPS/CNRS, 31326 Castanet-Tolosan, France; (b) Instituto de Ecología, Universidad Nacional Autónoma de
México, Mexico.
The evolution of spermatophyte plants involved fundamental changes in cell wall structure and function which
resulted from diversification of carbohydrates and proteins. Cell wall proteomics analyses identified a novel
family of proteins of yet unknown function, the DUF642 (Domain of Unknown Function 642) proteins. These
proteins were found to accumulate at high level in quickly growing Arabidopsis thaliana organs [1]. The cell wall
localization of these proteins was confirmed using RFP fusion proteins in Nicotiana benthamiana cells. To
investigate the evolution of this family, 154 gene sequences from 24 plant species were analyzed, and
phylogenetic inferences were conducted [2]. Orthologous genes were detected in spermatophyte species and
absent in non-seed known plant genomes. Protein sequences shared conserved motifs that defined the signature of
the family. Distribution of conserved motifs indicated an ancestral intragenic duplication event. Gene phylogeny
documented paleoduplication events originating three or four clades, depending on root position (A, B, C and D).
A glycosylphosphatidylinositol (GPI)-anchor site and one or two galactose-binding domains-like (GBDLs) could
be predicted for some DUF642 proteins. The B, C, and D clades grouped the predicted GPI-anchored proteins.
Interaction of an A. thaliana DUF642 protein with a cell wall polysaccharide fraction has been demonstrated in
vitro. A competition assay with cellulose prevented this interaction. The degree of diversification and the
conservation of the family suggest that DUF642 proteins are key components in seed plant cell wall evolution.
[1] Irshad et al. (2008) BMC Plant Biol., 8, 94; [2] Vásquez-Lobo, Roujol et al. (2012) Mol. Phylogenet. Evol., 63, 510-520.
P3-09
New cell wall polysaccharides in charophytic algae
O’Rourke C., Fry S.C.
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, UK.
Plants colonised land 460 million years ago and charophytic algae represent the closest living relatives of land
plants. The ability to live on land may depend on the presence of certain cell wall polysaccharides such as
xyloglucan, a hemicellulose exclusively found in land plants [1]. The cell wall polysaccharides of charophytic
algae are poorly characterised. We aimed to use biochemical techniques to characterise the cell wall
polysaccharides of charophytic algae in relation to early land plant phylogeny. Hydrolysis of Coleochaete scutata
and Chara vulgaris cell walls in 2 M TFA yielded predominantly GalA, Gal, Glc and Man residues and also some
Ara, Xyl and traces of Fuc and Rha. In addition, hydrolysis of Chara and Coleochaete cell walls revealed an
abundance of an unusual monosaccharide, 3-O-methyl-D-galactose, more commonly found in lycophyte cell
walls, an evolutionarily isolated phylogenetic group [2].
Coleochaete and Chara hemicellulose extracts were fractionated by anion-exchange chromatography into five
classes. A strongly anionic fraction from Chara hemicellulose was found to be rich in Glc, Xyl, Gal and Fuc,
suggestive of a xyloglucan-like polysaccharide. However, XEG was unable to produce diagnostic xyloglucan
oligosaccharides in either Coleochaete or Chara hemicelluloses. Xylanase and mannanase digestion of
Coleochaete and Chara hemicelluloses gave xylan- and mannan-oligosaccharides whose composition will be
reported. The foundations of land plant polysaccharides are likely to be found in charophyte cell walls.
[1] Popper and Fry (2003) Annals of Botany 91, 1-12; [2] Popper et al. (2001) Phytochemistry 57, 711-719.
We thank the Leverhulme Trust for financial support.
P3-10
Presence of HRGPs and CM8 proteins in Cucurbitacea plant genomes
Jose-Estanyol M., Puigdomènech P.
Centre de Recerca en Agrigenòmica (CRAG), CSIC-IRTA-UAB-UB, Edifici CRAG, Campus UAB, Bellaterra (Cerdanyola
del Vallés), 081993 Barcelona, Spain.
HRGPs cell wall proteins are involved in different processes associated to plant development. Different families
of these genes as extensins, PRPs and AGPs have been described in different monocotiledoneous and
dicotiledoneous plants. Sometimes the proline-rich domain appears present in multidomain proteins associated to
other proteins domains as: leucine-rich repeats (LRXs), a kinase protein domain (PERKs) or a hydrophobic
cysteine-eight motif domain (CM8) in HyPRPs. We have been studying the expression, regulation and cellular
localization of one these proteins, ZmHyPRP [1]. ZmHyPRP appears to be expressed mainly in the scutellum of
maize immature embryos. Its cellular localization has been studied by fusion of its coding region to GFP under the
control of a constitutive promoter by particle bombardment of onion bulb epidermal cells. Results have shown
localization of the fusion protein in vesicles lining the plasma membrane of the bombarded epidermal cells. The
distribution analysis of these protein families in Cucurbitacea plants genomes, as Cucumis melo [2] has been
undertaken. Members of these protein families have been identified in this plant by Blast analysis from
Arabidopsis described proteins [3, 4] and from gene ontology statistics in htpp://www.icugi.org and
http://melonomics.net. Our results have shown that HRGPs proteins in rapport to HRGPs-multidomain proteins
are underrepresented in C. melo genome with respect their described presence in the Arabidopsis one. For CM8
proteins these differences are less evident. Different causes that can explain these results will be discussed.
[1] Jose-Estanyol and Puigdomènech, (2012) Plant Mol. Biol., 80, 325-335; [2] Garcia-Mas et al. (2012) Proc. Natl. Acad.
Sci. USA, 109, 11872-11877; [3] Showalter et al. (2010) Plant Physiol., 153, 485-513; [4] Jose-Estanyol et al. (2004) Plant
Physiol. Biochem., 42, 355-365.
P3-11
High-throughput cell wall profiling of ripening in wine grapes (cv. Cabernet sauvignon) versus
table grapes (cv. Crimson seedless) reveals differences in polymer abundance
Moore J.P. (a), Zhang S.-L. (a), Yu G. (a), Joubert C. (a), Raath P. (a), Fangel J. (b), Willats W.G.T. (b), Vivier M. (a)
(a) Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, South Africa; (b)
Department of Plant Biology and Biotechnology, University of Copenhagen, Denmark.
Wine grapes are small, possess thick skins and fleshy pulp-tissue whereas table grapes are generally much larger,
are thin-skinned and have a crunchy texture [1]. Unlike wine grapes, which have been bred for concentrated flavor
and aroma in juice (must) for winemaking, table grapes are primarily grown for eating where texture and visual
appearance are important [1]. Table grapes are also exposed to hormone (mainly gibberellin) treatments [1]. No
comparative study has yet investigated the differences (or similarities) in wine grape [2] versus table grape
cultivars in relation to cell wall structure/composition. High-throughput cell wall profiling tools [3] were used to
assess changes during ripening, using monosaccharide analysis, CoMPP and FT-IR spectroscopy coupled with
chemometrics [4]. Patterns of cell wall polymer turnover were clearly consistent between cultivars following a
developmental profile; including galactans, galacto-gluco-mannans, gluco-mannans and xyloglucan components.
AGPs and extensins also showed turnover and deposition at the onset of ripening concurring with earlier studies.
Specific differences were evident for pectin (HG and esterification levels), xyloglucan and cellulose proportions at
similar stages. This study provides a useful framework to understand and investigate the molecular genetic (and
hormone responsive) parameters that govern cell wall assembly/turnover in ripening grapes.
[1] G.L. and L.L. Creasy (2009) Grapes, CABI Wallingford UK; [2] K.J. Nunan et al. (1998) Plant Physiol. 118, 783-792; [3]
E. Nguema-Ona et al. (2012) Carbohydrate Polymers, 88, 939-949.; [4] J.P. Moore et al. (2013) Planta., 237, 739-754.
P3-12
Kingdom-wide plant cell wall metaglycomics
Fangel J.U. (a), Mikkelsen M.D. (a), Domozych D.S. (b), Jacobsen N. (a), Harholt J. (a), Ahl L.I. (a), Ulvskov P. (a), Willats
W.G.T. (a)
(a) Department of Plant and Environmental Sciences, University of Copenhagen, Faculty of Life Sciences, Thorvaldsensvej
40, 1870 Frederiksberg, Denmark; (b) Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College,
Saratoga Springs, New York, 12866, USA.
Millions of years of evolutionary development have produced plant cell wall components and architectures that
are exquisitely adapted to serve varied functional requirements. However, our understanding of the diversity and
evolutionary history of cell walls remains limited. To address this, we have established a metaglycomic platform
that, analogous to metagenomics, is based on the analysis of diverse environmental samples. Our approach is
based on immuno-microarrays that enable the high-throughput profiling of cell walls from more than 350 species
representing the major green plant taxa, including chlorophyte algae, charophyte algae and land plants. Using this
platform we seek to map polysaccharide diversity and, by integrating this data with existing metagenomic and
transcriptomic resources, to gain insight into the forces that have driven cell wall evolution.
In contrast to metagenomics, metaglycomics is in its infancy - largely because rapidly sampling glycomes that
typically contain highly complex and heterogeneous polysaccharides is technically difficult. The system we have
developed is based on multiplexed sequential extraction of cell wall components, which are then printed as arrays
using adapted peizo-based non-contact robots. The arrays are probed with panels of monoclonal antibodies
(mAbs) and carbohydrate binding modules (CBMs) and the output provides semi-quantitative information about
epitope occurrence. We have also undertaken a parallel programme aimed at the production of applicationspecific mAb and CBM probe sets. This system is versatile and can be applied to many areas of plant
glycobiology, for example polysaccharide processing and functional studies, some of which will be discussed.
P3-13
The cell wall of tobacco and tomato pollen tubes contains fucosylated xyloglucan not found in
somatic cells
Dardelle F. (a), Lehner A. (a), Bardor M. (a), Rihouey C. (b), Causse M. (c), Lerouge P. (a), Driouich A. (a), Mollet J.-C. (a)
(a) Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale, UPRES EA 4358, IRIB, Normandy University,
University of Rouen, 76821 Mont-Saint-Aignan Cedex, France; (b) Laboratoire Polymères, Biopolymères, Surfaces, UMR
CNRS 6270, IRIB, Normandy University, University of Rouen, 76821 Mont-Saint-Aignan Cedex, France; (c) Génétique et
Amélioration des Fruits et Légumes, INRA UR1052, 84143 Montfavet Cedex, France.
In plant, fertilization relies on the delivery of the sperm cells carried by the pollen tube to the ovule. The proper
cell wall assembly is crucial for promoting the pollen tube growth. Xyloglucan (XyG) is a major cell wall polymer
known for maintaining the cell wall integrity allowing cell expansion. In Arabidopsis thaliana (Brassicaceae),
XyG of the pollen tube wall was shown to be highly fucosylated and O-acetylated, suggesting a specific role of
these two features in the pollen tube growth [1]. In somatic cells of Solanaceae, XyG is lacking the fucosyl
residue and instead is composed of arabinosylated XyG [2-5], due presumably to an adaptative and/or selective
diversification. Analyses by oligosaccharide mass profiling [6] of pollen tube XyG showed that in Nicotiana
tabacum the XyG was mainly fucosylated. In wild (Solanum pimpinellifolium and S. peruvianum) and selected (S.
lycopersicum) tomatoes, pollen tubes were composed of arabinosylated and fucosylated XyG with the highest
level of fucosylation found in wild species. In addition, bioinformatic analyses indicate that S. lycopersicum
genome [7] contains genes encoding proteins related to AtFUT1 and PsFT1, two characterized XyG
fucosyltransferases from A. thaliana [8] and Pisum sativum [9]. Our findings indicate that (a) pollen tubes have a
specific set of functional XyG fucosyltransferases, (b) the male gametophyte has not evolved at the same pace as
the vegetative sporophyte and (c) fucosylation of XyG may play a central role in the mechanical properties of the
cell wall and in the interaction with the cell wall of the transmitting tract cells during pollen tube growth.
[1] F. Dardelle et al. (2010) Plant Physiol., 153, 1563-1576; [2] W.S. York et al. (1996) Carbohydr. Res., 285, 99-128; [3]
I.M. Sims et al. (1996) Carbohydr. Res., 293, 147-172; [4] Z. Jia et al. (2003) Carbohydr. Res., 338, 1197-1208; [5] M.
Hoffman et al. (2005) Carbohydr. Res., 340, 1826-1840; [6] N. Obel et al. (2009) Mol. Plant 2, 922-932; [7] The tomato
genome consortium (2012) Nature, 485, 635-641; [8] R.M. Perrin et al. (1999) Science, 284, 1976-1979; [9] A. Faik et al.
(2000) J. Biol. Chem., 275, 15082-15089.
P3-14
Monoclonal antibodies for the dissection of sulphated fucans in cell walls of brown algae
Torode T. (a), Hervé C. (b), Knox J.P. (a)
(a) Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds LS2 9JT, UK; (b) UMR1739 Marine Plants
and Biomolecules, Station Biologique de Roscoff, Place Georges Teissier, 29688 Roscoff, France.
Cell walls of the brown algae contain a diverse range of polysaccharides, including alginates and sulphated
fucans, with medically- and industrially-useful bioactivities. The sulphated fucan family of polysaccharides is
highly complex in structural terms. These glycans, specific to the brown algae, are defined as fucose- and
sulphate-rich polysaccharides but extensive variations in sugar composition, structure and sulphate content are
observed. The roles of fucans in cell wall architectures and cell properties are not known in detail. Molecular
probes, such as monoclonal antibodies are powerful tools to locate, dissect and study complex
heteropolysaccharides such as the brown algal fucans. The presentation will describe progress in the generation of
monoclonal antibodies directed to the sulphated fucan family of polysaccharides and the development of
approaches to understand antibody recognition in relation to glycan sulphation. Cell wall extractions reveal
differences in the occurrence of fucan epitopes in the vegetative and reproductive tissues of Fucus vesiculosus.
Fluorescence imaging of fucan sulphation patterns in these tissues indicates significant developmental dynamics
for this class of algal polysaccharide.
P3-15
Enzymes Involved in the Processing of Coastal Algal Biomass
Ficko-Blean E., Michel G., Czjzek M.
Station Biologique de Roscoff, Roscoff, France.
One of the most appreciated roles of carbohydrates is in metabolism and many organisms devote a large
proportion of their genomes to genes encoding proteins involved in carbohydrate processing. The genome of the
the marine bacterium Zobellia galactanivorans has been sequenced and annotation of the genome has revealed a
remarkable number of predicted gene products that are expected to be dedicated to processing the highly varied
complex algal glycans found in the marine environment, such as cell wall processing enzymes and enzymes
involved in nutrient uptake. However, much of this knowledge is actually limited to the ‘in silico’ level, moreover
annotation is mostly done on the basis of terrestrial model organisms that are significantly different from those
belonging to the marine environment. Thus, the in-depth knowledge on the biochemical details of marine algal
and associated bacterial metabolisms lags well behind their terrestrial counterparts. Marine algal polysaccharides
display a huge chemical diversity and differ greatly in composition and structure, notably, all marine algae
produce sulfated polysaccharides, which are absent in land plants.
The structural and biochemical aspects of some of these enzymes will be presented in the context of their overall
biological function. The investigation into the carbohydrate-active enzymes from the prokaryotic bacterium Z.
galactanivorans is beginning to uncover highly specialized systems involved in the recognition and processing of
protective algal matrix polysaccharides.
Session 4 : Functions of Plant CW in planta
Growth, morphogenesis & development, Signaling & defense, Response to environment
P4-01
Transgenic Expression of Pectin Methylesterase Inhibitors in Arabidopsis and Tobacco limits
Tobamovirus spreading
Lionetti V. (a), Raiola A. (b), Fabri E. (a), Cervone F. (a), Bellincampi D. (a)
(a) Department of Biology and Biotechnologies “C. Darwin”, Sapienza University of Rome, Rome, Italy; (b) Department of
Land, Environment, Agriculture and Forestry, University of Padua, Legnaro (PD) Italy.
Viral infection is a complex process influenced by the balance of virus-encoded proteins and host factors which
support virus replication, cell-to-cell and long distance movement through the plant. Virus-encoded movement
proteins (MPs) are necessary to allow cell-to-cell spread through plasmodesmata (PD). The interaction of MP
with plant pectin methylesterase (PME) is required for Tobacco mosaic virus (TMV) local spreading in tobacco.
The viral exit out of the vascular system is also partly PME-dependent. Pectin demethylation directed by PME is
likely to be a source of methanol that has been recently found to facilitate TMV spreading by triggering PD
dilation. We here report that the expression of a PME inhibitor from Actinidia chinensis (AcPMEI) in Nicotiana
tabacum decreases the overall PME activity and increases the level of pectin methylesterification of the wall
without affecting plant morphology and development. After inoculation with TMV the transformed plants
expressing AcPMEI exhibited a significant delay of viral spreading. A reduced susceptibility against Turnip vein
clearing virus (TVCV) infection was also observed in Arabidopsis plants overexpressing the AtPMEI-2 inhibitor.
Overall, our results indicate PMEIs as efficient tools to limit Tobamovirus infection.
P4-02
WallNet - Exploring the Biosynthesis and Function of Rhamnogalacturonan II During Root
Development in Arabidopsis
Smyth K. (a), Ociepa P. (b), Mikolajek H. (b), Werner J.M. (b), Marchant A. (b)
(a) University of Greenwich, United Kingdom; (b) University of Southampton, United Kingdom.
Pectins are a functionally important class of complex polysaccharides found in the primary cell wall of plants.
There are 4 major forms of pectin: homogalacturonan, rhamnogalacturonan I, rhamnogalacturonan II (RGII) and
xylogalacturonan. RGII is a complex structurally conserved polysaccharide found in all vascular plants. RGII has
an important structural role in the primary cell wall with boron-mediated cross-linking of two RGII molecules
resulting in formation of a three-dimensional pectic network. Despite the importance of RGII, surprisingly few
genes required for its biosynthesis have been identified. Gene knockout approaches have been limited by the
lethality of many mutants, including those resulting in a loss of the Kdo nucleotide sugar activating enzyme,
CMP-Kdo synthase (KdsB). To overcome this lethality and study the effect of disrupting Kdo biosynthesis in the
sporophyte generation, an inducible transactivation system combining the ethanol inducible ALCR/alcA and
GAL4/VP16 expression system has been adopted. This provides both temporal and spatial control over expression
of a KdsB antisense sequence. Further work has been carried out to determine the developmental function of RGII
using 2β-deoxy-Kdo, a potent inhibitor of both the bacterial and Arabidopsis KdsB enzymes. Exogenous
application of 2β-deoxy-Kdo to Arabidopsis seedlings results in severe effects on root development which can be
partially rescued by addition of boron [1]. These approaches are allowing a detailed analysis of RGII function
during root development to be undertaken, focussing on elongation, root hair formation and lateral root
biogenesis.
[1] K.M. Smyth et al. (2013) Molecular Plant, Advance access doi:10.1093/mp/sst011.
P4-03
Twisting the golden angle: Arabidopsis phyllotaxis depends on cellulose synthase guidance
Landrein B. (a), Lathe R. (b), Bringmann M. (b), Vouillot C. (a), Ivakov A. (b), Boudaoud A. (a), Hamant O. (a), Persson S.
(b)
(a) Laboratoire de Reproduction de développement des plantes, INRA, CNRS, ENS Lyon, UCB Lyon 1, Université de Lyon,
46 Allée d’Italie, 69364 Lyon, Cedex 07, France;(b) Max Planck Institute of Molecular Plant Physiology, Germany.
Attempts have been made to understand the interplay between patterning and growth during morphogenesis in
Arabidopsis. This has largely been focused on the relationship between the spiral phyllotactic pattern, i.e. the
organogenesis pattern prescribed in the shoot apical meristem, and anisotropic growth. We observed a subtle
right-hand torsion of the stem in the cellulose synthase interacting (csi1) mutant. As cellulose deposition is largely
uncoupled from cortical microtubule guidance in this mutant, it is plausible that the cellulose fibers are produced
in a tilted fashion, which could result in a scenario prescribed in fiber-reinforced models of growing cylinders. We
indeed observed tilted fibers and Cellulose Synthase trajectories in the csi1 mutant cells. Strikingly, we also
identified a novel bimodal and robust phyllotactic pattern in the csi1 mutant. This phenotype was entirely
explained by the interplay between the chirality direction of organ initiation sequences in the meristem and the
right-handedness of stem torsion, which emerges as a result of the defects in cellulose microfibril orientations.
Interestingly, the csi1 torsion and phyllotactic phenotypes were entirely rescued in a csi1 prc1 background, in
which cellulose microfibrils are compromised. The relation between anisotropic growth and phyllotaxis was
further confirmed and generalized in the katanin and spiral2 mutant backgrounds. Thus, in wild type plants,
cortical microtubules actively prevent cellulose synthases from deviating from transverse trajectories in growing
cells and this relationship is prerequisite for spiral phyllotaxis.
P4-04
Cell wall microstructure of potato cortex tissue and correlations with bruise susceptibility upon
harvest and storage
Scharf R., Orfila C.
School of Food Science and Nutrition, University of Leeds, LS 2 9JT, Leeds, UK.
Potato tuber cortex is an interesting model for investigating the relationship between molecular microstructure and
mechanical properties, most particularly in relation to texture and bruising. Potatoes (Solanun tuberosum cv.Lady
Rosseta) were grown in replicate field plots and studied at different harvest and storage times. Tissue
microstructure was investigated using confocal fluorescence microscopy that allowed visualization of cell wall
polysaccharides. Cell wall and phenolic composition was analysed chemically using HPLC following new acid
hydrolysis procedure that involved sequential hydrolysis in TFA and H 2SO4. Cortex mechanical properties were
measured using a TA.XT2i Texture Analyser that allowed the tissue to be deformed under controlled conditions.
Bruising was performed using a falling bolt and the impact was recorded using a high-speed video camera. The
results show that cortex tissue consists of tightly packed hexagonal parenchyma-type cells measuring around 50100 mm in diameter. Potatoes harvested later in the season were more susceptible to bruising (x2). The cortex was
on average 11% tougher (p<0.05) requiring more work (J) to deform. This was associated with lower levels of
pectin methyl esterification in cortex cell walls. When potatoes were stored for 6-7 months, potatoes from later
harvests were more susceptible to bruising (x3) compared to potatoes harvested early (x1.5). Upon storage, the
cortex was 55% tougher (p<0.05) irrespective of harvest time and cortex cell walls appeared to have lower levels
of galacturonic acid, glucuronic acid and neutral sugars, including rhamnose, arabinose and galactose. At the same
time, total phenolics (5-caffeoylquinic, ferulic, p-coumaric and caffeic acids) significantly decreased with both
harvest and storage time. Therefore, the cell wall microstructure of the tissue may be implicated in determining
bruising susceptibility, independently of biochemical substrates of polyphenol oxidase.
P4-05
βIII-gal is involved in galactan reduction during phloem element differentiation in chickpea stems
Martín I. (a), Albornos L. (a), Hernández-Nistal J. (b), Labrador E. (a), Dopico B. (a)
(a) Departamento de Fisiología Vegetal, Centro Hispano Luso de Investigaciones Agrarias, Universidad de Salamanca. Pza.
Doctores de la Reina s/n. 37007 Salamanca, Spain. (b) Departamento de Fisiología Vegetal, Universidad de Santiago de
Compostela. Campus de Lugo, 27002 Lugo, Spain. [email protected].
βIII-Gal, a member of the chickpea β-galactosidase family, is the enzyme responsible for the cell wall autolytic
process [1]. This enzyme displays significant hydrolytic activity against cell wall pectins and its natural substrate
has been determined as a pectin arabinogalactan [2]. In the present work, the location of βIII-Gal was analysed in
epicotyls and radicles from 4- and 8-day-old etiolated seedlings and in the five stem internodes of 11-day-old
chickpea plants by using specific anti-βIII-Gal-antibodies. Our results revealed that besides its possible role in
early events during primary xylem and phloem fibre differentiation, βIII-Gal acts on the development of sieve
elements. The main location of βIII-Gal in primary phloem in the basal-most sections of the oldest epicotyls and
radicles is also observed on the five stem internodes of 11-day-old plants, where βIII-Gal was exclusively detected
in this tissue, except for a faint labelling in phloem fibres on the third internode.
This location is consistent with the reduction in galactan observed with LM5 antibodies during the maturation of
this tissue, with a marked decrease in differentiated phloem cell walls in the basal-most internodes, in contrast to
the presence of this epitope in the procambial zone.
Thus, βIII-Gal could act on its natural substrate, the neutral side chains of rhamnogalacturonan I, contributing to
cell wall reinforcement allowing phloem elements to differentiate, and conferring the necessary strengthening of
the cell wall to fulfil its function.
[1] B. Dopico et al. (1990) Physiol. Plant., 80, 629-635 ; [2] I. Martín et al. (2005) Plant Cell Physiol., 46, 1613-1622.
Acknowledgments: Project funding by the Spanish Ministerio de Ciencia e Innovación (MICINN) (BFU2009-08769), Fellowships: Lucía
Albornos, FPU grant from Spanish Ministerio de Educación; Lucía Izquierdo, FPI grant from MICINN.
P4-06
Knockout mutants of arabidopsis thaliana β-galactosidase (subfamily a1). modifications in their
cell wall polysacharides and enzymatic activities
Moneo M., Izquierdo L., Martín I., Dopico B., Labrador E.
Departamento de Fisiología Vegetal, Centro Hispano Luso de Investigaciones Agrarias, Universidad de Salamanca. Pza.
Doctores de la Reina s/n. 37007. Salamanca, Spain. [email protected].
The GH35 family of β-galactosidases of Arabidopsis thaliana have been divided in seven subfamilies [1]. The
largest subfamilie, a1, comprises six proteins localized in the cell wall, whose function is unknown. To study the
function of these cell wall β-galactosidases, we use knockout mutants of the genes encoding subfamily a1 βgalactosidase. In this communication we will discuss the results related to the mutant of Arabidopsis ecotype col0
in the locus At3g1375 (BGAL1), At3g52840 (BGAL2) and At5g56870 (BGAL4).
Cell walls of 10-days-old mutants and wild type plants were analysed to determine the changes that occur in the
structure and composition of the wall of the mutant by determining the neutral sugar content and the FTIR
(Fourier transform infrared spectroscopy) spectra. We also studied the enzymatic activities of cell walls, mainly
βgalactosidase and β-galactanase in mutants in relation to wild type. Since some of the mutants present higher βgalactosidase activity than expected, we analysed whether the absence of an Arabidopsis β-galactosidase is
compensated by increased expression of genes encoding other a1 subfamily enzymes.
Cell wall autolysis process is accomplished mainly by β-galactosidases in dicot plants and it has been related with
cell wall modifications during development. Thus, we check the possible involvement of the BGAL1, BGAL2
and BGAL4 in the cell wall autolytic process, by study whether cell walls from bgal1, bgal2 and bgal4 display
autolytic activities and the amount and composition of the hydrolysed sugars.
[1] Ahn et al., (2007). Phytochem, 68, 1510-1520.
Acknowledgments: Project funding by the Spanish Ministerio de Ciencia e Innovación (MICINN) (BFU2009-08769).
Fellowships: María Moneo, Programa Predoctoral de Formación de Personal Investigador no doctor (Basque Governement); Lucía
Izquierdo, FPI grant from MICINN.
P4-07
Characterization of transgenic arabidopsis plants overexpressing βIII-gal from Cicer arietinum.
Izquierdo L., Moneo M., Martín I., Dopico B., Labrador E.
Departamento de Fisiología Vegetal, Centro Hispano Luso de Investigaciones Agrarias (CIALE), Universidad de
Salamanca. Pza Doctores de la Reina s/n. Salamanca 37007 Spain. [email protected].
One of the members of the chickpea β-galactosidase family, βIII-Gal, displays significant hydrolytic activity
against cell wall pectins and its natural substrate is a pectic arabinogalactan attached to rhamnogalacturonan I [1].
Previous results point to a possible involvement of this protein in sieve elements development, where βIII-Gal
could act on pectic side chains contributing to cell wall reinforcement and allowing phloem elements
differentiation [2].
In the present work, transgenic Arabidopsis plants overexpressing βIII-Gal under the control of 35S CaMV
promoter have been generated as an approach to gain insight into the function of this protein and the biological
role of pectic galactan side chains. Nineteen T 2 lines were selected and screened for single insertion by Southernblot. Eight single insertion lines were determined and ten individual plants of each line were taken to T 3. All these
lines were screened for homozygosis and 6 individual lines were selected. RT-PCR studies carried out with the
homozygous transgenic lines showed that βIII-Gal transcript accumulation was higher in lines 1.10.5, 1.6.6 and
3.7.4, which were chosen to carry out the subsequent analyses.
Preliminary phenotypic studies revealed a significant developmental delay in transgenic line 1.10.5. Transformed
10-day-old plants show notable differences in total fresh weight per plant (2.72 mg) when compared to WT (4.54
mg). Analyses of cell wall composition and cell wall autolytic capacity of transformed plants are being conducted
in order to establish a relation of the observed phenotype to the action of βIII-Gal.
[1] I. Martín et al. (2005) Plant Cell Physiol., 46, 1613-1622; [2] I. Martín et al. (2013) Plant Cell Physiol., In press.
Acknowledgments: Project funding by the Spanish Ministerio de Ciencia e Innovación (MICINN) (BFU2009-08769).
Fellowships: María Moneo, Programa Predoctoral de Formación de Personal Investigador no doctor (Basque Governement); Lucía
Izquierdo, FPI grant from MICINN.
P4-08
Analysis of the interactions between mucilage composition and fatty acid content in flaxseed
Miart F. (a), Pageau K. (a), Fournet F. (a), Fontaine J.-X. (a), Bouton S. (a), Van Wuytswinkel O. (a), Thomasset B. (b),
Mesnard F. (a)
(a) EA3900 BIOPI Biologie des Plantes et Innovation, UFR des Sciences, 33 rue Saint Leu, F-80039 Amiens France; (b)
CNRS UMR6022 Génie Enzymatique et Cellulaire, Université de Technologie de Compiègne, BP 20529, F-60205
Compiègne Cedex France.
In flax (Linum usitatissimum), the seed coat epidermal cells produce a large amount of mucilage that is mainly
composed of polysaccharides [1]. It is a powerful tool for plant cell wall research, because it is easily accessible
and is of great interest for the identification of novel genes involved in synthesis, secretion and modification of
cell wall chemical components like pectins [2]. In Arabidopsis thaliana, a relation was demonstrated between
genes involved in mucilage biosynthesis and the fatty acid content in embryo [3]. To find out whether a similar
relation would occur in flaxseed, a phenotypic screening of the mucilage proportion on recombinant inbreed lines
from the crossing between Oliver (a winter “oil” flax) and Viking (a spring “fiber” flax) was performed to select
several lines with different proportions in mucilage and omega-3 contents. The epidermal cells of the seed coat of
these lines is being analysed by histochemical techniques and the carbohydrate composition of their mucilage is
being chemically characterized.
[1] Naran R. et al. (2008) Plant Physiol.148, 132-141; [2] Arsovski A. et al. (2010) Plant Signaling and Behavior.5, 1-6; [3]
Shi L. et al. (2012) Plant J.69, 37-46.
P4-09
Receptor-like kinases involved in feedback regulation from the secondary plant cell wall
Stoppel R. (a), Oikawa A. (a), Ebert B. (a), Scheller H.V. (a)
(a) Joint BioEnergy Institute, Feedstocks Division, Lawrence Berkeley National Laboratory, Emeryville, California 94608,
USA.
Plant cell walls are complex structures composed of polysaccharides that influence plant development and
differentiation and also comprise the most abundant biomaterial on earth with the potential to provide a source of
cheap sugars for industrial biotechnology. Intense research over the recent years has provided an understanding of
many aspects of cell wall biosynthesis and numerous genes and proteins involved in this process. It becomes
obvious that plants have sophisticated feedback mechanisms, which enable them to adapt the compositions of
their walls in response to environmental changes or biotechnological intervention. The major group of protein
classes that have been implicated in feedback from the cell wall are the receptor-like kinases (RLKs). Candidate
RLKs that may recognize small molecules such as peptides and saccharides were identified in a previous coexpression approach [1]. Using fusions with fluorescent proteins, we could show that all of them are localized in
the plasma membrane together with strongly co-expressed candidate ligands that could represent signals that
trigger signalling pathways. In order to obtain a better insight into the biosynthesis and regulation of the
secondary cell wall, we set up a screen for kinase-ligand interactions using chimeric RLKs containing a kinase
domain from wall-associated-kinase 1 (WAK1) fused with the candidate receptor domains from the identified
RLKs. Transient co-transformation of candidate ligands and RLK domain swap constructs in tobacco leaves along
with analyses of knockout mutants suggest that these proteins are factors in conserved regulatory pathways during
secondary wall development and can be crucial for the signalling perception.
[1] A. Oikawa et al. (2010) PLoS One, 5, e15481.
P4-10
Identification of 2 extensin-related proteins and their involvement in elongation of Arabidopsis
thaliana cells
Boron A.K. (a), Van Orden J. (a), Markakis M.N. (a), Mouille G. (b), Adriaensen D. (c), Verbelen J.-P. (a), Höfte H. (b),
Vissenberg K. (a)
(a) Laboratory of Plant Growth and Development, Biology Department, University of Antwerp, Groenenborgerlaan 171,
2020 Antwerpen, Belgium; (b) Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, INRA, Route de Saint-Cyr,
78026 Versailles cedex, France; (c) Laboratory of Cell Biology and Histology, Veterinary Sciences Department, University
of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium.
The synthesis and composition of cell walls is dynamically adapted in response to many developmental and
environmental signals. In this respect cell wall proteins controlling cell elongation are critical for decent cell
development. Transcription analysis identified 2 genes with a potential role in the cell expansion process of
Arabidopsis thaliana.
Phylogenetic analysis of the first gene (AtPRPL1) showed most resemblance with AtPRP1 and AtPRP3 proteins
that are involved in root hair growth and development; the second gene is suggested to encode a hydroxyprolinerich glycoprotein family protein containing a GPI-anchor. Phenotypic analysis of knock-out lines of both genes
revealed that cell elongation is impaired in root hairs and in dark-grown hypocotyls respectively.
Detailed gene expression patterns generated by analysis of promoter::GUS/GFP lines, locations of protein-GFP
fusions, the effect of overexpression on cell elongation and the effect of altered gene expression levels on cell wall
composition (FT-IR analysis) will be discussed together with the possible mechanisms of elongation control.
P4-11
An Arabidopsis thaliana class III peroxidase is involved in the regulation of cell expansion
Raggi S. (a), Ferrarini A. (b), Cervone F. (a), Ferrari S. (a)
(a) Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Sapienza Università di Roma Italy ; (b) Dipartimento di
Biotecnologie, Università degli Studi di Verona Italy.
Cell wall strongly influences plant growth. To investigate the role of cell wall pectin, and in particular of
homogalacturonan (HGA), in this process, we have analyzed Arabidopsis plants expressing a fungal
polygalacturonase, which degrades de-esterified HGA. These plants, like mutants affected in HGA biosynthesis,
show reduced growth and cell adhesion. To better understand the basis of this phenotype, we have performed a
transcript profile analysis of these plants and identified genes up-regulated in response to altered pectin. One of
these genes, PER71, encoding a class III peroxidase, was further studied. Characterization of loss- and gain-offunction plants indicate that PER71 negatively regulates cell expansion, and is required for the production of
reactive oxygen species in response to cell wall stress or to microbial elicitors. These data suggest that this
peroxidase gives a major contribution to the reinforcement of the cell wall both under physiological conditions
and in response to biotic stress, and therefore play an important role in plant growth and development.
P4-12
Regulation of pectin methylesterases (PMEs) by subtilases (SBTs) and pectin methylesterases
inhibitors (PMEIs) during root growth
Sénéchal F. (a), Graff L. (b), Surcouf O. (c), Guérineau F. (a), Marcelo P. (d), Assoumou Ndong Y. (a), Fournet F. (a),
Mareck A. (c), Rayon C. (a), Mouille G. (e), Stinzi A. (b), Höfte H. (e), Lerouge P. (c), Schaller A. (b), Pelloux J. (a)
(a), EA3900-BIOPI, UPJV, 33 Rue St Leu, F-80039 Amiens, France; (b), Universität Hohenheim, Institut für Physiologie
und Biotechnologie der Pflanzen (260) D-70593 Stuttgart, Germany; (c), EA4358-Glyco-MEV, IFRMP 23, Université de
Rouen, F-76821 Mont-Saint-Aignan, France; (d), ICAP, UPJV, 1-3 Rue des Louvels, F-80037 Amiens, France; (e), IJPB,
UMR1318 INRA-AgroParisTech, Bâtiment 2, INRA Centre de Versailles-Grignon, Route de St Cyr (RD 10), F-78026
Versailles, France.
In Arabidopsis thaliana, homogalacturonan (HG), the main pectic constituent can be modified by the activity of
cell wall enzymes, which belong to large multigene families. More particularly, the degree of methylesterification
(DM) of HG can be modified by pectin methylesterases (PMEs), whose activity can be regulated by endogenous
PMEIs (pectin methylesterase inhibitors). In most organisms, two types of protein structure have been reported for
PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an Nterminal extension (PRO part), which shows similarities with PMEI (PMEI domain, Pfam04043) [1]. It was
shown that the PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating
PME activity through a post-translational mechanism. Recent results suggest that subtilases (SBTs) play a key
role in this process [2]. Following the identification of co-expressed PME, PMEI and SBT, we report, using
biochemistry and functional genomics approaches, on the possible role of the interplay of these three proteins in
the fine tuning of HG structure, and its role in controlling root growth in Arabidopsis.
[1] Pelloux J. et al. (2007) Trends Plant Sci., 12, 267-277 ; [2] Wolf S. et al. (2009) Plant J., 58, 361-375.
P4-13
The impact of Arabidopsis ecotype on aphid feeding involves cell wall differences
Wattier C. (ab), Pau-Roblot C. (a), Pelloux J. (a), Cherqui A. (b), Rustérucci C. (a)
(a) EA 3900 BIOPI - Biologie des Plantes et Innovation, Université de Picardie Jules Verne, 80039 Amiens, France;(b) FRE
3498 CNRS-EDYSAN - Ecologie et Dynamique des Systèmes Anthropisés, Université de Picardie Jules Verne, 80039 Amiens,
France.
Aphids are phloem feeding insects that insert their stylets through the plant cell wall layers to reach the sieve
elements. During stylets progression in the apoplasm, most cells are briefly punctured intracellularly for probing.
Aphid infestation induces plant defence responses including cell wall modifications. Both feeding behavior and
physiology of the green peach aphid Myzus persicae are compared during the infestation on two ecotypes of
Arabidopsis thaliana. Clearly Columbia ecotype is less suitable to M. persicae than Wassilewskija one. The
antixenosis and the antibiosis effects on M. persicae are discussed in regards to cell wall composition and cell wall
enzymes activities related to aphid-plant interactions.
P4-14
Cell wall metabolism of Miscanthus ecotypes in response to cold acclimation
Le Gall H., Fontaine J.-X., Domon J.-M., Fliniaux O., Molinié R., Gillet F., Pelloux J., Mesnard F., Rayon C.
EA 3900-BIOPI, Université de Picardie Jules Verne, 80039 Amiens, France.
Miscanthus is a potential energy crop grass with several advantages in biomass production. However, it can be
damaged by late frost when shoots emerge too early in the spring and during the first winter after planting. The
effects of cold acclimation on metabolome and cell wall were investigated in different Miscanthus ecotypes (frostsensitive, frost-resistant or intermediate).
Within this context, a 1H NMR metabolomic study was performed. Multivariate data analysis revealed a clear
separation between the different samples (ecotype and condition). The metabolites corresponding to the
discrimination were identified usind 2D NMR spectra.
In ambient temperature controls, each clone displayed different glucuronoarabinoxylan (GAX) contents and
degree of arabinose substitution on the xylan backbone [1]. These variations indicate that the clones have different
genetic backgrounds leading to different cell wall compositions, and the differences might explain their different
response to frost. During cold acclimation, an increase in ß-glucan content was observed in all genotypes. Uronic
acid level increased in the frost sensitive genotype but decreased in the frost tolerant genotype in response to cold.
A large increase in CAD activity under cold stress was displayed in each clone, but it was largest in the frosttolerant clone. The marked increase in PAL activity, observed in the frost-tolerant clones under cold acclimation,
could suggest a reorientation of the products towards the phenylpropanoid pathway or aromatic synthesis.
[1] JM Domon et al. (2013) Phytochem., 85, 51-61.
P4-15
Cold acclimation induces cell wall modifications during pea plant development
Baldwin L. (a), Domon J.-M. (a), Klimek J.F. (b), Fournet F. (a), Sellier H. (c), Gillet F. (a), Pelloux J. (a), Lejeune-Hénaut
I. (c), Carpita N.C. (b), Rayon C. (a)
(a) EA 3900-BIOPI, Université de Picardie Jules Verne, 80039 Amiens, France; (b) Department of Botany & Plant
Pathology, Purdue University, 915 West State Street, West Lafayette, IN 47907-2054, United States. (c) INRA USTL UMR
1281, Laboratoire de Génétique et d’Amélioration des Plantes, Estrées-Mons BP50136, 80203 Péronne, France.
Pea (Pisum sativum) cell wall metabolism was investigated in response to chilling in a frost-sensitive genotype
‘Terese’ and a frost-tolerant genotype ‘Champagne’. Stipules of cold-acclimated and non-acclimated control
plants were harvested at different times in both genotypes. For both genotypes, cold induced mostly changes in
the content of xylose, arabinose, galactose and galacturonic acid residues in the cell wall. A lower level in
xyloglucan and an increased amount in xylogalacturonan was observed in Champagne compared with Terese. A
substantial amount of pectin associated unbranched (1→5)-α-arabinans and branched arabinans were observed in
Champagne. The rise in level of branched and unbranched (1→4)-β-galactan only occurred in Champagne.
