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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 βgalactosidase 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. 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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