Furthermore, greater JIM7 labeling was observed in Champagne compared to Terese, indicating cold acclimation
induced an increase in the degree of methylesterification. No significant changes in pectin remodeling enzymes
were observed except a decrease in polygalacturonase enzyme activity in both genotypes at the end of cold
acclimation. Cold acclimation induced strong increase in the amount of (1→4)-β-xylan the frost-sensitive Terese,
indicating that cold precociously induced secondary cell wall biosynthesis in the frost sensitive genotype,
prematurely halting cell growth. This indicates that the increase of pectin matrix in Champagne during cold
acclimation functions in maintaining the integrity with the other polysaccharides of the cell wall under frost.
P4-16
A comprehensive analysis of secondary cell wall formation-associated genes
Endo S. (a), Naramoto S. (a), Saito C. (a) Fukuda H. (a)
(a) Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan.
To understand further the mechanism of secondary cell wall formation, we need to identify more key players
regulating secondary cell wall formation. Our systematic gene expression analysis displayed a number of
uncharacterized genes that are expressed tightly coupled with secondary wall formation [1–3]. To characterize
roles of these genes as well as genes for putative sugar modifying enzymes in secondary wall formation, we
produced transgenic Arabidopsis plants harbouring each of 101 genes. Each gene is expressed under the control of
CaMV 35S promoter. The over-expression of 68 among the 101 genes affected seedling development in T1
generation. The phenotypes were categorized into several groups such as “low transformation efficiency”, “root
growth defect”, “shoot growth defect”, “high stomatal density” and “vascular patterning defect”. Further studies
are going to address the question if the phenotypes are coupled with some changes in cell wall structure.
[1] T. Demura et al. (2002) Proc. Natl. Acad. Sci. USA, 99, 15794-15799 ; [2] M. Kubo et al. (2005) Genes Dev., 19, 18551860 ; [3] K. Ohashi-Ito et al. (2010) Plant Cell, 22, 3461-3473.
P4-17
Suppressor screening of the REDUCED WALL ACETYLATION 2 mutant of Arabidopsis
thaliana
Fimognari L., Stranne M., Nafisi M., Sakuragi Y.
Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg 1871,
Denmark.
There is a growing interest in reducing the degree of the cell wall acetylation as the acetylation of cell wall
polysaccharides is an important parameter in the lignocellulosic biofuel production [1-3]. However, the
consequence of such modification on the plant fitness is poorly understood.
Previously it has been shown that the T-DNA insertion in the REDUCED WALL ACETYLATION 2 ( RWA2)
gene in Arabidopsis thaliana results in the increased resistance against the necrotic fungal pathogen Botrytis
cinerea [4]. The rwa2 mutants exhibited ca. 20% reduction in the acetylation content in pectin and xyloglucan as
well as in xylan. Consequently the rwa2 mutants exhibited increased permeability due to collapsed trichomes and
conditional growth impairments.
The aim of this project is to gain new insights into the underlining molecular link between the reduced wall
acetylation phenotype to the collapsed trichomes and the conditional growth impairments. To this end, the rwa2-3
seeds have been mutagenized with ethyl methanesulfonate and T2 generation seeds that suppress the rwa2
phenotypes have been screened.
[1] MJ Selig et al. (2009) Cellulose, 16, 711-722 ; [2] S. Helle et al. (2003) Enzyme Microb Tech, 33, 786–792 ; [3] D. KleinMarcuschamer et al. (2010) Biomass Bioenerg, 34, 1914-1921 ; [4] Y Manabe et al. (2011) Plant Physiol, 155, 1068-78.
P4-18
Changes in Cell Wall-Bound Hydroxycinnamates in Maize Stems Infested with Sesamia
nonagrioides Lefèbvre
Santiago R. (a), Hadj-Boussada K. (a), Barros-Rios J. (a), Fornalé S. (b), Malvar R.A. (a)
(a) CSIC-Misión Biológica de Galicia, Department of Maize Breeding and Genetics. PO-Box 28, P.C. 36080, Pontevedra,
Spain; (b) Centre for Research in Agricultural Genomics (CRAG) Consortium CSIC-IRTA-UAB-UB, Edifici CRAG, Campus
UAB 08193 Bellaterra (Cerdanyola del Vallés) Barcelona, Spain.
Cell wall fortification through oxidative cross-linking of cell wall polymers inhibits insect larvae access to
nutrients by increasing tissues toughness and indigestibility. Several reports have pointed out the role of the wallbound hydroxycinnamates as a maize constitutive defense mechanism against corn borers. However, if maize
plants react by changing the cell wall phenolic composition in response to corn borer injury has not been
extensively studied. The aim of our work was to evaluate the changes in strengthening related traits of maize
stalks after being infested with Mediterranean corn borer larvae (MCB, Sesamia nonagrioides Lef.). Ferulate and
p-coumarate monomers, diferulates, and lignin concentration and composition were determined in two maize
inbred lines (CO125 and PB130) selected on the basis of their respective high and low hydroxycinnamate
concentrations in stem pith tissues. After fifty-five days from infestation, the inbred line PB130 increased the
concentration of ferulate monomers and diferulates. Both inbred lines showed a significant reduction on pcoumarates after damage; however, no significant effect was observed for lignin concentration or composition
measured as syringyl to guaiacyl (S/G) ratio. These results provide direct evidence of the induced response in the
hydroxycinnamate content after insect injury. Further research is needed in order to elucidate the integration
process of these hydroxycinnamates into the cell wall after borers attack.
P4-19
Remodeling Arabidopsis cell wall uncouples resistance to pathogens from tradeoffs
Miedes E. (a), Riviére M.-P. (a), Sánchez-Vallet A. (a), Sánchez-Rodríguez C. (a), Ranocha P. (b), Bartel X. (c), Marco
Y. (c), Goffner D. (b) , Hahn M.G. (d), Molina A. (a)
(a) Centro de Biotecnología y Genómica de Plantas (UPM-INIA). Universidad Politécnica Madrid. Madrid, SPAIN; (b)
Unité Mixte de Recherche Centre National de la Recherche Scientifique Univ Toulouse III. Castanet Tolosan, FRANCE; (c)
Centre National de la Recherche Scientifique Instiut National de la Recherche Agronomique. Castanet Tolosan, FRANCE;
(d) Complex Carbohydrate Research Center. Athens, GA, USA.
The understanding of the dynamics and evolution of plant defensive responses is of fundamental importance as
the impact agricultural yield. In particular, the characterization of trade-offs associated to broad-spectrum, durable
resistance in crops needs further attention. The contribution of plant cell wall to this type of resistance was
analysed in Arabidopsis thaliana by determining the susceptibility/resistance of 20 cell wall mutants (cwm) to
different types of pathogens. We identified a significant number of cwm that uncoupled resistance to pathogens
from seed or/and biomass production, further indicating that wall remodelling might be an efficient strategy to
overcome trade-offs associated to pathogen resistance. The characterization of the genetic and molecular bases of
the resistance in the cwm mutants revealed that novel, previously uncharacterised signalling pathways controlled
their defensive responses. The relevance of cell wall-mediated resistance to pathogens was also supported by the
finding that key components of Arabidopsis thaliana defensive mechanisms, such as the ERECTA Receptor-like
Protein Kinase or the heterotrimeric G protein (AGB1)), modulated the immune response by regulating cell wall
integrity. Mutants impaired in these genes showed a mis-regulation of cell wall-associated genes and alterations in
cell wall composition/structure compared with those of wild-type plants.
[1] F. Llorente et al. (2005) Plant J., 43, 165-180; [2] C. Sánchez-Rodríguez et al. (2009) Mol. Plant Microbe. Interact., 22,
953-963.
P4-20
Agricultural conditions affect bast fiber formation and cell wall development in hemp
Fernandez-Tendero E. (abe), Day A. (b), Augier L. (a), Legros S. (c), Deyholos M. (d), Hawkins S. (b), Chabbert B. (ef)
(a) Fibres Recherche Développement, F-10901 Troyes, France ; (b) Université Lille Nord de France, Lille 1 UMR INRA
1281, SADV, F-59650, Villeneuve d'Ascq cedex, France ; (c) CETIOM, F-10901 Troyes, France ; (d) Department of
Biological Sciences, University of Alberta, Edmonton, Canada; (e) INRA, UMR614 Fractionnement des AgroRessources et
Environnement F-51100 Reims, France ; (f) Université de Reims Champagne-Ardenne, UMR 614, Fractionnement des
AgroRessources et Environnement F-51100 Reims, France.
Hemp (Cannabis sativa L.) is receiving increasing interest as an industrial crop due to the development of a new
range of industrial applications based on plant fibers (both core and bast fibers). Fiber yield and quality can be
greatly modified by physicochemical processing and are also influenced by both genotype and environment. In
order to gain a better understanding of how these factors impact on hemp fibers, two commercial hemp varieties
were grown under different agricultural conditions and analyzed for fiber content, cell size and cell wall
composition. Our results show that irrigation and high seed density impact on secondary fiber, but not primary,
formation, as well as on different morphological characters and cell wall composition of primary and secondary
fibers. All of these parameters can potentially modify the performances and industrial quality of the harvested
fibers. Initial transcriptomics (Fluidigm) of different targeted genes has also provided detailed information on how
different environmental conditions affect gene expression, primary and secondary metabolism, cell wall
biosynthesis and remodeling in both inner (xylem-rich) and outer (bast-fiber rich) hemp stem tissues. Altogether,
these results will lead to a better knowledge of the molecular events involved in fiber development contributing to
hemp-breeding programs aimed at improving the production of high quality fibers for different industrial uses.
P4-21
Complementation of a family 3 α-L-arabinofuranosidase/β-xylosidase repression by the increase of
family 51 isozyme expression
Tateishi A. (ab), Yoshimoto K. (a), Takeuchi R. (a), Inoue H. (ab)
(a) Graduate School of Nihon University, Fujisawa, Kanagawa, 252-0880, Japan; (b) College of Bioresource Sciences,
Nihon University, Fujisawa, Kanagawa, 252-0880, Japan.
cDNA clones (SlArf/Xyl1-4) encoding α-L-arabinofuranosidase/β-xylosidase belonging to glycoside hydrolase
family 3 were obtained from tomato (Solanum lycopersicum) fruit. Divergent expression patterns of the isolated
genes were shown. Among them, expression of SlArf/Xyl2 was high in early stage of developmental tissues, such
as 5 days after anthesis fruit. Antisense tomato plants which suppressed SlArf/Xyl2 expression were created,
however, no obvious changes in the activities, monosaccharide composition or fruit phenotype were observed in
the fruit. A corresponding recovery of LeARF1 expression was observed in transgenic fruit; LeARF1 is a putative
α-L-arabinofuranosidase belonging to glycoside hydrolase family 51 and it expressed during fruit development
stage [1]. Activation of SlArf/Xyl4 and LeXYL2 [1], both of which belong to family 3 and their expressions also
correspond to that of SlArf/Xyl2, were not observed. Increment of family 51 α-L-arabinofuranosidase expression,
rather than same family, caused a recovery of the activity in SlArf/Xyl2-suppressed fruit.
[1] A. Itai et al. (2003) J. Exp. Bot., 54, 2615-2622.
P4-22
Cellulose biosynthesis is altered in wood of poplar subjected to climate change
Afif D., Richet N., Cabané M.
Université de Lorraine, Ecologie et Ecophysiologie Forestières, UMR 1137, Vandoeuvre les Nancy, F-54506, France and
INRA, Ecologie et Ecophysiologie Forestières, UMR 1137, Champenoux, F-54280, France.
Trees have to cope with increasing levels of carbon dioxide and ozone in the troposphere. Both gases are
considered as greenhouse gases involved in global warming (climate change). Wood is an important raw material
for several uses particularly as feedstock for bioenergy fuel. One important component of wood is cellulose, a
large sink for carbon assimilated by trees. The biosynthesis and deposition of cellulose in wood are highly
controlled during the development. Wood formation and composition are affected by environmental factors.
Elevated CO2 and ozone had opposite effects on biomass production. The first one increases the biomass
production since the CO2 fixation is enhanced, while ozone reduces the biomass production. The purpose of this
work is to estimate the effects of elevated CO 2 and / or ozone on cellulose biosynthesis. Plants of hybrid poplar
(Populus tremula x alba clone INRA 717-1-B4) have been exposed in controlled chambers to either filtered-air
(control), elevated CO2 (800µl l-1), ozone (200nl l-1) or a combination of the two gases. Trees were bent in order to
induce tension wood formation on the upper side of the stem. Tension wood was characterized by a higher content
in cellulose compared to opposite wood. Cellulose biosynthesis was analysed at different levels ; (i) activity of
enzymes which supply UDPG, (ii) transcript abundance of all members of the CesA gene family and (iii)
cellulose content. First results showed that ozone alters cellulose biosynthesis more than elevated CO 2 whatever
the tissue, tension or opposite wood. Under ozone treatment, CesA genes involved in secondary cell wall
formation and those in primary cell wall displayed opposite expression profiles.
P4-23
Nostresswall: novel information on the effect of drought stress on the cell wall
Chabi M. (a), Le Gall H. (b), Durand-Tardif M. (c), Balzergue S. (d), Tokarski C. (e), Blervacq A.S. (a), Day A. (a), Fliniaux
O. (b), Gillet F. (b), Domon J.-M. (b), Rayon C. (b), Fontaine J.-X. (b), Goulas E. (a), Grec S. (a), Neutelings G. (a), Rolando
C. (e), Bendahmane A. (d), Sibout R. (c), Pelloux J. (b), Mesnard F. (b), Hawkins S. (a), Lucau-Danila A. (a)
(a) Université Lille Nord de France, Lille 1 UMR INRA 1281, SADV, F-59650, Villeneuve d'Ascq cedex, France; (b)
University Jules Verne Picardie, BIOPI – Biologie des Plantes et Innovation, UFR des Sciences, 33 rue St Leu, 80039
Amiens cedex, France; (c) INRA/CNRS – URGV, 2, rue Gaston Crémieux, CP5708, 91057 Evry cedex, France; (d) INRA
UMR 1318, Institut Jean-Pierre Bourgin, Saclay Plant Sciences, F-78000 Versailles, France ; (e) Université Lille Nord de
France, Lille 1 MSAP - Miniaturisation pour la Synthèse, l'Analyse et la Protéomique, USR Lille1/CNRS n°3290, F-59650
Villeneuve d'Ascq cedex, France.
A major transformable plant product is the plant cell wall composed of cellulose, hemicelluloses and, in certain
tissues, lignin. This resource (lignocellulosics) is transformed into biofuels and is used in bio-based materials. In
the NoStressWall project, we aim to produce comprehensive data via multi-scale –omics analyses on the impact
of drought stress – with a major focus on the cell wall - in two plant species (flax and Brachypodium). We have
chosen these 2 species because we wish to evaluate the impact of drought stress directly on two major types of
valorisable cell wall structure: flax for the use of their long cellulose fibres in composite materials and textiles and
Brachypodium as a model bio-fuel species system. The NoStressWall project aims to: i) generate and integrate
large amounts of transcriptomic, metabolomic and proteomic data together with analyses of cell wall structure and
modifications induced by drought stress, ii) use a reverse genetics screen to identify specific mutants in available
flax and Brachypodium chemical mutant populations, and iii) functionally characterize selected mutants. The
proposed project is essentially fundamental but as drought stress impacts directly on flax fiber quality and
Brachypodium biomass production, our results on these species will be of direct interest to breeders, farmers and
end-users of fibers (composite materials, textiles) and biomass. The overall project strategy will be presented and
initial results discussed.
P4-24
Evaluation of the role of the β-Galactosidase gene Faβgal4 on strawberry fruit softening
Paniagua C. (a), García-Gago J.A. (a), Blanco-Portales R. (b), Muñoz-Blanco J. (b), Waldron K.W. (c), Quesada M.A. (a),
Mercado J.A. (a)
(a) Dep. Biología Vegetal, Universidad de Málaga, Spain; (b)Dep. Bioquímica y Biología Molecular, Universidad de
Córdoba, Spain; (c) Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
Softening during ripening of fleshy fruits is mainly due to the disassembly of cell walls induced by cell wallmodifying enzymes. In strawberry, Fragaria × ananassa Duch, solubilisation of the pectin polysaccharides and
the loss of galactose from pectin side chains are the main processes occurring during ripening [1]. β-Galactosidase
activity increases in strawberry fruits during ripening. Three different β-gal genes have been described in
strawberry fruit although only one of them showed a ripening-specific expression pattern [2]. Our research group
has isolated a novel β-gal gene, Faβgal4, which expression is enhanced during strawberry ripening. To assess the
role of this gene in softening, we have obtained transgenic strawberry plants expressing an antisense sequence of
this gene. Silencing of Faβgal4 in transgenic fruits was close to 80%. Phenotypic analyses were carried out in
transgenic plants during three consecutive growing seasons, using non-transformed plants as control. In two
transgenic lines showing a significant Faβgal4 silencing, fruits were slightly firmer than control at ripening.
However, most transgenic fruits were smaller than control and deformed. Cell walls from these transgenic ripe
fruits were isolated and sequentially extracted to obtain different cell wall fractions. An increase in the content of
pectins covalently bound to the cell wall, extracted with sodium carbonate, as well as an increase on pectins in the
hemicellulosic fractions, extracted with KOH, was observed in both transgenic lines. Neutral sugars were
analysed by gas chromatography with flame ionisation detection. The amount of galactose was higher in all cell
wall fractions in transgenic fruits when compared with controls, being the differences higher in the hemicellulosic
fraction. The results obtained suggest that Faβgal4 participates in the solubilisation of covalently bound pectins
during the ripening process reducing strawberry fruit firmness.
[1] Redgwell et al. (1997) Planta, 203,174-181 ; [2] Trainotti et al. (2001) Journal of Experimental Botany, 361,1635-1655.
This research was supported by FEDER EU Funds and the Ministerio de Educación y Ciencia of Spain (grant reference AGL2011-24814).
P4-25
Significance of grass cell wall feruloylation for wall growth and sugar remobilization
Buanafina M.M. de O., Iyer P.R.
Department of Biology, 208 Mueller Laboratory, Pennsylvania University, University Park, PA 16802, USA.
Cell walls are complex structures that serve several important functions in the life of plants. The cell walls of
grasses are distinctive in composition, with xylans usurping the roles of some of the other matrix polysaccharides.
Xylan consists of a backbone of 1,4-linked β-D-xylopyranose residues, containing arabinose as the major sidechains. A proportion of the arabinose side-chains are further substituted with acidic residues, notably ferulic acid
(FA). Peroxidase-mediated oxidative coupling of FA residues function to cross-link xylans [1] presumably with
significant effects on the physical characteristics of the cell wall including extensibility, digestibility, cell wall
sugar remobilization and resistance to pests. Interestingly, as cell expansion ceases, arabinosyl substitutions
decline, coinciding with a significant increase in ferulate deposition [2].
We have demonstrated previously the expression of an Aspergillus niger ferulic acid esterase gene (faeA) in
Festuca arundinacea [3] and the potential of the recombinant FAEA to reduce the level of cell wall ferulates. Our
findings also indicated the positive effect of FAEA on increasing digestibility.
Using transgenic plants with reduced levels esterified cell wall ferulates by the expression of FAEA, we report on
the possible role of feruloylation on cell wall sugar remobilization on senescence and on oligosaccharides chain
lengths in tall fescue cell walls. Additionally, we assess the impact of wall ferulates on leaf growth and insect
resistance.
[1] Buanafina, M M de O (2009). Molecular Plant. 2:861-872; [2] Carpita, N. C. Plant Physiol 1984, 76 (1), 205-212; [3]
Buanafina et al. (2010). Plant Biotechnology J. 8:316-331.
P4-26
Nanostructural differences in pectic polymers isolated from strawberry fruits with low expression
levels of pectate lyase or polygalacturonase genes
Posé S. (a), Paniagua C. (a), Kirby A.R. (b), Mercado J.A. (a), Morris V.J. (b), Quesada M.A. (a)
(a) Dep. Biología Vegetal, Universidad de Málaga, 29071, Málaga, Spain; (b) Institute of Food Research, Norwich Research
Park, Colney, Norwich NR4 7UA, UK.
Our research group has obtained transgenic strawberry plants expressing antisense sequences of either a pectate
lyase (APEL lines) [1] or a polygalacturonase gene (APG lines) [2]. Both genes encode ripening-specific endopectinases with a common target, deesterified homogalacturonans, but each enzyme act by a different mechanism
and pH range. Ripe fruits from both transgenic genotypes were significantly firmer than control, being APG fruits
on average 25% firmer than APEL fruits. Cell wall analysis of both transgenic genotypes indicated that pectin
fractions extracted with CDTA and sodium carbonate were significantly modified in transgenic fruits [2,3]. To
gain insight in the role of these pectinases in pectin disassembly during ripening, CDTA and Na 2CO3 pectins have
been analyzed by atomic force microscopy (AFM). APEL and APG CDTA pectins had similar contour lengths
but both were significantly longer than control. Similarly, APG carbonate chains were longer than control,
showing APEL carbonate chains an intermediate length. Furthermore, transgenic pectins displayed a more
complex branching pattern and a higher number of micellar aggregates, especially in the sodium carbonate
fractions of APG samples. Acid hydrolysis of carbonate pectins reduced the number of micellar aggregates. AFM
analyses confirm that the inhibition of both pectinases reduces pectin disassembly, and also suggest that each
pectinase acts on specific pectin domains. Particularly, polygalacturonase silencing induces more significant
pectin modifications, nicely correlated with the firmer phenotype of APG fruits, than the down-regulation of
pectate lyase.
[1] Jimenez-Bermudez et al. (2002) Plant Physiol., 128, 751-759 ; [2] Quesada et al. (2009a) Plant Physiol., 150, 10221032 ; [3] Santiago-Domenech et al. (2008) J. Exp. Bot., 59, 2769-2779.
This research was supported by FEDER EU Funds and the Ministerio de Educación y Ciencia of Spain (Grant ref AGL2011-24814).
P4-27
Biotechnological Potential of Carbohydrate Binding Modules from Oomycetes
Martinez T. (abcd), Lafitte C. (a), Dumas B. (a), O’Donohue M. (bcd), Dumon C. (bcd), Gaulin E. (a)
(a) UMR 5546 UPS/CNRS Laboratoire de recherche en sciences végétales (LRSV), France; (b) INRA, UMR792, Ingénierie
des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; (c) Université de Toulouse; INSA, UPS, INP; LISBP,
135 Avenue de Rangueil, F-31077 Toulouse, France; (d) CNRS, UMR5504, F-31400 Toulouse, France.
Oomycetes are fungal-like microorganisms evolutionary distant from true fungi, and comprising major plant
pathogens. Oomycetes express proteins able to interact with plant cell wall polysaccharide like cellulose, through
the presence of carbohydrate-binding module belonging to family 1 of the CAZy database (CBM1) [1]. Fungal
CBM1-containing proteins are implicated in cellulose degradation whereas in oomycetes, the Cellulose Binding
Elicitor Lectin (CBEL), a well-characterized CBM1-containing protein from Phytophthora parasitica, is
implicated in cell wall integrity, adhesion to cellulosic substrates and most importantly in induction of plant
immunity responses [2,3]. In this study we characterized the cellulose binding of CBEL and evaluated the
potential of CBM1-containing proteins from oomycetes for future biotechnological applications.
[1] FV. Mateos et al. Mol (1997) Plant Microbe Interact., 10(9),1045-1053 ; [2] E. Gaulin et al. (2002) J. Cell. Sci., 115(23),
4565-4575 ; [3] E. Gaulin et al. (2006) Plant Cell, 18(7),1766-1777.
P4-28
Role of cell wall matrix polysaccharides in rice growth and development
Yokoyama R., Kido N., Horie S., Nishitani K.
Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai,
980-8578, Japan.
The morphological diversity of plants mirrors the diversity of cell types, each of which adopts a specific shape
and plays a role that is peculiar to plant species. The cell wall plays several critical roles in determining the cell
type, thereby having key roles in regulating growth and differentiation in each plant species.
The cell walls from commelinoid monocotyledons, which include cereals, have a number of major structural and
compositional differences from those of dicotyledonous species. The primary cell walls of commelinoid
monocotyledonous plants have relatively little xyloglucan (XyG), and the predominant glycan that cross-links the
cellulose microfibrils is instead glucuronoarabinoxylan (GAX) and (1,3)(1,4)-beta-D-glucan (MLG). Moreover,
compared with the pectin-abundant wall of dicotyledonous plants, the cereal cell wall contains less pectin and
higher amounts of phenylpropanoids, which form extensive interconnecting networks.
We specifically aimed at elucidating roles of MLGs, XyGs and GAXs in rice plant because these polysaccharides
have been considered to play essential roles in regulation of rice growth. For this purpose, we generated
transgenic rice plants, in which expression of the genes involved in biosynthesis or degradation of these
polysaccharides is modified. The results showed that defects in these polysaccharides exert various effects on rice
growth and development. The unique roles of each polysaccharide in rice plant and further investigation will be
discussed.
P4-29
Link between C/N imbalance perception and plant Cell Wall biosynthesis
Li Z., Verger S., Clément G., Höfte H., Mouille G.
Institut Jean-Pierre Bourgin, UMR1318 INRA/AgroParisTech, Route de St Cyr, 78026 Versailles, France.
The ability of the plant to control the balance between mineral nutrition and photosynthesis is a key parameter for
crop yield and must be coordinated so that cells can optimize their metabolism in response to environmental or
developmental signals. This C (carbon) and N (nitrogen) nutrient balance perception and signaling mechanism is
poorly understood. Our project uses an original approach to provide new insights into the mechanisms of the
perception of the C/N balance and the allocation of C to reserve and/or cell wall-associated C-sinks. It has been
previously observed that Arabidopsis mutants with an altered pectin composition also have a dramatically altered
perception of the C/N balance [1, 2]. This is a feature specific for pectin mutants since no such changes appear to
occur in mutants for other cell wall components such as cellulose or xyloglucan.
The link between the pectin biosynthetic pathway and C/N balance response is not yet understood. So we took
advantage of mutants deficient for the pectic polysaccharide homogalacturonan (quasimodo1 & 2) and a
collection of suppressor mutants in which the perception of the C/N balance is restored to various extents. Then
we combine state of the art technologies in metabolomics and cell wall analysis with the genetic and molecular
analysis of the suppressor mutants to obtain new insights into the link between the metabolism of the cell wall and
the perception of the C/N balance. Our strategy leads to the identification of key actors and steps involved in this
mechanism that will be detailed.
[1] Gao P. et al. (2008) PLoS One 3. DOI: e138710.1371/journal.pone.0001387 ; [2] Mouille G. et al. (2007) Plant Journal
50:605-614. DOI: 10.1111/j.1365-313X.2007.03086.x.
P4-30
Arabidopsis Thaliana P4H3 is involved in anoxia tolerance
Petrakis N., Klepadlo M., Lakehal A., Kalaitzis P.
Mediterranean Agronomic Institut of Chania, Deparment of Horticultural Genetics and Biotechnology, Greece.
Plant prolyl-4-hydroxylases (P4Hs) catalyze the proline hydroxylation, a major post-translational modification of
hydroxyproline rich glycoproteins, a very important superfamily of cell wall proteins which includes
arabinogalactan proteins (AGPs), extensins and proline-rich proteins. In Arabidopsis there are 13 P4Hs genes
while several of them are differentially expressed during abiotic stress [1]. In our study, we investigated the role
of Arabidopsis P4H3 in response to anoxia using P4H3 knock out and overexpression lines. The knock out and
overexpression lines exhibited lower and higher survival rates compared to the wild type, respectively. The
molecular basis of this response was investigated by monitoring the enzymatic activity as well as the gene
expression levels of the fermentation pathway. In addition, the expression of hypoxia-related genes such as HRE1,
HRE2, SUS1 and SUS4 was determined. Western blot analysis revealed alterations in the content of AGP-bound
epitopes during hypoxia and anoxia while the physical interaction of P4H3 with several AGP proteins which are
up-regulated under hypoxia and anoxia was demonstrated using a yeast two hybrid approach.
[1] F. Vlad et al. (2007) Phys Plantarum, 130 (4), 471-83.
P4-31
The role of cell wall components of root border cells and border-like cells in root protection
Plancot B., Jaber R., Gotté M., Durand C., Nguema-Ona E., Vicré-Gibouin M., Driouich A.
Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV)- EA 4358 et GRR-VASI de Haute Normandie.
PRES Normandie Université. Université de Rouen. 76821 Mont Saint Aignan, Cedex. France.
Root tips of most plant species produce a large number of cells programmed to separate from the root cap and to
be released into the external environment. These living cells are named either border cells (for instance in
legumes), or border-like cells (for instance in Brassicaceae species). Border cells are released as individual cells,
while border-like cells remain attached to each other, via their intact cell walls, forming a block of several layers
of cells. Both border cells (BC) and border- like cells (BLC) are metabolically active, and secrete different classes
of metabolites and proteins in the rhizosphere. It has been shown that many cell wall components including matrix
polysaccharides such as pectic xylogalacturonans (XGA), and glycoproteins such as arabinogalactan proteins
(AGPs) and extensins, are incorporated into the walls of these cells, and/or secreted into the rhizosphere.
A number of recent studies have shown that many of these cell wall polysaccharides and glycoproteins play an
active role in the rhizosphere, either by attracting symbiotic rhizobia, or by protecting the root tips against
soilborne pathogens. AGPs for instance, were shown to play important roles in protecting the root against soil
pathogens. XGA are abundantly secreted by border-like cells, probably to slow-down the ingress the soilborne
pathogens. When stimulated by elicitors such flg22, BC and BLC produce reactive oxygen species (ROS); exhibit
callose deposition, over-express extensin and accumulate ER-bodies. Together, these findings indicate that cell
wall components secreted by root BC and BLC are fundamental for root protection within the rhizosphere [1,2].
[1] A. Driouich et al. (2010) Border cells versus border-like cells: are they alike? J. Exp. Botany 61, 3827-3831; [2] A.
Driouich et al. (2012) Unity is strength: the power of border cells and border like cells in relation with plant defense;
Secretions and Exudates in Biology Systems. Vivanco JM and Baluska F, 91-108, Springer.
P4-32
The role of receptor like kinases in Arabidopsis plant cell wall integrity maintenance
McKenna J. (a), Boutrot F. (b), van der Does D. (b), Zipfel C. (b), Hamann T. ( ac)
(a) Imperial College London, London, UK; (b) The Sainsbury Laboratory, Norwich, UK; (c) Norwegian University of
Science and Technology, Trondheim, Norway.
Plant cells need to maintain the functional integrity of their cell walls during cell morphogenesis and pathogen
defense. Evidence has accumulated that plants have evolved a dedicated cell wall integrity (CWI) maintenance
mechanism, which initiates compensatory responses if cell wall damage (CWD) occurs. The available data
implicate reactive oxygen species- (ROS) and jasmonic acid (JA)- based signaling processes as well as
downstream responses like active inhibition of root growth and ectopic lignin deposition in the CWD response1.
While experimental data support the involvement of the Receptor Like Kinase (RLK) THE in plant CWI
maintenance, the contributions of other RLKs implicated in CWI maintenance remain to be determined2-6. In
parallel, it is also unclear whether (and to what extent) genes originally implicated in the response to pathogen
infection are involved in CWI maintenance. To further clarify the function of THE in CWI maintenance and
determine the contributions of other RLKs implicated, we investigated systematically how loss of function alleles
in THE, FEI1, FEI2, HERK1, HERK2, BAK1, BKK1, BIK1, PEPR1, PEPR2 and a novel LRR-RLK tentatively
named LRI for LRR-RLK REQUIRED FOR ISOXABEN RESPONSE affect the response to CWD caused by
cellulose biosynthesis inhibition. We found that most of the RLKs investigated (including the ones originally
implicated in pathogen response) affect only particular aspects of the response to CWD. Our results also show that
THE and LRI represent key elements, required for mediating all the responses to CWD examined in Arabidopsis.
[1] L. Denness et al. (2011) Plant Phys., 156, 1364-1374; [2] Hématy et al. Curr Biol. (2007) 17(11), 922-31; [3] Wolf et al.
(2012) Annual Rev Plant Biol 63, 381–407; [4] Boisson-Dernier et al. (2011) J Exp Bot 62, 1581–91; [5] Lindner et al.
(2012) Cur Opinion Plant Biol 659–669; [6] Ringli (2010) Plant Phys. 153, 1445–52.
P4-33
Understanding cell wall re-modelling during the symbiotic interaction
ectomycorrhizal fungus Tuber melanosporum and Corylus avellana
between
the
Balestrini R. (a), Sillo F. (a), Faccio A. (a), Bonfante P. (a), Willats W.G.T. (b), Fangel J.U. (b)
(a) Istituto per la Protezione delle Piante del CNR and Dipartimento di Scienze della Vita e Biologia dei Sistemi, UniTO; (b)
Section for Plant Glycobiology, Department of Plant Biology and Biotechnology, University of Copenhagen.
It has been reported that the symbiotic ectomycorrhizal fungi have a small set of genes coding for secreted
enzymes putatively involved in the degradation of plant cell wall polysaccharides [1, 2]. Within the context of the
Tuber melanosporum genome sequencing project, genes coding for putative plant cell wall degrading enzymes
(CDWEs) have been identified and manually annotated; several of them were found to be up-regulated during the
symbiosis, suggesting a role in plant cell wall degradation and facilitation of the fungal progression through the
pectin-rich middle lamella, when the fungus develops inside plant tissues. Gene expression data (microarray,
RNAseq, qRT-PCR) on T. melanosporum and Corylus avellana ectomycorrhizae (ECM) have shown that two
fungal putative endoglucanases (TmelCMC3 and TmelEG), a pectate lyase B (TmelPLB), a GH28
polygalacturonase (TmelPGN1) and a rhamnogalacturonase (TmelRghA) were up-regulated. qRT-PCR
experiments have demonstrated that, in agreement with the RNAseq data, a gene encoding a rhamnogalacturonan
acetylesterase (TmelRgaE) is also highly up-regulated in ECM, although the array data did not indicate any upregulation. In addition, we have employed glycan microarray technology [3] to analyse the impact of fungal
colonization on plant cell wall composition in the ectomycorrhizae, compared with uncolonized halzenut roots.
Preliminary data suggest that cell walls are affected by the presence of the fungus and the observed changes are
consistent with gene expression data, which have shown an up-regulation of enzymes involved in pectin
degradation. To support glycoarray data, in situ immunolabelling experiments are ongoing by using monoclonal
antibodies with specificity for plant cell-wall components.
[1] F. Martin et al. (2008) Nature, 452, 88-92; [2] F. Martin et al. (2010) Nature, 464, 1033-1038; [3] I. Moller et al. (2007)
Plant J., 50, 1118-1128.
P4-34
Genome-wide characterisation of mechanical stress responsive miRNAs from xylem tissues of
different poplar genotypes
Gourcilleau D., Lesage-Descauses M.-C., Millet N., Chartrin A., Rogier O., Laurans F., Déjardin A., Pilate G., Leplé J.-C.
INRA, UR588 AGPF, 2163 avenue de la Pomme de Pin, CS40001, 45075 Orléans cedex 2, France.
miRNAs are small non-coding RNAs 20-24 nucleotides in length that can direct the cleavage or inhibit the
translation of target gene transcripts. Since their discovery, miRNAs have been involved in various biological
processes but our knowledge for their involvement in regulating wood formation is still scarce. In poplar, tension
wood formation in response to gravitropic stimuli is largely used as a model system to study wood formation [1].
Up to now, Populus mechanical stress miRNAs have been identified from a single genotype, Populus cv
Nisqually-1, the clone used for the Populus genome sequencing [2]. In order to discover new miRNAs and/or
genotype specific expressed miRNAs, the content in small RNAs from tension wood (TW) and opposite wood
(OW) tissues have been characterized using next-generation sequencing. 1708 distinct putative miRNAs
sequences were identified from 12 sequenced libraries (3 poplar genotypes, TW/OW, 2 biological replicates).
Only 95 miRNAs correspond to previously characterized mature miRNAs and a few of newly identified
sequences are only detected from some genotypes. Some conserved as well as newly identified miRNAs present
tissue-specific and/or genotype-specific expression patterns. These results together with prediction of gene targets
may help us to infer transcriptional networks associated with regulation of wood formation. This work was
supported by the ANR project “TreeForJoules” ANR-10-KBBE-0007.
[1] G. Pilate et al. (2004) New Phytol., 164, 63-72 ; [2] S.F. Lu et al. (2005) Plant Cell, 17 (8), 2186-2203.
P4-35
Lignin as a contributor to virulence rather than defence ? — novel insights from parasitic plant
haustoria
Pielach A. (a), Allison G.G. (b), Popper Z.A. (a)
(a) Botany and Plant Science and Ryan Institute for Environmental. Marine and Energy Research, School of Natural
Sciences, National University of Ireland, Galway, Ireland; (b) Aberystwyth University, IBERS, Gogerddan, Aberystwyth
SY23 3EE, United Kingdom.
Parasitic plants produce organs called haustoria to attach to host plants, infiltrate their vasculature and absorb
nutrients. Haustoria of several parasite species attached to resistant hosts are often encapsulated by a lignified
interfacial layer assumed to be the main mechanism of host defence. While the precise structure, composition and
assembly of encapsulation layers are poorly characterised, interfacial lignins have frequently been suggested as a
crucial component.
The interfaces of Rhinanthus minor (a hemiparasitic herb of species-rich meadows) with a grass host,
Arrhenatherum elatius, and a eudicot non-host, Plantago lanceolata, were investigated using histology,
immunocytochemistry and Raman spectroscopy. Lignin-like substances were present in the cell walls of
haustorial interfacial parenchyma and an interfacial extramural layer, where they co-localised with xyloglucans
and arabinogalactan proteins. They were also found at the contact surface of non-infective haustoria appressed to
pots (lab-grown plants).
These findings suggest that at least some of the interfacial polyphenolic substances previously assumed to
contribute to host defence are synthesised by and beneficial to the parasite. Consequently, reinterpretation of
interfacial lignin’s role in parasitic plant-host interactions is needed.
P4-36
Biochemical and histochemical characterization of the polysaccharides present in the G-layer of
wild type and AGP-modified transgenic poplars
Guedes F.T.P. (a), Laurans F. (a), Assor C. (b), Takeuchi M. (a), Boizot N. (a), Vigouroux J. (b), Lesage-Descauses M.-C.
(a), Leplé J.-C. (a), Déjardin A. (a), Pilate G. (a)
(a) INRA, UR0588 Amélioration, Génétique et Physiologie Forestières, F–45075 Orléans cedex 2, France ; (b) INRA UR
1268, PVPP, rue de la Géraudière, BP 71627, 44316 Nantes, France.
In hardwood trees, tension wood formation is a remarkable adaptive mechanism that makes possible for the tree to
re-orient its axes. In poplar, tension wood fibers harbor an extra cell wall layer named G-layer, responsible for the
peculiar mechanical properties of tension wood. In this layer, highly crystalline cellulose is embedded in a
polysaccharide matrix, whose composition may be very important for tension wood properties. Therefore, we
investigated the polysaccharide distribution during G-layer differentiation. In addition to glucose, biochemical
analyses of isolated G-layers reveal the presence of xylose, rhamnose, arabinose and galactose. Arabinose and
galactose may be present on rhamnogalacturonan I (RGI) side chains or in specific glycosylated proteins such as
arabinogalactan protein (AGP). The last possibility could be verified by a reactivity test with the Yariv reagent
and using AGP specific antibodies. Using 186 specific antibodies directed against the major cell wall
polysaccharides, we observed that the changes in polysaccharides localization between tension and normal wood
only occurred within the G-layer, which composition varies throughout the maturation process. Most of the 30
antibodies that label exclusively the G-layer recognize AGP or RGI epitopes. These polysaccharides may be able
to take a gel-like structure that potentially acts on the G-layer cellulose microfibrils to create the strong force
characteristic of tension wood [1]. In order to verify this hypothesis, we produced and analyzed transgenic poplars
altered for the expression of a fasciclin-like AGP. All these results will be discussed in detail in this
communication.
[1] B. Clair et al. (2008) Biomacromolecules, 9, 494-498.
This project is partly funded by the French ANR Blanc Project "Stress in Trees".
P4-37
COBRA-LIKE 2 regulates cellulose deposition in Arabidopsis seed coat mucilage
Harpaz-Saad S. (a), Ben-Tov D. (a), Tzfadia O. (b), Kieber J.J. (c)
(a) The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University, Rehovot 76100, Israel ;
(b) Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel; (c) University of North Carolina,
Biology Department, Chapel Hill, NC, 27599, USA.
A common adaptation in angiosperms is the differentiation of the maternally derived seed coat epidermal cells
into mucilage secretory cells (MSCs). Recent studies, establish cellulose as an essential component of seed
mucilage, deposited into a set of rays radiating the seed upon mucilage secretion and anchoring the pectic
component of seed mucilage to the seed surface (1-3). We hypothesized that there are biologically relevant
coexpression relationships that will be revealed only when analyzing tissue or condition specific data sets.
Preliminary coexpression analysis during the course of seed development, suggested COBRA-LIKE 2 (COBL2),
which has not been assigned any function so far, to be involved in the regulation of cellulose deposition in MSCs.
Indeed, two independent T-DNA insertion lines, cobl2-1 and cobl2-2, were identified as seed mucilage mutants.
Seed mucilage of both lines display substantially reduced rays of cellulose along with increased solubility of the
pectic component, as previously described for seed mucilage mutants disrupted in cellulose deposition. Moreover,
MSCs of both cobl2 alleles display compromised radial cell walls and collumela structures and ∼30% reduction
in seed crystalline cellulose, as compared to wild-type.
[1] S. Sullivan et al.(2011) Plant Physiol 156, 1725; [2] S. Harpaz-Saad et al.(2011) Plant J 68, 941; [3] V. Mendu et al.
(2011) Plant Physiol 157, 441.
P4-38
VIGS-induced Silencing of three putative tomato prolyl 4-hydroxylases causes alterations in plant
growth
Fragostefanakis S., Sedeek K.E.M., Raad M., Zaki M.S., Kalaitzis P.
Mediterranean Agronomic Institut of Chania, Deparment of Horticultural Genetics and Biotechnology, Greece.
Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs)
that is catalyzed by prolyl 4-hydroxylases (P4Hs). The tomato genome comprises ten unigenes encoding putative
P4Hs. We used Tobacco Rattle Virus (TRV)-based virus induced gene silencing (VIGS) system to investigate the
role of three putative P4Hs in tomato growth and whether are involved in the protein content of HRGPs such as
extensins and arabinogalactan proteins (AGPs). Eight-days old tomato seedlings were infected with the
appropriate TRV vectors and plants were allowed to grow under standard conditions for six weeks. Lower P4H
transcript levels were associated with lower hydroxyproline content in root and shoot tissues. SlP4H-silenced
plants exhibited longer roots and shoots with larger leaves. In addition, the leaves of SlP4H1- and SlP4H9silenced plants exhibited higher epidermal cell number, while the leaves of SlP4H7-silenced plants comprised of
more expanded cells. These results indicate that P4Hs are implicated in leaf growth altering the programmes of
cell division and expansion. In addition, western blot analysis revealed that silencing of SlP4H7 and SlP4H9
resulted in reduced levels of both JIM13-bound AGP epitopes as well as and JIM11-bound extensin epitopes,
while silencing of SlP4H1 resulted in the reduction of the content of AGP proteins. Collectively these results
indicate that P4Hs are implicated in distinct roles in plant growth programmes.
P4-39
Dissection of the root growth response to phosphate starvation in Arabidopsis thaliana
Péret B. (a), Deschamps S. (a), Arnaud C. (a), Bonnot C. (a), Clément M. (a), Maes L. (a), Thibaud M.-C. (a), Creff A. (a),
Mouille G. (b), Nussaume L. (a), Desnos T. (a)
(a) Laboratoire de Biologie du Développement des Plantes (LBDP), SBVME, UMR7265 CEA/CNRS/AMU, CEA cadarache,
13108 St Paul-lez-Durance Cedex, France ; (b) Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre
de Versailles-Grignon, Route de St Cyr (RD10) 78026 Versailles Cedex, France.
When on a low-Pi medium, the Arabidopsis root growth is rapidly inhibited [1]. In the root tip, this is
accompanied by differential expression of several cell wall remodelling genes as well as by a strong change of the
Fourier-Transform InfraRed (FTIR) microspectroscopy profile. By using the Arabidopsis natural variation we
have identified LPR1, and its paralogue LPR2, involved in the root response to low-Pi. They both encode for a
multicopper oxidase, the activity of which reduces root groxth when seedlings are on a low-Pi medium [2,3]. The
LPR1 protein is located in the endoplasmic reticulum and genetically interacts with PDR2, a P5-type ATPase [4].
These results provide strong evidence for the involvement of the endoplasmic reticulum at the root tip in sensing
and/or responding to Pi deficiency. We isolated 85 new EMS mutants with a long primary root on a low-Pi
medium, including 18 new lpr1 alleles. In some of these mutants grown in low-Pi, the FTIR profile resembles the
one of WT seedlings grown in high-Pi.
In order to get more insights into this pathway, we screened a chemical library in order to find drugs altering the
Pi signalling. We found two drugs restoring root growth of WT seedlings in low-Pi.
[1] Péret et al. (2011) Trends Plant Sci.16:442-50; [2] Reymond et al. (2006) Plant Cell and Environ. 29:115-125; [3]
Svistoonoff et al. (2007) Nature Genetics39:792-796; [4] Ticconi et al. (2009) Proc. Natl. Acad. Sci. USA106:14174-14179.
P4-40
Xylem development and composition as a target to breed for better performances under drought ?
Durand-Tardif M. (a), Bensussan M. (a), Ducamp A. (a), Guillebaux A. (a), Moquet F. (b)
(a) Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech INRA Centre de Versailles-Grignon Route de St-Cyr
(RD10) 78026 Versailles Cedex France ; (b) GAUTIER SEMENCES Route d'Avignon 13630 Eyragues France.
Considering the evolution of climate towards more frequent occurrences of extreme events, particularly water
deficit episodes; the demand for sustainable energy and biofuel production from lignocellulose; the evolution of
agricultural practices towards low inputs management, including water, our team is studying the impact of xylem
formation and composition on biomass production, under fluctuating water supply.
The AtESKIMO1 (AtESK1 or AtTBL29) gene has first been identified because its inactivation results in freezing
tolerance without acclimation. Results acquired independently by Xin and Browse and by our team show that
knockout Atesk1 plants have a regular development but are dwarf. We have shown that Atesk1 has a stressed
phenotype: it shows a reduced rosette area and a reduced water content, it has a low evapo-transpiration and its
transcriptome is similar to a plant that experiences a drought stress. Moreover its xylem is collapsed (irregular
xylem or irx phenotype), the hydraulic conductance of the Atesk1 root system is dramatically reduced and xylem
chemical is affected.
We used esk1 as a “stress-ready” genotype. We have generated an EMS mutagenised population from Atesk1.
Several suppressor lines, which produce larger plants than the Atesk1 mutant have been identified, most of them
showing a regular xylem and some still showing an irx phenotype. Suppressor genes are expected to directly
suppress the irx phenotype of the xylem, or to allow better growth of plants despite the collapsed xylem. As far as
we know, this biological material is unique and original. Mutations causative for the suppressed phenotypes are
currently identified thanks to whole genome sequencing. The suppressed lines are going to be characterised in
depth for the functionality of their xylem vessels: chemical composition, cytology and hydraulic properties. In
addition, specific functional analyses will be performed on each BEEM gene depending on its annotation and
previous characterisation.
P4-41
Cellular originalities of the oil palm fruit abscission zone and abscission process
Tranbarger T.J. (a), Roongsattham P. (a)*, Fooyontphanich K. (a),Pizot M. (a), Jantasuriyarat C. (b), Suraninpong P. (c),
Collin M. (a), Morcillo F. (d), Verdeil J.L. (e)
(a) IRD, UMR DIADE, 911 Avenue Agropolis BP 64501, 34394 Montpellier cedex 5, France ; (b) Department of Genetics, Faculty of Science, Kasetsart
University, Bangkhen Campus, 50 Phahonyothin Road, Jatujak, Thailand; (c) Institute of Agricultural Technology, Walailak University, Thasala District,
Nakhon Si Thammarat 80160; (d) CIRAD, UMR DIADE, (e) CIRAD, UMR AGAP, MRI-PHIV; F-34398 Montpellier, France ; *current address Department
of Biology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand, 90112.
The oil palm fruit abscission zone (AZ), situated between the pedicel and mesocarp tissues, consists of multiple
cell layers that contain high amounts of unmethylated pectin and polygalacturonase (PG) enzyme activity [1, 2, 3].
To determine whether the specificity of cell separation is due to cellular characters of the AZ, we combined
molecular, histochemical, electron microscopy and immunolocalization approaches to examine the events during
AZ development and function during oil palm fruit shedding. AZ cells accumulate large amounts of intracellular
pectic substances during development, which are lost from the cells after separation. During early fruit
development the AZ contains numerous plasmodesmata (PD) that interconnect cells within each layer, but very
few between cell layers. In the AZ of ripening fruit, PD are less frequent, wider and mainly locate in the middle of
adjacent cells almost exclusively within AZ cell layers, suggesting an importance for intra AZ layer cell-cell
communication. The nuclei within AZ layer cells are clearly aligned, and remained aligned after cell separation
takes place, again suggesting AZ layer cell interconnectivity is important for cell separation to occur. Polarized
vesicle accumulation and secretory activity occurs at the tip of AZ cells during development that result in
striations to form in the cell walls between cells of different layers, providing a large target surface where
separation could take place. The antibodies JIM5, JIM7, and LM7, which detect pectin with different methylesterification status, were used to characterize the pectin in the AZ cell walls [4]. The JIM5 epitope increased in
the AZ layer cell walls prior to separation, and then a dramatic polarized increase in both JIM5 and JIM7 epitopes
is observed on the separated AZ cell surfaces. A total of 11 Glycosyltransferases (GTs), 7 pectin
methyltransferases (PMTs), 3 pectic methylesterases (PMEs) and 14 PGs were expressed in the AZ with 2 GTs, 1
PMT, 1 PME and 5 PGs that decreased and 5 PGs that increased specifically in the AZ cell layers during
abscission. The results obtained from diverse approaches allow an integrated view of the fruit abscission process
in oil palm and provide evidence for original mechanisms that underlie oil palm fruit shedding.
[1] Henderson J et al. (2001). Phytochemistry 56: 131-139; [2] Roongsattham P (2011) Cell separation processes that underlie fruit abscission and shedding
in oil palm (Elaeis guineensis Jacq.). UNIVERSITÉ MONTPELLIER 2, Montpellier, France; [3] Roongsattham P et al. (2012). BMC Plant Biology 12:
150 ; [4] Clausen MH et al. (2003). Carbohydr Res 338: 1797-1800.
P4-42
Overexpression of the grapevine PGIP1 in tobacco results in remodeling of the leaf
arabinoxyloglucan network in the absence of fungal infection
Nguema-Ona E. (ab), Moore J.P. (a), Fagerström A. (cd), Fangel J.U. (d), Willats W.G.T. (d), Hugo A. (e), Vivier M.A. (a)
(a) Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, South Africa; (b)
Glyco-MEV Laboratoire, Université de Rouen, Mont Saint Aignan, France; (c) Energy Biosciences Institute, University of
California, Berkeley, CA, USA; (d) Department of Plant Biology and Biotechnology, University of Copenhagen, Denmark;
(e) Department of Microbiology, Stellenbosch University, South Africa.
Overexpression of Vitis vinifera polygalacturonase-inhibiting protein 1 (Vvpgip1) protects tobacco plants against
Botrytis cinerea [1]. Additional roles for VvPGIP1, beyond the classical endopolygalacturonase (ePG) inhibition
mechanism, contribute to the protection observed [2]. Genetic and biochemical datasets point to the cell wall and
in particular, the xyloglucan component of the transgenic VvPGIP1 lines as being involved in the resistance
process [2]. A profiling analysis [3] of healthy leaves, from wild-type and transgenic plants, was used to elucidate
the role of wall-associated processes in PGIP-derived resistance pre-infection. CoMPP analysis revealed subtle
changes in pectin and cellulose components; and marked changes in the hemicellulose matrix, which showed
reduced binding in transgenic leaves of VvPGIP1 expressing plants [4]. Enzymatic oligosaccharide fingerprinting,
optimized for tobacco arabinoxyloglucans, revealed that XEG-soluble polysaccharides were altered in relative
abundance for certain oligosaccharides in transgenic lines [4]. VvPGIP1 overexpression therefore results in cell
wall remodeling of the cellulose-xyloglucan network in tobacco in advance of potential infection.
[1] D.A. Joubert et al. (2007) Transgenic Res., 15, 687-702 ; [2] E. Alexandersson et al. (2011) BMC Notes., 4, 493 ; [3] E.
Nguema-Ona et al. (2012) Carbohydrate Polymers., 88, 939-949 ; [4] E. Nguema-Ona et al. (2013) BMC Plant Biol. 13, 46.
P4-43
Identification of miRNA/targets and signalization networks linked to gravitropism in poplar
Richet N. (a), Lesage-Descauses M.-C. (a), Millet N. (a), Déjardin A. (a), Pilate G. (a), Coutand C. (b), Leplé J.-C. (a)
(a) INRA, UR 588 AGPF, 2163, avenue de la Pomme de pin, CS 40001 Ardon, 45075 Orléans Cedex 2, France;(b) INRA,
UMR 547 PIAF, 234 avenue du Brézet, 63100 Clermont-Ferrand, France.
Gravity is one of the most important environmental parameter affecting plant growth and development. In
response to such mechanical stimulus trees constantly remodel their position by developing a specialized wood
called reaction wood. In angiosperms trees, such as poplars, reaction wood is named tension wood (TW) and is
localized on upper side of branches and inclined stems [1]. In this study the objective is to identify specific
molecular actors involved in the gravitropic response in wood. Firstly, young hybrid poplars were bent during one
month in order to identify miRNAs and their potential gene targets by RNA degradome analysis [2]. In a second
step, with the support of isotropic medium device, we analysed short-term molecular response after bending the
trees for 30 min. This second experiment enables us to identify earlier actors and signalisation networks
associated with this tropism. Together, these degradome analyses (short and long term experiments) will provide
us a set of candidate genes and miRNAs for functional analysis in poplar.
[1] G. Pilate et al. (2004) Lignification and tension wood. CR Biol. 327: 889-901; [2] M.A. German et
al. (2009) Nat. Protoc. 4: 356-362.
This project was supported by the ANR project “TROPIC” ANR-11-BSV7-0012.
P4-44
Cell wall-related aspects of the effect of brassinosteroids on Arabidopsis hypocotyl gravitropism
Suslov D. (a), Vandenbussche F. (b), Funke N. (c), Ruprecht C. (c), Ivakov A. (c), Vissenberg K. (d), Persson S. (c), Van Der
Straeten D. (b)
(a) St. Petersburg State University, St. Petersburg, Russia; (b) Department of Physiology, Ghent University, Ghent, Belgium;
(c) Max-Planck-Institute for Molecular Plant Physiology, Potsdam, Germany; (d) University of Antwerp, Antwerp, Belgium.
Brassinosteroids were found to be negative regulators of gravitropism in etiolated Arabidopsis hypocotyls acting
via changes in cell wall mechanics [1]. On horizontal Petri plates treatment with 24-epibrassinolide (EBL, 100
nM) resulted in hypocotyls that lay flat on the agar surface. Their cell walls showed increased in vitro extension in
creep tests at pH 6, suggesting that the hypocotyl cell walls could be too weak to support their weight against
gravity. EBL did not influence the wall monosaccharide composition but led to disordered cellulose microfibril
orientation in the outer epidermal wall as revealed with the Pontamine S4B dye. This could explain the effect of
EBL on gravitropism, because wild type plants treated with oryzalin (250 nM) and pom2-4 mutants, both having
disordered microfibrils, also demonstrated a decrease in the percentage of standing hypocotyls.
Brassinazole (BRZ, 1 µM), an inhibitor of brassinosteroid biosynthesis, increased the percentage of hypocotyls
growing upright to 100%, versus 70-80% in the untreated control, while decreasing their in vitro extension. The
positive effect of BRZ on gravitropism could thus result from increasing the wall mechanical strength. BRZ
stimulated gravitropism independently of cellulose orientation, as it increased the percentage of standing
hypocotyls in oryzalin-treated wild type and pom2-4 plants to 100%. The BRZ effect on gravitropism was
accompanied by a decrease in crystalline cellulose and mannose, and an increase in non-cellulosic glucose, which
is consistent with changes in cell wall mannans, xyloglucans and/or cellulose crystallinity. Thus the opposite
effects of EBL and BRZ on gravitropism are mediated by different mechanisms affecting the wall mechanics.
[1] F. Vandenbussche et al. (2011) Plant Physiol., 156, 1331-1336.
P4-45
Characterisation of two putative galactosyl transferases potentially involved in arabinogalactanprotein biosynthesis
Hernandez-Sanchez A.M. (a), Lampugnani E.R. (b), Doblin M.S. (a), Bacic A. (ac)
(a) ARC Centre of Excellence in Plant Cell Walls, School of Botany, The University of Melbourne, Parkville, VIC, Australia;
(b) Plant Cell Biology Research Centre, School of Botany, The University of Melbourne, Parkville, VIC, Australia; (c) Bio21
Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia.
Arabinogalactan-proteins (AGPs) are plasma membrane-cell wall proteoglycans implicated in many aspects of
plant development, including cell expansion and division1. AGP biosynthesis is complex and involves numerous
post-translational modifications including extensive glycosylation of their protein backbones. The glycosyl
transferase (GT) 31 family members are thought to be involved in the biosynthesis of the AGP β-(1,3)-galactan
backbone2. Here, we describe the characterisation of two GT 31 family members, At1g74800 (GALT2) and
At1g77810 (GALT9), and their involvement in AGP biosynthesis in the model plant Arabidopsis thaliana. Both
GALT2 and GALT9 are localised in the Golgi, as predicted for GTs involved in the formation of the β-(1,3)galactan, and are expressed in vegetative and reproductive tissues, including pollen grains. Plants which lack
either GALT2 or GALT9 function have reduced AGP levels in flowers (~10%) whereas plants that over-express
GALT2 display changes in the AGP content in leaves and a swollen trichome socket cell phenotype. This result is
consistent with the role of AGPs in cell expansion. The characterisation of these mutants and over-expressing
plants, as well as other data related to GALT function, will be discussed with respect to the involvement of
GALT2 and GALT9 in AGP biosynthesis.
[1] J. Yang et al. (2007) Plant J., 49, 629-640 ; [2] Y. Qu et al. (2008) Plant Mol. Biol., 68, 43-59.
P4-46
Arabinogalactan proteins of border cells: at the frontier between root and microbes
Vicré-Gibouin M. (a), Nguema-ONa E. (a), Cannesan M.A. (a), Durand C. (a), Plancot B. (a), Laval K. (b), Lerouge P. (a),
Follet-Gueye M.-L. (a), Driouich A. (a)
(a) Laboratoire Glycobiologie et Matrice Extracellulaire Végétale, EA 4358, IFRMP 23, Université de Rouen, 76821 Mont
Saint Aignan, France ; (b) Laboratoire BioSol Esitpa, 76134 Mont-Saint-Aignan, France.
The plant root system is a vital organ that serves to anchor the plant in the soil and to absorb nutrients and water.
At the root tip, the root cap produces a population of living, metabolically active cells that are released into the
rhizosphere as border cells or border-like cells [1-5]. Due to their particular position at the interface between root
and soil, root border cells play a major role in the interaction with soilborne microorganisms [3,6,7].
Homogalacturonan was found to be essential in root border/ border-like cells organization [8]. However, little is
currently known on the structure and function of the cell wall components of such root cells.
We investigated the sugar composition of the cell wall of the root cap in two species: Pisum sativum, which
makes “classical” border cells, and Brassica napus, which makes atypical border-like cells. Cell walls of both
border cells and border-like cells were found to be enriched in arabinogalactan proteins [7,9]. The analysis of their
carbohydrate moieties, linkages and electrophoretic characteristics revealed i) significant structural differences
between B. napus and P. sativum root cap arabinogalactan proteins and ii) a cross-link between these
glycoproteins and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on
the behaviour of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots [7]. We
demonstrated that arabinogalactan proteins are involved in the control of early infection of roots by A. euteiches.
Our findings highlight a novel role for these proteoglycans in root-microbe interactions.
[1] Hawes MC et al. (2000) Trends Plant Sci 5: 128–133 ; [2] Vicré M et al. (2005) Plant Physiol 138: 998-1008 ; [3]
Driouich A. et al. (2007) Trends Plant Sci 12: 14-19 ; [4] Driouich A. et al. (2012) In Signaling and communication in Plants.
Edited by F. Baluska/ J. Vivanco. pp 91-107 ; [5] Cannesan MA et al. (2011) Ann Bot 108:459-469. [6] Driouich A et al.
(2010) J Exp Bot 2010, 61: 3827-3831 ; [7] Cannesan MA et al. (2012) Plant Physiol 2012, 159: 1658-1670 ; [8] Durand C et
al. (2009) Plant Physiol 2009, 150: 1411-1421 ; [9] Nguema-Ona E et al. (2012) Annals of Botany, 110: 383-404.
P4-47
Different roles of lignification in leaves and stems of poplars subjected to abiotic stresses
Cabané M. (a), Afif D. (a), Richet N. (a), Pollet B. (b), Tozo K. (c), Lapierre C. (b)
(a) Université de Lorraine, Ecologie et Ecophysiologie Forestière , UMR 1137, B.P. 70239, F-54506 Vandœuvre lès Nancy,
France; INRA, Ecologie et Ecophysiologie Forestières, UMR 1137, Champenoux, F-54280, France ; (b) AgroParisTech,
UMR 1318 78850 Thiverval-Grignon, France; INRA, UMR 1318, 78850 Thiverval-Grignon, France ;(c) Département de
Botanique, Faculté de sciences, Université de Lomé, B.P. 1515 Lomé, Togo.
Lignins, major components of wood, provide strength, rigidity and hydrophobicity to plant cell walls and are
consequently essential for plant development. Their production and deposition into the cell wall are highly
regulated. Abiotic stresses are also known to modulate lignification. We investigated the effects of elevated
carbon dioxide and/or ozone on lignification in leaves and stems of young poplars. Ozone modified lignin
biosynthesis in leaves and stems. Leaves displayed a rapid stimulation supporting the idea of a defence response
whereas stems responded late by a decrease of the lignin pathway suggesting an adjustment response to carbon
availability. However, the lignin to cellulose ratio was enhanced in stems probably to ensure mechanical support
despite radial growth reduction. Under elevated CO2, lignification was enhanced in leaves and stem as a
consequence of increased carbon supply. Both constraints, ozone and elevated CO2, increased lignification of the
cell wall but the underlying mechanisms and roles were different.
P4-48
Role of jasmonic acid during floral organ abscission and seedling growth
Patterson S. (a), Kim J. (b), Binder B. (c)
(a) Department of Horticulture University of Wisconsin, Madison, USA; (b) USDA, Beltsville, Maryland, USA; (c)
Department of Biochemistry University of Tennessee, Knoxville, USA.
Abscission is a developmentally regulated process regulating detachment of an organ from the main body of the
plant. During cell separation, changes in cell wall composition, morphology and plant cell wall signalling occur.
We have characterized numerous delayed floral organ abscission mutants in Arabidopsis and identified several
novel genes associated with cell separation as well as identified new roles for previously characterized genes. We
propose that abscission can be divided into 4 phases: Phase1, preabscission, where abscission zones (boundaries)
are established in early development, Phase 2 where abscission cells acquire competence to respond abscission
signals, Phase 3 where abscission cells are activated and cell wall loosening occurs, and Phase 4 where cell repair
is observed in the post abscission trans-differentiation phase. Recently we observed that the JA receptor COI1 is
important in regulation of this process during floral organ abscission. We will present an overview of floral organ
abscission in Arabidopsis and characterization of the novel mutant coi1-37. In coi1-37 floral organ abscission is
delayed, pollen dehiscence is blocked and leaves display curling. Responses to both jasmonic acid and ethylene in
coi1-37 combined with additional characterization of aos and ein2-1 provide evidence of crosstalk between
ethylene and JA during the abscission process. Additional observations on coi1 seedlings treated with ethylene
demonstrating interactions between the two hormones will also be presented.
J. Kim et al. (2013) PlosONE April 2013 | Volume 8 | Issue 4 | e60505.
P4-49
Anti-fungal peptides are produced in Heliophila coronopifolia (Brassicaceae) root border-like cells
and associate with pectin-rich mucilage as a preformed defence mechanism
Weiller F. (ab), Moore J.P. (a), Young P. (a), de Beer A. (a), Driouich A. (b), Vivier M. (a)
(a) Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, South Africa;(b)
Glyco-MEV Laboratoire, Université de Rouen, Mont Saint Aignan, France.
The South African endemic Brassicaceae Heliophila coronopifolia possesses four plant defensin-encoding genes
(HcAFP1-HcAFP4) that are differentially expressed in vegetative and reproductive tissue, and are involved in
defence against fungal infections [1]. Videomicroscopy, using live-cell-imaging technology, was used to
characterise border-like cells [2] of H. coronopifolia under cultivation. Border-like cells were shown to possess a
cellulose cell wall, contained viable nuclei and were embedded in a layer of mucilage. The mucilage was shown
to be of a pectin-rich nature (very similar to that found in Arabidopsis thaliana, see [3]) as evidenced by
dissolution of the sheath and dissociation of the border-like cells when treated with pectinase. Quantitative realtime PCR was used to measure the expression of all four defensin-encoding genes (HcAFP1-HcAFP4) in root tip
cells. A polyclonal antibody raised against HcAFP2 (but cross-reactive with all four defensins) confirmed
peptides were present in root tip tissue, mucilage and border-like cells. This study provides a direct link between
defensins (functioning in preformed defence) and border-like cells, both embedded in a protective pectin mucilage
sheath, during normal plant growth and development.
[1] A. de Beer and M.A. Vivier (2011) BMC Res Notes 4, 459; [2] A. Driouich et al. (2010) J. Exp. Bot., 61, 3827-3831; [3]
C. Durand et al. (2009) Plant Physiol., 150, 1411-1421.
P4-50
Comparative analysis of the complexes formed by PGIP-2 from P. vulgaris and four fungal
polygalacturonases by Small Angle X-Ray Scattering (SAXS) and site-directed mutagenesis
Andreani F. (a), Benedetti M. (a), Sicilia F. (b), Leggio C. (b), Di Matteo A. (c), Federici L. (d), De Lorenzo G. (a), Pavel
N.V. (b), Cervone F. (a)
(a) Dipartimento di Biologia e Biotecnologie “C. Darwin”, Sapienza Università di Roma, Italia; (b) Dipartimento di
Chimica, Sapienza Università di Roma, INFM CRS-SOFT, c/o, Italia; (c) Dipartimento di Chimica e Dipartimento di Scienze
Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, Italia; (d) Dipartimento di Scienze Biomediche, Universita` di
Chieti ‘‘G. D’Annunzio’’, Italia.
Fungal endo-polygalacturonases (PGs) degrade the α-1,4-galacturonosyl bonds of homogalacturonan and
facilitate the growth of hyphae within the plant tissue. Many fungi secrete PGs during the invasion and, in some
cases, these enzymes contribute significantly to their pathogenicity or virulence. Against fungal PGs, plants have
evolved many specific polygalacturonase inhibiting proteins (PGIPs) that contribute to the plant defense.
Overexpression of PGIPs slows down the fungal infection and favours the accumulation of oligogalacturonides,
endogenous elicitors of the plant defences. The crystallographic structures of several PGs and of PGIP2 from
Phaseolus vulgaris (PvPGIP2) are available but the structural requirements of the PG-PGIP interaction still remain
obscure. We report here a comparative analysis of the complexes formed by PvPGIP2 and the PGs produced by
four different phytopathogenic fungi (Fusarium phyllophylum, Aspergillus niger, Colletotrichum lupini and
Fusarium verticillioides), through SAXS analysis and site-directed mutagenesis. This study highlights the residues
involved in the specific interactions and paves the way to the design of novel PGIPs with improved inhibition
capabilities.
P4-51
The ethylene precursor ACC stimulates G-layer formation in fibers of hybrid aspen
Felten J. (a), Lesniewska J. (ab), Delhomme N. (c), Love J. (a), Mellerowicz E. (a), Rüggeberg M. (d), Gorzsás A. (e),
Sundberg B. (a)
(a) Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural
Sciences, SE-901 83 Umeå, Sweden;(b) Department of Botany, Institute of Biology, University in Bialystok, Swierkowa 20 b,
15-950 Bialystok, Poland; (c) Umeå Plant Science Center, Department of Plant Physiology, Umeå University, SE-901 83
Umeå, Sweden; (d) Swiss Federal Institute of Technology Zurich (ETH Zurich), Institute for Building Materials, CH-8093
Zurich, Switzerland; (e) Department of Chemistry, Umeå University, SE-901 83 Umeå, Sweden.
The phytohormone ethylene has the potential to regulate secondary growth of plants. We demonstrated previously
that application of exogenous ethylene, or of its in planta precursor 1-aminocyclopropane-1-carboxylic acid
(ACC), stimulates xylem growth in stems of hybrid aspen (Populus tremula x tremuloides) in an ethylenesignalling dependent way [1]. More recently, we observed that ACC application has the potential to induce the
formation of gelatinous-layer-(G-layer)-like structures in hybrid aspen fibers. G-layers are rich in cellulose and
normally form in tension wood (TW) fibers (denoted G-fibers). In ACC-treated (upright) wildtype trees G-fibers
formed all around the stem while they were nearly absent in ACC-treated ethylene insensitive plants. ACCinduced G-layers had an ultrastructure similar to TW G-layers, as observed by transmission electron microscopy.
The G-layers reacted positively to anti-galactan antibodies (LM5) and negatively to anti-xylan antibodies (LM10).
Furthermore, FTIR microspectroscopic analysis confirmed that ACC-induced G-fibers had a chemical fingerprint
resembling TW G-fibers. Finally, the cellulose microfibril angle (MFA) was reduced from 25° in the xylem of
untreated trees to 5° in ACC-treated trees, which is a MFA similar to that observed in TW G-layers. These data
confirm that ACC-treatment induces genuine G-layers in hybrid aspen. RNA-Seq data from ACC treated wildtype
and ethylene insensitive stems as well as TW is now analysed to decipher how ACC induces G-layer formation at
a molecular level downstream of the ethylene signalling cascade.
[1] J Love et al. (2009) PNAS, 106, 5984-5989.
P4-52
Unravelling the function and expression pattern of Arabinogalactan Proteins in the reproductive
tissues of Arabidopsis thaliana
Pereira A.M. (a), Nobre S. (a), Costa M. (a), Masiero S. (b), Sprunck S. (c), Coimbra S. (a)
(a) Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Portugal; (b)2 Dipartimento di Bioscienze,
Universitá degli Studi di Milano, Italy; (c) Cell Biology & Plant Biochemistry, University of Regensburg, Germany.
Arabinogalactan proteins (AGPs) are heavily glycosylated proteins ubiquitously expressed and identified in
Angiosperms and also in algae. In Angiosperms, several evidences suggest their involvement in different
reproductive key processes leading to double fertilization. AGPs are also prominent candidates to regulate and
support the pollen tube growth through the pistil tissues[1]. Here we describe the different distribution of specific
AGP genes throughout the Arabidopsis female reproductive tissues and point out their possible roles in distinctive
reproductive processes. Different AGP genes are shown to be expressed along the pathway followed by the
pollen tube during its journey to reach the egg and the central cell inside the embryo sac. The specific and
differential presence of these proteins was observed in the stigmatic cells, the transmitting tissue, the funiculus,
the integuments that surrounds the embryo sac and in the female gametophytic cells. The expression pattern of
these AGPs in the female reproductive tissues brings new and important evidences for selecting specific AGPs
that are involved in sexual plant reproductive processes, being of great significance for this field of study. At the
present we are characterizing some AGP mutants showing defects in reproductive processes. agp4 mutants reveal
a high percentage of ovule and seed abortion. agp4 knock-out plants’ seeds show defects in the shape and size of
the cells that are part of the embryo sac and seed’s endothelium. agp25 RNAi knock-down mutants show similar
defects that are now being studied.
[1] S. Coimbra et al. (2007) J. Exp. Bot., 58-16, 4027-4035 ; [2] Wuest et al. (2010) Curr. Biol., 20, 506-512.
P4-53
Plant stress signaling: The Arabidopsis EUL protein interacts with fasciclin-like arabinogalactan
proteins
Van Hove J., De Tender C., Van Damme E.J.M.
Ghent University, Dept. Molecular Biotechnology, Laboratory of Biochemistry and Glycobiology, Coupure Links 653,
9000 Ghent, Belgium.
The family of proteins related to the Euonymus lectin is of interest because this carbohydrate-binding domain is
present in all terrestrial plants and its expression is induced when the plant is subjected to stress situations, such as
drought and increased salt concentrations. Our current research aims at unravelling the physiological role of the
Arabidopsis EUL protein. The EUL lectin from Arabidopsis thaliana consists of one EUL domain preceded by an
unknown N-terminal domain and represents the most widespread form of EUL lectins. Although this EUL protein
is synthesized in the cytoplasmic compartment, it was shown to follow a non-conventional route for secretion,
resulting in lectin secretion from plant cells and export to the plasmamembrane/cell wall. To discover interaction
partners for this protein, the tandem affinity purification (TAP) approach was used. The EUL domain was N- or
C-terminally fused to a TAP tag and transformed into Arabidopsis suspension cells. The list of candidate
interactors yielded a subset of fasciclin-like arabinogalactan proteins (FLAs). Fasciclins are known to play a role
in protein-protein interactions at the cell surface while arabinogalactans are important cell wall constituents. The
highly galactosylated glycosylation pattern of these FLAs fits well with the sugar binding specificity of the
Arabidopsis EUL lectin determined by glycan array analysis. The interaction between the Arabidopsis lectin and
FLAs was also confirmed by bimolecular fluorescence complementation assays. Thus it can be expected that the
perceived interaction between the lectin and fasciclin-like arabinogalactan proteins is mediated by proteincarbohydrate interactions. These findings open the debate whether lectins can play a role in the signal transduction
pathways originating from the cell wall through interaction with arabinogalactans.
P4-54
The Powdery Mildew Resistance5 (PMR5) gene: the intersection of cell wall synthesis and disease
resistance
Chiniquy D., Cherk Lim C., Somerville S.
Plant and Microbial Biology Department, University of California, Berkeley, CA and Energy Biosciences Institute, Berkeley,
CA, USA.
Powdery mildew is a biotrophic fungus that infects many agriculturally important crop species worldwide,
including: wheat, barley, strawberry, and grapevines. The powdery mildew fungus must penetrate the plant cell
wall using cell wall degrading enzymes to get to the plant cell, and as a biotrophic fungus, it must also complete
its life cycle without triggering the plant’s elaborate defense system. The Arabidopsis thaliana powdery mildew
resistant (PMR) mutants are resistant to the powdery mildew Golovinomyces cichoracearum, and one of these
mutants, pmr5, carries a mutation in a gene member of the TBL/DUF231 family, which includes several genes
involved in cell wall biosynthesis and modification. Of all the Arabidopsis plants carrying mutations in members
of this family (with 46 members in Arabidopsis), only a mutation in the PMR5 gene results in resistance to
powdery mildew. This resistance does not appear to trigger any known defense signaling pathways. The pmr5
mutant cell wall is deficient in acetate and has altered pectic sugar composition, suggesting that it too mediates
cell wall alterations as seen with other TBL/DUF231 member proteins. We are investigating what elements of the
cell wall are modified by PMR5 and how this modification leads to increased disease resistance.
P4-55
Degradation and synthesis of β-glucans by a Magnaporthe oryzae endotransglucosylase
Takahashi M. (a), Yoshioka K. (b), Imai T. (b), Miyoshi Y. (c), Yoshida K. (d), Yamashita T. (e), Takeda T. (a)
(a) Iwate Biotechnology Research Center; (b) Kyoto University; (c) Kyoto Prefectural University; (d) The Sainsbury
Laboratory; (e) Iwate University, Japan.
A Magnaporthe oryzae enzyme, endotransglucosylase (ETG), was identified to be involved in 1,3-1,4-β-glucan
degradation after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatographytandem mass spectrometry. A recombinant ETG prepared by overexpression in M. oryzae exhibited endo-typical
depolymerization of polysaccharides containing β-1,4-linkages, in which 1,3-1,4-β-glucan was the best substrate.
When cellooligosaccharides were used as the substrate, the ETG generated reaction products with both shorter
and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides into
β-1,4-linked glucans were observed after incubation with ETG. These results indicate that ETG cleaves the
glycosidic bond of β-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule
functioning as an acceptor substrate. Furthermore, ETG treatment caused increased extension of heat-inactivated
wheat coleoptiles. The result suggests that ETG functions to loosen plant cell wall by cleaving the 1,3-1,4-βglucan tethers between cellulose microfibrils, which leads to enhance invasion of M. oryzae to rice.
On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a
maximum length of 26 glucose units. Thus, ETG functions to depolymerize and polymerize β-glucans, depending
on the size of the acceptor substrate [1].
[1] Takahashi et al. (2013) J. Biol. Chem., in press.
Session 5 : Advanced Understanding of CW Structure, Biosynthesis & Function
Bioinformatic & omic approaches, Computational & biophysical approaches
P5-01
Transmembrane structural organization of plant cellulose synthase proteins
Davis J.K., Sethaphong L., Voynov M., Smirnov A.I., Yingling Y., Haigler C.H.
North Carolina State University, Center for Lignocellulose Structure and Formation, USA.
Many essential aspects of plant cellulose synthase (CESA) structure and function are still unknown. One
important unsolved problem is how these proteins accomplish transfer of glucose from UDP-glucose in the
cytosol to elongating cellulose polymers in the apoplast. Computational modeling predicts structures for eight
transmembrane helices (TMHs) in CESA proteins, but the insertion of these predicted domains in the membrane
has not been verified experimentally. The recently-published crystal structure of bacterial cellulose synthase
catalytic subunit (BcsA; Morgan et al. 2013) has provided invaluable insight into the general biochemical
mechanisms of cellulose synthesis and translocation. However, sequence and structure alignments of plant CESA
proteins with BcsA present some ambiguities, particularly with regard to the putative TMHs of the CESA
proteins. We are investigating the structure and organization of the transmembrane regions of a CESA protein
involved in the synthesis of Arabidopsis thaliana secondary cell walls. Our approach is to employ electron
paramagnetic resonance (EPR) spectroscopy to examine the spatial arrangement and local environment of CESA
transmembrane helices in vitro. To accomplish this, we designed a suite of expression constructs encoding
peptides that span CESA protein TMHs. The peptides are designed for site-directed spin labeling (SDSL)
derivatization at specific positions, based on computational modeling of CESA TMHs and sequence alignment
with BcsA. Using Pichia pastoris as a heterologous expression system, we identified cell lines that accumulate
high levels (mg/L culture) of CESA peptides. The TMH-spanning peptides were then solubilized from
microsomal membrane fractions and brought to a high degree of purity by chromatographic methods. Ultimately
the purified peptides will be derivatized with spin labels for EPR, which will enable us to infer distances between
derivatized sites and the local electrostatic environment of the derivatized residues. These experiments are
expected to establish the presence and composition of a putative transmembrane pore in an individual plant
CESA.
Morgan, J.L.W, et al. (2013) Crystallographic snapshot of cellulose synthesis and membrane translocation. Nature 493, 181187. Research support was provided by the Center for Lignocellulose Structure and Formation (award no. DE–SC0001090), a DOE
Energy Frontiers Research Center.
P5-02
Integrative analysis of Transcriptome, Translatome and Proteome data from root cell types of
Arabidopsis thaliana
Rajasundaram D. (ab), Klie S. (b), Persson S. (b), Selbig J. (ab)
(a) Institute of Biochemistry and Biology, University of Potsdam, Germany; (b) Max Planck Institute of Molecular Plant
Physiology, Potsdam, Germany.
The study of plant cell walls is still limited by the paucity of our knowledge of cellulose biosynthesis related
genes despite the amount of high-throughput gene expression data available. Recently, considerable effort was
undertaken to derive information which also captures the spatiotemporal dynamics of gene expression in complete
cellular systems. A particular example is a gene expression compendium generated for different developmental
stages as well as root cell types of Arabidopsis thaliana. Other studies extended our view beyond the
transcriptome by monitoring cell specific abundances of genes and gene-products on the level of the translatome
and proteome. Clearly, such a ‘multi-omics’ data set poses challenges for data integration and so far, there have
been no clear results pertaining to the degree of similarity of patterns obtained for genes and gene-products on
different system levels. As such, it has been repeatedly reported that particularly on the level of the transcriptome
and proteome only weak linear relationships are present considering patterns of protein abundances and
corresponding gene-expression levels. By including the intermediate system level of the translatome, an analysis
of the cell wall related biology is presented. Particularly, classical computational approaches relying on pairwise
similarities (i.e. relevance networks utilizing Pearson’s correlation coefficient) are extended by projection-based
approaches, such as canonical correlation analysis (CCA). Briefly, we employ CCA to obtain linear combinations
of gene co-expression patterns which are maximally correlated with their respective protein abundances. Finally,
our approach allows identifying and confirming key genes associated with production of cell wall polymers.
[1] J. Petricka et al.(2012) Proc Natl Acad Sci., 109 (18): 6811-6818; [2] Mustroph et al.(2009) Proc Natl Acad Sci.,
44(106):18843-18848; [3] M. Brady et al.(2007) Sci., 318(5851):801-806.
P5-03
The N-Terminus of PaRBOH1 from Picea abies is a Phosphorylation Target
Nickolov K. (ab), Häggman H. (b), Kärkönen A. (a)
(a) Dept. of Agriculture, University of Helsinki, Finland;(b) Dept. of Biology, University of Oulu, Finland.
Lignin biosynthesis during secondary plant cell wall formation is a process of oxidative polymerization of
monolignol radicals activated by H2O2-requiring peroxidases and/or O2-requiring laccases. One possible source
of apoplastic reactive oxygen species (ROS) are NADPH oxidases, known as respiratory burst oxidase homologs
(RBOHs) in plants. RBOHs are a regulatory merging node in the ROS generation network by integrating
phosphorylation and Ca2+-signaling, a function determined by multiple serine, threonine and tyrosine amino acid
residues as well as by Ca2+-binding EF-hand motifs in their N-terminal domain. RBOHs are also a known target
for Ca2+-dependent protein kinases. The full-length sequence of a NADPH oxidase PaRBOH1 has been isolated
previously from an extracellular lignin-producing tissue culture line of Picea abies (L.) Karst.
We performed in silico analysis of PaRBOH1 to predict possible post-translational modifications. In order to
determine whether the predicted phosphorylation events really happen, the initial 400 amino acids of the Nterminal part have been heterologously expressed in E. coli with an additional 6xHis-tag at the C-terminus. The
purified peptide has then been used as a substrate in radioactive in vitro kinase assays with several types of spruce
protein extracts as kinase sources, and Ca2+ supplementation or depletion. We have been able to purify 33Plabelled PaRBOH1 N-terminal peptide from the kinase reaction mix, which indicates that the peptide was indeed
subjected to phosphorylation(s). Currently we are conducting non-radioactive kinase reactions followed by mass
spectrometry analysis in order to resolve the phosphorylation number and sites in the peptide.
P5-04
Genome-wide atlas of lignin and lignan genes in the fibre crop Linum usitassimum
Neutelings G. (a), Blervacq A.S. (a), Hano C. (b), Hawkins S. (a)
(a) Université Lille Nord de France, Lille 1 UMR INRA 1281, SADV, F-59650, Villeneuve d'Ascq cedex, France; (b)
LBLGC, UPRES EA 1207, Université d'Orléans, France.
Flax (Linum usitatissimum) is a very ancient crop cultivated for seeds and fibers. The extremely long primary bast
fiber cells are used to make textiles and composite materials. Their secondary cell walls are very rich in crystalline
cellulose and contain unusually low amounts of lignin that can however have a strong impact on final product
quality. A better understanding of lignin biosynthesis in flax is therefore necessary to improve fiber quality. The
monomeric units (monolignols) of the lignin polymer can also form optically active dimers known as lignans. The
dimerization of 2 coniferyl alcohol units results in the formation of pinoresinol, which is converted to lariciresinol
and then secoisolariciresinol. The diglycoside (SDG) is the major lignan in this species and mostly accumulate in
the seed (SDG accounts for up to 2% of the dry weight) but also in the stem [1]. We believe that the accumulation
of a wide range of glycosylated and non-glycosylated phenolics (lignans and neo-lignans) may be related to the
hypolignification of the flax bast fibers as has already been observed when monolignol genes are down-regulated
in model plants.
We took advantage of the recently published genome sequence [2] to identify and analyse the gene structures of
69 gene models potentially encoding enzymes included in the 10 protein families involved in monolignol
biosynthesis. We determined transcript abundance in different tissues and organs to identify those genes
implicated in lignin metabolism during plant growth but also to search for genes that could be specifically
expressed under different stress conditions.
[1] R. Huis et al. (2012) Plant Physiol., 158, 1893-1915 ; [2] Z. Wang et al. (2012) Plant J., 72, 461-473.
P5-05
Identification and study of Brachypodium distachyon COMT TILLING mutants
Ho-Yue-Kuang S. (ab), Dalmais M. (c), Bouvier d’Yvoire M. (a), Antelme S. (a), Chateigner-Boutin A.-L. (b),
Lapierre C. (a), Jouanin L. (a), Sibout R. (a)
(a) Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique de Versailles, Versailles, France ; (b)
Biopolymères Interactions Assemblages, Institut National de la Recherche Agronomique de Nantes-Angers, Nantes, France ;
(c) URGV, Unité de Recherche en Génomique Végétale, Université d'Evry Val d'Essonne, INRA, Evry, France.
Plant cell wall forms a network composed of polysaccharides, proteins and lignins in some specialized tissues.
Grass cell walls are different from dicots : they contain high amount of hydroxycinnamic acids linked to
arabinoxylans or lignins. The presence of these acids may impact polysaccharides properties and lignin
deposition. In vitro experiments suggest that the caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT)
that is involved in the syringyl units biosynthesis in lignins may participate to the methoxylation of caffeic acid to
produce ferulic acid (FA) in the cytosol prior being transported into the Golgi apparatus. In Brachypodium
distachyon, COMTs are encoded by a multigenic family. We are studying the COMT gene family to determine
whether the same genes are responsible for both lignin and FA biosynthesis and whether specific isoforms are
required in the different plant tissues. Phylogenetic analysis shows that Bradi3g16530 is similar to other grass
COMTs involved in lignin biosynthesis. Q-RT-PCR showed that Bradi3g16530 is expressed in stems, leaves,
roots, grains and endosperms. TILLING was used to find allelic series of COMT mutants. Thus fifteen mutated
lines were identified for Bradi3g16530. The line 7549 showed a 50% decrease in the lignin S/G ratio, the
accumulation of 5-OHG residues characteristics of a COMT deficiency and a 20% decrease in the amount of pcoumaric acid but no change in FA. The mutated COMT may have a residual activity or another enzyme may be
involved in the biosynthesis of FA. Enzymatic assays are currently performed on the recombinant mutated
proteins to test these hypotheses.
P5-06
Biochemical and physiological characterization of fut4 and fut6 mutants defective in
arabinogalactan-protein fucosylation in Arabidopsis
Liang Y., Pattathil S., Xu W-L., Basu D., Venetos A., Faik A., Hahn M.G., Showalter A.M.
Department of Environmental and Plant Biology (Y.L., W.-L.X., D.B., A.V., A.F., A.M.S.), Molecular and Cellular Biology
Program (Y.L., D.B., A.F., A.M.S.), Ohio University, Athens, Ohio 45701; Complex Carbohydrate Research Center (S.P.,
M.G.H.), Department of Plant Biology (M.G.H.), University of Georgia, Athens, Georgia 30602, USA.
Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins (HRGPs) present in
plant cell walls. AGPs are characterized by arabinose/galactose-rich side chains, which define their interactive
molecular surface. Fucose residues are found in some dicot AGPs, and AGP fucosylation is developmentally
regulated. We previously identified Arabidopsis (Arabidopsis thaliana) FUT4 and FUT6 genes as AGP-specific
fucosyltransferases (FUTs) based on their enzymatic activities when heterologously expressed in tobacco
(Nicotiana tabacum) BY2 suspension-cultured cells [1]. Here, the functions of FUT4 and FUT6 and the
physiological roles of fucosylated AGPs were further investigated using Arabidopsis fut4, fut6 and fut4/fut6
mutant plants. All mutant plants showed no phenotypic differences in vegetative and reproductive growth
compared to wild type plants under physiological growth conditions. However, roots of wild type and fut4 mutant
plants contained terminal fucose epitopes, which were absent in fut6 and fut4/fut6 mutant plants as indicated by
eel lectin staining. Monosaccharide analysis showed fucose was present in wild type leaf and root AGPs, but
absent in fut4 leaf AGPs and in fut4/fut6 double mutant leaf and root AGPs, indicating FUT4 was required for
fucosylation of leaf AGPs while both FUT4 and FUT6 contributed to fucosylation of root AGPs. Glycome
profiling of cell wall fractions from mutant roots and leaves showed distinct glycome profiles compared to wild
type plants, indicating fucosyl residues on AGPs may regulate intermolecular interactions between AGPs and
other wall components. The current work exemplifies the possibilities of refinement of cell wall structures by
manipulation of a single or a few cell wall biosynthetic genes.
P5-07
A model for cell wall loosening via defect migration in cellulose microfibrils
Lipchinsky A.
Saint-Petersburg State University, Russia.
Consideration of the microfibril structural features in conjunction with the current body of knowledge about the
molecular mechanisms underlying polymer rheology suggests that conformational defects on the microfibril
surface could be the key players that promote the disruption of hydrogen bonds and van der Waals interactions at
the microfibril-matrix interface in a high-stress environment. In this context, the following three assumptions are
discussed and justified: (i) microfibril-matrix interfaces cause steep stress gradients on the microfibril surface, (ii)
stress gradients drive the motion of conformational defects along the microfibril surface towards the microfibrilmatrix interfaces, and (iii) the approaching of defects to the microfibril-matrix interfaces facilitates the
dissociation of matrix polysaccharides from cellulose microfibrils. A number of issues pertaining to the function
of wall loosening proteins, in particular expansins, seem to find a natural explanation within the framework of the
model considered.
P5-08
Identification of Grass Cell Wall Synthesis Genes from Correlations between Gene Expression
and Cell Wall Composition in Rice
Lin F. (a), Manisseri C. (b), Fagerstrom A. (d), Williams B. (c), Chiniquy D.M. (bc), Peck M.L. (a), Saha P. (a), VegaSanchez M. (bc), Fangel J.U. (d), Willats W.G.T. (d), Scheller H.V. (b), Ronald P.C. (bc), Bartley L.E. (abc)
(a) Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA; (b) Joint BioEnergy
Institute, Emeryville, CA 94608 and Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; (c) Department of
Plant Pathology and The Genome Center, University of California, Davis, CA 95616, USA;(d) Department of Plant and
Environmental Sciences, University of Copenhagen, Denmark.
Grass cell walls serve as animal feed and are a potential sustainable feedstock for biofuel production.
Glycosyltransferases (GTs) and acyltransferases (ATs) perform cell wall synthesis but the roles of many GT and
AT families/members remain undetermined. We are identifying the correlations between the putative cell wall
synthesis genes and cell wall components to establish testable hypotheses of gene function. In this study, we took
30 samples from different organs and different developmental stages of rice (Oryza sativa) and measured
monosaccharide components, hydroxycinnamates, lignin, enzymatic digestibility and cell wall epitopes. We also
used qPCR to measure the expression of grass-diverged genes that are putatively involved in cell wall synthesis.
We analyzed the data with both Pearson’s and Gini correlations, the latter of which is a hybrid of parametric and
non-parametric methods. We identified 107 significant correlations between cell wall components and GTs and
ATs in whole data set (q<0.05, correlation coefficient>0.6). For example, among expected correlations, we found
a correlation between p-coumarate and the expression of a recently characterized p-coumarate monolignol
transferase gene. We will also discuss a number of novel correlations that provide leads for better understanding
cell wall synthesis in grasses and improving cell walls for economic uses.
P5-09
Spin-probe EPR Study of Local Polarity and Viscosity of Cotton Cellulose Nanodomains and the
effects of Solvents
Voinov M.A., Marek A.A., Smirnov A.I.
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695-8204, USA.
Cellulose fibres of natural origin such as cotton contain both crystalline and amorphous nanodomains with
strikingly different physicochemical properties. While understanding interactions of these domains with chemical
compounds is important for the technological use of this abundant and renewable biomaterial, the presence and
characterization of the amorphous phase in cellulose may indicate some abnormalities in its synthesis. Thus, from
both technological and biological perspectives detailed characterization of the cellulose fibres represents an
important problem. Here we describe the use of spin probe EPR method to study local polarity and viscosity of
cellulose nanodomains. The method is based on introduction of EPR-active “spin probes” and is somewhat
analogous to fluorescence methods although the EPR probes are generally smaller and their chemical structure is
more diverse. Finally but not lastly, EPR is capable of providing different and, in some cases, unique analytical
data than the competing techniques. Two types of cotton fibres – from G. hirsutum and G. Barbadense (provided
by Prof. Haigler, NCSU) were loaded with hydrophobic probe Tempo and more hydrophilic probe Tempol using
a series of polar and non-polar solvents. The EPR spectra indicated the presence of multiple compartments with
different probe solubility, dynamics, and polarity. Loading cotton with a specific solvent following on by an
introduction of a small nitroxide through a gaseous phase resulted in different distribution of the probe in cellulose
domains/compartments. Remarkably, cotton nanodomains were found to retain locally polarity of the solvents
used for the probe introduction many days after the experiment. Least-squares simulation analysis of EPR data at
9.5 and 94 GHz allowed us to obtain detailed data on local probe motion in cotton nanodomains.
Supported as a part of the Center for LignoCellulose Structure and Formation under DOE Award DE-SC0001090.
P5-10
Distribution of coniferin in differentiating xylems of normal and compression woods using
MALDI imaging mass spectrometry
Yoshinaga A. (a), Kamitakahara H. (b), Takabe K. (a)
(a) Laboratory of Tree Cell Biology, Division of Forest and Biomaterials Science, Graduate School of Agriculture,
KyotoUniversity, Japan; (b) Laboratory of the Chemistry of Biomaterials, Division of Forest and Biomaterials Science,
Graduate School of Agriculture, Kyoto University, Japan.
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was applied to detect
monolignol glucosides in differentiating xylems of normal and compression woods in several Japanese softwoods.
MALDI-IMS using transverse sections of normal wood in Japanese cypress (Chamaecyparis obtusa) showed that
coniferin (m/z 365, [M+Na]+) was mainly distributed in tracheids during secondary wall formation. This result
agreed well with our previous data on coniferin distribution as revealed by Raman microscopy (Morikawa et al.
2010[1]). Since MALDI-TOF MS spectrum and collision induced dissociation (CID) spectrum of sucrose were
almost similar to that of coniferin, it was found to be difficult to distinguish coniferin and sucrose using MALDIIMS. To distinguish coniferin and sucrose in MALDI-IMS, vapor treatment of osmium tetroxide was applied to
MALDI-IMS. After vapor treatment of osmium tetroxide, peak of coniferin shifted to m/z 399, indicating that two
hydroxyl groups were introduced to C=C double bond in coniferin. In contrast, sucrose showed no shift after the
treatment. Treatment of differentiating xylem in normal wood in Japanese cypress confirmed that coniferin was
mainly distributed in tracheids during secondary wall formation before active lignification. In compression wood,
no obvious distribution of coniferin was seen in differentiating xylem, suggesting other mechanism that
monolignols or monolignol glucosides are not pooled and synthesized and efficiently transported to cell walls.
[1] Y. Morikawa et al. (2010) Holzforschung, 64, 61-67.
P5-11
Regulatory mechanisms of UDP-glucose 4-epimerase isoforms
Steinschauer V., Krol M., Seifert G.J.
(a) University of Natural Resources and Life Science, Vienna; Department of Applied Genetics and Cell Biology, Muthgasse
18, 1190 Vienna, Austria.
UDP-D-glucose 4-epimerase (UGE) is required for normal cell wall biosynthesis. Multiple UGE isoforms play
non-redundant role in growth and cell wall strucuture . To assess the respective contributions of transcriptional
and post-transcriptional control we have shuffled cis-regulatory elements between the key UGE isoforms UGE1,2 and -4 and to complement uge4 single mutants and uge1 uge2 uge4. We have assessed the functional
significance of potential protein phosphorylation sites as well as the presence of serine-rich terminal domains.
Furthermore the functional requirement of cytosolic localization of UGE was addressed. We find that both
transcriptional regulation as well as protein properties are essential for full biological UGE in vivo function. Golgi
- targeted UGE was found biologically inactive suggesting insufficient levels of UDP-Glc in this compartment.
[1] Rösti, J., Barton, C. J., Albrecht, S., Dupree, P., Pauly, M., Findlay, K., Roberts, K., and Seifert, G. J. (2007) Plant Cell
19, 1565-1579.
FUNDING: This work is supported by the FWF (The Research Fund). Grants: P21782-B12, I1182-B22
P5-12
Analytical tools to follow in vitro xylan synthesis
Blanc N.F. (a), Mortimer J. (a), Rubtsov D. (a), Gilbert H.J. (b), Bolam D. (b), Dupree P. (a)
(a) Department of Biochemistry, University of Cambridge, UK, CB2 1QW ; (b) Institute for Cell and Molecular Bioscience,
University of Newcastle, UK, NE2 4HH.
Xylan is the major hemicellulose in flowering plants’ cell walls and its synthesis can be followed in vitro using
stem microsomes. By providing a fluorescently-labelled xylooligosaccharide as acceptor, it is possible to track the
activity of glycosyl transferases (GTs) through detection and characterisation of newly formed oligosaccharides.
This can be achieved using DNA-sequencer Assisted Saccharide High-throughput analysis (DASH), a capillary
electrophoretic method developed in our lab which separates sugars by size, charge and linkage. In vitro
biosynthetic modifications to the acceptor are also identified with the use of xylan-specific glycosyl hydrolases of
known activity.
In vitro xylan synthesis can give us insights into the mechanisms of action of the xylan synthase complex in
several species such as Arabidopsis and willow. Moreover, by comparing assays in the presence or absence of a
specific GT it may be possible to draw conclusions regarding the enzyme’s activity and role.
P5-13
Purification of a truncated Δ68-AtFUT1 expressed in Hi5 insect cells for crystallographic study
Cicéron F., Chazalet V., Lerouxel O., Breton C.
CERMAV-CNRS, University of Grenoble, Grenoble, France.
Glycosyltransferases (GTs) represent an ubiquitous group of enzymes that catalyses the synthesis of glycosidic
linkages. At present, the crystal structure of 105 different GTs have been solved providing structural information
for 38 GT families, but none of these known structures describes GTs related to plant cell wall biosynthesis [1]. In
order to fill that gap, we perform the purification of a truncated form of Arabidopsis Fucosyltransferase 1 (Δ68AtFUT1), a well characterized enzyme involved in xyloglucan biosynthesis, transferring L-Fucose from GDP-LFuc onto the galactose residue of Galacto-Xyloglucan [2]. Δ68-AtFUT1 enzyme harbours the replacement of 68
amino-acids at the N-terminus by a His 6-tag sequence used for purification. Truncated AtFUT1 was shown to
remain active when expressed soluble in the medium culture of Hi5 insect cells. A two-step purification including
nickel affinity and gel permeation led to purification of homogenous and monodisperse Δ68-AtFUT1 protein, at a
minimum yield of 2.5 mg protein per liter of culture medium, and production appears suitable for crystallographic
study. As Arabidopsis AtFUT1 belongs to CAZy family GT37, it is expected to adopt a characteristic GT-B fold,
and efforts are underway to obtain crystal of Δ68-AtFUT1.
[1] C. Breton et al. (2012) Curr Opin Struct Biol., 22, 540-549 ; [2] G. Vanzin et al. (2002) P.N.A.S, 99, 3340-3345.
P5-14
Identification of cell wall proteins in flax
Day A. (a), Goulas E. (a), Rolando C. (b), Jamet E. (c), Hawkins S. (a), Tokarski C. (b)
(a) Université Lille Nord de France, Lille 1 UMR INRA 1281, SADV, 59655, Villeneuve d'Ascq cedex, France; (b) USR
CNRS 3290, MSAP University Lille 1, 59655, Villeneuve d'Ascq cedex, France; (c) LRSV, UPS/CNRS,Castanet-Tolosan,
France.
In order to improve our understanding of cell wall biology in flax (Linum usitatissimum L.) we are currently
developing proteomics that will complement previously developed transcriptomics and metabolomics [1]. We
have firstly focused on the extraction and identification of cell wall proteins (CWPs) in flax stems. Sequential salt
(CaCl2, LiCl) extractions were used to obtain fractions enriched in cell wall proteins from the stem of 60-day-old
flax plants. High-resolution FT-ICR mass spectrometry analysis and the use of recently published genomic data
[2] allowed the identification of 11,912 peptides corresponding to a total of 1,418 different proteins [3]. Prediction
of subcellular localization using TargetP, Predotar and WoLF PSORT led to the identification of 152 putative flax
CWPs that were classified into 9 different functional classes previously established for Arabidopsis thaliana.
Examination of different functional classes revealed the presence of a number of proteins known to be involved
in, or potentially involved in cell wall metabolism in plants. This data has recently been added to WallProtDB
(http://www.polebio.lrsv.ups-tlse.fr/WallProtDB/). We are also using a proteomics approach as part of the French
NoStressWall project to investigate the impact of drought stress on flax and Brachypodium distachyon plants with
a special focus on cell wall metabolism. Recent results on the effects of modifications to different cell wall
extraction protocols will be discussed in the context of improving protein identification in flax cell walls.
[1] Huis et al. (2012) Plant Physiol., 158, 1893-1915; [2] Wang et al. (2012) Plant J., 72, 461-473; [3] Day et al. (2013)
Proteomics. 13: 812-825.
P5-15
Deciphering the Golgi proteome in cellulose-deficient cells
de Castro M. (ab), Irar S. (b), García L. (a), Largo A. (a), Caparrós-Ruiz D. (b), Acebes J.L. (a), García-Angulo P. (a)
(a) Área de Fisiología Vegetal, Facultad de CC Biológicas y Ambientales, Universidad de León, E-2407, León, Spain; (b)
Centre de Recerca en Agrigenómica (CRAG), Consorci CSIC-IRTA-UAB,Cerdanyola del Vallés, Barcelona, Spain.
Habituation of maize culture cells to dichlobenil (an inhibitor of the cellulose biosynthesis) causes a reduction in
their cellulose content counteracted by a modified arabinoxylan network, in which an overall alteration of the
metabolism of hemicelluloses seems to be involved.
It is known that important metabolic enzymes are Golgi-resident proteins. Thus, taking the advantage of what can
offer us cellulose deficient cells with a modified metabolism leading to changes in their hemicellulosic networks,
in the present work, Golgi-enriched fractions from non-habituated (Snh) and habituated to 1.5 and 6 µM
dichlobenil of DCB (Sh1.5 and Sh6) maize cell suspensions were obtained, in order to detect differences in their
“Golgi proteomic picture”. Then, these fractions were subjected to 2-D electrophoresis, and proteins differentially
expressed among cell lines were subsequently sequenced. First of all, results showed that several proteins, as
enolase 1, chaperonine 60 and 1-aminocyclopropane-1-carboxylate oxidase 1, were upregulated in habituated
cells and may play an important metabolic role in cellulose-deficient maize cells, since were coincident with
previous proteomic analysis carried out in the total proteome from maize habituated cells [1]. Caffeoyl-CoA Omethyltransferase 1 (CCoAOMT) was downregulated in habituated cells. This protein had been suggested to be
replaced in dichlobenil-habituated cells by COMT [1] and its downregulation had been related to qualitative and
quantitative changes in lignin composition in alfalfa [2]. Expression of ascorbate peroxidase was repressed in
habituated cells, supporting previous results of its activity in the overall proteome of such cell line. Interestingly,
an alpha-1,4-glucan-protein synthase (UPTG) responsible for UDP-forming was also downregulated in the
habituated cell lines. Therefore these results shed light on the dichlobenil-habituation process but also on the
synthesis of heteroxylans.
[1] Mélida et al. (2010) Mol. Plant, 3: 842-85 ; [2] Guo et al. (2001) Plant Cell, 13: 73-88.
P5-16
Computational study of lignin-protein non-covalent interactions
Pandey J.L., Watts H.D., Kubicki J.D., Richard T.L.
Pennsylvania State University, USA.
Lignin is primarily comprised of the 4-hydroxyphenylpropanoid monomers coniferyl alcohol (MG), sinapyl
alcohol (MS) and p-coumaryl alcohol (MH). The β-O-4 linkage is the predominant linkage found in lignin. MG
dimers with β-O-4 linkage were used as lignin proxies in this computational study to evaluate potential
intermolecular interactions between lignin and proteins. β-O-4 dimers have two chiral centers and occur as four
possible stereoisomers: (R,S)-β-O-4, (R,R)-β-O-4, (S,R)-β-O-4 and (S,S)-β-O-4. Density Functional theory (DFT)
energy minimization method M052X/6-31+G(d) was used to determine favorable conformers of lignin and
protein proxies as well as favorable interactions between these proxies. This method calculates the putative ππ interactions and H-bonds between proxies. The goal of this study is to develop efficient methods to study ligninprotein interactions in silico and ultimately to use these methods to design short peptide markers that interact
strongly with lignin in plant cell walls. Strongly interacting peptides can be used to functionalize AFM tips or
localize other tags for lignin visualization.
P5-17
Screening for Arabidopsis seed coat mutants using an EMS population
Vasilevski A. (a), Ahad A. (b), Günl M. (a), Bolger A. (c), Haughn G. (b), Usadel B. (ac)
(a) Forschungszentrum Jülich, IBG2 Plant Sciences, Jülich, Germany ; (b) Department of Botany, University of British
Columbia, Vancouver, Canada; (c) Institute for Biology 1, RWTH Aachen University Aachen, Germany.
Relatively little is known about synthesis and regulation of pectin biosynthesis. One way to gain insight into
pectin synthesis and modification is to use model systems. One such system is the Arabidopsis seed mucilage,
which is produced in the Arabidopsis seed coat epidermal cells and consists of rhamnogalacturonan (RGI), which
is easily extracted, and contains only small amounts of cellulose and xyloglucan [1], [2], [3].
Mutants with seed coat mucilage defects were identified by screening 1700 M2 EMS plants for alterations in
sugar composition. Mutants with altered mucilage composition are expected to have changes in pectin
biosynthesis and/or modification. More than 10 mutants with heritable and profound changes were identified, and
according to the observed chemical alteration divided in five phenotypic groups. Candidates within each
phenotypic group were tested for complementation. Furthermore, all candidate lines were analysed for changes in
seed surface morphology and mucilage release. Three lines from different phenotypic groups, along with the Col2 wild type as a reference genome, were sequenced using deep sequencing. Using SNPs detection tools, we
confirmed the sensitivity of our approach by identifying a known mucilage biosynthetic gene RHM2/MUM4. In
the other two sequenced lines we detected candidate genes for mucilage synthesis/modification and currently we
are working on the confirmation of the phenotype by looking in the potential knock-out lines. The rest of the
candidate lines are cleaned up by crosses to the Col-2 WT and are prepared for Hiseq Illumina sequencing.
[1] B. Usadel et al. (2004) Plant Physiol., 134(1), 286-95; [2] T.L. Western et al. (2004) Plant Physiol., 134(1), 296-306; [3]
A.A. Arsovski et al. (2010) Plant Signal Behav., 5(7), 796-801.
P5-18
Identification of proteins involved in cell wall assembly and remodeling by a proteomic approach
on Brachypodium distachyon grain.
Francin-Allami M. (a), Merah K. (ab), Albenne C. (b), Sibout R. (c), Pavlovic M. (a), Lollier V. (a), Rogniaux H. (a), Guillon
F. (a), Jamet E. (b), Larré C. (a)
(a) INRA, UR1268, BIA, 44316 Nantes, France; (b) LRSV, UPS/CNRS, 31326 Castanet-Tolosan, France ; (c) INRA,
UMR1318, IJPB, 78026 Versailles, France.
A major part of the daily caloric intake of human societies around the world is derived from cereal grain. The
major components in domesticated cereal grains are starch and proteins. Cell wall polysaccharides only account
for about 3-8% of grain but have major effects on the use of grain. Cell walls are mainly composed of
polysaccharides (pectins, cellulose and hemicelluloses) and smaller proportion of glycoproteins.
Except for cellulose, precursor oligosaccharides are synthesized in the Golgi apparatus, then transported to the cell
wall where they are presumably assembled to larger polysaccharides. In addition, the polysaccharide remodeling
occurs to meet the plant needs during its different stages of development. The actors required for these processes
are poorly understood, especially in cereal plants. Glycosyl hydrolases (GH) may play a crucial role in the
reorganization of cell wall polysaccharides.
To identify the enzymes involved in the assembly and remodeling mechanisms of cereal cell wall, we performed a
proteomic analysis of grain cell wall from the cereal plant model Brachypodium distachyon. Grains were collected
at three stages of development and subjected to cell wall fractionation. Then cell wall proteins (CWPs) were
extracted by successive steps using CaCl 2 and LiCl buffers, according to a protocol derived from [1].
Identification of proteins was performed using mass spectrometry (LC-MS/MS). The results revealed CWPs
potentially involved in remodeling or re-arrangement of cell wall polysaccharides. These proteins will be
compared with other sets of data obtained from other B. distachyon organs.
[1] Feiz et al. (2006) Plant Methods 27, 2-10.
P5-19
Elastic properties of the growth-controlling outer cell walls of maize coleoptile epidermis
Lipchinsky A., Sharova E.I., Medvedev S.S.
Saint-Petersburg State University, Russia.
The effects of tensile stress and temperature on cell wall elasticity have been investigated in the outer cell walls of
coleoptile epidermis of 4- and 6-day-old Zea mays L. seedlings. The change in tensile stress from 6 to 40 MPa
caused the increase in cell wall elastic modulus from 0.4 to 3 GPa. Lowering the temperature from 30 to 4 °C
resulted in instantaneous and reversible cell wall elongation of 0.3-0.5 ‰. At a given temperature and stress level
the wall elastic modulus of 6-day-old seedlings tended to be 30% higher than that of 4-day-old plants. The
relationship between cell wall elasticity and mechanical stress indicated that the stress distribution within the cell
wall is highly uneven. Analysis of the effect of temperature on wall elastic strain showed that structural
differences between crystalline and amorphous load-bearing polymers were not the only cause of the uneven
stress distribution. We suggest that the uneven stress distribution is partially related to the stress gradient between
inner and outer layers of the cell wall.
A. Lipchinsky et al. (2013) Acta Physiol. Plant. doi:10.1007/s11738-013-1255-4.
P5-20
Tensile test of plant cell wall analogs thin films using image stereocorrelation
Assor C. (ab), Sabatier L. (b), Cathala B. (a), Aguié-Béghin V. (c), Arnould O. (b)
(a) UR 1268, PVPP, INRA, rue de la Géraudière, BP 71627, 44316 Nantes, France ; (b) LMGC, Université Montpellier 2,
CNRS UMR5508, CC048 place Eugène Bataillon, 34090 Montpellier, France; (c) INRA, UMR614 Fractionnement des
AgroRessources et Environnement, Reims, France.
The plant cell wall can be the optimal scale of investigation for understanding the properties and the variability of
vegetal organs structure. At the molecular scale, the wall constituents’ organisation might have a strong influence
on theses properties. Primary cell walls are separated into monocotyledons (cereals) and dicotyledons (fleshy
fruits) depending on the type of molecules involved (cellulose, hemicelluloses, pectin, etc.). These polymers
structures and concentration within the cell wall change during the organ development (i.e., growth and
maturation). Cell wall models built from commercial polymers have been used in the literature to study the
influence of these components and their organisation on cell wall mechanical properties. However these analogs,
although they’re interesting, don’t take into account the molecule modification with time (growth and maturation)
and space (organisation within the tissue) or the change of the hydration state of the wall with time.
We aim to characterize mechanical properties of thin films made of hemicelluloses extracted from organ with an
original method that permit molecular structure to be preserved (Assor et al., 2013) [1]. The first step of our work
consists in developing a specific static tensile test adapted to cell wall analogs These tests are made in a
temperature and humidity controlled environment. Due to their low thickness (around some tenth of micrometre at
best), these films are particularly difficult to handle and the mechanical measurements are thus coupled with an
image stereo-correlation device. It allows us to measure the strain fields at the surface of the sample, even if it is
not perfectly plane, and thus to check the real loading of the film. Finally, this measurement technique allows us
to measure the elastic longitudinal modulus of the film together with its Poisson’s ratio without any effect of the
tensile machine stiffness or occurrence of slippage in the loading grips.
[1] Assor, C. ; Quémener, B. ; Vigouroux, J. ; Lahaye, M. Fractionation and structural characterization of LiCl-DMSO
soluble hemicelluloses from tomato. Carbohydrate Polymers, 2013, Vol 94, Issue 1, 46-55.
P5-21
Profiling of the Plant Cell Wall Proteome: Structural Diversity and Evolutionary Origins of Plant
N-Glycoproteins
Ruiz-May E. (a), Sørensen I. (a), Howe K.J. (b), Zhang S. (c), Thannhauser T.W. (b), Domozych D.S. (d), Rose J.K.C. (a)
(a) Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA; (b) USDA-ARS, Robert W. Holley Center for
Agriculture and Health, Ithaca, NY 14853, USA; (c) Institute of Biotechnology, Ithaca, NY 14853, USA; (d) Department of
Biology and Skidmore Microscopy Imaging Center, Skidmore College, Saratoga Springs, NY 12866, USA.
The plant cell wall and associated apoplastic environment is host to a complex cocktail of proteins with diverse
functions. However, the cell wall proteome is still relatively poorly characterized compared with those of
intracellular compartments, particularly with respect to the post-translational modifications of the constituent
proteins. For example, a large proportion of cell wall resident proteins are N-glycosylated, and yet little is known
about the details of the glycosylation sites, the structural heterogeneity of the N-glycans or their functional
significance. Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the
study of N-glycoproteins and has been used to characterize a broad range of eukaryotic N-glycoproteomes.
Moreover, the use of multiple lectins with different affinities has been reported to provide a significant benefit for
the analysis of plant N-glycoproteins. However, it has yet to be determined whether certain lectins, or
combinations of lectins are optimal for plant N-glycoproteome profiling, or whether specific lectins show
preferential association with particular N-glycosylation sites or N-glycan structures. We have used three mannosebinding lectins, concanavalin A, snowdrop lectin and lentil lectin, to profile the N-glycoproteome of tomato
(Solanum lycopersicum) fruit pericarp. Through coupling LAC with a shotgun proteomics strategy and an
orthogonal hydrophilic interaction chromatography analysis, we identified many hundreds of N-glycoproteins, and
their N-glycosylation sites, as well as the N-glycan structures of a subset of the proteome. The use of multiple
lectins substantially increased N-glycoproteome coverage and while there were no discernible differences in the
structures of N-glycans, or the charge, isoelectric point (pI) or hydrophobicity of the glycopeptides that
differentially bound to each lectin, differences were observed in the amino acid frequency at the -1 and +1
subsites of the N-glycosylation sites.. More recently, we used the same analytical pipeline to evaluate the Nglycoproteome of Penium margaritaceum, a desmid belonging to the charophycean green algae (CGA), which are
believed to be the immediate ancestors of land plants. The goal was to determine whether patterns of Nglycosylation are conserved across 450 million years of plant evolution and results will be presented.
P5-22
Specific features of cell wall proteomes of monocots: a focus on Brachypodium distachyon as a
model plant and on sugarcane as a source of biomass for cellulosic bioethanol
Douché T. (a), Calderan-Rodrigues M.J. (b), Burlat V. (a), Valot B. (c), Zivy M. (c), Labate C. (b), Jamet E. (a)
(a) LRSV, UPS/CNRS, 31326 Castanet-Tolosan, France;(b) Laboratório Max Feffer de Genética de Plantas, ESALQ,
Universidade de São Paulo, Piracicaba, Brazil; (c) PAPPSO, Génétique Végétale, INRA/CNRS, Gif sur Yvette, France.
Monocot primary cell walls exhibit specific features as compared to dicots, notably the presence of mixed (1,3)
(1,4)-- D-glucans, arabinoxylans (AXs), and phenolic compounds like ferulic and coumaric acids, but only a
little proportion of pectins. In secondary walls, there are more complex AXs in monocots. Their arabinosyl side
chains can be cross-linked with lignin through feruloyl esters. Sugarcane is expected to become an important
resource of raw material for production of cellulosic ethanol. However, the conversion process is not
economically viable yet, mostly due to the high cost of the chemical pre-treatments and the enzymatic hydrolysis
required to deconstruct cell walls. A better knowledge of the cell wall proteome should help identifying new
candidates playing roles in the remodeling of the cell wall structure. Cell walls of growing organs of B.
distachyon, i.e. leaves and culms, and of sugarcane cell suspension cultures, have been purified and cell wall
proteins (CWPs) have been extracted with CaCl2 and LiCl solutions according to [1]. Identification of proteins
has been done using mass spectrometry (LC-MS/MS). Specific profiles of CWPs have been found with a
prevalence of some glycoside hydrolase (GH) families and of oxido-reductases [2,3]. Three GHs have been
immunolocalized in B. distachyon tissues, among which a GH18 shown to accumulate in cell corners [3].
[1] Feiz et al. (2006) Plant Methods 27, 2-10; [2] Douché et al. (2013) Proteomics (accepted with revisions); [3] CalderanRodrigues et al. (2013) Proteomics (submitted).
P5-23
Contribution of hemicellulose network to plant cell wall properties
Videcoq P. (a), Assor C. (b), Arnould O. (b), Barbacci A. (a), Lahaye M. (a)
(a) UR 1268, PVPP, INRA, rue de la Géraudière, BP 71627, 44316 Nantes, France ; (b) LMGC, Université Montpellier 2,
CNRS UMR5508, CC048 place Eugène Bataillon, 34090 Montpellier, France.
Plant cell walls are made of interacting networks of polysaccharides (cellulose, hemicelluloses, pectins) and some
structural proteins. Cellulose-xyloglucan networks are considered to be the main source of structural strength in
primary cell wall. Little is known about the contribution of each polysaccharide network to mechanical properties.
The aim of this work is to evaluate the role of hemicellulose, especially xyloglucans, by monitoring plant cell wall
mechanical properties during targeted enzymatic degradation and establishing links with xyloglucan chemical
composition and structure.
Spatially homogeneous parenchyma from contrasted texture Golden Delicious (Go) and Granny Smith (Gr) apples
were sampled and vacuum infused with enzymes in buffer solution aimed at maintaining and homogenising turgor
pressure and limiting oxidation. Glucanases specific from xyloglucan and/or cellulose backbone and glycoside
hydrolases (α-fucosidase, and β-galactosidase) were used to study the impact of xyloglucan and its side chains on
parenchyma mechanical properties by dynamic mechanical analysis.
The results emphasize the key roles of side chains in the regulation of mechanical properties and are interpreted as
resulting from direct modifications of polysaccharides conformations and interactions or indirect regulation of
endogenous enzymes involved in the remodelling of networks assemblies. This study explores how xyloglucan
can be related to the mechanical function emerging at the tissue scale and provides insight into structure-function
relationships during plant organ development.
P5-24
Membrane Assembly and Interactions of Individual CesA Transmebrane Helices by Site-directed
Spin-labelling EPR
Li L., Voynov M.A., Smirnov A.I.
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27695-8204, USA.
Over the last decade site-directed spin-labelling (SDSL) EPR emerged as a powerful biophysical method for
studying structure and local dynamics of proteins and other biomolecules without their crystallization. The
method is based on a covalent modification of a protein side chain with small (molecular volume similar to
phenylalanine) nitroxide molecular tags and analyzing EPR signals for effects of rotational dynamics, polarity, or
dipolar interactions that are determined by distances to other nitroxides. Here we employ SDSL EPR to study
membrane insertion and helix-to-helix interactions of selected transmembrane helices (TMHs) of CesA in order to
verify CesA computational and homology models. A series TMH 4 and 5 with single point Cys mutations and no
connecting loop were prepared using solid-state peptide synthesis, covalently modified with an EPR-active side
chains at selected positions of the peptide sequence, and inserted into bilayers prepared from long-chain (DOPC),
short chain (DLPC), or mixed DOPC/DLPC lipids. Formation of α-helices by these two TMHs was verified by
CD. Local polarity experienced by the nitroxide-labeled side chains was measured based on the exquisite
sensitivity of EPR parameters (i.e., g-factor and Aiso) to dielectric and hydrogen-bonding effects. Further, we
investigated effects of a membrane-spanning α-helical WALP23 peptide on TMHs’ membrane insertion and
helix-to-helix interactions detected directly from EPR spectra. Polarity and depth parameter Φ profiles for bilayers
of the same lipid compositions were separately calibrated using WALP23. One of the conclusions of this work is
that the specific lipid composition of the bilayers and the presence of other membrane-spanning helices appear to
be an important requirement for proper insertion of individual CesA TMHs in lipid bilayers.
Supported as a part of the Center for LignoCellulose Structure and Formation under DOE Award DE-SC0001090.
P5-25
Genome-wide studies of multigene families involved in Eucalyptus phenylpropanoid metabolism
and lignin branch pathway
Carocha V. (acd), Soler M. (a), Hefer C. (b), Cassan-Wang H. (a), Myburg A. (b), Fevereiro P. (cf), Paiva J.A.P. (de), GrimaPettenati J. (a)
(a) LRSV, Univ. Toulouse III, UPS, CNRS, BP 42617, Auzeville, 31326 Castanet Tolosan, France; (b) BCBU, Dep. of
Genetics, FABI, Univ. Pretoria, Pretoria, 0002, South Africa; (c) ITQB, Av. República, Quinta do Marquês, 2780-157
Oeiras, Portugal; (d) IICT/MNE; Palácio Burnay - Rua da Junqueira, 30, 1349-007 Lisboa, Portugal; (e) IBET, Av.
República, Quinta do Marquês, 2781-901 Oeiras, Portugal; (f) DBV, FCUL, Campo Grande, 1749-016 Lisboa, Portugal.
The biosynthesis of lignin monomers comprises ten enzymatic reactions catalyzed by enzymes encoded by
members of multigene families. Lignin, one of the main components of plant secondary cell walls, is a major
obstacle for improving industrial processes from several segments of the wood industry such as pulp and paper
production. Eucalyptus grandis and E. globulus are major fiber sources for those industries and therefore have a
substantial economic importance. The recent availability of the genome of Eucalyptus grandis provides research
opportunities to understand the genomic architecture of those multigene families.
We performed a genome-wide survey of the ten E. grandis multigene families, and conducted comparative
phylogenetic analyses by including bona fide lignin-biosynthetic genes from other species. We surveyed the
expression profiles of all families’ members through RNAseq in six E grandis tissues and, for a subset of putative
bona fide lignin-biosynthetic genes, by RT-qPCR in a large panel of 16 E. globulus tissues.
All total, we identified 174 genes scattered all over the eleven E. grandis chromosomes. The four largest families
(COMT, CAD, CCoAOMT and PAL) revealed to be extensively impacted by tandem gene duplication, which
explains their significant expansion in comparison to other plant species. The combination of phylogeny and
expression profiling analyses allowed us to define a Eucalyptus lignin toolbox featuring 29 members exhibiting
strong, preferential expression in highly lignified tissues. All ten multigene families are represented in the
toolbox.
P5-26
Both ChIP-SEQ and in planta gene modification indicate a function of PtaMYB221in lignin
biosynthesis and secondary cell wall formation in poplar
Lakhal W., Boizot N., Lesage-Descauses M.C., Leplé J.C., Laurans F., Lainé V., Millet N., Ferrigno P., Pilate G.,
Déjardin A.
INRA, UR0588, Amélioration, Génétique et Physiologie Forestières, F-45075 Orléans Cedex 2, France.
Poplar wood or secondary xylem is composed of vessels, fibers and parenchyma rays. Their secondary cell wall
(SCW) consists of a number of layers sequentially deposited during cell differentiation. The formation of the
SCW is controlled by a number of transcription factors (TF) that coordinate the expression of many genes
involved in the biosynthesis, assembly, and deposition of the different SCW components. We investigated the
function of one of them, PtaMYB221, a poplar ortholog of EgMYB1[1], known as a transcriptional repressor of
the lignin biosynthetic pathway in Eucalyptus. Toward this end, we developed two complementary approaches,
Chromatin Immuno-Precipitation followed by next-generation DNA sequencing (ChIP-SEQ) and in planta gene
modification using genetic engineering. Using ChIP-SEQ with an antibody specific to PtaMYB221, we
determined at the genome scale the set of PtaMYB221binding sites in differentiating secondary xylem of poplar
bent trees: 488 putative targets of PtaMYB221 were identified including 15 genes potentially involved in lignin
biosynthesis, 3 genes related to pectin degradation, 1 gene with a possible role in xylan biosynthesis, 1 gene
involved in cellulose biosynthesis, 1 gene involved in xyloglucan biosynthesis and numerous TFs related to SCW
development. Over-expression of native PtaMYB221 induced in transgenic poplars important sylleptic branching
but had no effect on tension wood formation. On the reverse, dominant repression of PtaMYB221 resulted in
poorly lignified fibers and parenchyma rays. We conclude that PtaMYB221 is important for lignin biosynthesis
regulation during wood differentiation.
[1] S. Legay et al. (2007) Plant Science, 173(5), 542-549.
This work has been funded by different projects from Région Centre, INRA AIP Bioressources REGEX and Plant-KBBE TreeForJoules.
P5-27
Isolation of Golgi proteomes from grasses
González Fernández-Niño S. (a), Vega-Sanchez M. (a), Carpita N.C. (c), Lao J. (a), Smith-Moritz A.M. (a), Petzold C. (a),
Bacic A. (d), Ronald P.C. (b), Heazlewood J.L. (a)
(a) Joint BioEnergy Institute and Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, CA
94720, USA; (b) Department of Plant Pathology, University of California, Davis, CA 95616, USA; (c) Department of Botany
& Plant Pathology and Bindley Bioscience Center, Purdue University, IN 47907-2054, USA; (d) ARC Centre of Excellence
in Plant Cell walls, School of Botany, University of Melbourne, Melbourne VIC 3010, Australia.
Many aspects of grass cell walls are distinct from those of the better-studied dicots. In order to advance our
understanding of grass cell wall biosynthesis and protein modification, we are focusing our efforts in genetically
tractable model species such as rice (Oryza sativa) and maize (Zea mays). The development of methodologies to
better elucidate the protein composition of organelles actively involved in the synthesis of matrix components of
the cell wall (e.g. Golgi apparatus), is an important issue; especially for species that have a potential role in the
biofuel industry. Using Free-Flow Electrophoresis (FFE) combined with mass spectrometry (MS) in Arabidopsis
thaliana we achieved the successful purification of Golgi cisterns, obtaining a population of proteins that represent
the Golgi proteome of this model dicot. However, to date there is little information of this nature in any type of
grass. Both rice and maize are food crops and represent model monocots, and are thus good candidates for
proteomic analyses. Findings in these species can be extrapolated to other grass species of importance to biofuels
research, such as switchgrass. We have been able to separate and analyze proteins from the Golgi apparatus of
both rice and maize, confirming its purification by quantitative proteomic assays and immunoblotting.
P5-28
MYB46-mediated transcriptional regulation of secondary wall biosynthesis in plants
Kim W.-C. (ac), Kim J.-Y. (a), Ko J.-H. (b), Han K.-H. (acd)
(a) Department of Horticulture, (c) DOE-Great Lakes Bioenergy Research Center; Michigan State University, East Lansing,
MI 48824; (b) Department of Plant and Environmental New Resources, Kyung Hee University, Yongin-si, Korea; (d)
Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, Korea.
The secondary cell wall is a defining feature of xylem cells and allows them to resist both gravitational forces and
the forces of the tension associated with the transpirational pull on their internal columns of water. Also,
secondary walls constitute the majority of plant biomass. Formation of secondary wall requires coordinated
transcriptional regulation of the genes involved in the biosynthesis of cellulose, hemicellulose, and lignin [1-2].
Transcription factor MYB46 and its paralog, MYB83, have been shown to function as a master switch for the
secondary wall biosynthetic program in Arabidopsis thaliana [3-4]. Overexpression of MYB46/MYB83 results in
ectopic deposition of secondary walls in the cells that are normally parenchymatous, while suppression of its
function reduces secondary wall thickening [3-6]. Recently, we have discovered that MYB46 directly regulates
the expression of all of the three cellulose synthases (CESA4, CESA7 and CESA8) in Arabidopsis plants [7]. We
then demonstrated that MYB46 is an obligate component of the transcriptional regulatory complex for functional
expression of the three secondary wall-associated CESAs. Furthermore, we obtained experimental evidence that
MYB46 also directly regulates key genes in hemicellulose and lignin biosynthesis. Based these observations, we
hypothesize that MYB46 activates its target genes in multiple layers of the transcriptional network, which results
in coordinated regulation of the biosynthesis of secondary walls. We are testing this hypothesis by identifying and
functionally characterizing the components of the MYB46-mediated transcriptional regulatory network.
Understanding of the regulatory program may lead to better strategies to biotechnologically control and uncouple
the biosynthetic pathways for the three major components and, hence, pathway-specific engineering of biomass
quality and quantity.
[1] T. Demura and Z. Ye (2010) Curr. Opin. Plant Biol., 13, 299-304; [2] H.-Z. Wang and R. Dixon (2012) Mol. Plant, 5,
297-303 ; [3] R. Zhong et al. (2007) Plant Cell, 19, 2776-2792; [4] J. Ko et al. (2009) Plant J., 60, 649-665; [5] R.L.
McCarthy et al. (2009) Plant Cell Physiol., 50, 1950-1964; [6] J. Ko et al. (2012) Mol. Plant, 5, 961-963;[7] W. Kim et al.
(2013) Plant J., 73, 26-36.
P5-29
Cell Wall Glycan Dynamics During Primary to Secondary Wall Development in Poplar Stems
Pattathil S. (ab), Avci U. (ab), Cooke R. (b), Hahn M.G. (ab)
(a) Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Rd., Athens, GA 30602, USA; (b)
BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
Understanding plant cell wall structure and biosynthesis is an important aspect of ligno-cellulosic bioenergy
research. Cell walls are dynamic in their composition and structure, varying considerably amongst different
organs, cells, and developmental stages. Poplar, a source of woody biomass that is rich in secondary walls, is one
of the main target feedstocks for sustainable biofuel production. Here, we employ a comprehensive suite of cell
wall glycan-directed monoclonal antibodies to examine variations in the cell wall glycomes of 10-week-old poplar
stem internodes. We employed Glycome Profiling and in situ immunolocalization to comprehensively monitor the
overall presence, extractability, and distributions among cell types of most major cell wall glycan-epitopes in the
first 25 stem internodes transitioning from a young to a mature developmental stage. The overall extractability and
cellular distribution of diverse glycan-epitopes varied across all internodes (INs). From IN-1 through IN-25,
Xyloglucan (XG) epitope extractability in 1M KOH extracts increased, but subsets of XG epitopes exhibited a
variable extractability pattern in oxalate extracts. XG was observed to interact minimally with lignin throughout
internode development. Xylan-lignin interactions were observed from the IN3 through IN25 stages. Early
internodes showed higher abundance of pectic epitopes. Pectic-arabinogalactan epitopes interacted with lignin at a
higher degree in developmental stages IN1 through IN11. Such analyses are instrumental in comparing cell wall
variations during organ development of native and genetically modified feedstocks for bioenergy production.
Acknowledgements: The glycome profiling was supported by the BioEnergy Science Center administered by Oak Ridge national
Laboratory and funded by a grant (DE-AC05-00OR22725) from the Office of Biological and Environmental Research, Office of Science,
United States, Department of Energy. The generation of the CCRC series of plant cell wall glycan-directed monoclonal antibodies used in
this work was supported by the NSF Plant Genome Program (DBI-0421683 and IOS-0923992).
P5-30
Transcriptomic identification of arabinogalactan protein polysaccharide biosynthetic enzymes
Stonebloom S. (ab), Ebert B. (b), Barry K. (c), Scheller H.V. (b)
(a) University of Copenhagen, Denmark; (b) Lawrence Berkeley National Laboratory, California, USA; (c) Department of
Energy Joint Genome Institute, California, USA.
Arabinogalactan proteins (AGPs) are a group of plant cell wall glycoproteins consisting of heavily arabinogalactosylated hydroxyproline-rich peptides, which are often anchored to the plasma membrane with GPIanchors. The arabinogalactan polysaccharides attached to AGPs are structurally complex and diverse. AGPs have
been implicated in numerous biological functions in plant growth and development, most recently as a possible
periplasmic reservoir for Ca2+ ions. Functional studies of AGPs have been hindered by a lack of understanding of
AGP glycan biosynthesis. Few enzymes involved in the production of AGP glycans have been identified to date.
To identify AGP glycan biosynthetic enzymes we performed a transcriptomic study of AGP gum secretion in
Meryta sinclairii, a plant native to New Zealand. Like Gum Acacia, M. sinclairii secretes an arabingalactan gum
following wounding1. Through this transcriptome analysis we identified 34 up-regulated glycosyltransferase-like
genes as good candidates for genes possessing AGP glycan biosynthetic activities. The role of candidate AGP
glycan biosynthetic genes was tested by transiently silencing the Nicotiana benthamiana ortholog of each gene
using Virus Induced Gene Silencing and analyzing AGP glycan structure in silenced tissues.
[1] Sims and Furneaux.(2003) Carbohyd Polym. 52, 423-431.
P5-31
A 3-D model of the grass primary cell wall and its enzymatic degradation
Vetharaniam I. (a), Kelly W. (a), Attwood G. (a), Harris P. (b)
(a) AgResearch Limited, New Zealand; (b) School of Biological Sciences, The University of Auckland, New Zealand.
Understanding the physical disintegration of plant material and the underlying enzymatic processes are key to
improving cell wall (lignocellulose) digestibility. The interactions involved have a complex interdependence that
cannot be adequately understood by experiment alone, but rather require a quantitative and integrative approach to
link experimental data. Computer simulation provides such a tool, and as a proof of principle we have developed a
novel 3-D, agent-based model of the non-lignified primary grass cell wall and simulated its digestion by enzymes.
The model represents cellulose, hemicelluloses (arabinoxylans, 1,3;1,4-β-glucans, and xyloglucans) and pectin at
the level of their constituent monosaccharides, and can contain arbitrary combinations of different enzymes
(varying in number, size and catalytic activity). It accounts for steric effects (such as hindrance to diffusing
enzymes and accessibility of target bonds) and predicts breakdown products resulting from enzyme activity.
The model can be parameterised to represent walls in different cell types and taxa, and can include a variety of
enzymes. Thus it can be applied to a range of systems where cell walls are degraded and to cell-wall modification
by endogenous enzymes. Additionally it can be manipulated to explore other types of plant cell walls and
hypotheses of how their component polymers are linked, offering opportunities in plant physiology and, in
particular, the study of the conformation of cell-wall polymers.
P5-32
Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis
Using a Combination of Bioinformatics Approaches
Voxeur A. (a), André A. (a), Breton C. (b), Lerouge P. (a)
(a) Laboratoire Glyco-MEV, EA 4358, IRIB, University of Rouen, Mont-Saint-Aignan, France ; (b) CERMAV-CNRS,
University of Grenoble 1, Grenoble, France.
Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked
homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that
is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the
plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation [1]. In this
study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved
in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane
proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding
enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study
results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the
CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of αgalacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both
homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the
transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were
retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B
like fold and a fucosyltransferase signature. In order to validate this approach, the involvement of some of these
candidates in RGII biosynthesis is currently investigated.
[1] M.A. O'Neill et al. (2004) Annu. Rev. Plant Biol., 55, 109-139.
P5-33
Elucidation of a protein interaction network in homogalacturonan biosynthesis
Sakuragi Y. (a), Have Lund C. (a), Stenbæk A. (a), Atmodjo M.A. (b), Bromley J.R. (a), Werweries M. (a), Mohnen D. (b)
(a) Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg 1871, Denmark; (b)
Complex Carbohydrate Research Center, Department of Biochemistry and Molecular Biology, University of Georgia, 315
Riverbend Rd, GA 30602, USA.
Pectins are highly complex polysaccharides that have essential roles in plant growth and development. Growing
evidence supports the notion that protein-protein interactions (PPIs) occur widely among proteins involved plant
cell wall polysaccharide biosynthesis [1]. However, direct experimental evidence is still limited.
In order to gain further insights into PPIs in cell wall biosynthesis, we focused on GAUT1 as a model system.
GAUT1 is a homogalacturonan:galacturonosyltransferase involved in homogalacturonan biosynthesis and has
been shown to interact with GAUT7, its homolog [2]. Three complementary screening strategies were employed:
i) protein co-expression analysis among GAUT1 homologs; ii) Renilla luciferase complementation assay among
candidate interacting proteins identified by transcriptional co-expression analysis; iii) cDNA library screening
using a split ubiquitin system. The results show that, in addition to GAUT7, GAUT5 and GAUT6 interact with
GAUT1 and act as molecular anchors in the Golgi apparatus. Furthermore, proteins involved in vesicle transport,
cell wall biosynthesis, and protein degradation were identified to interact with GAUT1. These results suggest that
a protein interaction network involving GAUT1 exists and controls homogalacturonan biosynthesis and secretion.
[1] Oikawa A et al (2013) Trends Plant Sci 18:49; [2] Atmodjo MA et al (2011) Proc Natl Acad Sci USA 108 :20225.
P5-34
The Eucalyptus grandis R2R3-MYB transcription factor family
Soler M. (a), Camargo E. (a), Carocha V. (abc), Wang H. (a), San Clemente H. (a), Savelli B. (a), Hefer C. (d), Myburg A.
(ef), Paiva J.A.P. (bc), Grima-Pettenati J. (a)
(a) LRSV Laboratoire de Recherche en Sciences Végétales, UMR5546, Université Toulouse III /CNRS, BP 42617, Auzeville,
31326 Castanet Tolosan, France; (b) Instituto de Investigação Científica e Tropical (IICT/MCTES) Palácio Burnay - Rua da
Junqueira, 30, 1349-007 Lisboa; (c) Instituto de Biologia Experimental e Tecnológica (IBET) Av. da República, Quinta do
Marquês, 2781-901 Oeiras, Portugal; (d) Bioinformatics and Computational Biology Unit, (e) Department of Genetics, (f)
Forestry and Agricultural Biotechnology Institute (FABI); University of Pretoria, Private Bag X20, Pretoria, 0028, South
Africa.
Eucalyptus species grow very fast and produce high yields of biomass, representing the main wood industrial
plantations in the world. To improve wood properties related to bioethanol production, we are focusing our efforts
towards the identification of genes regulating the biosynthesis of secondary cell wall (SW) polymers in
Eucalyptus. The R2R3-MYB superfamily contains many members known to regulate the phenylpropanoid
metabolism, including SW formation and lignin biosynthesis. In Eucalyptus we have already characterized two
R2R3-MYB factors (EgMYB2 and EgMYB1) acting respectively as activator and repressor of SW formation [1,
2]. Here, taking profit of the new release of the E. grandis genome (www.phytozome.net), we performed a
genome-wide survey of the R2R3-MYB superfamily. The phylogenetic comparison of this family with
Arabidopsis, rice, poplar and grapevine showed that three subgroups are expanded in woody species (Eucalyptus,
poplar and grapevine), whereas five subgroups are completely absent in the herbaceous plants Arabidopsis and
rice. Tandem duplications seem to disproportionately affect these subgroups, suggesting diversification of specific
functions in woody plants. In contrast, subgroups containing key genes regulating lignin biosynthesis and
secondary cell wall formation are more conserved across all the species analyzed and generally not affected by
tandem duplication events. RNAseq and microfluidic qPCR analyses in Eucalyptus revealed preferential transcript
abundance in xylem for EgMYB1, EgMYB2 and orthologs of Arabidopsis genes regulating lignin biosynthesis
and SW formation. Moreover, transcript abundance is higher in the cambium-enriched fraction of many genes
belonging to woody expanded subgroups and to subgroups absent in Arabidopsis and rice. Transgenic plants over
expressing some of these genes are currently being analyzed to better understand their roles.
[1] M. Goicoechea et al. (2005) Plant J., 43, 553–567; [2] S. Legay et al. (2010) New Phytol., 188, 774–786.
P5-35
Cell wall diversity in forage maize: biochemical properties and genetic complexity
Torres A.F., van der Weijde T., Deschesne A., Dolstra O., Visser R.G.F., Trindade L.M.
Wageningen UR Plant Breeding, Wageningen University and Research Centre P.O. Box 386, 6700 AJ Wageningen, The
Netherlands.
Genetic studies are ideal platforms for assessing the extent of genetic diversity and genomic complexity
controlling cell wall biochemical properties in lignocellulosic crops. They also represent an attractive tool for
studying the complex relationship between cell wall composition and biomass processing quality, i.e.
saccharification efficiency for cellulosic ethanol production. Through the exhaustive characterization of a forage
maize doubled haploid (DH) population, we reveal the “highly” heritable variation in cell wall compositional
content, polymeric ultrastructure and industrial quality potentially available within this model grass species. We
also dissect the genetic mechanisms controlling cell wall biochemical diversity and report on the finding of
multiple chromosomal regions, named quantitative trait loci (QTL’s), associating diverse cell wall characteristics
with cell wall digestibility and saccharification properties. The results show that in addition to lignin content and
composition the degree of hemicellulose substitution plays an important role in cell wall saccharification
efficiency. Overall, our results confirm that different cell wall compositional profiles, combining the synergistic
effects of variation in multiple cell wall characters, can lead to phenotypes with improved bio-processing
amenability. The large extent of malleable cell wall diversity found in forage maize, and presumably present in
other relevant bioenergy species, also validates classical breeding strategies as means towards the optimization of
lignocellulosic biomass for the production of bioenergy and other bio-commodities.
P5-36
Transcriptomic dynamics during tension wood differentiation in Eucalyptus globulus
Paiva J.A.P. (ab), Carocha V. (ab), Oliveira J. (c), Graça C. (ab), Pera S. (ab), Ladouce N. (d), Marsaud N. (e), Bouchez O.
(e), San-Clemente H. (d), Bessa F. (a), Quilhó T. (a), Araújo C. (f), Leal L. (f), Freitas A.T. (c), Rodrigues J.C. (a), GrimaPettenati J. (d)
(a) IICT/MCTES Palácio Burnay - Rua da Junqueira, 30, 1349-007 Lisboa, Portugal; (b) IBET Av. da República, Quinta do
Marquês, 2781-901 Oeiras, Portugal; (c) INESC-ID/IST, R. Alves Redol 9, 1000 Lisboa, Portugal; (d) LRSV, Université
Toulouse III, UPS, CNRS, BP 42617, Auzeville, 31326 Castanet-Tolosan, France; (e) Plateforme Génomique, Génopole
Toulouse/Midi-pyrénées, INRA Auzeville, Chemin de Borderouge-BP 52627, 31326 Castanet-Tolosan, France; (f) ALTRI
FLORESTAL SA, Quinta do Furadouro, 2250-582 Olho Marinho, Portugal.
Wood complex anatomical, chemical and physical properties are determined upon composite ontogenetic
processes [1]. In addition to the highly organized constitutive expression, several layers of gene expression
regulation mechanisms occur in crosstalk mode upon environmental stimuli, creating a highly dynamic
transcriptome. One remarkable example is angiosperms trees’ response to gravitropic stimulus leading to the
formation of tension wood (TW) whose cell walls exhibit new and characteristic properties. TW is therefore an
excellent model to study the regulation underlying secondary cell wall formation and to infer how this regulation
impacts
the
final
properties
of
wood.
In
the
frame
of
the
projects
microEGo
(https://sites.google.com/site/microegopublic/; FCT PTDC/AGR-GLP/098179/2008) and P-KBBE Treeforjoules
(http://tfj.lrsv.ups-tlse.fr/; ANR-2010-KBBE-007-01 and FCT P-KBBE/AGR_GPL/0001/2010), the transcriptome
dynamics established 1 and 3 weeks after induction of TW in grown- field E. globulus trees was accessed by NGS
technology (Illumina Hi-Seq 2000 GA), and annotated by mapping the PE-reads (100bp) against the E. grandis
genome sequence (www.phytozome.org) using Top-hat. Tension and opposite differentiating xylem tissues were
collected from three E. globulus distinct genotypes, bent for 1 to 3/4 weeks, and chemically and anatomically
characterized. Among the 93 genes differentially expressed identified by Cufflinks, more than a half (53 genes)
were shown to be differentially expressed (P<0.05) between tension (3/4 weeks) and opposite wood (1 week or
3/4 weeks).
[1] J.A.P. Paiva et al. (2008) New Phytologist, 178, 283-301.
P5-37
Mesoporosity in developing wood cell walls: a new step towards understanding of maturation
stress generation in trees
Chang S.-S. (a), Quignard F. (b), Clair B. (ac)
(a) Laboratoire de Mécanique et Génie Civil (LMGC), CNRS, Université Montpellier 2, cc 048, Place E. Bataillon, 34095
Montpellier, France ; (b) Institut Charles Gerhardt Montpellier, UMR 5253 CNRS-UM2-ENSCM-UM1, 8 rue de l’Ecole
Normale, 34296 Montpellier cedex 5, France ; (c) CNRS, UMR Ecologie des Forêts de Guyane (EcoFoG), Campus
Agronomique, BP 701, 97387 Kourou, French Guyana.
Trees are able to maintain their verticality or definite angles thanks to their ability to generate asymmetrical
tensile stress in the newly formed wood layers. The mechanism of stress generation during maturation is still not
fully understood but matrix is suspected to play a major role. To progress in the understanding of active stress
generation in tension wood, the mesoporosity (pore size from 2 to 50 nm) of the cell wall and its evolution during
maturation of poplar tension wood and normal wood were measured by nitrogen adsorption-desorption isotherm.
Variations in the thickness of the gelatinous layer (G-layer) were also measured to clarify whether the
mesoporosity change simultaneously with the deposition of the G-layer in tension wood. Results show a high
mesoporous texture near the cambial zone both in tension wood and normal wood. During maturation,
mesoporosity decreases with the lignification process up to nearly disappear in normal wood whereas a new
mesoporous system appears in tension wood with the building of the G-layer. Pores in G-layers have different
pore shape compare to cambial zone and their size increase during maturation process. This increase in pore size
could be due to the G-layer matrix swelling allowing to propose new hypothesis on the mechanism of stress
generation in trees.
Session 6 : Uses of plant CW and derived products
Food, feed, chemicals & fuel, Renewable biomedical & smart materials
P6-01
Structure of Pectin in Citrus Fruits and Functionality
Kaya M. (a), Sousa A.G. (b), Crépeau M.-J. (a), Sørensen S.O. (b), Ralet M.-C. (a)
(a) INRA, UR1268 Biopolymères Interactions Assemblages, 44300 Nantes, France ; (b) CP Kelco ApS., Ved Banen 16, DK4623 Lille Skensved, Denmark.
The structure of pectin is based on a backbone of polygalacturonic acid, which is called homogalacturonan regionHG (1). Besides, segments consisting of alternating sequences of L-rhamnosyl and D-galacturonosyl residues,
ramified with side chains of arabinans, arabinogalactans and galactans, are called Rhamnogalacturonan I - RGI
(2). Commercial pectin is mainly derived from citrus peels. Pectin exhibit functional properties like gelling,
thickening, and emulsifying that are widely used in food industry. The project aims to find out the variation of
pectin components depending on plant sources and processing in relation with functional properties.
In this study, dried citrus fruit peels (orange, grapefruit, lime and lemon) were used as raw material for pectin
extraction. The peels were subjected to HCl, HNO 3 and H2C2O4 extraction conditions. The pHs were 1.5, 2.1, and
4.6 respectively. Pectins were obtained on laboratory scale (HCl and H 2C2O4) and industrial scale (HNO3).
The extraction conditions affected intrinsic viscosity and molecular weight of pectins significantly. In regard to
monosaccharide composition, GalA/ rhamnose ratio (mol %) of pectins extracted by oxalic acid was higher than
the others. Additionally, the degree of branching (Ara + Gal/ Rha mol %) was higher in pectins extracted by
hydrochloric acid and oxalic acid. Alterations in the composition and structure of the pectins due to the conditions
of extraction will be reported.
[1] Thibault, J.-F., Renard, C. M. G. C., Axelos, M. A. V., Roger, P., & Crepeau, M. J. (1993). Studies of the length of
homogalacturonic regions in pectins by acid- hydrolysis. Carbohydrate Research, 238, 271–286; [2] Albersheim, P., Darvill,
A. G., O’Neill, M. A., Schols, H. A., Voragen, A. G. J. (1996). In J. Visser & A. G. J. Voragen (Eds.), Pectins and Pectinases
(pp 47–56).
P6-02
A Lignocellulosic analysis of Setaria viridis L. a developing model for the panoicoideae clade C4
grasses
Petti C., Tateno M., Debolt S.
Plant Physiology, Department of Horticulture, Agricultural Science Center North, University of Kentucky, Lexington, KY,
USA.
Currently, the feedstock for biofuel production is shifting from corn-based material to low-grade lignocellulosic
biomass from perennial C4 grasses, principally in the Panicoideae clade [1]. The Panicoideae comprises of many
agriculturally important grasses includes Zea mays L. (maize), Panicum virgatum L. (switchgrass), Sorghum
bicolor (L.) Moench (Sorghum), Miscanthus x Giganteus and Saccharum officinarum L. (sugar cane), all species
that are characterized by large and complex genomes [2]. A model organism for the Panidoideae clade that
displays manageable complexity is needed. Setaria viridis L. has many suitable traits as a model for the
panoicoideae clade [3]. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor (CBI)
response and saccharification dynamics of S. viridis are compared with annual and perennial bioenergy crops as a
baseline study into the applicability for translational research. In general, a lower CBI potency was observed for
isoxaben and morlin, but not for DCB. The potential for forward genetics is explored to generate and characterize
mutants to aid in translational studies.
[1] C. Byrt et al. (2011) J. Integrat. Plant Biol., 53, 120-135; [2] M. Devos et al. (1997) Plant Mol. Biol., 35, 3-15; [3] T.
Brutnell et al. (2010) Plant Cell, 22, 2537-2544.
P6-03
Possible implications of RGI side chains on HM pectin gelling capabilities
Sousa A.G. (ab), Sørensen S.O. (a)
(a) R&D Department, CP Kelco, Ved Banen 16, DK-4623 Lille Skensved. Denmark; (b) Department of Plant and
Environmental Sciences, 'Plantglycobiology', University of Copenhagen, Thorvaldsensvej 40, 1. floor, DK-1871
Frederiksberg C Denmark.
Citrus pectins are complex polysaccharides composed of a α-(1,4)-linked D-galacturonic acid (GalA) region
designated homogalacturonan (HG) or smooth region. Other regions consist of alternating sequences of the
repeating diglycosyl units [→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→]1 that are partly substituted at O-4 and/or O-3
positions of α-L-Rhap residues. The substitutions are neutral sugars of different types, i.e. (1→5)-α-L-arabinans
and (1→4)-β-D-galactans. These domains constitute the rhamnogalacturonan-I (RGI) or hairy region 2.
Pectin has many applications in food science and nutrition as a gelling and stabilizing agent. It can be obtained
from a wide range of raw materials, using a variety of extraction procedures. A novel extraction process based on
oxalic acid has been tested at pilot plant scale and compared with the most common industrial process using nitric
acid.
Fine pectin structure analysis has revealed a different monosaccharide composition of RGI side chains. In fact,
arabinose content in oxalic acid extracted pectins was higher compared to the nitric acid extracted pectins. The
use of enzymes specific for either arabinan or galactan will allow the assessment of the implications on varying
neutral sugar composition in the gelling capabilities of pectins. Rheological and detailed HPSEC studies were
performed on branched and unbranched pectins while maintaining the same degree and pattern of
methylesterification, galacturonic acid content and intrinsic viscosity.
[1] W.G.T. Willats et al. (2006) Trends in Food Science & Technology., 17(3), 97-104; [2] B.M. Yapo et al. (2007)
Carbohydrate Polymers, 69(3), 426-435.
P6-04
Modifying the phenylpropanoid flux in C3’H-RNAi maize plants
Fornalé S. (a), Capellades M. (a), Rigau J. (a), García L. (b), Encina A. (b), Caparrós-Ruiz D. (a)
(a) Center for Research in Agricultural Genomics (CRAG) Consortium CSIC-IRTA-UAB-UB, Spain; (b) Facultad de
Ciencias Biológicas y ambientales, Universidad de León, Spain.
Lignin is probably the major determinant of cell walls recalcitrance to saccharification and its genetic reduction
would improve the yields of fermentable sugars for biofuel production or the digestibility of feedstocks. Among
the possible targets for downregulation, p-coumaroyl-CoA 3’-hydroxylase (C3’H) is a strategically placed lignin
gene that catalyzes the first reaction leading to the synthesis of the two major lignin subunits (G and S monomers)
that account for more than 90% of total lignin in angiosperms.
We produced three maize C3’H-RNAi plants having different degree of residual gene expression and their
macroscopic phenotype varies from wild-type to severely compromised. Thus, the most repressed lines showed
reduced plant growth, male sterility and a huge accumulation of anthocyanins, similarly to what already reported
in the case of the Arabidopsis ref8 null mutant [1] and the highly C3’H-repressed alfalfa [2] and poplar [3] plants.
In all the maize C3’H-RNAi plants, lignin content was reduced by about 20% and lignin monomeric composition
was changed. As a consequence of C3’H repression, higher levels of H subunits are synthesized mainly at
expenses of S monomers, leading to a reduction of the S/G ratio. These changes lead to an increase of cell wall
degradability despite a decrease of cellulose content. In addition, transgenic plants accumulate higher levels of
soluble antioxidants. Altogether these results highlight the viability of modulating the expression of C3’H to
obtain plants with improved digestibility or energetic values without compromising plant fitness or biomass.
[1] N. Abdulrazzak et al. (2005) Plant Phys., 140, 30-48 ; [2] M.S. Srinivasa et al. (2005) Proc. Natl. Acad. Sci. USA, 102,
16573-16578; [3] H.D. Coleman et al. (2008) Proc. Natl. Acad. Sci. USA, 105, 4501-4506.
P6-05
Innovative materials for the visual detection of xylanase
Moreau C., Beury N., Cathala B.
INRA, UR1268 Biopolymères, Interactions et Assemblages, 44300 Nantes, France.
Lignocellulosic materials are abundant and renewable resources that can be used for the production of fuel ethanol
and industrially-relevant chemicals. Among technologies envisioned to transform the raw material, enzymatic
bioconversion offers an environmentally-friendly pathway. However, the improvement of enzymatic biorefining
requires a constant effort for discovering new activities in order to improve biomass fractionation and
transformation. Recently, we demonstrated that multilayer thin films based on polysaccharides from plant cell
walls are suitable biomaterials for the detection of enzyme [1-3]. Indeed, by controlling the film thickness and
architecture at the nanoscale, a structural color appears due to interference phenomena. When the film is
submitted to enzyme that can hydrolyse biopolymer, the thickness of the film is decreased inducing a colour
change visible at naked eyes. Here, we present the elaboration of new enzymatic detector based on thin films
composed of wheat arabinoxylan. Films were built by spin-coating deposition of polymer layer followed by a
cross-linking process with melamine-formaldehyde resin. Deposition parameters were optimized to obtained
nanometric films that allow the appearance of structural colour. The cross-linking reaction was controlled by
tuning the resin/arabinoxylan ratio to afford stable films in aqueous solution but still sensitive to enzyme
degradation. This new device allows a simple, fast and easy to handle detection of the xylanase activities and will
be useful for high throughput screening assay for new enzyme source discovery.
[1] C. Cerclier et al. (2011) Adv. Mater., 23, 3791-3795 ; [2] A. Guyomard-Lack et al. (2012) Eur. Phys. J., Special Topics,
213, 291-294 ; [3] A. Dammak et al. (2013), Holzforschung, in press.
P6-06
spa1, a Brachypodium cell wall mutant with a high yield of sacharification suitable for biofuel
production
Timpano H. (ab), Sibout R. (ab), Devaux M.-F. (c), Looten R. (c), Alvarado C. (c), Martin M. (ab), Lapierre C. (ab), Badel E.
(d), Citerne S. (ab), Vernhettes S. (ab), Höfte H. (ab), Guillon F. (c), Gonneau M. (ab)
(a) INRA, UMR1318, IJPB, Saclay Plant Sciences, F-78000 Versailles, France; (b) AgroParisTech, IJPB, RD10, F-78000
Versailles, France; (c) INRA UR1268 Biopolymers, Interactions Assemblies, F-44316 Nantes, France; (d) INRA Site de
Crouël chemin de Beaulieu, 63039 Clermont-Ferrand cedex 2 – France.
Energy-rich lignocellulosic biomass of plant cell walls can be broken down to fermentable sugars to produce
bioethanol. However, the complex structure of plant cell walls makes them very resistant to degradation and
improving the ease and yield of cell wall saccharification represents a major technological challenge.
Brachypodium distachyon is an excellent gramineae model to identify genes important for energy grasses. We
identified in the INRA-Versailles Brachypodium collection, a line with a still undescribed impaired growth status
combining floppiness and brittleness. This line contains less crystalline cellulose but more lignin and an increase
in xylan content resulting in a reduced mechanical strength and a high friability of the straw. We used the
IMATOR enzymatic reactor to follow in real time the physical evolution of straw particle size during degradation
and the release of sugar. Sugar release was about four fold more than that measured with the WT line and this
increase came with a slight reduction of average particle size. The deconstruction yield of the cell wall
polysaccharides in this line suggests that usual step of chemical pretreatment could be avoided.
[1] M.W. Bevan, et al. (2010) Current opinion in biotechnology, 21, 211-7 ; [2] International Brachypodium Initiative (2012)
Nature, 463, 763-8; [3] M.F. Devaux, et al. (2006) Journal of Food Engineering, 77, 1096-1107.
P6-07
Screening of sugars and phenolics released during pretreatment of miscanthus, maize and sugar
cane bagasse for potential added value products from c4 crops
Gomez L.D. (a), Bird S. (a), Vanholme R. (b), Simister R. (a), Boerjan W. (b), McQueen-Mason S.J. (a)
(a) Department of Biology, University of York, York YO10 5DD, UK ; (b) Department of Plant Systems Biology, VIB,
Technologiepark 927, 9052, Gent, Belgium.
The viability of cellulosic fuels hangs on the reduction of the costs involved in the conversion processes. An
approach to reach a realistic cost for conversion of biomass into large volumes/low cost products is by identifying
added value products that can be obtained during the production of biomass derived fuels. This biorefinery
concept involves the benefits of reducing the cost of the overall process, replace petroleum derived materials and
chemicals, and reduce waste streams. Pretreatments have been considered for a long time as the key to take the
biochemical conversion of biomass to levels which are compatible with industrial applications. A great deal of
research has been put into the development of different pretreatments over the last 10 years and the literature is
rich in diverse and creative approaches to increase the efficiency of pretreatments in terms of increased fuel
production after fermentation. An interesting new approach to evaluate pretreatmens is the preservation of the
valuable chemicals in the pretreatment liquors in order to add value to the process from products obtained by
fractionation of the biomass. In the present work we characterise the sugars and lignin derivatives present in the
pretreatment liquor of maize, miscanthus and sugar cane bagasse under a range of acid and alkaline pretreatments.
This allows a detailed picture of the conditions necessary for favouring a certain type of compounds and also a
horizontal comparison of the compounds generated between species.
P6-08
Preparing nano-cellulose for high-performance composites
Ulvskov P. (a), Cassland P. (b), Jørgensen B. (a), Harholt J. (a), Fangel J. (a), Dinesen M. (a), Margueijo V. (c), Whale E. (d),
Willats W.G.T. (a), Shilton S. (c), Hofmann Larsen F. (e)
(a) Dep. Plant & Environmental Sciences, University of Copenhagen, Denmark; (b) Novozymes, Denmark; (c) Chemical &
Process Engineering, University of Strathclyde, UK; (d) Cellucomp, UK; (e) Dep. Food Science, University of Copenhagen,
Denmark.
Plant based composite materials can in principle replace, or compete effectively with, glass or carbon fiber based
composites. The strength of cellulose compares very favorably with that of carbon fibers, for example. The reason
why cell wall based high performance composites are still in their infancy is that much development effort has
gone into the use of intact fiber cells from flax, hemp, cotton etc. and only recently was it realized that the cell
wall should be reconstructed from first principles and the elementary building blocks if the full potential of
cellulose should be exploited.
We will present data on the use of enzymes for the release of nano-cellulose from primary walls. This has proven
to be non-trivial and the results challenge the prevailing tethering glycan cell wall model. Our results will be
discussed in the light of the contrasting view of Scheller and Ulvskov [1] who argued that load bearing structures
comprising direct contacts between cellulose micro-fibrils have received too little attention.
[1] Scheller H.V. and Ulvskov P. (2010) Ann. Rev. Plant Biology 61: 263-289.
P6-09
The effect of maize cell wall composition on the optimization of dilute-acid pretreatments and
enzymatic saccharification
Torres A.F., Van der Weijde T., Dolstra O., Visser R.G.F., Trindade L.M.
Wageningen UR Plant Breeding, Wageningen University and Research Centre P.O. Box 386, 6700 AJ Wageningen, The
Netherlands.
At the core of cellulosic ethanol research are innovations that increase the commercial and environmental
performance of the industry. Along developments in pretreatment, enzyme and fermentation technologies,
enhancing the yield and composition of biomass has the potential to improve the cost-efficiency of ethanol
production. In our study, stem samples from eight maize genotypes contrasting in cell wall composition were
subjected to a series of thermal dilute-acid pretreatments of increasing severity and evaluated for glucose yields
after enzymatic saccharification. The biochemically diverse genotype-set displayed significant differences in
fermentable sugar yields at all processing conditions evaluated. We reveal, nevertheless, that the mechanisms
controlling biomass conversion efficiency vary in relation to the processing conditions employed. At highlysevere pretreatments, biomass conversion efficiency was primarily influenced by the inherent efficacy of the
thermochemical process, and maximum glucose yields were obtained from cellulosic feedstocks harboring the
highest cellulose contents per dry gram of biomass. When applying mild dilute-acid pretreatments, however,
maximum glucose yields were observed for genotypes combining high stem cellulose content, reduced cell wall
lignin and highly substituted hemicelluloses. These results ultimately suggest that cellulosic feedstocks combining
the synergistic effects of variation in multiple cell wall characters, can influence the selection of processing
conditions towards more sustainable and cost-efficient alternatives. We also re-enforce the notion that the
development of superior lignocellulosic feedstocks requires an in-depth knowledge of the bioprocessing
technologies used in the industry, as well as their effect on the chemical and physical integrity of plant cell walls.
P6-10
Plant cell wall bioinspired foam derived from cellulose nanocrystals stabilized Pickering emulsions
Winter H.T. (a), Bizot H. (b), Capron I. (b), Cathala B. (b)
(a) University of Freiburg, Chair of Forest Biomaterials, Werthmannstraße 6, 79085 Freiburg, Germany; (b) INRA, UR1268
Biopolymères, Interactions et Assemblages, 44300 Nantes, France.
Complex porous materials have attracted high research attention in a broad range of applications including
chemical, environmental/energetical, optical, electronical, medical, and biotechnological applications for their
ability to obtain architectures with homogeneous and defined pore sizes. Plant cell wall can be of great inspiration
for such organized materials. We recently demonstrated the ability of cellulose nanocrystals (CN) to stabilize
oil/water interface to form highly stable Pickering emulsions [1,2]. These emulsions were used to template
homogeneous cellular solid foams by removal of water and adequately selected oil through freeze-drying [3]. We
report here the formation of bioinspired composite foam containing CN, pectin and synthetic lignin. The foam
was obtained by a sequential pathway similar to those previously reported for the synthesis of bacterial
cellulose/pectin/synthetic lignin composite [4]. Firstly, the CN were used to stabilize oil in water emulsions.
Thereafter, the emulsion was creamed by centrifugation and the droplets were dispersed in a pectin solution
containing peroxidase. Addition of calcium induced the formation of pectin gel embedding the oil droplets. Then
coniferyl alcohol was added to the matrix in order to diffuse inside the structure and polymerize. Finally, oil and
water were removed by freeze drying to obtain a dry composite foam. All the steps were followed by scanning
electron microscopy and Fourier transform infrared spectroscopy.
[1] I. Kalashnikova et al. (2011) Langmuir, 27, 12, 7471-7479 ; [2] I. Kalashnikova et al. (2012) Biomacromolecules, 13, 1,
267-275 ; [3] I. Capron et al. (2013) submitted ; [4] J.P Touzel et al. (2003) J. Agri. Food. Chem, 51, 981-986.
P6-11
Role of the cell wall in maintaining tissue integrity in canned navy beans
Chu J., Orfila C.
School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK.
Extensive heat processing is required to ensure the palatability and digestibility of legume seeds, including beans.
Navy beans (Phaseolus vulgaris) canned in tomato sauce are a popular food in the UK diet. The canning process
(heat at 126oC for 21 minutes) has a dramatic softening effect on the bean (99.7% decrease in toughness (Nm-2)
compared to the blanched bean, as measured by a mechanical compression test). However, the tissue holds its
integrity and the cotyledon cells remain adhered to each other. We have observed the changes to the cell wall that
occur during canning by immunofluorescence microscopy and have investigated pectin solubility from the bean
into the surrounding medium (tomato sauce). The microscopy results indicated that cotyledon cells walls swell up
slightly following canning. Non-methyl esterified homogalacturonan (labelled by JIM5) was still abundant in both
the primary cell wall and the middle lamella, while methyl esterified homogalacturonan (labelled by JIM7)
appeared to have been largely lost during the canning process. In addition, there was little loss of the JIM5 epitope
into the surrounding sauce as measured by ELISA. Therefore, it appears that canning has a dramatic effect the
mechanical properties of the cotyledon tissue but does not appear to affect the solubility or functionality of acidic
homogalacturonan, which is likely to be involved in maintaining cell adhesion after canning. Further work is
under way to characterise bean pectin in more details in relation to composition, molecular size and its interaction
with other cell wall components.
P6-12
Modification of dicot secondary cell wall formation by monocot NST transcription factor
Sakamoto S. (a), Yoshida K. (b), Mitsuda N. (a), Ohme-Takagi M. (ac)
(a) Bioproduction Reseach Institute, National Institute of Advanced Industrial Science and Technology (AIST),Japan; (b)
Technology Center, Taisei Co. Ltd., Japan; (c) Institute of Environmental Science and Technology (IEST), Saitama
University, Japan.
Secondary cell wall accumulates into wood which has a large potential for bioenegy production. We found before
that NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) transcription factors are master
regulators of secondary wall formation in sclerenchyma cells except for vascular vessel in Arabidopsis thaliana
[1][2]. The nst1 nst3 double mutant showed loss of secondary walls there and the phenotype of it was rescued by
Oryza sativa NST (OsNST) orthologue as well as Arabidopsis thaliana NST3 (AtNST) itself when they were
driven by AtNST promoter. The stem strength of OsNST-complemented plant was higher than that of wild type
and AtNST-complemented plants. As reflecting this observation, lignin-like substance was ectopically
accumulated in stem pith of OsNST-complemented plant. In addition, xylose and lignin contents in OsNSTcomplemented plant were higher than those of wild type and AtNST-complemented plant, while galactose,
arabinose, and rhamnose contents were lower. The transcriptional activation ability of OsNST for reporter gene
driven by NST binding sites was higher than that of AtNST, while that of OsNST fused with Gal4 DNA binding
domain for reporter gene driven by Gal4 binding sites was similar to that of AtNST. These results suggest that
higher preference of OsNST to NST binding site resulted in enhanced production of secondary cell wall in plant.
Furthermore, glucose productivity by the enzymatic saccharification from OsNST-complemented plant was
higher than that from wild type plant, indicating that the genetic modification with the OsNST driven by AtNST
promoter is one of the breakthrough technologies for the enhancement of woody material from plant resource.
[1] N. Mitsuda et al. (2005) Plant Cell 17, 2993-3006 ; [2] N. Mitsuda et al. (2007) Plant Cell 19, 270-280.
P6-13
The effect of carbohydrate-binding modules (CBMs) on plant cell wall properties: an in vivo
approach
Leijon F. (a), Mélida H. (a), Larsson T. (b), Berthold F. (b), Melzer M. (c), Guerriero G. (ad), Gomez L. (e), Sundqvist G. (a),
Bolam D. (f), McQueen-Mason S. (e), Bulone V. (a)
(a) Division of Glycoscience, Royal Institute of Technology (KTH), Stockholm, Sweden;(b) INNVENTIA AB, Stockholm,
Sweden; (c) Department of Structural Cell Biology, IPK Gatersleben, Germany; (d) Present address: Centre de Recherche
Public Gabriel Lippmann, Luxemburg;(e) Department of Biology, University of York, York, United Kingdom; (f) Institute for
Cell and Molecular Biosciences, Newcastle University, Newcastle, United Kingdom.
Carbohydrate-binding modules (CBMs) are functional domains in carbohydrate-active enzymes that promote the
association of the enzymes with their substrates. These domains have received considerable attention in light of
the pivotal role that they play in the hydrolysis of polysaccharides and their potential to manipulate cell wall
structures. An interesting approach to enhance the effect of current pretreatments for biofuel production from
plant biomass is the exploitation of the interactions between pure CBMs and cell wall carbohydrates to reduce the
recalcitrance of the cell wall to degradability. Our work aims at overexpressing CBMs in planta in order to
elucidate their effect on cell wall structure and degradability. Selected CBMs, representing families 3, 17 and 28,
were expressed in tobacco BY2 cell suspension cultures using Agrobacterium-mediated transformation.
Microscopic observations of the CBM-transformed cells showed striking changes in cell morphology. Moreover,
observations of the cell wall ultrastructure by transmission electron microscopy revealed unique features in each
of the CBM-overexpressing lines. Saccharification tests showed an increase in the cell wall degradability of the
CBM3 transformants while the rest of the lines were unaffected. In order to understand the basis of the cell wall
and saccharification alteration via CBMs we have performed a systematic cell wall analysis using a series of tools
such as mass spectrometry coupled to gas chromatography, X-ray diffraction and NMR spectroscopy.
P6-14
A new substrate for measurement of cellulase (endo-1,4-beta-glucanase)
McCleary B., Mangan D., Draga A., Ivory R.
Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.
A new substrate and assay procedure for the measurement of cellulase (endo-1,4-β-glucanase) is described. This
procedure involves the use of benzylidene blocked, 2-chloro-4-nitrophenyl cellotriose. A range of blocked and
non-blocked cello-oligosaccharides have been evaluated as general and specific substrates for cellulase, including
modified cellobiose, cellotriose and cellotetraose. Of these, the cellotetraose based substrates are preferable, but
derivatisation of cellotetraose is more difficult and yields are much lower than that obtained with cellotriose. The
value of substrates, particularly benzylidene-blocked 2-chloro-4-nitrophenyl-cellotriose (BClPNP-G3) in the
routine assay of cellulase enzymes from different sources and with different substrate, sub-site requirements, has
been studied. The effect of, and optimization of, β-glucosidase in the substrate mixtures will be described as well
as the stability of the substrate and substrate β-glucosidase mixtures in solution.
P6-15
Cell wall composition in wheat straw: Interactions with nitrogen status
Baldwin L. (a), Ahl L.I. (b), Mravec J. (a), Willats W.G.T. (a), Schjørring J.K. (a)
(a) Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40,
DK-1871 Frederiksberg C, Denmark; (b) Department of Geography and Geology, Faculty of Science, University of
Copenhagen, Rolighedsvej 23, DK-1958 Frederiksberg C, Denmark.
The aim of this work is to reveal and overcome bottlenecks in biomass productivity, quality and resource-use
efficiency. For that, wheat has to be developed into a dual purpose crop with and increased straw biomass, an
increased density of plant cell wall and a reduced content of problematic alkali element and silicates. The
improvement of straw yield and quality must be achieved without compromising grain yield. The supply of
nitrogen has a decisive influence on the yields of both grain and straw biomass. However, very little information
is available on how the composition and structure of the cell walls are affected by increasing N status. The final
aim of this work is to develop nitrogen strategies to ensure high straw quality and productivity.
For the first year of the study, winter wheat was grown in field in different conditions. Three levels of nitrogen
and the influence of growth regulator application were tested. Different harvest times were chosen along the plant
life cycle. These samples are used to make a first characterization of plant cell wall composition and structure
according to the nitrogen level. Nitrogen and carbon levels are quantified by IR-MS. Cell wall components
structure profile are characterized with a carbohydrate microarray technique, the CoMPP. Cell wall structure will
also be understood by microscopy studies with specific cell wall antibodies and staining. The analyses will be
complemented by cellulose quantification, lignin analysis, protein content and saccharification performance.
This study is part of the B21st, Biomass for the 21 st century project. This project is a Danish initiative for the integrated development of
biomass and conversion technologies for biobased fuels and chemicals.
P6-16
Bioethanol production from hydrothermally pretreated stover biomass in maize genotypes
Barros-Rios J. (a), Romaní A. (b), Garrote G. (b), Ordas B. (a)
(a) Misión Biológica de Galicia (CSIC), Apartado 28, E-36080, Pontevedra, Spain ;(b) Department of Chemical
Engineering, Faculty of Science, University of Vigo (Campus Ourense), As Lagoas, 32004 Ourense, Spain; and CITI (Centro
de Investigación, Transferencia e Innovación) – University of Vigo, Tecnopole, San Cibrao das Viñas, Ourense, Spain.
Stover samples from twelve maize genotypes were pretreated in aqueous media under non-isothermal conditions
to reach maximum temperatures (TMAX) in the range 210-225 °C in order to assess the effects of the
pretreatment severity on the fractionation of stover and on the susceptibility of processed samples toward
enzymatic hydrolysis. Both the fraction of cellulose susceptible to hydrolysis and the hydrolysis rate increased
with the severity of the pretreatments. However, the overall bioethanol yield decreased for substrates pretreated at
TMAX above 220 °C because of hemicellulose degradation (mainly arabinan) in the solid fraction. All genotypes
showed high cellulose to glucose conversions (above 87%). The wide genetic variation observed for total plant
biomass yield (ton/ha) was key to finding profound differences in total bioethanol production between extreme
maize genotypes evaluated (1,300 vs. 2,400 L/ha). The presence of lignin and acetyl groups in the spent solids
explained from 40 to 55 percent of the total genetic variation observed in bioconversion of corn stover into
ethanol, suggesting that additional biochemical and anatomical factors influence recalcitrance to sugar release in
corn stover.
P6-17
Antibacterial activity of pectin against Staphyloccocus aureus and Escherichia coli clinical isolates
El-Khatib S. (a), Daoud Z. (b), Baydoun E. (c), Abdel-Massih R.M. (a)
(a) Department of Biology, Faculty of Sciences, University of Balamand, El-Koura, Lebanon ;(b) Faculty of Medicine,
University of Balamand, El-Koura, Lebanon ;(c) Department of Biology, American University of Beirut, Beirut, Lebanon.
The amount, structure and chemical composition of pectin differs between plant sources, within a plant over time
and even in different parts of a plant. Pectin is a natural part of human diet, and it is nowadays considered as a
food supplement. However its role as an antimicrobial agent is still not clear. The antibacterial activity of pectin is
affected by the source of the pectin extract, the pH used, and the molecular weight of pectin. In this study, the
antibacterial activity of citrus pectin (high molecular weight and high methoxyl pectin) was investigated against
twenty clinical isolates of Staphylococcus aureus, sixteen strains of Extended spectrum beta-lactamase (ESBL)
Escherichia coli exhibiting different profiles of resistance, and against reference strains. The molecular weight of
pectin was monitored by size exclusion chromatography and enzyme treatment analysis.
A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC). The greatest antibacterial activity was observed with pectin at pH 5. The MIC
values against S. aureus ranged between 0.39 – 3.125 mg/ml and the MBC values ranged between 3.125 – 12.5
mg/ml. Pectin exhibited lower antibacterial activity against E. coli with MICs of 25 and 50 mg/ml and MBCs
ranging between 25 and 50 mg/ml. The data obtained in this study demonstrate that pectin showed potentially
potent antibacterial properties against Gram-positive bacteria.
P6-18
Generating barley plants with modified straw by suppressing HCT and C3H
Magama F. (a), Stephens J. (b), Halpin C. (a)
(a) Division of Plant Sciences, University of Dundee at the JHI, Invergowrie, Dundee, UK;
Institute,Invergowrie, Dundee, UK.
(b) The James Hutton
Non-food plant biomass has received a great deal of attention as a sustainable feedstock for biofuels. However,
the properties of lignin, a major secondary cell wall component, are linked with cell wall recalcitrance to
deconstruction to yield fermentable sugars from cellulose and hemicellulose. Manipulation of lignin biosynthesis
genes in Arabidopsis and other dicots species have effected changes in lignin quantity and composition resulting
in improvements in cellulose extraction, Kraft pulping and forage digestibility with or without necessarily
affecting plant growth and development. My work focuses on the RNAi suppression of barley HCT and C3H, the
first committed enzymes in the biosynthesis of the predominant monomers of lignin. Barley is used as a model for
grass bioenergy crops and as an economically important crop in the UK. Suppression of HCT and C3H produced
pleiotropic effects on barley plant growth and development including sterility, reductions in plant height, and
reductions to seed count and seed weight. Straw lignin content was determined by the Klason method and was
reduced by 4-20% of control levels in HCT RNAi plants and 10-50% of control levels in C3H RNAi plants. This
reduction in lignin was generally correlated with endogenous protein levels in the transgenic lines determined by
immunoblotting assays with specific barley polyclonal antibodies. A limited saccharification investigation showed
up to 153% increase in sugar release in the severe downregulated plants. A clear understanding of the effects of
RNAi down-regulation of HCT and C3H as well as other barley lignin genes presents an exciting opportunity to
optimise novel strategies for manipulating lignin and associated cell wall polymers in favour of biofuel
production.
P6-19
Phenotyping barley straw for agronomic and biofuel-related traits
Grussu D. (a), Oakey H. (a), Comadran J. (b), Shafiei R. (a), Uzrek N. (b), Hooper M. (a), Simister R. (c), Waugh R. (ab),
Gomez L. (c), McQueen Mason S. (c), Halpin C. (a)
(a) Division of Plant Sciences, University of Dundee at the JHI, Invergowrie, Dundee, UK; (b) The James Hutton Institute,
Invergowrie, Dundee, UK ;(c) CNAP, Department of Biology, University of York, Heslington, York, UK.
Dwindling fossil fuels, climate change and political unrest have all served to drive forward research into
producing alternative renewable fuels. Second generation biofuels that would use non-food plant biomass as the
raw material have received a great deal of attention. Our work has specifically targeted barley straw as the
potential feedstock. Barley is both a model grass with a genetically tractable diploid genome and an important
commercial cereal crop. To evaluate the potential of cereal straw as a biofuel feedstock, we have been working
with a large collection of 850 elite 2-row spring barley genotypes grown in a polytunnel in each of 2 successive
years. Phenotypic measurements were recorded for each genotype and correlated against each other to elucidate
possible links between key traits. This data was also used in a genome wide association scan to pinpoint regions
and genes which may be involved in the control of these traits. In addition to this, data was obtained from
national field trials for a subset of the genotypes. This provided scores for key agronomic traits such as lodging,
brackling, necking and leaning and allowed these to be similarly correlated with the phenotypic measurements
from the polytunnel. Results showed that there was no significant overall correlation between the important
biofuel production trait, saccharification, and phenotypes that would have an impact on crop health or yield. In
particular, there was no correlation between saccharification yield and lodging. QTL analysis showed some
genomic loci that influenced both specific stem strength elements and saccharification but others that were
independent for each trait. This means that we can breed to improve straw saccharification without necessarily
impacting on field performance.
P6-20
eQTL Analysis on Spring Barley using RNA sequencing
Kam J. (a), Oakey H. (a), Bayer M. (b), Comadran J. (b), Waugh R. (b), Halpin C. (a)
(a) Division of Plant Sciences, University of Dundee at the JHI, Invergowrie, Dundee, UK ; (b) The James Hutton
Institute,Invergowrie, Dundee, UK.
We aim to identify regulatory elements that control the expression of lignin biosynthesis genes that influence
saccharification efficiency, a major target for second generation biofuel production. To achieve this, we have
measured the expression levels of known barley lignin biosynthesis genes across a population of 2-row spring
barley varieties for which genotyping data is available and that are suitable for GWAS. This allows us to map
expression level as a quantitative trait and to search for genomic regions that regulate the expression level of
individual lignin biosynthesis genes. These regions, identified by co-locating Single Nucleotide Polymorphism
(SNP) markers, are known as expression Quantitative Trait Loci (eQTLs).
To generate the data needed for this study, barley global expression data was obtained from the 2 nd internode
(Zadok 35) of 144 spring barley genotypes using RNA sequencing (RNAseq). We will use the RNAseq data to
identify eQTL that act in cis (i.e. that are in the lignin genes themselves) or in trans (i.e. distinct genes at a
different location, for example transcription factors acting on lignin biosynthesis genes). Thus, this approach may
identify novel regulators of lignin or secondary cell wall biosynthesis, revealing more about the genetics of cell
wall assembly, and potentially providing new genes for useful manipulation.
In addition to global transcript abundance, RNAseq also yields information on transcript sequence polymorphism,
potentially enriching current SNP information for these genotypes. The combination of mapping based on both
SNPs in gene sequences and gene expression variation should be an extremely powerful tool for refining genomic
loci tightly correlated with traits (such as saccharification yield) and discovering the underlying causal genes.
P6-21
Analysis of the cinnamyl alcohol dehydrogenase (CAD) gene family and downregulation of
HvCAD2 gene by RNAi in barley (Hordeum vulgare)
Maluk M. (a), Daly P. (a), Stephens J. (b), Lyon J. (b), Lapierre C. (c), Gomez L. (d), Waugh R. (b), Halpin C. (a)
(a) Division of Plant Sciences, University of Dundee at the JHI, Invergowrie, Dundee, UK ; (b) The James Hutton Institute,
Invergowrie, Dundee, UK; (c) UMR 1318 INRA-AgroParisTech, Institut National de la Recherche Agronomique 78026
Versailles, France; (d) Department of Biology, University of York, Wentworth Way, Heslington, York YO10 5DD, UK.
Cinnamyl alcohol dehydrogenase (CAD) is the final enzyme in the lignin biosynthesis pathway; CAD mutants and
transgenic plants have changes to lignin content/structure/composition that make cell walls more digestible. Here
we described the CAD multigene family in barley and other grasses and identify HvCAD2 as the principal CAD
involved in lignin biosynthesis. The HvCAD2 gene was downregulated using RNAi via Agrobacterium-mediated
transformation to produce transgenic barley plants. These plants showed reduced enzyme activity and had an
orange stem phenotype in the T1 generation. The CADRNAi barley lines had similar or slightly reduced Klason
lignin content relative to the control plants but the lignin structure and composition were altered (lower
thioacidolysis yield, reduced S/G ratio, lower amount of H and S units, increased sinapaldehyde accumulation,
reduced p-coumaric esters and ferulic acid abundance relative to the control plants). Changes in lignin in barley
CADRNAi lines led to moderately improved sugar release. Because lignin plays a major role in culm strength and
pathogen resistance, the influence of down-regulation of CAD in barley transgenic plants on that features was
characterized. There was no detrimental impact on straw strength and pathogen resistance observed in the barley
CADRNAi lines. This study demonstrates that it is possible to downregulate the CAD gene in order to change
lignin structure and composition in plants to improve their digestibility, without adverse impacts on plant growth,
fertility and pathogen resistance.
P6-22
Reversing Forward: Genome-Wide Mutation Screening for Improved Saccharification Efficiency
in Barley
Shafiei R. (a), Wilson Y. (a), Gomez L. (a), Hooper M. (a), Simister R. (c), Uzrek N. (c), Oakey H. (a), Waugh R. (ab),
McQueen-Mason S. (c), Halpin C. (a)
(a) Division of Plant Sciences, University of Dundee at the JHI, Invergowrie, Dundee, UK ; (b) The James Hutton Institute,
Invergowrie, Dundee, UK; (c) CNAP, Department of Biology, University of York, Heslington, York, UK.
Cell wall degradability and lignin content of barley straw are two main features that influence saccharification
(SA) efficiency and subsequently impact sustainability of second generation biofuel production. The framework
of our gene discovery pipeline is based on combinations of technologies, including high throughput phenotyping
and genome-wide association scans (GWAS). A direct approach to establish function of candidate genes
identified is to develop a TILLING (Targeting Induced Local Lesions IN Genomes) population, a reverse
screening platform. Subsequently, mutations are identified through “TILLING by sequencing”. To this end, barley
Optic cv. seeds were mutagenized with ethyl methanesulphonate (EMS). Around 2500 M2 plants were grown in
controlled conditions and leaf tissues were harvested and pooled in a 3 dimensional fashion for DNA extraction
and deconvolution purposes. DNA pools were screened for mutations in candidate genes using NGS sequencing,
and deconvolution used to work back to single plants.
In parallel, employing a forward approach, an Optic mutant population (M5) was screened for their SA efficiency
and stem physical features. Consequently, 16 mutants were identified with significantly improved SA efficiency.
The straw of the mutant population was analysed for mechanical properties. Despite noticeable variation in straw
phenotypes and SA efficiencies, many of the mutant straws were as physically strong as Optic, suggesting that SA
properties can be improved without compromising straw strength. The mutations are being mapped and their
potential use for plant breeding is being investigated.
P6-23
High value functional products from DDGS
Kosik O. (a), Charalampopolous D. (b), Rastall R. (b), Gibson G. (b), Frazier R. (b), Lovegrove A. (a), Shewry P.R. (ab)
(a) Department of Plant Biology and Crop Science, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK ; (b) Department
of Food Science, University of Reading, Whitenights Campus, Reading, Berks,RG6 6AH, UK.
Dried distillers grains and syrup (DDGS) is a co-product of ethanol production from wheat or corn grain. DDGS,
consists of soluble and insoluble dietary fibre, (ca 40-50% w/w, mainly arabinoxylan, cellulose, and β-glucans)
and proteins (ca 30-40% w/w), but lacks the rigid structure of lignocellulosic materials. Prebiotics are ‘nondigestible food ingredients that have a beneficial effect on health through their selective metabolism by bacteria in
the intestinal tract (1). The current commercial prebiotics, available are fructans (inulin, fructooligosaccharides)
and galactans however hydrolysis products of AX, arabinoxylooligosaccharides (AXOS) are emerging prebiotics
(2). Relatively mild processing of DDGS could lead to the production of large amounts of bioactive
carbohydrates. And thus there is the opportunity of producing AX and AXOS from DDGS at a commercial scale.
Moreover, wheat genotypes influence the content of AX (3), transgenesis and mutagenesis can be used to make
specific changes in AX structure notably the proportions of xylose residues that are monosubsituted and
disubstituted with arabinose (4). Wheat lines commonly used for distilling, as well as DDGS from these wheat
lines (produced at lab-scale by Scottish Whisky Research Institute (SWRI)) will be used to identify the extent to
which AX and AXOS vary in their amount and composition. The arabinose:xylose ratios of these fractions, their
proportions of mono- and di-substituted xylose residues and their molecular weight, as well as the mixed-linked
glucan (MLG) content, will be determined.
[1] Gibson et al. (2004) Nutr. Res. Rev., 17, 259-275 ; [2] Cloetens et al. (2010) Brit. J. Nutr., 103, 703-713 ; [3] P.R. Shewry
et al. (2010) J.Agric. Food Chem., 58, 9291-9298; [4] Anders et al. (2012) PNAS, 109(3), 989-993.
P6-24
Biotechnological Potential of Carbohydrate Binding Modules from Oomycetes
Martinez T. (abcd), Lafitte C. (a), Dumas B. (a), O’Donohue M. (bcd), Dumon C. (bcd), Gaulin E. (a)
(a) UMR 5546 UPS/CNRS Laboratoire de recherche en sciences végétales (LRSV), France; (b) INRA, UMR792, Ingénierie
des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France ; (c) Université de Toulouse; INSA, UPS, INP; LISBP,
135 Avenue de Rangueil, F-31077 Toulouse, France ; (d) CNRS, UMR5504, F-31400 Toulouse, France.
Oomycetes are fungal-like microorganisms evolutionary distant from true fungi, and comprising major plant
pathogens. Oomycetes express proteins able to interact with plant cell wall polysaccharide like cellulose, through
the presence of carbohydrate-binding module belonging to family 1 of the CAZy database (CBM1) [1]. Fungal
CBM1-containing proteins are implicated in cellulose degradation whereas in oomycetes, the Cellulose Binding
Elicitor Lectin (CBEL), a well-characterized CBM1-containing protein from Phytophthora parasitica, is
implicated in cell wall integrity, adhesion to cellulosic substrates and most importantly in induction of plant
immunity responses [2,3]. In this study we characterized the cellulose binding of CBEL and evaluated the
potential of CBM1-containing proteins from oomycetes for future biotechnological applications.
[1] FV. Mateos et al. Mol (1997) Plant Microbe Interact., 10(9),1045-1053 ; [2] E. Gaulin et al. (2002) J. Cell. Sci., 115(23),
4565-4575 ; [3] E. Gaulin et al. (2006) Plant Cell, 18(7),1766-1777.
P6-25
The effect of functional carbohydrates on the intestinal tract of broiler chickens and pigs
Tian L., Schols H.A., Gruppen H.
Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands.
There is an immense and fast growing pressure on global availability of nutrients as countries become more
affluent and world’s population continues to grow. This cannot be solved anymore by only increasing the number
of production animals. Improvement of feed and food efficiency in domestic animals is mandatory to ensure the
worldwide demand for food. To achieve that goal the world is depending upon new technologies in intensive
animal food production systems that ensure a more efficient way for meat production.
In recent years, researchers have made improvements of feed efficiency which is attributed to a higher genetic
potential, better estimation of nutrient requirements and more knowledge of feedstuffs to more accurately meet
the requirement and prevent unnecessary nutrient losses. Minor attention has been paid to the improvement of
efficiency by directly influencing the nutrient digestion in the intestinal tract and influencing the physiology of
uptake in the gastrointestinal track of livestock.
It is known from earlier studies that carbohydrates may have a large influence on quantitative food intake, nutrient
absorption, and weight gain. However, the relation between the chemical structure of complex cell wall
polysaccharides and oligomeric fragments derived therefrom and their biofunction is not yet clear.
In this project, we try to identify specific oligo- and polysaccharide structures originating from oat grains and
defatted soybeans, playing a role in the health and the efficient feed conversion in broiler chickens and pigs.
P6-26
Screening a barley multi-parent population: Searching for cell wall traits relevant for
bioconversion in commercially bred barely lines
Günl M. (a), Voiniciuc C. (a), Usadel B. (ab)
(a) Forschungszentrum Jülich, IBG-2: Plant Siences, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany; (b) RWTH Aachen,
Institute for Biology I, Worringer Weg 1, 52056 Aachen, Germany.
More than 10 million tons of barley were harvested in 2010 in Germany alone and barley presents one of the
major crops worldwide. In addition to the grain, substantial amounts of barley straw are generated. However, the
straw is mostly left on the field to provide the soil with nutrients, but it is estimated that about one third of the
straw can be removed from the field, without affecting soil quality. This lignocellulosic biomass material will
provide a valuable feedstock for biorefineries. However, effects that inhibit the conversion to biofuels and other
biomaterials need to be reduced to a minimum.
As part of this project a double haploid barley (Hordeum vulgare L.) MAGIC (“Multiparent Advanced Genetic
InterCross”) population, which has been created from eight commercially bred barley varieties, is analyzed for
alterations in cell wall carbohydrate composition to identify traits that determine their suitability for
bioconversion. The MAGIC population shows a large degree of genetic diversity and allows precise mapping of
genetic loci. The cell walls of the MAGIC population founder lines have been characterized in detail. In particular
cell wall fractionation, monosaccharide compositional analyses and sugar linkage analyses has been used for the
characterization of cell wall structures [1].
[1] C. E. Foster et al. (2010) J. Vis. Exp., 11 (37).
P6-27
Identification of QTL for saccharification in maize stover
Legay S. (a), Griveau Y. (a), Bird S. (b), Courtial A. (c), Barrière Y. (c), Gomez L. (b), McQueen Masson S. (b), Mechin V.
(a), Reymond M. (a)
(a) INRA, Institut Jean-Pierre Bourgin, 78026 Versailles, France; (b) CNAP, Department of Biology, University of York,
Heslington, York YO10; 5YW, UK; (c) INRA, Unité de Génétique et d’Amélioration des Plantes, 86600 Lusignan, France.
With the worldwide increasing demand of energy and global climate change issues, understanding how to get the
best value out of biomass is becoming an important issue. Lignocellulosic biomass is a complex and intricate
matrix composed of 3 main polymers: lignin, cellulose and hemicellulose that are able to interact directly or, in
the case of grasses, through hydroxycinnamic acids. The high level of complexity of the cell wall renders it
recalcitrant to enzymatic hydrolysis, which is required for sugar release, initial step towards bioethanol
production. Mutants of the lignin biosynthesis pathway genes have proven to impact degradability of
lignocellulosic biomass (ref). Variation for degradability is also observed amongst natural accessions but up to
now little is known about the genetic determinism responsible for this variation.
As a European initiative the SUNLIBB project (Sustainable Liquid Biofuels from Biomass Biorefinery; FP7)
focusses on the overall steps of the biofuel production chain from feedstock improvement to the integrated
production of biofuels and added value products. As part of this project, our objective is to improve biomass
quality in grasses for biorefineries and biofuel production. To tackle this goal, genes involved in degradability and
saccharification will be identified using natural variation in maize. 119 maize recombinant inbred lines were
screened for their sugar release potential using a high throughput saccharification robot (university of York). QTL
for saccharification potential were then detected. Candidate genes underlying major QTL as well as colocalization
with QTL for digestibility and cell wall composition, previously detected in maize, will be presented.
P6-28
A promising biorefinery approach for sugar beet pulp, alternative to its use for biofuel production
Leijdekkersa M. (b), Schols H.A. (a), Gruppen H. (a)
(a) Wageningen University, Laboratory of Food Chemistry, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands ;(b)
Cosun Food Technology Centre, P.O. Box 1308, 4700 BH, Roosendaal, The Netherlands.
Sugar beet pulp is a large volume by-product of the sugar industry. The main constituent monosaccharides in
sugar beet pulp are glucose (from cellulose), galacturonic acid and arabinose (both present in pectin). So far,
studies towards valorization of sugar beet pulp primarily focussed on production of monosaccharides for biofuel
production. However, the economic feasibility remains challenging. Instead of serving as carbon source for
fermentation, galacturonic acid and arabinose are also interesting molecules for further conversion into building
blocks which can be subsequently transformed into high-value biobased chemicals or materials. Moreover, the
remaining cellulose after saccharification may be an interesting component for application as biobased fiber or
nanocomposite material and the recalcitrant oligosaccharides have prebiotic potential.
In our study, a novel biorefinery concept for sugar beet pulp was investigated. An enzymatic saccharification
process was optimized to release the maximum amounts of monomeric galacturonic acid and arabinose with
limited concomitant degradation of cellulose, using conditions that are feasible for industrial upscaling. The
recalcitrant oligosaccharides that were obtained after hydrolysis were characterized using mass spectrometry [1].
Additionally, the in vitro fermentation characteristics of these oligosaccharides were determined, providing
information on the fermentability of the various types of oligosaccharides, short chain fatty acid production and
induced changes in the composition of colonic microbiota.
[1] A.G.M. Leijdekkers et al. (2013) Bioresour. Technol., 128, 518-525.
P6-29
Manipulation of lignin 4CL and CCR genes to improve biofuel production from cereal straw
Zwirek M. (a), Kam J. (a), Grussu D. (a), Stephens J. (b), Halpin C. (a)
(a) Division of Plant Sciences, University of Dundee at the JHI, Invergowrie, Dundee, UK; (b) The James Hutton Institute,
Invergowrie, Dundee, UK.
Cereal straw has the potential to be used as a raw material for biofuel production. However, the lignin content and
composition of straw affects the efficiency of fermentable sugar release (saccharification) from the cell wall,
thereby reducing the potential biofuel yield. Manipulation of lignin to improve saccharification efficiency is a
main focus of bioenergy research. In this study, two genes of the lignin pathway in barley: 4-coumarate CoA
ligase (4CL) and cinnamoyl CoA reductase (CCR) were characterised and the effect of suppression of those genes
on barley phenotype was determined. In barley, these genes are present within gene families of several members
that differ in spatial expression patterns. Three 4CL and four CCR gene family members are predominantly
expressed in highly lignified tissues, such as stem and roots. Transgenic barley plants were produced using RNAi
constructs designed with conserved regions of 4CL or CCR cDNA sequences. The transgenic lines show
consistent reductions of 4CL or CCR protein across generations. In addition, plants have changes in lignin
content and significantly improved saccharification when compared to control plants. The changes in lignin
content in suppressed 4CL and CCR lines have been measured using the Klason method. Results indicate that
downregulation of 4CLs and CCRs genes affects the ratio of two lignin fractions, acid soluble lignin (ASL) and
acid insoluble lignin (AIL). Although the ratio of ASL to AIL contents is changed, the combined content of both
fractions remains lower than in controls and the transgenic plants have reduced lignin overall. An increase in the
proportion of ASL vs. AIL could be an advantage for industrial applications. Manipulation of both genes in barley
has the potential to increase the saccharification and digestibility of the straw and highlights the prospects for
doing the same in other energy crops.
P6-30
Identifying novel genes to improve lignocellulosic biomass for biofuel applications
Marriott P.E. (a), Gomez L.D. (a), Sibout R. (b), McQueen-Mason S.J. (a)
(a) Department of Biology, University of York, York YO10 5DD, UK ; (b) Institut Jean-Pierre Bourgin, INRA Centre de
Versailles-Grignon, 78026 Versailles, France.
Lowering the costs of producing bioethanol from lignocellulosic biomass is required for its commercialization.
One approach of doing so is to produce crops that are more susceptible to hydrolysis in order to reduce preprocessing and enzyme inputs. To this end, we are using a forward genetic approach with the objective of
identifying novel genes that increase the digestibility of plant cell walls. We have screened a chemically
mutagenised population of the model grass Brachypodium distachyon (produced by INRA, Versailles) for
improved saccharification with industrial cellulases. This revealed 12 mutant lines with heritable increases in
saccharification. Characterization of these 12 mutants revealed a range of different alterations in cell wall
composition, including significant changes in lignin, hemicellulose, cellulose and ferulic acid content and also in
lignin composition. These results show that saccharification can be significantly improved through a number of
distinct modifications of the cell wall, giving the potential for combining more than one of these modifications in
biofuel crops to obtain even higher ethanol yields. Furthermore, the mutations seem to have little effect on plant
phenotype and mechanical strain tests revealed that none of the mutants showed a reduction in stem strength or
stiffness compared to WT, an important trait for crop field performance. Current work is focussed on mapping the
responsible mutations and characterisation of the mutated genes. This will enable subsequent examination of
orthologous genes in the relevant cereal and grass crops used for biofuel production.
P6-31
Impact of textile processing on cotton fibre polysaccharide composition
Runavot J.-L. (a), Hernandez-Gomez M.C. (b), Knox J.P. (b), Goubet F. (a), Meulewaeter F. (a)
(a) Bayer CropScience NV, Innovation Center, Technologiepark 38, 9052 Zwijnaarde, Belgium ; (b) Centre for Plant
Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
Cotton fibres have been widely studied as a model for cell elongation because of their ability to elongate a single
cell up to several centimetres. Due to its favourable properties cotton is as well the main source of natural fibre for
the textile industry. But despite its importance for textile applications, little is known about the impact of
industrial processing on cotton fibre composition. Improved knowledge of the biochemical composition of fibres
along the process would potentially allow the textile industry to optimize cotton processing.
Mature fibre is mainly composed of cellulose but it also comprises other polysaccharides. These polysaccharides
are known to be present during fibre development and have been shown to be decreasing during the secondary
cell wall phase [1,2] due to the large production of cellulose. Non-cellulosic polysaccharides are suggested to be
removed during the chemical treatments of textile processing, but this has not yet been studied in detail.
During this study we determined the non-cellulosic polysaccharide composition of cotton fibres from samples
gathered during textile processing. This approach revealed that harsh chemical treatments from textile processing
impact the polysaccharide content of fabrics differently and that some of the non-cellulosic polysaccharides are
(partially) retained after these treatments.
[1] H. Tokumoto et al. (2002) Plant Cell Physiol., 43, 411-418; [2] M. C. Meinert and D. P. Delmer (1977) Plant Physiol.,
59, 1088-1097.
P6-32
De-esterified homogalacturonan negatively affects saccharification efficiency
Ferrari S., Francocci F., Bastianelli E., Verrascina I., Lionetti V., De Lorenzo G., Bellincampi D., Cervone F.
Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Sapienza Università di Roma, Rome, Italy.
Production of biofuels from lignocellulosic biomasses is strongly limited by the recalcitrance of the cell walls to
enzymatic saccharification. Pectin composition is a potentially interesting target to engineer plants or select novel
varieties with improved for biofuel production. We have previously observed that transgenic plants constitutively
expressing a fungal polygalacturonase (PG) show reduced levels of de-esterified homogalacturonan (HGA) and
have increased saccharification efficiency, but diplay growht defects. Here we show that the controlled expression
of pectic enzymes in Arabidopsis thaliana increases saccharification efficiency without negative effects on plant
growth. Furthermore, Arabidopsis mutants for genes that affect HGA biosynthesis or methylation also display
increased saccharification. Analysis of a core collection of Arabidopsis accessions also showed a negative
correlation between levels of de-esterified HGA and saccharification efficiency. Taken together, these data
indicate that pectin esterification has a major impact on the conversion of lignocellulosic biomasses into biofuels,
and genetic variation in HGA composition could be exploited to breed more efficient energy crops.
P6-33
Mechanisms and enzymes for lignocellulose deconstruction from a marine wood borer
McQueen-Mason S. (a), Besser K. (a), Kern M. (a), Elias L. (a), Eborall W. (a), Gomez L. (a), Malyon G. (b), McGeehan J.
(b), Streeter S. (b), Schnorr K. (c), Cragg S. (b), Bruce N. (a)
(a) Centre for Novel Agricultural Products, Biology Department, University of York, UK; (b) Department of Biological
Sciences, University of Portsmouth, UK; Novozymes A/S, Denmark.
Limnoria quadripunctata is a wood-boring isopod that lives on a diet of lignocellulose in the marine environment.
This animal is unusual in having a digestive tract that is free of microbial life. This is in contrast to other woodeating animals that depend, at least in part, on the assistance of gut microbes for the digestion of this recalcitrant
material. We have been investigating the digestive system of this animal to identify the mechanisms and enzymes
involved in the digestive process and to understand why no microbes live in digestive system. The digestive
system of L. quadripunctata is essentially a two-vessel bioreactor. Wood particles are tightly packed into a large
linear hindgut, which is lined with cuticle indicating that is not involved in secretory of absorptive processes. A
four-lobed hepatopancreas is connected to the hindgut, but wood particles are prevented from entering it by the
presence of a physical filter. The hepatopancreas is lined by secretory cells and produces a range of digestive
enzymes dominated by glycosyl hydrolases (GHs). The most abundant GHs are processive cellobiohydrolases
from the GH7 family. Such GH7 cellulases were previously only known in wood-degrading fungi and protist
symbionts from termite digestive systems. We will present detailed structural and biochemical characterisation of
a GH7 from L. quadripunctata. One unusual feature of this enzyme is its stability in up to 4M NaCl. This high salt
stability appears to be a common feature in the GHs from this animal and we will discuss the structural
characteristics associated with this trait. We will present data on the chemical processes occurring in the hindgut
and how these prevent microbial life and potentially necessitate the presence of a protective cuticle between gut
contents and the living tissues.
P6-34
Genetic analyses of the maize nested association mapping population reveal quantitative trait loci
and candidate genes contributing to phenylpropanoid abundance or saccharification yield
Penning B.W. (ab), Sykes R.W. (c), Babcock N.C. (d), Dugard C.K. (b), Held M.A. (e), Klimek J.F. (b), Shreve J. (f), Fowler
M. (c), Gamblin D. (g), Ziebell A. (c), Davis M. (c), Decker S.R. (c), Filley T.R. (g), Mosier N.S. (h), Springer N.M. (i),
Thimmapuram J. (f), Weil C.F. (d), McCann M.C. (a), Carpita N.C. (ab)
(a) Department of Biological Sciences, (b) Department of Botany & Plant Pathology, (d) Department of Agronomy, (f) Cyber
Center, College of Agriculture, (g) Department of Earth, Atmospheric and Planetary Sciences, (h) Department of
Agricultural & Biological Engineering; Purdue University, West Lafayette, IN 47907, USA; (c) Bioscience Center, National
Renewable Energy Laboratory, Golden, CO, USA; (e) Department of Chemistry & Biochemistry, Ohio University, Athens,
OH, USA; (i) Department of Plant Biology, University of Minnesota, St. Paul Minneapolis, MN, USA.
Lignocellulosic biomass is a renewable and sustainable source of partially reduced carbon for conversion to liquid
transportation fuels and bio-based products. Considerable effort has been devoted in recent years to reduce the
recalcitrance of biomass to digestion by cellulolytic hydrolases and other enzymes, thought to be a consequence of
lignin abundance. We used pyrolysis molecular-beam mass spectrometry of lignocellulosic cell wall materials
from maize stems as a high throughput method to establish quantitative trait loci (QTL) for the abundance of pcoumaric acid, and guaiacyl and syringyl lignin, in a population of Intermated B73 x Mo17 (IBM) recombinant
inbred lines in a multi-year study. In parallel, a high throughput enzymatic hydrolysis and assay for glucose,
xylose and total sugar in the same materials was used to establish QTL for saccharification yield. We identified
six QTL for lignin abundance, one QTL for p-coumarate abundance, and ten QTL for high glucose or xylose
release during saccharification. None of the QTL are in common, indicating that the genes contributing to lignin
abundance and saccharification yield are different. We interpret these results to mean that factors other than lignin
abundance, such as carbohydrate and lignin architecture, side-group substitutions, or other types of modifications
and interactions, exhibit genetic variance and constitute many additional determinants of recalcitrance. Genomewide association screens (GWAS) for lignin abundance and saccharification yield in a 282-member Association
Panel of maize inbreds and landraces greatly narrowed the lists of candidate genes with relevant single-nucleotide
polymorphisms (SNPs) between the B73 and Mo17 parents. Large differences in transcript abundance of cell
wall-related alleles in developing stem tissues were also observed between B73 and Mo17, providing an
additional independent list of candidate genes within each QTL interval that may contribute to the two traits.
This work was supported by the National Science Foundation “Hy-Bi”, an Emerging Frontiers in Research and Innovation (EFRI) program,
Award No. 0938033, by the U. S. Department of Energy Feedstock Genomics Program, Office of Biological and Environmental Research,
Office of Science, and by the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center
funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Award Number DE-SC0000997.
P6-35
Biomimetic thin films model of plant cell wall for enzymatic hydrolysis studies
Dammak A. (a), Moreau C. (a), Beury N. (a), Gouider L. (a), Cousin F. (b), Jean B. (c), Azzam F. (a), Bonnin E. (a),
Cathala B. (a)
(a) UR1268 Biopolymères Interactions Assemblages, INRA, F-44316 Nantes, France ; (b) Laboratoire Léon Brillouin,
CEA/CNRS Saclay, 91191 Gif-sur-Yvette cedex, France ; (c) Centre de Recherche sur les Macromolécules Végétales
(CERMAV-CNRS) BP 53, 38041 Grenoble Cedex, France.
Better understanding of the mechanisms contributing to enzymatic hydrolysis of plant cell walls is of critical
importance for the conversion of lignocellulosic biomass to biofuels. Investigation of the effect of the
organization of cell wall polymers on the enzyme action remains a challenge. We recently report the elaboration
of multilayered nanometric thin film displaying semi-reflective properties that can be used as enzymatic assay for
the detection of biopolymer degrading enzymes [1,2,3]. Due to the complexity of cell wall organization, cellulosic
films with controlled and tunable architecture are valuable biomimetic substrates for studying enzyme hydrolytic
action [4]. This would provide insight into relationship between substrate properties and enzymatic degradation
process. In this work, two cellulose nanocrystal/xylogucan (CN-XG) films with different architectures were
obtained by the Layer-by-Layer method. As revealed by the growth pattern, CN layers are adsorbed as a single or
a double layer depending on the concentration of CN dispersion. Neutron reflectivity is used to determine volume
fractions of XG and CN in the two cases. The impact of architecture difference on enzymatic degradation process
was described using Quartz Crystal Microbalance with Dissipation (QCM-D) to monitor the changes in frequency
and energy dissipation during enzyme action.
[1] C. Cerclier et al. (2010). Langmuir., 26, 17248-17255 ; [2] C. Cerclier et al.(2011). Adv. Mater.,23, 3791-3795 ; [3] A.
Dammak et al. (2013). Holzforschung., just accepted; [4] A.Guyomard-Lack et al.(2012). Eur. Phys. J., Special Topics., 213,
291-294.
P6-36
Identification of efficient glucanases and their application in marine biomass fermentation
Zwikowics C., Jamrow T., Meineke T., Ellinger D., Voigt C.A.
Phytopathology and Biochemistry, Biocenter Klein Flottbek, University of Hamburg, Hamburg, Germany.
The demand on renewable energy is growing. Cellulosic biomass and marine biomass such as seaweed have high
potential as feed stock for bioethanol production (second and third generation biofuels). Several species of
seaweed grow fast and are highly effective in biomass production. In general, marine biomass has no competition
to the food industry. Our research is focused on the cell wall polymer callose in plant and laminarin in algae.
Therefore, our study is aimed on screening and identification of efficient glucanases for saccharification of these
polymers and subsequent fermentation to bioethanol. We could produce several (1,3)-ß-glucanases of different
species in E.coli, followed by purification for activity assays with different glucans. In the screening, we
identified effective endo- and exo-glucanases from both, bacteria and fungi. Highest activities of these glucanases
were observed between pH 5 and 6. Some glucanases showed substrate specificity for laminarin from Laminaria
digitata and callose from Euglena gracilis. Subsequent application of the most effective glucanases in the
fermentation of biomass from the brown algae Fucus vesiculosus showed an increase of bioethanol yield.
Additionally, acid pretreatment of the biomass could further improve saccharification and bioethanol production
compared to base treatment. In conclusion, the combination of the right pretreatment and application of effective
(1,3)-ß-glucanases is required for optimizing ethanol production from marine biomass.
P6-37
Effects of pectin composition on cooking properties of potato tubers
Kortstee A. (a), Suurs L. (a), Huang J.H. (b), Schols H.A. (b), Visser R. (a), Trindade L. (a)
(a) Wageningen UR Plant Breeding, the Netherlands; (b) Wageningen UR Food Chemistry, the Netherlands.
Pectin is a complex polysaccharide and an integral part of the primary cell wall of plants, contributing to cell
strength and cell adhesion. These biological functions determine some of the potato tuber quality traits. In this
study we have analyzed the cooking behavior of transgenic potato lines differing in pectin composition. The
transgenic potato lines express enzymes which modify pectin including: rhamnogalacturonan lyase (RGL), βgalactosidase (β-gal), UDP-glucose 4-epimerase (UGE), endo-1,4-β-D-galactanase (eGAL), endo-α—1,5-Larabinase (eGARA) and pectin acetyl esterase (PAE). Monosaccharide composition analysis of the cell wall
fraction showed that pectin composition was altered in tubers of the transgenic lines. Effects of the introduced
enzymes were in the pectin side chains arabinan and galactan and in the (methyl) esterification of the pectin
backbone. Tubers harvested in two consecutive years, 2011 and 2012 were analyzed for agronomic traits e.g.
fresh weight, percentage of dry mass, amount of cell wall material, starch and sugars. Texture after cooking was
assessed visually [1] and by texture analyzer [2]. The variation in determined cooking properties could be partially
explained by the variation in length and complexity of the pectic side chains but not by amount of dry mass, cell
wall content or starch. In lines with decreased galactan content (β-gal, eGAL) changes in cooking properties of the
potato tubers were observed, including increased firmness. Tubers of the line with decreased acetylated pectin
(expressing a PAE gene) were also more firm. Future experiments will include a detailed analysis of the fine
structure of pectin for a better understanding of its functions.
[1] van Marle (1997) PhD thesis, Wageningen; [2] H. Ross et al. (2010) J. Sci. Food Agric., 90, 1527-1532.
P6-38
Characterization of genes involved in phenylopropanoid and lignin biosynthesis: towards
improving cell walls for biofuels
Sundin L., Vanholme R., Goeminne G., Boerjan W.
Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium.
Lignin is a fundamental constituent of the secondary cell wall [1] and is a troublesome obstacle in the refining of
lignocellulosic biomass into valuable products like biofuels, because it protects cellulose against enzymatic
breakdown. The chemical breakdown of lignin by pretreatments is very costly, hence there is large interest in
modifying lignin in plants. Our research is focused on engineering plant cell walls to ease their conversion into
biofuels. The goal of the presented research project is to discover new genes that encode enzymes involved in the
lignification pathway. First, 21 candidate genes were selected based on their expression characteristics in a
systems biology project completed within the group. The corresponding mutant lines were screened by phenolic
profiling followed by a comprehensive cell wall analysis for the most promising candidates. We show that
TRANSALDOLASE2 is involved in lignin biosynthesis [2]. The inflorescence stem of the transaldolase2 mutant
has lower levels of oligolignols (small lignin oligomers) as compared to wild type. Furthermore, the senesced
inflorescence stem of these mutants displayed a reduced amount of lignin, changes in the lignin composition and
the biomass was more susceptible to enzymatic hydrolysis.
[1] W. Boerjan et al. (2003) Annu. Rev. Plant Physiol. Plant Mol. Biol., 54, 519–546; [2] R. Vanholme et al. (2012) Plant
cell.,24, 3506-29.
P6-39
Modifying Secondary Cell Wall Biosynthesis to Develop Improved Bioenergy Crops
Lau J. (a), Petersen P.D. (ab), Yang F. (a), Loqué D. (a), Scheller H.V. (ac)
(a) Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; (b) Department of Plant
Biology and Biotechnology, University of Copenhagen, DK-1871 Frederiksberg, Denmark; (c) Department of Plant &
Microbial Biology, University of California, Berkeley, CA 94720, USA.
Plant biomass for bioenergy purposes is composed largely of secondary cell walls, about a third of which is
hemicellulose. In angiosperms, hemicelluloses are mainly composed of xylans, which are polymers of pentoses
that are less desirable than hexoses for fermentation. In our previous study, we have generated transgenic
Arabidopsis plants where xylan is synthesized normally in vessels but not in interfascicular fibers [1]. The
engineering of reduced xylan can be combined with increased deposition of C6 sugars in the fibers that results in
transgenic plants that have reduced xylan and increased cellulose deposition in the fibers. This was accomplished
by overexpressing a fiber-specific transcription factor under control of a downstream promoter. These plants
exhibit normal growth and development. Effects on cell wall density, monosaccharide composition and
saccharification efficiency will be reported. Our results demonstrate a method to generate bioenergy crops with
improved properties for saccharification and production of more easily fermentable sugars.
[1] Petersen, Lau et al. (2012) Biotechnol Biofuels 5, 84.
P6-40
Genome wide analysis reveals WAK and PME implications in textural default of apple fruit
Mikol S. (abc), Bruneau M. (abc), Celton J.-M. (abc), Orsel M. (abc), Laurens F. (abc), Renou J.-P. (abc)
(a) INRA, UMR1345 Institut de Recherche en Horticulture et Semences, SFR 4207 QUASAV Angers, France; (b)
AgroCampus-Ouest, UMR1345 Institut de Recherche en Horticulture et Semences, F-49045 Angers, France; (c) Université
d’Angers, UMR1345 Institut de Recherche en Horticulture and Semences, F-49071 Beaucouzé, France.
Fruit maturation involves many physiological and biochemical changes including cell wall modifications. Some
cultivars show undesirable ripening process leading to mealiness, which is characterized by texture deterioration
resulting in soft, grainy and dry fruit.
Based on sensorial analysis, six genotypes were divided into mealy and unmealy fruits. Establishment of
mealiness was monitored over four time points during fruit maturation and conservation. Microscopic analysis
revealed a progressive loss of cell to cell adhesion, while biochemical analysis pointed out specific alteration of
pectin in mealy fruit. To understand molecular bases of mealiness development, we used a newly developed
microarray chip containing 120 000 probes specific to both sense and antisense transcripts. Several genes were
identified as potential key regulators including one PME. Interestingly WAK antisense transcripts were also
modified, which might indicate novel regulatory mechanisms through PTGS of these potential signaling genes.
Using this multidisciplinary approach, we suggest an original model to partially explain the development of
mealiness in apple.
P6-41
Generation of a Trichoderma reesei Mutant that Produces Higher Levels of Cell Wall-Degrading
Enzymes
Takeda T., Takahashi M., Nakano Y.
Iwate Biotechnology Research Center, 22-174-4 Narita Kitakami Iwate 024-0003, Japan.
Cellulose, composed of β-1,4 linked glucosyl units, is the most abundant naturally produced biopolymer on earth,
and can be utilized as a sustainable and renewable energy resource in place of fossil fuel. Establishing conditions
for the efficient degradation of cellulose will contribute to the enhanced use of bioethanol, a biobased alternative
to gasoline, that will enhance biomass recycling and reduce carbon dioxide emissions.
The genus Trichoderma comprises species of filamentous fungi that secrete high levels of cell wall-degrading
enzymes including endoglucanases, cellobiohydrolases, β-glucosidases and xylanases. The combined actions of
these enzymes result in a synergistic increase in the efficiency of substrate hydrolysis. Plant cell wall is mainly
composed of β-glucans, which can be converted into glucose by the actions of hydrolytic enzymes. Increasing the
productivity of hydrolytic enzymes with high specific activities through biotechnological modification will lead to
the efficient glucose production from plant cell wall components. To enhance the ability to degrade cell wall
components by the T. reesei hydrolytic enzymes, we first introduced β-glucosidase gene (Mocel3A) of
Magnaporthe oryzae and then induced gene mutations by UV irradiation. The transformant T. reesei
overexpressing Mocel3A showed higher activity toward pNPG and cellobiose, indicating the increase in βglucosidase activity. Furthermore, a mutant T. reesei made from the transformant by UV irradiation exhibited
increased levels of glucose production from oligosaccharides and plant cell wall components compared with the
transformant. Analyses of proteins secreted by the T. reesei strains indicated that a mutant T. reesei produced
higher levels of hydrolytic enzymes. Our goal is to identify a gene that causes to enhance the production of
hydrolytic enzymes.
Author index
A
Abdel-Massih R.M.
Abramson M.
Acebes J.L.
Acet T.
Adler S.
Adriaensen D.
Afif D.
Ageeva M.A.
Aguié-Béghin V.
Ahad A.
Ahl L.I.
Aigner N.
Albenne C.
Albornos L.
Allison G.G.
Alonso-Simón A.
Alvarado C.
Álvarez J.M.
Amakorova P.
Amanda D.
Amanda D.-C.
Amorim M.I.
Amos R.
Anderson C.T.
André A.
Andreani F.
Antelme S.
Aoki S.
Araújo C.
Arnaud C.
Arnould O.
Assor C.
Assoumou Ndong Y.
Atanassov I.
Atkinson R.
Atmodjo M.A.
Attwood G.
Atwell S.
Auer M.
Augier L.
Avci U.
Awano T.
Azzam F.
Bacete L.
182
52
79, 102, 161
23
61
128
134, 147
77
53, 78, 163
162
119, 181
44
6, 117, 162
126
63, 141
102
106, 176
102
35
39
16
34, 115
16
21
169
148
33, 156
77
171
143
163, 165
141, 163, 165
93, 129
88
91, 101
16, 170
169
39
63, 116
133
60, 72, 168
25, 91, 92
191
B
Baba K.
Babcock N.C.
77
190
Bacic A.
Badel E.
Bain M.
Baldwin L.
Balestrini R.
Balzergue S.
Baratiny D.
Barbacci A.
Bardor M.
Barrière Y.
Barron C.
Barros-Rios J.
Barry K.
Bartel X.
Bartley L.E.
Bashline L.
Bastianelli E.
Basu D.
Batth T.S.
Baydoun E.
Bayer M.
Beahan C.T.
Bellincampi D.
Bellini C.
Ben-Tov D.
Bendahmane A.
Benedetti M.
Bensussan M.
Berger A.
Berthet S.
Berthold F.
Bessa F.
Besser K.
Beury N.
Binder B.
Biot E.
Bird S.
Biswal A.K.
Bittebière Y.
Bizot H.
Blacklock L.
Blanc N.F.
Blancaflor E.B.
Blanco M.F.
Blanco-Portales R.
Blervacq A.S.
38
15, 16, 39, 86, 96,
98, 146, 167
176
15
131, 181
140
135
82
42, 165
120
50, 187
8, 52, 62
52, 132, 181
168
38, 133
157
19
189
22, 156
109
182
183
15
36, 124, 189
35
142
33, 135
148
143
30
87
25, 180
171
190
176, 191
147
86
177, 187
16
67, 71
178
88
64, 159
75
14
135
135, 155
Blokhina O.
Boerjan W.
Boizot N.
Bolam D.
Bolger A.
Bolin J.T.
Bonfante P.
Bonnin E.
Bonnot C.
Boron A.K.
Bosch M.
Bou Daher F.
Bouchabké-Coussa O.
Bouchet B.
Bouchez O.
Boudaoud A.
Bouton S.
Boutrot F.
Bouvier d’Yvoire M.
Brandizzi F.
Breton C.
Bringmann M.
Bromley J.R.
Brown D.
Bruce N.
Bruenig L.
Brummell D.
Bruneau M.
Buanafina M.M. de O.
Buffetto F.
Bulone V.
Burgert I.
Burlat V.
Burr S.
Burton R.A.
Busse-Wicher M.
99
47, 65, 111, 177,
192
141, 166
159, 180
162
4
140
50, 68, 69, 72, 105,
191
143
128
51, 60
42
33, 87
58
171
125
35, 92, 12
36, 38, 139
156
17
160, 169
125
7, 104, 170
7
190
21
101
193
136
70, 80
47, 100, 108, 180
10, 44
106, 164
78
45, 90, 117
7
C
Cabané M.
Calderan-Rodrigues M.J.
Camargo E.
Cannesan M.A.
Caparrós-Ruiz D.
Capellades M.
Capron I.
Cardenas C.L.
Carocha V.
Carpita N.C.
134, 147
164
170
146
161, 175
175
178
72
166, 170, 171
4, 131, 167, 190
Carr P.
Cass C.
Cassan-Wang H.
Cassin A.
Cassland P.
Catchmark J.
Catena M.
Cathala B.
Causse M.
Celton J.-M.
Cervone F.
Cézard L.
Chabbert B.
Chabi M.
Chabout S.
Chang S.-S.
Chantreau M.
Charalampopolous D.
Chartrin A.
Chateigner-Boutin A.-L.
Chazal R.
Chazalet V.
Chebli Y.
Cheng K.
Cherk Lim C.
Cherqui A.
Chiniquy D.
Chong S.-L.
Chormova D.
Chou Y.H.
Chu J.
Cicéron F.
Ciesielski P.
Citerne S.
Clair B.
Clay R.
Clément G.
Clément M.
Coimbra S.
Collin M.
Colombo J.
Comadran J.
Cooke R.
Cornuault V.
Correa J.
Cosgrove D.J.
Costa M.L.
88
51
166
98
177
73, 74, 80
50
163, 176, 178, 191
120
193
38, 124, 129, 148,
189
33, 62
53, 78, 102, 133
135
110
172
102
185
140
58, 106, 156
62
160
42
17
150
130
150, 157
65, 82
6
45
179
160
4
61, 176
172
60
138
143
34, 115, 149
144
44
183
168
59, 80
116
8, 9, 21, 70, 80
34, 115, 149
Costa R.
Cottyn B.
Courtial A.
Cousin F.
Coutand C.
Cragg S.
Creff A.
Crépeau M.-J.
Crowell E.F.
Crowley M.
Czjzek M.
51
82
187
191
145
190
143
30, 68, 80, 174
86
4
121
D
Dalessandro G.
Dalmais M.
Dalüge N.
Daly P.
Dammak A.
Daoud Z.
Dardelle F.
Davis J.K.
Davis M.
Davis R.H.
Day A.
de Beer A.
De Caroli M.
de Castro M.B.
De Lorenzo G.
De Tender C.
Debolt S.
Decker S.R.
Déjardin A.
Del Gado E.
del Rocio Cisneros Castillo L.
Delhomme N.
Demailly H.
Demont-Caulet N.
Dempere A.
Demura T.
Denance N.
Derba-Maceluch M.
Derbyshire P.
Deschamps S.
Deschesne A.
Desnos T.
Desprez T.
Devaux M.-F.
Dewhirst R.A.
Deyholos M.K.
90
33, 156
5
184
191
182
14, 120
44, 154
190
72
133, 135, 160
148
90
79, 102, 161
38, 148, 189
150
174
190
140, 141, 145, 166
44
6
149
35, 93
82
54
89
38
25, 65, 82
95
143
171
143
86
50, 62, 176
88
89, 96, 105, 133
Di Matteo A.
Díaz-Moreno S.M.
Dima O.
Dinesen M.
Doblin M.S.
Dolstra O.
Domon J.-M.
Domozych D.S.
Doñas D.
Dong R.
Dong W.
Dopico B.
Douché T.
Douglas C.J.
Draeger C.
Draga A.
Drakakaki G.
Driouich A.
Ducamp A.
Ducrot P.-H.
Dugard C.K.
Dumas B.
Dumon C.
Dumont M.
Dunand C.
Dupree P.
Durand C.
Durand S.
Durand-Tardif M.
148
100, 108
47
177
15, 16, 86, 96, 146
171, 178
76, 105, 130, 131,
135
20, 28, 46, 115,
119, 164
14
72
75
126, 127
164
29, 112
43
180
108
30, 98, 120, 139,
146, 148
143
82
190
137, 185
137, 185
87
106
7, 14, 24, 25, 40,
46, 64, 66, 71, 97,
159
139, 146
11, 62
135, 143
E
Ebert B.
Eborall W.
Eeckhout S.
Eggert D.
Ehrhardt D.
Eichenberger C.
Ekengren S.
El-Khatib S.
Elias L.
Ellinger D.
Ellis B.
Encina A.
Endler A.
Endo S.
14, 101, 128, 168
190
114
5
19
43
100
182
190
37, 191
25
79, 175, 102
32, 107
131
Endres S.
Escudero V.
Esquivel-Rodriguez J.
Estephan M.-B.
Estevez J.M.
Ezcurra I.
15
38
105
78
22
25
F
Fabri E.
Faccio A.
Fagerstedt K.
Fagerström A.
Faik A.
Fan B.
Fang L.
Fangel J.U.
Farrant J.
Federici L.
Felekis D.
Felten J.
Fernandez-Tendero E.
Ferrari S.
Ferrarini A.
Ferrigno P.
Fevereiro P.
Ficko-Blean E.
Filley T.R.
Fimognari L.
Fincher G.B.
Fliniaux O.
Fock-Bastide I.
Fode B.
Follet-Gueye M.-L.
Fontaine J.-X.
Fooyontphanich K.
Fornalé S.
Foulon L.
Fournet F.
Fowler M.
Fragostefanakis S.
Francin-Allami M.
Francocci F.
Francoz E.
Franková L.
Fratzl P.
Frazier R.
Freeman J.
Freitas A.T.
124
140
99
144, 157
22, 156
43
74
28, 119, 140, 144,
157, 177
30
148
43
149
133
129, 189
129
166
166
121
190
39, 132
45, 117
130, 135
61
23
146
127, 130, 135
144
132, 175
53
92, 93, 98, 127,
129, 131
190
142
106, 162
189
106
28
10
185
24
171
Fry S.C.
Fu J.
Fukuda M.
Fukuhara Y.
Fullerton C.
Funke N.
6, 28, 79, 84, 85,
88, 90, 99, 118
7
111
111
91
145
G
Gaillard C.
Galindo L.
Gamblin D.
Gamboa de Buen A.
García L.
García-Angulo P.
García-Gago J.A.
Garnier C.
Garrote G.
Gaulin E.
Geitmann A.
Gendrot G.
Georgelis N.
Geserick C.
Gheysen G.
Ghosh S.
Gianzo C.
Gibson G.
Gidley M.
Gigli-Bisceglia N.
Gilbert H.J.
Gille S.
Gillet F.
Giuliani A.
Gloazzo Dorsz J.
Glöckner A.
Goacher R.E.
Goeminne G.
Goffner D.
Gomez L.D.
Gondolf V.M.
Gonneau M.
González Fernández-Niño S.
Gorshkova T.A.
Gorzsás A.
Gotté M.
Goubet F.
Gouider L.
Goulas E.
Gourcilleau D.
81
96
190
117
161, 175
79, 102, 161
135
69
181
137, 185
42
87
21
15
65
4
94, 110
185
81
38
70, 159
17
130, 131, 135
71
22
37
61
192
38, 133
177, 180, 183, 184,
187, 188, 190
101
86, 176
64, 167
73, 77, 105
149
139
189
191
135, 160
140
Grabber J.H.
Graça C.
Graff L.
Grec S.
Greiner S.
Gretz M.
Greve M.
Grienenberger E.
Griffiths J.S.
Grima-Pettenati J.
Gritsch C.
Griveau Y.
Gro Rydahl M.
Grossniklaus U.
Gruppen H.
Grussu D.
Gu F.
Gu J.
Gu Y.
Guedes F.T.P.
Guénin S.
Guérineau F.
Guerriero G.
Guiderdoni E.
Guillebaux A.
Guillon F.
Günl M.
Gutierrez L.
51
171
129
102, 135
37
90
14
112
5
100, 166, 170, 171
24
187
80, 109
43
10, 186, 187
183, 188
108
73
19, 32, 85, 97
141
35, 98
105, 129
180
87
143
8, 11, 50, 58, 62,
70, 80, 106, 162,
176
66, 162, 186
35, 93
Harding S.E.
Harholt J.
Harpaz-Saad S.
Harris P.
Hashimoto-Sugimoto M.
Haughn G.W.
Have Lund C.
132
90
155
38, 60, 72, 133,
156, 168
44, 68, 84, 154
91, 101
182, 183, 184, 188
36, 139
40, 125
53
167
94
155
116
16, 64
111
33, 35
Höfte H.
H
Hadj-Boussada K.
Haeger A.
Häggman H.
Hahn M.G.
Haigler C.H.
Hallett I.
Halpin C.
Hamann T.
Hamant O.
Hambardzumyan A.
Han K.-H.
Handford M.
Hano C.
Hansen B. Ø.
Hao Z.
Hara H.
Hara-Nishimura I.
Hawkins S.
Hayashi T.
Heazlewood J.L.
Hefer C.
Heikkinen S.L.
Heilmann I.
Held M.A.
Hernandez-Gomez M.C.
Hernández-Nistal J.
Hernandez-Sanchez A.M.
Hervé C.
Higaki A.
Hijazi M.
Himmel M.E.
Himmeldirk K.
Hishiyama S.
Ho Y.Y.
Ho-Yue-Kuang S.
Hobson N.
Hocq L.
Hoffmann L.
Hofmann Larsen F.
Holland C.
Holman H.-Y.
Hongo S.
Hooper M.
Horie S.
Howe K.J.
Huang J.H.
Huang S.-C.
Hugo A.
Hyung-Rae K.
79
28, 111, 119, 177
142
169
35
5, 107, 162
170
102, 133, 135, 155,
160
77
14, 50, 64, 101, 167
100, 166, 170
53
20
17, 22, 75, 190
74, 189
126
146
29, 120
93
6
4
22
111
15, 86
156
89
93
117
177
37, 86, 93, 109,
110, 128, 129, 138,
176
85
64
35
183, 184
137
164
192
9, 80
144
105
I
Iba K.
Ibragimova N.N.
Ilc T.
Imai T.
Ingram G.
35
77
15
151
39
Inoue H.
Irar S.
Islam A.
Itakura M.
Iurlaro A.
Ivakov A.
Ivory R.
Iyer P.R.
Izquierdo L.
134
161
78
77
90
125, 145
180
136
126, 127
J
Jaber R.
Jacobsen N.
Jam M.
Jamet E.
Jamrow T.
Jantasuriyarat C.
Jean B.
Jensen J.K.
Johansen I.E.
Johnson K.
Johnson N.
Johnston S.L.
Jönsson L.
Joo M.
Jordá L.
Jørgensen B.
Jose-Estanyol M.
Jouanin L.
Joubert C.
Jung H.-J.G.
139
119
29
6, 105, 117, 160,
162, 164
191
144
191
23
28
39, 96
23
101
65
109
38
111, 177
118
33, 82, 87, 111, 156
119
52
K
Kaida R.
Kajita S.
Kalaitzis P.
Kallas Å.
Kam J.
Kamimura N.
Kamitakahara H.
Kaneko S.
Kärkönen A.
Karlen S.
Karp A.
Katayama Y.
Kato K.
Kaya M.
Keegstra K.
Kelly W.
77
111
138, 142
25
183, 188
111
158
71
99, 155
51
24
111
89
174
97
169
Kern M.
Kesten C.
Kido N.
Kieber J.J.
Kiefer-Meyer M.-C.
Kieliszewski M.
Kiemle S.
Kiemle S.N.
Kihara D.
Kiharaf D.
Kim H.
Kim H.R.
Kim J.
Kim J.-Y.
Kim S.-J.
Kim S.H.
Kim W.-C.
Kirby A.R.
Kitazawa K.
Klepadlo M.
Klie S.
Kliebenstein D.
Klimek J.F.
Knox J.P.
Ko J.-H.
Koh P.L.
Kolbeck A.
Kondo M.
Konishi M.
Koo B.-W.
Kortstee A.
Kosik O.
Kotake T.
Koutaniemi S.
Kovensky J.
Kozlova L.V.
Kračun S.K.
Krishnamoorthy P.
Kroeger J.
Krol M.
Kubicki J.D.
Kumar M.
Kunieda T.
Kusumi K.
Kusunose T.
L
190
32, 107
137
142
98
22, 75
90
70
105
4
51, 72
4
147
167
17
9
167
136
71
138
154
39
131, 190
29, 35, 43, 59, 60,
63, 74, 80, 95, 120,
189
167
98
37
33, 35
35
9
192
25, 185
71
82
76
77
109
20
42
159
84, 161
88
33, 35
35
111
L’Enfant M.
Labate C.
Labrador E.
Ladouce N.
Lafitte C.
Lahaye M.
Lai V.
Lainé V.
Lakehal A.
Lakhal W.
Lamport D.T.A.
Lampugnani E.R.
Landrein B.
Lao J.
Lapidot S.
Lapierre C.
Largo A.
Largo-Gosens A.
Larre C.
Larson E.R.
Larsson T.
Lathe R.
Lau J.
Laurans F.
Laval K.
Le Gall H.
Le Gall S.
Le Moigne M.-A.
Le-Bris P.
Le-Lam-Thuy Phan J.
Leal L.
Lebris P.
Lee C.M.
Lee K.
Lee K.J.D.
Lefebvre V.
Legay S.
Léger F.
Leggio C.
Legros S.
Lehner A.
Lei L.
Leijdekkersa M.
Leijon F.
Lejeune-Hénaut I.
Lemoine J.
Lenucci M.S.
76, 105
164
126, 127
100, 171
137, 185
42, 67, 72, 76, 165
90
166
138
166
34
96, 98, 146
125
50, 167
52
33, 50, 62, 63, 82,
87, 111, 147, 156,
176, 184
161
102
58, 106, 162
20
180
125
193
140, 141, 166, 193
146
130, 135
67
29
87
117
171
111
9
35
59
93
187
33, 62
148
133
87, 98, 120
32, 85, 97
187
180
131
71
90
Leplé J.-C.
Lerouge P.
Leroux C.
Leroux O.
Lerouxel O.
Leru A.
Lesage-Descauses M.-C.
Lesniewska J.
Levesque-Tremblay G.
Li L.
Li S.
Li Z.
Liang Y.
Liepman A.
Lilley K.S.
Lin F.
Lindeboom J.
Ling C.
Lionetti V.
Lipchinsky A.
Liu L.
Liu Q.
Liu X.
Liu Y.
Livingstone M.
Liwanag A.J.M.
Llewellyn D.J.
Lloyd C.
Lollier V.
Looten R.
Lopez-Fernandez F.
Lopez-Sanchez P.
Loqué D.
Loudet O.
Love J.
Lovegrove A.
Lu F.
Lucau-Danila A.
Lucenius J.
Lund C.H.
Lunn J.
Lyon J.
87, 140, 141, 145,
166
87, 93, 98, 105,
120, 129, 146, 169
98
63, 114
160
106
140, 141, 145, 166
149
107
165
19, 32, 85, 97
138
22, 156
90
46
80, 157
19
54
36, 124, 189
157, 163
17
104
22
112
15
101
104
95
162
50, 176
97
81
50, 101, 193
30
149
24, 59, 185
51
135
25
104
103
184
M
Maaheimo H.
Madhavi Latha G.
Maes L.
Magama F.
82
65
143
182
Magnenet V.
Makowski L.
Malm E.
Maluk M.
Malvar R.A.
Malyon G.
Manabe Y.
Manfield I.
Mangan D.
Manisseri C.
Mansfield S.D.
Mansoori N.Z
Mansouri K.
Maranas J.K.
Marcello P.
Marchant A.
Marco Y.
Mareck A.
Marek A.A.
Margueijo V.
Marion-Poll A.
Markakis M.N.
Marriott P.E.
Marsaud N.
Martens H.J.
Marti L.
Martín I.
Martin M.
Martinez T.
Martinez Y.
Masai E.
Masiero S.
Master E.R.
Matsuda K.
Mazel J.
Mazumder K.
McCann M.C.
McCleary B.
McConville M.
McCormick H.
McGeehan J.
McKenna J.
McNair G.R.
McQueen Mason S.J.
Mechin V.
Medvedev S.S.
Meineke T.
42
4
47
184
52, 132
190
39
59
180
18, 37, 157
4, 25, 51, 72, 89,
107
86, 107
84
9, 43, 80
92, 93, 129
124
38, 133
93, 105, 129
158
177
30
128
188
171
39
38
126, 127
176
137, 185
106
111
34, 149
61
89
87
60
190
180
86
60
190
36, 139
4
177, 180, 184, 183,
187, 188, 190
187
163
191
Mélida H.
Mellerowicz E.J.
Melzer M.
Ménard D.
Merah K.
Mercado J.A.
Mesnard F.
Metzger T.H.
Meulewaeter F.
Meyerowitz E.
Meynard D.
Miart F.
Michel G.
Miedes E.
Mikkelsen D.
Mikkelsen M.D.
Mikkonen K.S.
Mikol S.
Mikolajek H.
Mikshina P.V.
Milani P.
Millet N.
Mitchell R.A.C.
Mitsuda N.
Miyoshi Y.
Moerschbacher B.
Mohanty S.S.
Mohnen D.
Mokshina N.E.
Molina A.
Molinari M.
Molinié R.
Moller I.E.
Mollet J.-C.
Moneo M.
Mongelard G.
Moore J.P.
Moquet F.
Morcillo F.
Moreau C.
Moreno A.
Moreno I.
Moriwaki K.
Morreel K.
Morris G.A.
Morris R.
Morris V.J.
Mort A.
100, 102, 180
25, 65, 82, 92, 149
180
95
162
135, 136
127, 130, 135
10
74, 189
40
87
109, 127
121
36, 38, 133
81
28, 119
53
193
124
73, 77
40
140, 145, 166
24
179
151
10
16
16, 170
105
36, 38, 133
78
130
98
14, 87, 98, 120
126, 127
35
30, 119, 144, 148
143
144
176, 191
14
14
35
47
69, 79
45
136
7
Mortimer J.C.
Mosier N.S.
Mouille G.
Mravec J.
Muller K.
Muñoz-Blanco J.
Muranaka T.
Mutić J.
Mutwil M.
Myburg A.
7, 14, 40, 64, 66,
159
190
35, 61, 63, 93, 110,
128, 129, 138, 143
105, 109, 181
107
135
111
68
116
100, 166, 170
N
Nafisi M.
Nakano T.
Nakano Y.
Nakashima J.
Naramoto S.
Narciso J.O.
Naumann M.
Naylor D.
Negi J.
Nelson B.J.
Neutelings G.
Newbigin E.
Ng A.
Nguema-Ona E.
Nguyen Phan C.T.
Nickolov K.
Nielsen E.
Nikolic J.
Nikolovski N.
Nishimura M.
Nishitani K.
Nobre S.
Noirot M.
Nonaka M.
North H.
Novak O.
Nussaume L.
39, 132
35
194
75
131
96
5, 18, 37
17
35
43
135, 155
96, 98
64
30, 139, 144, 146
84
155
108
68
7, 46
33, 35
33, 35, 137
34, 149
61
77
30
35
143
O
O’Donohue M.
O’Rourke C.
Oakey H.
Obayashi H.
Ochs J.
Ociepa P.
Ohme-Takagi M.
Oikawa A.
137, 185
28, 118
183, 184
77
46
124
179
128
Okahisa-Kobayashi Y.
Okazawa A.
Olek A.T.
Oliveira J.
Ordas B.
Orellana A.
Orfila C.
Orsel M.
63
111
4
171
181
14, 101
125, 179
193
P
Padmakshan D.
Paës G.
Pageau K.
Paiva J.A.P.
Palmer R.
Pandey J.L.
Paniagua C.
Park E.
Park J.Y.
Park S.
Park Y.B.
Parsons H.T.
Patil P.
Pattathil S.
Patterson S.
Pau-Roblot C.
Paul L.N.
Pauly M.
Pavel N.V.
Pavlovic M.
Peaucelle A.
Peck M.L.
Pedersen H.L.
Pellny T.K.
Pelloux J.
Penning B.W.
Pera S.
Pereira A.M.
Pereira L.G.
Perera C.
Péret B.
Persson S.
Pesquet E.
Petersen P.D.
Petrakis N.
Petrascu I.
51
78
92, 127
166, 170, 171
95
161
135, 136
108
51
9
9, 80
109
7
60, 156, 168
147
76, 130
4
17
148
162
93
157
109
24
35, 76, 92, 93, 98,
105, 129, 130, 131,
135
190
171
149
34
91
143
19, 20, 32, 107,
116, 125, 145, 154
24, 95
193
138
16
Petrova A.A.
Petti C.
Petzold C.J.
Pielach A.
Pilate G.
Pinto Paiva J.
Pinzon D.
Piro G.
Pizot M.
Plancot B.
Plasencia A.
Pogorelko G.
Pollet B.
Pomar F.
Popper Z.A.
Posé S.
Poulain D.
Pouliquen C.
Prakash R.
Prashant Mohan-Anupama P.
Preis I.
Puigdomènech P.
73
174
109, 167
141
140, 141, 145, 166
100
96
90
144
139, 146
100
36, 45
147
102
63, 114, 115, 141
136
30
67
101
65
52
118
Q
Quéméner B.
Quesada M.A.
Quignard F.
Quilhó T.
Quilichini T.D.
72, 76, 105
135, 136
172
171
29
R
Raad M.
Raath P.
Raggi S.
Raimundo S.
Raiola A.
Rajasundaram D.
Ralet M.-C.
Ralph J.
Ralton J.
Ranocha P.
Rastall R.
Ratke C.
Rautengarten C.
Ray S.
Rayon C.
Reboul R.
Reimer R.
Remoroza C.
142
119
129
114
124
154
30, 58, 70, 71, 80,
105, 174
51, 72
86
38, 106, 133
185
25
14, 101
72
4, 76, 105, 129,
130, 131, 135
15
5
10
Ren Y.
Renou J.-P.
Revilla G.
Reymond M.
Ricardi M.M.
Ricci E.J.
Richard T.L.
Richet N.
Rigau J.
Rihouey C.
Ringli C.
Riviére M.-P.
Roach M.J.
Robert P.
Roberts A.W.
Roberts E.
Rodrigues J.C.
Rogier O.
Rogniaux H.
Rogowsky P.
Rolando C.
Romaní A.
Ronald P.C.
Rondeau-Mouro C.
Roongsattham P.
Ropartz D.
Rose J.K.C.
Rouau X.
Roujol D.
Rubtsov D.
Rüggeberg M.
Ruiz-May E.
Runavot J.-L.
Ruprecht C.
Rustérucci C.
39
193
94, 110
187
22
68
161
134, 145, 147
175
120
43
38, 133
89
62
68, 84, 90
68
171
140
11, 67, 71, 106, 162
87
135, 160
181
50, 64, 157, 167
8
144
11, 30, 70, 71
164
50
6, 117
46, 66, 159
10, 149
164
189
116, 145
130
S
Sabatier L.
Sadowski P.
Saeys Y.
Saez-Aguayo S.
Saha P.
Saito C. Fukuda H.
Sakamoto S.
Sakata Y.
Sakuragi Y.
163
46
47
30
157
131
179
77
39, 104, 132, 170
Saland E.
Salgado Salter J.
Sallé C.
Sampedro J.
Samuels L.
San Clemente H.
Sánchez-Rodríguez C.
Sánchez-Vallet A.
Santiago R.
Sarkar P.
Sato K.
Saulnier L.
Savatin D.V.
Savelli B.
Saxe F.
Scavuzzo-Duggan T.
Schaffer R.
Schaller A.
Scharf R.
Scheller H.V.
Schjørring J.K.
Schnorr K.
Schols H.A.
Schröder R.
Schroeder J.I.
Schuetz M.
Schultink A.
Seal C.E.
Sedbrook J.
Sedeek K.E.M.
Segonzac C.
Seifert G.J.
Selbig J.
Sellier H.
Sénéchal F
Serimaa R.
Sethaphong L.
Shafiei R.
Sharma V.
Sharova E.I.
Shewry P.R.
Shilton S.
Shimada T.
Shirley N.J.
Shliaha P.
Shoseyov O.
Showalter A.M.
6
22
30
94, 110
4, 25, 29
100, 170, 171
19, 20, 38, 133
38, 133
52, 132
63, 116
35, 111
8, 11, 24, 50, 62
38
170
10
84
91
129
125
14, 39, 50, 64, 101,
104, 128, 157, 168,
193
181
190
10, 186, 187, 192
91, 101
35
25
17
34
51
142
36
23, 159
154
131
93, 105, 129
25
84, 154
183, 184
50, 64
163
24, 59, 95, 185
177
33, 35
117
46
52
22, 156
Shreve J.
Sibout R.
Sicilia F.
Sillo F.
Simister R.
Simmons T.J.
Slabaugh E.
Smirnov A.I.
Smith B.
Smith R.
Smith R.S.
Smith-Moritz A.M.
Smyth K.
Soler M.
Somerville C.
Somerville S.
Sopeña S.
Sorek N.
Sørensen I.
Sørensen S.O.
Sorieul M.
Sormani R.
Soulhat C.
Sousa A.G.
Sousa C.
Sparks J.A.
Springer N.M.
Sprunck S.
Srinastava A.
Srivastava V.
Steadham J.
Steenkeste K.
Stein A.
Steinschauer V.
Stenbæk A.
Stephens J.
Stinzi A.
Stokes J.R.
Stonebloom S.
Stoppel R.
Stott K.
Stranne M.
Streeter S.
Strnad M.
Suliman M.
Sun L.
Sundberg B.
190
33, 87, 111, 135,
156, 162, 176, 188
148
140
177, 183, 184
99
84
154, 158, 165
91
25
43
64, 167
124
100, 166, 170
18
150
38
18
30, 164
174, 175
66
110
33
174, 175
115
75
190
149
54
47, 100
45
69
90
159
104, 170
182, 184, 188
129
81
101, 168
101, 128
64
39, 132
190
35
106
58
10, 149
Sundin L.
Sundqvist G.
Suraninpong P.
Surcouf O.
Suslov D.
Suurs L.
Sykes R.W.
Szemenyei H.
192
47, 180
144
93, 105, 129
145
192
190
18
T
Tabuchi A.
Takabe K.
Takabe K.
Takahashi M.
Takahashi-Schmidt J.
Takeda T.
Takeuchi M.
Takeuchi R.
Talbot M.
Tamura M.
Tan L.
Tanaka R.
Tang Y.
Tateishi A.
Tateno M.
Temple H.
Ten E.
Tenhaken R.
Tenkanen M.
Thannhauser T.W.
Thibaud M.-C.
Thimmapuram J.
Thomasset B.
Thompson D.S.
Tian L.
Tierney M.L.
Tietze A.A.
Timpano H.
Tobimatsu Y.
Tokarski C.
Tony P.
Torode T.
Torres A.F.
Tosi P.
Tozo K.
Tran J.
Tranbarger T.J.
Tranquet O.
Trebus R.
21
91
92, 93, 103, 158
151, 194
25, 99
151, 194
141
134
104
111
16
92
75
134
174
14, 101
54
15
53, 65, 82
164
143
190
127
78
186
20
45
86, 176
51
135, 160
61
29, 120
171, 178
24, 59, 95
147
30
144
80
103
Trindade L.M.
Tryfona T.
Tsai A.Y.-L.
Tsuji Y.
Tsumuraya Y.
Tsuyama T.
Tucker M.R.
Tuomainen P.
Turbant A.
Turner S.
Turumtay H.
Tzfadia O.
86, 107, 171, 178,
192
7, 24, 64, 66, 71, 97
61
111
71
103
117
82
92
88
65, 111
142
U
Uehara I.
Ulvskov P.
Umezu S.
Usadel B.
Utz D.
Uzrek N.
77
28, 111, 119, 177
7
66, 67, 103, 162,
186
94
183, 184
V
Vain T.
Väisänen E.
Valdivia E.R.
Valot B.
van Cutsem P.
Van Damme E.J.M.
van de Meene A.M.L.
Van de Wouwer D.
van der Does D.
van der Meene A.
Van Der Straeten D.
van der Weijde T.
Van der Weijde T.
Van Hove J.
Van Orden J.
Van Wuytswinkel O.
Vandenbussche F.
Vanholme B.
Vanholme R.
Várnai P.
Vasilevski A.
Vázquez-Lobo A.
Vega-Sanchez M.E.
Velasquez S.M.
Veličković D.
Venetos A.
Verbelen J.-P.
86
99
94, 110
164
109
150
16
65
36, 139
15
145
171
178
150
128
92, 127
145
47, 65, 111
47, 177, 192
34
67, 162
117
50, 64, 157, 167
22
11
156
128
Verdeil J.L.
Verger S.
Verhertbruggen Y.
Vermerris W.
Vernhettes S.
Verrascina I.
Vetharaniam I.
Viane R.L.L.
Vicré-Gibouin M.
Vidal Melgosa S.
Videcoq P.
Vigouroux J.
Vilaplana F.
Virkki L.
Vissenberg K.
Visser R.G.F.
Vivier M.A.
Vogler H.
Voigt C.A.
Voiniciuc C.
Voinov M.A.
Vouillot C.
Voxeur A.
Voynov M.A.
144
110, 138
64, 109
54
86, 176
189
169
114
30, 139, 146
109
42, 69, 165
62, 72, 141
100
82
128, 145
171, 178, 192
119, 144, 148
43
5, 18, 37, 191
5, 66, 186
158
125
6, 87, 169
154, 165
W
Wadouachi A.
Wagenknecht M.
Wagner E.
Wagner T.
Waldron K.W.
Wallace I.
Wan Y.
Wang H.
Wang Y.
Ward J.
Wattier C.
Watts H.D.
Waugh R.
Weber A.
Webster G.
Weil C.F.
Weiller F.
Werner J.M.
Werweries M.
Whale E.
Wightman R.
Wilkerson C.G.
Wilkinson M.D.
76
10
21
97
135
19
24
100, 170
33, 87
59
130
161
183, 184
43
59
190
148
124
170
177
40
17, 23, 51
24, 59
Willats W.G.T.
Williams B.
Wilson S.M.
Wilson Y.
Winter H.T.
Winters A.
Withers S.
Wolf S.
Worden N.
Wormit A.
28, 30, 80, 109,
114, 119, 140, 144,
157, 177, 181
157
15, 16, 90, 98
184
178
51
51
37
108
103
X
Xiao C.
Xiong G.
Xu W-L.
Xue H.
Xue J.
21
17
156
23
60
Y
Yamaguchi M.
Yamashita T.
Yanagisawa S.
Yang B.
Yang F.
Yasukawa C.
Yennawar N.
Ying R.
Yingling Y.G.
Yokoyama R.
York W.S.
Yoshida K.
Yoshimi Y.
Yoshimoto K.
Yoshinaga A.
Yoshioka K.
Yoshiura K.
Young P.
Young Z.
Yu G.
Yu H.
Yu X.
89
151
35
67
193
77
21
8
44, 84, 154
35, 137
60
151, 179
71
134
93, 158
151
91
148
36
119
100
64
Z
Zabotina O.
Zaki M.S.
Zarra I.
Zemelis S.
Zeng W.
Zerzour R.
36, 45
142
94, 110
17
15, 96
42
Zhang S.
Zhang S.-L.
Zhang T.
Zhang X.J.
Zhang Z.
Zhou J.
Zhou Y.
164
119
8, 9
70
64
15
58
Zhu Y.
Ziebell A.
Zimmer J.
Zipfel C.
Zivy M.
Zwikowics C.
Zwirek M.
51
190
44
36, 38, 139
164
191
188
Table of contents
Abstracts from oral presentations listed by session and order of talks...............................................................1
Session 1 : Plant Cell Wall Architecture; Structure, Interactions & Cross-Links of CW Components.................3
CesA dimers are the fundamental building blocks of a plant cellulose synthase complex................................................................................................................4
Cellulose ultrastructure is influenced by the GPI-anchored protein COBL4.......................................................................................................................................4
Structure and biochemistry of (1,3)-β-glucan....................................................................................................................................................................................5
Cellulose and an AGP play distinct roles in mediating the adherence of seed coat mucilage...........................................................................................................5
AGP31, an arabinogalactan protein, as a network-forming protein in A. thaliana cell walls..............................................................................................................7
Wall glycoproteins and membrane glycolipids act as ‘chaperones’ promoting rhamnogalacturonan-II-borate cross-linking............................................................7
Half of the xyloglucan in cell walls of tissue cultures is linked to pectin via a highly branched arabinan.........................................................................................8
Distinct domains of glucuronoxylan may create interfaces for interaction with cellulose and lignin................................................................................................8
The impact of arabinoxylan structure on the properties of cell walls in wheat grain endosperm.....................................................................................................9
Direct visualization of polysaccharide interactions in plant primary cell walls by AFM.....................................................................................................................9
Monitoring large-scale ordering of cellulose in intact plant cell walls using sum frequency generation (SFG) spectroscopy..........................................................10
Determining the structure of cellulose and hemicellulose in the primary plant cell wall using small angle neutron scattering [SANS].........................................10
Orientation analysis of cellulose in complex plant tissues by X-ray diffraction...............................................................................................................................11
Modification of sugar beet pectin by pectin acetylesterases and structural elucidation of the modified pectins by novel LC-MS approaches...............................11
MALDI mass spectrometry imaging (MSI) as a new and promising method to investigate polysaccharides in wheat grains..........................................................12
Session 2 : Dynamics of plant CW Components : from Biosynthesis to Remodelling.........................................14
Nucleotide sugar transporters are involved in the biosynthesis of rhamnose- and arabinose-containing cell wall polysaccharides..............................................15
Characterization of a Arabidopsis UMP / UDP-Gal, UDP-Rha and UDP-Ara antiporter family...........................................................................................................15
Genetic analysis of de novo and salvage pathways of nucleotide sugars for plant cell walls.........................................................................................................16
Investigation of the mechanism of (1,3;1,4)-β-D-glucan biosynthesis and assembly in Poaceae spp.............................................................................................16
Sub-cellular-location of the Cellulose Synthase-Like CslF and CslH genes in members of the Poaceae..........................................................................................17
Pectin as a domain of cell wall polysaccharides and proteoglycans and biological function of pectin biosynthetic GAUT1 and the GAUT gene family.................17
The wall polysaccharide O-acetylation machinery...........................................................................................................................................................................18
Homogalacturonan biosynthesis and methylesterification in Arabidopsis depend on a family of plant-specific Golgi-associated proteins....................................18
Phosphorylation as a key mechanism in (1,3)-β-glucan biosynthesis..............................................................................................................................................19
Control of cellulose deposition: COBRA vs. MONGOOSE..................................................................................................................................................................19
Internalization of cellulose synthase complexes is mediated via clathrin-based endocytosis.........................................................................................................20
Regulation of cellulose synthesis through clathrin-mediated endocytosis......................................................................................................................................20
Phosphoinositides regulate cell wall deposition in Arabidopsis leaves............................................................................................................................................21
Root hair cell wall organization is sensitive to the expression of VTI13, a SNARE required for trans-Golgi network function in Arabidopsis..................................21
Expansin interactions with plant cell walls......................................................................................................................................................................................22
Activation tag screening reveals the functions of polygalacturonases in cell wall expansion and plant development...................................................................22
New molecular players of the glyco-network control the polarized growth at a single plant cell level...........................................................................................23
Functional identification of a hydroxyproline-O-galactosyltransferase specific for arabinogalactan-protein biosynthesis in Arabidopsis......................................23
Cellular and genetic dissection of FASCICLIN LIKE ARABINOGALACTAN PROTEIN4.........................................................................................................................24
Transcriptional regulation of xylan biosynthesis.............................................................................................................................................................................24
Differences and similarities of xylan synthesis in dicots and grasses.............................................................................................................................................25
Non-cell autonomous post-mortem lignification of xylem vessels...................................................................................................................................................25
Lignin deposition in xylem relies on non-lignifying neighbouring cells............................................................................................................................................26
PtxtXyn10A regulates cellulose microfibril angle in aspen wood.....................................................................................................................................................26
Session 3 : Evolution & Diversity of plant CW..................................................................................................28
The algal origins of hemicellulose biosynthesis...............................................................................................................................................................................29
Novel hemicellulose-remodeling transglycanases from charophytes: towards the evolution of the land-plant cell wall................................................................29
Cell-wall deposition during asymmetric cell division in the brown algal fucoid zygote...................................................................................................................30
Building the pollen wall - Interplay of biosynthetic pathways, ABC transporters, and sporopollenin assembly..............................................................................30
Natural variation in the polysaccharide components of Arabidopsis seed coat epidermal cells......................................................................................................31
Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls against desiccation.....................................................................31
Session 4 : Functions of Plant CW in planta......................................................................................................32
The IC proteins; new players of the cellulose synthase complex.....................................................................................................................................................33
Dissecting the molecular mechanism underlying intimate relationship between cellulose microfibrils and cortical microtubules.................................................33
Spatiotemporal secretion of PEROXIDASE36 responsible for seed coat mucilage extrusion in Arabidopsis thaliana......................................................................34
Identification of laccases involved in lignin formation in Brachypodium distachyon.......................................................................................................................34
Periplasmic AGP-Ca2+ is an essential component of the calcium oscillator that regulates plant growth and development..........................................................35
AGP6 and AGP11 biological mode of action in Arabidopsis pollen and pollen tube growth.............................................................................................................35
Disruption of PME activity alters hormone homeostasis and triggers compensatory mechanisms controlling adventitious rooting..............................................36
Versatile roles of PME and pectin in plant development..................................................................................................................................................................36
The Arabidopsis LRR-RLK LRI is a novel regulator of cell wall integrity sensing..............................................................................................................................37
New aspects of cell wall integrity signalling revealed through post-synthetic modifications..........................................................................................................37
A receptor protein links cell wall surveillance with brassinosteroid signalling.................................................................................................................................38
The Rab GTPase SMG1 and its effector PMR4 are involved in signaling cascade and the regulation of CWI in plants....................................................................38
The triple mitogen-activated protein kinases ANPs regulate immunity triggered by oligogalacturonides and orchestrate signaling and ROS formation in
mitochondria, plastids and nucleus.................................................................................................................................................................................................39
SignWALLing: Signals derived from Arabidopsis cell wall activate specific resistance to pathogens..............................................................................................39
Cell wall acetylation is important for surface permeability and resistance to Botrytis cinerea.......................................................................................................40
The role of DEFECTIVE KERNEL1 (DEK1) in mechano-sensitive growth of plant cells......................................................................................................................40
The molecular basis of cell wall stiffness at the shoot apical meristem: a specific role of the CSLD gene family...........................................................................41
Session 5 : Advanced Understanding of CW Structure, Biosynthesis & Function..............................................42
Another brick in the [cell] wall.........................................................................................................................................................................................................43
Regulation of plant cell growth through cell wall mechanics...........................................................................................................................................................43
Analysis of mechanical properties of cell walls by MEMS-based Cellular Force Microscopy............................................................................................................44
Coarse-grained simulation of cell wall load-bearing network structure...........................................................................................................................................44
Modeling mesoscale structure and mechanics of the secondary wood cell wall.............................................................................................................................45
Structural comparison of the cytosolic domain of cellulose synthases in bacteria and plants........................................................................................................45
New insights into xyloglucan biosynthesis: functional organization of a multi-enzyme complex....................................................................................................46
The role of genes from the cellulose synthase superfamily in xylan biosynthesis..........................................................................................................................46
The plant Golgi apparatus – insights from quantitative proteomics................................................................................................................................................48
Membrane trafficking dynamics during cell wall development, polar expansion and cell adhesion in the model unicellular green alga, Penium margaritaceum 48
Quantitative proteomics reveals that plant plasma membrane microdomains are involved in molecular transport, stress responses and callose biosynthesis. .49
Candidate Substrate-Product Pairs (CSPP): structural characterization of plant metabolites and its relevance for lignin pathway engineering............................49
Session 6 : Uses of plant CW and derived products...........................................................................................51
Dry separation of ground maize stems provides fractions with distinct enzymatic degradation patterns......................................................................................52
Increasing C6 cell wall sugar content by engineering the accumulation of a low recalcitrance polysaccharide in plants...............................................................52
Cell-wall compositional analysis of the energy crop Miscanthus as a means to optimise its application as a feedstock for bioenergy and biorefining.................53
Altering plant lignins for improved processing................................................................................................................................................................................53
Modifications of cell wall properties by production of recombinant resilin composites in transgenic plants...................................................................................54
Effect of cell wall diferuloylation on agronomic fitness and silage quality in maize........................................................................................................................54
Wood and cereal hemicelluloses as future bio-based oxygen barrier materials..............................................................................................................................55
New ultra-thin nanostructured films based of lignin and cellulose with variable optical properties................................................................................................55
Development of flexible lignin nanotubes for the smart delivery of therapeutic agents.................................................................................................................56
Abstracts from poster presentations listed by session and order of posters......................................................57
Session 1 : Plant Cell Wall Architecture; Structure, Interactions & Cross-Links of CW Components...............59
Isolation and Structural Characterisation of Rhamnogalacturonan in Ginseng Roots......................................................................................................................60
Revisiting the cell wall of the wheat endosperm.............................................................................................................................................................................60
New light on the structure and function of the wheat arabinogalactan peptide..............................................................................................................................61
Epitope detection chromatography: a novel methodology to dissect the complexity of plant cell walls........................................................................................61
In situ analysis of cell wall polysaccharides in stems of Miscanthus species...................................................................................................................................62
Characterization of monoclonal antibodies against xylan epitopes.................................................................................................................................................62
Towards a New Screen for Detecting Changes in Plant Cell Wall Composition and Structure Using Time-of-Flight Secondary Ion Mass Spectrometry.................63
Effect of the mutation laurina on the monosaccharide composition of cell walls in Coffea Arabica seeds from the fertilization to the harvest.............................63
FTIR monitoring of cell wall compositional changes in maize stems at different developmental stages.........................................................................................64
Lignin structural analysis by Raman spectroscopy: from purified molecules to plant cell walls......................................................................................................64
Exploring architectural cell wall heterogeneity: celery collenchyma as case study........................................................................................................................65
Cell wall composition changes with maturation of bamboo Phyllostachys bambusoides................................................................................................................65
The Arabidopsis primary cell wall contains an unusual xylan, which is synthesised by a small set of glycosyltransferases...........................................................66
Compositional and structural characterization of cslf6 rice mutant................................................................................................................................................66
Understanding the role of O-acetylated matrix polysaccharides in woody plants...........................................................................................................................67
Modifying plant cell walls by expression of cell wall degrading enzymes........................................................................................................................................67
Structural characterisation of grass glucuronoarabinoxylan...........................................................................................................................................................68
Cell wall analysis of barley stems in modern cultivars, landraces and wild varieties......................................................................................................................68
The screening of cell wall polysaccharides composition and structure in plant collections.............................................................................................................69
Application of polysaccharide analysis using carbohydrate gel electrophoresis (PACE) for detecting novel mucilage mutants.....................................................69
Freeze fracture electron microscopy provides evidence for a cuticular layer on the surface of leaves of the moss Physcomitrella patens...................................70
Diffusion of molecular probes in cell wall mimicking matrixes........................................................................................................................................................70
Size, conformation and interactions of polysaccharides..................................................................................................................................................................71
Plant and fungal Pectin Methylesterase diffusion in pectin gels : a multiscale approach................................................................................................................71
New insight into fine structure of red wine RGII..............................................................................................................................................................................72
What is the conformation of MLG in the grass cell wall?.................................................................................................................................................................72
Deciphering the structure of oligosaccharides by a new tandem mass spectrometry method based on photo-activation in the VUV range.................................73
Specific interaction of β-galactosyl Yariv reagent with β-1,3-galactan............................................................................................................................................73
Lignin-directed monoclonal antibodies............................................................................................................................................................................................74
Hemicellulose fine structure in apple as investigated by enzymatic profiling.................................................................................................................................74
Spatial structure of rhamnogalacturonans I....................................................................................................................................................................................75
Adsorption of cell wall polysaccharides to model cellulose substrates............................................................................................................................................75
The effect of water-soluble polysaccharides on enzymatic digestibility and crystal orientation of Gluconacetobacter xylinus cellulose.......................................76
Cell walls and cotton fibre development..........................................................................................................................................................................................76
Long term exposure to microgravity triggers the downregulation of cell wall related genes and corresponding modifications in cell wall architecture in
Arabidopsis roots.............................................................................................................................................................................................................................77
Identification of the pI 4.6 extensin peroxidase gene in tomato.....................................................................................................................................................77
Structural characterisation of xyloglucans using electrospray ionisation mass spectrometry........................................................................................................78
Specific inhibition of pectin remodelling enzymes...........................................................................................................................................................................78
Movements of radiocesium and radioiodine in forest trees.............................................................................................................................................................79
Maize glucuronoarabinoxylans taking part in elongation growth....................................................................................................................................................79
Interactions between cell wall hydration and mechanical behaviour in plant cell walls and bacterial cellulose composites..........................................................80
Modelling progression of fluorescent probes in bio-inspired lignocellulosic assemblies..................................................................................................................80
Hydrodynamics characterisation of plant cell wall polysaccharides and their assemblies - Protein-like oligomerisation of carbohydrates....................................81
The biosynthesis and molecular weight distribution of hemicelluloses in cellulose-deficient maize cells: an example of metabolic plasticity..............................81
Water: the forgotten solvent...........................................................................................................................................................................................................82
Production and fine characterisation of new monoclonal antibodies against rhamnogalacturonan I..............................................................................................82
What’s new in the microscopy observation of the mature wheat ultrastructure.............................................................................................................................83
Molecular interactions and mechanical properties of bacterial cellulose composites.....................................................................................................................83
In vivo and in vitro polymerization of lignin by plant laccases........................................................................................................................................................84
Structural study of O-acetyl-glucuronoxylan in Arabidopsis thaliana reveals spatial distribution of acetyl substituents................................................................84
Session 2 : Dynamics of plant CW Components : from Biosynthesis to Remodelling.........................................85
Genetic and computational testing of the function of the region between cellulose synthase (CESA) transmembrane helices (TMH) 5 and 6 in Arabidopsis
thaliana............................................................................................................................................................................................................................................86
An XET activating factor (XAF) in plant cells....................................................................................................................................................................................86
Transglycanase activity detected with pectic polysaccharides: a novel α-arabinan : xyloglucan endotransglycosylase (AXE) activity ?.......................................88
Functional Analysis of Complexes with Mixed Primary and Secondary CESAs................................................................................................................................88
The Arabidopsis Cellulase KORRIGAN1 associates with the Cellulose Synthase Complex...............................................................................................................89
Identification of potential protein and/or lipid components involved in (1,3;1,4)-β-D-glucan biosynthesis in Italian ryegrass (Lolium multiflorum) suspensioncultured cells (SCCs)........................................................................................................................................................................................................................89
Role of rhamnogalacturonan-II in pollen germination and pollen tube elongation..........................................................................................................................90
MiR397 overexpression, a strategy to engineer laccase activity and lignification in model plants and in crop plants and trees...................................................90
The reaction of vitamin C with reactive oxygen species in the apoplast.........................................................................................................................................91
Role of IRX3 cysteine modifications in the function of the cellulose synthase complex (CSC)........................................................................................................91
Fasciclin-like Arabinogalactan Proteins: Influence of FLA mis-expression on cell wall biochemistry and biomechanics in flax and poplar.....................................92
VND-INTERACTING PROTEIN2 is controlled by ubiquitin-mediated proteolysis................................................................................................................................92
Heat stress differentially affects XET activity in organs of durum wheat plantlets..........................................................................................................................93
Mixed-linkage glucan biosynthesis in Physcomitrella patens expressing a CSLH gene from rice....................................................................................................93
Regulation of kiwifruit softening by differential cell wall modifications...........................................................................................................................................94
Temporal deposition of β-1,4-galactan in the early stage of the development of Poplar gelatinous layer......................................................................................94
Activity, localization and function of xylanases in differentiating poplar xylem..............................................................................................................................95
Pectins demethylesterification and seed development...................................................................................................................................................................95
A role for pectin methylesterases (PMEs) in dark-grown hypocotyl elongation in Arabidopsis........................................................................................................96
Xylan deposition and lignification in differentiating xylem in Mallotus japonicus under tension stress..........................................................................................96
Xyloglucan β-glucosidase in Arabidopsis.........................................................................................................................................................................................97
Characterisation of Nucleotide Sugar Transporters in Grapevine (Vitis vinifera L.).........................................................................................................................97
Relationship between microtubules and secondary cell wall in xylem vessels................................................................................................................................98
Cell walls in developing wheat and rice grains................................................................................................................................................................................98
The Tale of GalT14 and its possible role in AGP glycan biosynthesis in Arabidopsis thaliana.........................................................................................................99
Pectin-related gene expression in flax (Linum usitatissimum): role of pectinesterases (PME) in bast fiber development..............................................................99
Fucosylation of Arabidopsis arabinogalactan proteins...................................................................................................................................................................100
Cellulose Synthase Interactive 3 Coordinates with Cellulose Synthase Interactive 1 in the regulation of Cellulose Biosynthesis................................................100
A GT47 family glycosyl transferase from Nicotiana pollen mediates the synthesis of (1,5)-α-L-arabinan when expressed in Arabidopsis thaliana.....................101
AtPME48 encodes a pectin methylesterase involved in Arabidopsis pollen grain germination.....................................................................................................101
Plasma membranes of developing xylem of Norway spruce in monolignol transport studies.......................................................................................................102
Insights into the site of cleavage by GH12 and GH16 enzymes acting on poaceaen xyloglucan..................................................................................................102
Functional characterization of a CELLULOSE SYNTHASE-LIKE B (CSLB) protein from Populus trichocarpa....................................................................................103
New Transcription factors regulating lignified secondary cell wall formation in Eucalyptus.........................................................................................................103
Transglycosylases in softening fruit..............................................................................................................................................................................................104
Biosynthesis of pectic galactan: glycosyltransferases and UDP-galactose transporters. .............................................................................................................104
Identification of lignin mutants by forward and reverse genetics in a flax EMS population..........................................................................................................105
Maize primary cell walls accumulate a lignin-like polymer in response to a lacking on cellulose.................................................................................................105
Trehalose-6-Phosphate - part of the regulatory mechanism coordinating cellulose biosynthesis with carbon metabolism?........................................................106
Uncovering protein-protein interactions in glucuronoxylan biosynthesis......................................................................................................................................107
Pectin methylesterase and pectin remodelling differ in the fibre walls of two Gossypium species with very different fibre properties.......................................107
Biochemical characterization of an Arabidopsis pectin methylesterase AtPME3 and a pectin methylesterase inhibitor..............................................................108
Cell wall of gelatinous type: gene expression analysis to study development and function.........................................................................................................108
Focus on cell wall biogenesis in wheat grain using sub-cellular proteomic approach ..................................................................................................................109
A cell wall peroxidase required for correct mucilage release in Arabidopsis seed coat................................................................................................................109
ICs and their Interactors – new Cellulose Synthesis related Proteins............................................................................................................................................110
Identification and analysis of the highly esterified mucilage mutant defective in seed mucilage and embryo development.......................................................110
Endomembrane Trafficking and Polysaccharide Deposition..........................................................................................................................................................111
The Role of CSLD Proteins During Polarized Cell Wall Deposition in Arabidopsis..........................................................................................................................111
Investigating cell wall biosynthesis in the secretory pathway using Free Flow Electrophoresis....................................................................................................112
Reciprocal small-molecule probes localize polyionic structural carbohydrates in plant and fungal cell walls ..............................................................................112
Inducible overexpression of the transcription factor SWN5 is sufficient to activate secondary cell wall synthesis in Brachypodium...........................................113
Characterization of suppressors of cell adhesion defective mutants.............................................................................................................................................113
Analysis of transgenic Arabidopsis thaliana with pinoresinol reductase gene derived from a soil bacterium, Sphingobium sp. SYK-6........................................114
Expression of xylanase in lignin mutants......................................................................................................................................................................................114
VASCULAR-RELATED UNKNOWN PROTEIN 1 regulates secondary wall formation via hormone regulatory pathways in Arabidopsis...........................................115
Investigation of a KNAT7-BLH-OFP transcription factor complex involved in regulation of secondary cell wall biosynthesis in Arabidopsis thaliana...................115
Session 3 : Evolution & Diversity of plant CW................................................................................................116
Arabinogalactan proteins in seaweeds: taxonomic and seasonal variation...................................................................................................................................117
Ceratopteris richardii (C-fern): a model for investigating adaptive modification of vascular plant cell walls................................................................................117
WallEvo: a comprehensive online resource for plant cell wall evolution studies...........................................................................................................................118
Distribution of arabinogalactan proteins and pectins in cork oak female flower...........................................................................................................................118
Structural organization of plant cell walls is fairly conserved through evolution...........................................................................................................................119
Evolutionary transcriptomic study of cell wall related genes using RNA-seq and genome-wide phylogenetic analysis................................................................119
Xylans in Plantago Species............................................................................................................................................................................................................120
The DUF642 cell wall proteins: a new family of plant carbohydrate binding proteins ?................................................................................................................120
New cell wall polysaccharides in charophytic algae......................................................................................................................................................................121
Presence of HRGPs and CM8 proteins in Cucurbitacea plant genomes.........................................................................................................................................121
High-throughput cell wall profiling of ripening in wine grapes (cv. Cabernet sauvignon) versus table grapes (cv. Crimson seedless) reveals differences in
polymer abundance.......................................................................................................................................................................................................................122
Kingdom-wide plant cell wall metaglycomics................................................................................................................................................................................122
The cell wall of tobacco and tomato pollen tubes contains fucosylated xyloglucan not found in somatic cells............................................................................123
Monoclonal antibodies for the dissection of sulphated fucans in cell walls of brown algae...........................................................................................................123
Enzymes Involved in the Processing of Coastal Algal Biomass......................................................................................................................................................124
Session 4 : Functions of Plant CW in planta....................................................................................................126
Transgenic Expression of Pectin Methylesterase Inhibitors in Arabidopsis and Tobacco limits Tobamovirus spreading...............................................................127
WallNet - Exploring the Biosynthesis and Function of Rhamnogalacturonan II During Root Development in Arabidopsis............................................................127
Twisting the golden angle: Arabidopsis phyllotaxis depends on cellulose synthase guidance......................................................................................................128
Cell wall microstructure of potato cortex tissue and correlations with bruise susceptibility upon harvest and storage...............................................................128
βIII-gal is involved in galactan reduction during phloem element differentiation in chickpea stems.............................................................................................129
Knockout mutants of arabidopsis thaliana β-galactosidase (subfamily a1). modifications in their cell wall polysacharides and enzymatic activities..................129
Characterization of transgenic arabidopsis plants overexpressing βIII-gal from Cicer arietinum..................................................................................................130
Analysis of the interactions between mucilage composition and fatty acid content in flaxseed...................................................................................................130
Receptor-like kinases involved in feedback regulation from the secondary plant cell wall...........................................................................................................131
Identification of 2 extensin-related proteins and their involvement in elongation of Arabidopsis thaliana cells...........................................................................131
An Arabidopsis thaliana class III peroxidase is involved in the regulation of cell expansion.........................................................................................................132
Regulation of pectin methylesterases (PMEs) by subtilases (SBTs) and pectin methylesterases inhibitors (PMEIs) during root growth.......................................132
The impact of Arabidopsis ecotype on aphid feeding involves cell wall differences......................................................................................................................133
Cell wall metabolism of Miscanthus ecotypes in response to cold acclimation.............................................................................................................................133
Cold acclimation induces cell wall modifications during pea plant development..........................................................................................................................134
A comprehensive analysis of secondary cell wall formation-associated genes.............................................................................................................................134
Suppressor screening of the REDUCED WALL ACETYLATION 2 mutant of Arabidopsis thaliana....................................................................................................135
Changes in Cell Wall-Bound Hydroxycinnamates in Maize Stems Infested with Sesamia nonagrioides Lefèbvre.........................................................................135
Remodeling Arabidopsis cell wall uncouples resistance to pathogens from tradeoffs...................................................................................................................136
Agricultural conditions affect bast fiber formation and cell wall development in hemp ...............................................................................................................136
Complementation of a family 3 α-L-arabinofuranosidase/β-xylosidase repression by the increase of family 51 isozyme expression..........................................137
Cellulose biosynthesis is altered in wood of poplar subjected to climate change.........................................................................................................................137
Nostresswall: novel information on the effect of drought stress on the cell wall..........................................................................................................................138
Evaluation of the role of the β-Galactosidase gene Faβgal4 on strawberry fruit softening...........................................................................................................138
Significance of grass cell wall feruloylation for wall growth and sugar remobilization..................................................................................................................139
Nanostructural differences in pectic polymers isolated from strawberry fruits with low expression levels of pectate lyase or polygalacturonase genes...........139
Biotechnological Potential of Carbohydrate Binding Modules from Oomycetes............................................................................................................................140
Role of cell wall matrix polysaccharides in rice growth and development....................................................................................................................................140
Link between C/N imbalance perception and plant Cell Wall biosynthesis....................................................................................................................................141
Arabidopsis Thaliana P4H3 is involved in anoxia tolerance...........................................................................................................................................................141
The role of cell wall components of root border cells and border-like cells in root protection.......................................................................................................142
The role of receptor like kinases in Arabidopsis plant cell wall integrity maintenance..................................................................................................................142
Understanding cell wall re-modelling during the symbiotic interaction between the ectomycorrhizal fungus Tuber melanosporum and Corylus avellana.........143
Genome-wide characterisation of mechanical stress responsive miRNAs from xylem tissues of different poplar genotypes.......................................................143
Lignin as a contributor to virulence rather than defence ? — novel insights from parasitic plant haustoria.................................................................................144
Biochemical and histochemical characterization of the polysaccharides present in the G-layer of wild type and AGP-modified transgenic poplars...................144
COBRA-LIKE 2 regulates cellulose deposition in Arabidopsis seed coat mucilage.........................................................................................................................145
VIGS-induced Silencing of three putative tomato prolyl 4-hydroxylases causes alterations in plant growth................................................................................145
Dissection of the root growth response to phosphate starvation in Arabidopsis thaliana.............................................................................................................146
Xylem development and composition as a target to breed for better performances under drought ?..........................................................................................146
Cellular originalities of the oil palm fruit abscission zone and abscission process........................................................................................................................147
Overexpression of the grapevine PGIP1 in tobacco results in remodeling of the leaf arabinoxyloglucan network in the absence of fungal infection.................147
Identification of miRNA/targets and signalization networks linked to gravitropism in poplar........................................................................................................148
Cell wall-related aspects of the effect of brassinosteroids on Arabidopsis hypocotyl gravitropism...............................................................................................148
Characterisation of two putative galactosyl transferases potentially involved in arabinogalactan-protein biosynthesis..............................................................149
Arabinogalactan proteins of border cells: at the frontier between root and microbes..................................................................................................................149
Different roles of lignification in leaves and stems of poplars subjected to abiotic stresses.........................................................................................................150
Role of jasmonic acid during floral organ abscission and seedling growth....................................................................................................................................150
Anti-fungal peptides are produced in Heliophila coronopifolia (Brassicaceae) root border-like cells and associate with pectin-rich mucilage as a preformed
defence mechanism......................................................................................................................................................................................................................151
Comparative analysis of the complexes formed by PGIP-2 from P. vulgaris and four fungal polygalacturonases by Small Angle X-Ray Scattering (SAXS) and sitedirected mutagenesis....................................................................................................................................................................................................................151
The ethylene precursor ACC stimulates G-layer formation in fibers of hybrid aspen....................................................................................................................152
Unravelling the function and expression pattern of Arabinogalactan Proteins in the reproductive tissues of Arabidopsis thaliana.............................................152
Plant stress signaling: The Arabidopsis EUL protein interacts with fasciclin-like arabinogalactan proteins..................................................................................153
The Powdery Mildew Resistance5 (PMR5) gene: the intersection of cell wall synthesis and disease resistance...........................................................................153
Degradation and synthesis of β-glucans by a Magnaporthe oryzae endotransglucosylase...........................................................................................................154
Session 5 : Advanced Understanding of CW Structure, Biosynthesis & Function............................................156
Transmembrane structural organization of plant cellulose synthase proteins..............................................................................................................................157
Integrative analysis of Transcriptome, Translatome and Proteome data from root cell types of Arabidopsis thaliana.................................................................157
The N-Terminus of PaRBOH1 from Picea abies is a Phosphorylation Target..................................................................................................................................158
Genome-wide atlas of lignin and lignan genes in the fibre crop Linum usitassimum....................................................................................................................158
Identification and study of Brachypodium distachyon COMT TILLING mutants.............................................................................................................................160
Biochemical and physiological characterization of fut4 and fut6 mutants defective in arabinogalactan-protein fucosylation in Arabidopsis..............................160
A model for cell wall loosening via defect migration in cellulose microfibrils................................................................................................................................161
Identification of Grass Cell Wall Synthesis Genes from Correlations between Gene Expression and Cell Wall Composition in Rice.............................................161
Spin-probe EPR Study of Local Polarity and Viscosity of Cotton Cellulose Nanodomains and the effects of Solvents...................................................................162
Distribution of coniferin in differentiating xylems of normal and compression woods using MALDI imaging mass spectrometry................................................162
Regulatory mechanisms of UDP-glucose 4-epimerase isoforms....................................................................................................................................................163
Analytical tools to follow in vitro xylan synthesis..........................................................................................................................................................................163
Purification of a truncated Δ68-AtFUT1 expressed in Hi5 insect cells for crystallographic study..................................................................................................164
Identification of cell wall proteins in flax.......................................................................................................................................................................................164
Deciphering the Golgi proteome in cellulose-deficient cells..........................................................................................................................................................165
Computational study of lignin-protein non-covalent interactions..................................................................................................................................................165
Screening for Arabidopsis seed coat mutants using an EMS population.......................................................................................................................................166
Identification of proteins involved in cell wall assembly and remodeling by a proteomic approach on Brachypodium distachyon grain.....................................166
Elastic properties of the growth-controlling outer cell walls of maize coleoptile epidermis..........................................................................................................167
Tensile test of plant cell wall analogs thin films using image stereocorrelation............................................................................................................................167
Profiling of the Plant Cell Wall Proteome: Structural Diversity and Evolutionary Origins of Plant N-Glycoproteins.......................................................................168
Specific features of cell wall proteomes of monocots: a focus on Brachypodium distachyon as a model plant and on sugarcane as a source of biomass for
cellulosic bioethanol......................................................................................................................................................................................................................168
Contribution of hemicellulose network to plant cell wall properties..............................................................................................................................................169
Membrane Assembly and Interactions of Individual CesA Transmebrane Helices by Site-directed Spin-labelling EPR.................................................................169
Genome-wide studies of multigene families involved in Eucalyptus phenylpropanoid metabolism and lignin branch pathway...................................................170
Both ChIP-SEQ and in planta gene modification indicate a function of PtaMYB221in lignin biosynthesis and secondary cell wall formation in poplar................170
Isolation of Golgi proteomes from grasses....................................................................................................................................................................................171
MYB46-mediated transcriptional regulation of secondary wall biosynthesis in plants..................................................................................................................171
Cell Wall Glycan Dynamics During Primary to Secondary Wall Development in Poplar Stems......................................................................................................172
Transcriptomic identification of arabinogalactan protein polysaccharide biosynthetic enzymes..................................................................................................172
A 3-D model of the grass primary cell wall and its enzymatic degradation...................................................................................................................................173
Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches..................173
Elucidation of a protein interaction network in homogalacturonan biosynthesis..........................................................................................................................174
The Eucalyptus grandis R2R3-MYB transcription factor family......................................................................................................................................................174
Cell wall diversity in forage maize: biochemical properties and genetic complexity.....................................................................................................................175
Transcriptomic dynamics during tension wood differentiation in Eucalyptus globulus.................................................................................................................175
Mesoporosity in developing wood cell walls: a new step towards understanding of maturation stress generation in trees.........................................................176
Session 6 : Uses of plant CW and derived products.........................................................................................177
Structure of Pectin in Citrus Fruits and Functionality.....................................................................................................................................................................178
A Lignocellulosic analysis of Setaria viridis L. a developing model for the panoicoideae clade C4 grasses..................................................................................178
Possible implications of RGI side chains on HM pectin gelling capabilities....................................................................................................................................179
Modifying the phenylpropanoid flux in C3’H-RNAi maize plants....................................................................................................................................................179
Innovative materials for the visual detection of xylanase.............................................................................................................................................................180
spa1, a Brachypodium cell wall mutant with a high yield of sacharification suitable for biofuel production.................................................................................180
Screening of sugars and phenolics released during pretreatment of miscanthus, maize and sugar cane bagasse for potential added value products from c4
crops..............................................................................................................................................................................................................................................181
Preparing nano-cellulose for high-performance composites..........................................................................................................................................................181
The effect of maize cell wall composition on the optimization of dilute-acid pretreatments and enzymatic saccharification.......................................................182
Plant cell wall bioinspired foam derived from cellulose nanocrystals stabilized Pickering emulsions...........................................................................................182
Role of the cell wall in maintaining tissue integrity in canned navy beans...................................................................................................................................183
Modification of dicot secondary cell wall formation by monocot NST transcription factor............................................................................................................183
The effect of carbohydrate-binding modules (CBMs) on plant cell wall properties: an in vivo approach......................................................................................184
A new substrate for measurement of cellulase (endo-1,4-beta-glucanase)..................................................................................................................................184
Cell wall composition in wheat straw: Interactions with nitrogen status.......................................................................................................................................185
Bioethanol production from hydrothermally pretreated stover biomass in maize genotypes.......................................................................................................185
Antibacterial activity of pectin against Staphyloccocus aureus and Escherichia coli clinical isolates...........................................................................................186
Generating barley plants with modified straw by suppressing HCT and C3H................................................................................................................................186
Phenotyping barley straw for agronomic and biofuel-related traits...............................................................................................................................................187
eQTL Analysis on Spring Barley using RNA sequencing.................................................................................................................................................................187
Analysis of the cinnamyl alcohol dehydrogenase (CAD) gene family and downregulation of HvCAD2 gene by RNAi in barley (Hordeum vulgare).....................188
Reversing Forward: Genome-Wide Mutation Screening for Improved Saccharification Efficiency in Barley..................................................................................188
High value functional products from DDGS...................................................................................................................................................................................189
Biotechnological Potential of Carbohydrate Binding Modules from Oomycetes............................................................................................................................189
The effect of functional carbohydrates on the intestinal tract of broiler chickens and pigs..........................................................................................................190
Screening a barley multi-parent population: Searching for cell wall traits relevant for bioconversion in commercially bred barely lines....................................190
Identification of QTL for saccharification in maize stover..............................................................................................................................................................191
A promising biorefinery approach for sugar beet pulp, alternative to its use for biofuel production............................................................................................191
Manipulation of lignin 4CL and CCR genes to improve biofuel production from cereal straw.......................................................................................................192
Identifying novel genes to improve lignocellulosic biomass for biofuel applications.....................................................................................................................192
Impact of textile processing on cotton fibre polysaccharide composition.....................................................................................................................................193
De-esterified homogalacturonan negatively affects saccharification efficiency............................................................................................................................193
Mechanisms and enzymes for lignocellulose deconstruction from a marine wood borer..............................................................................................................194
Genetic analyses of the maize nested association mapping population reveal quantitative trait loci and candidate genes contributing to phenylpropanoid
abundance or saccharification yield..............................................................................................................................................................................................194
Biomimetic thin films model of plant cell wall for enzymatic hydrolysis studies...........................................................................................................................195
Identification of efficient glucanases and their application in marine biomass fermentation........................................................................................................195
Effects of pectin composition on cooking properties of potato tubers...........................................................................................................................................196
Characterization of genes involved in phenylopropanoid and lignin biosynthesis: towards improving cell walls for biofuels.......................................................196
Modifying Secondary Cell Wall Biosynthesis to Develop Improved Bioenergy Crops....................................................................................................................197
Genome wide analysis reveals WAK and PME implications in textural default of apple fruit.........................................................................................................197
Generation of a Trichoderma reesei Mutant that Produces Higher Levels of Cell Wall-Degrading Enzymes.................................................................................198
Author index....................................................................................................................................................199
Table of contents.............................................................................................................................................213