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Acknowledgements:<br />
Institut National de la Recherche<br />
Agronomique – INRA<br />
• Centre de Versailles-Grignon<br />
• Département Santé des Plantes et<br />
Environnement (SPE)<br />
• Département de Biologie<br />
Végétale (BV)<br />
• Département Caractérisation et<br />
Elaboration des Produits Issus de<br />
l’Agriculture (CEPIA)<br />
Région Ile-de-France<br />
« action financée par la Région Ile-de-<br />
France »<br />
DIM ASTREA<br />
« Agrosciences Territoires Ecologie<br />
Alimentation »<br />
IFR 87<br />
« La plante et son environnement »<br />
SFBV<br />
Société Française de Biologie Végétale<br />
EFOR<br />
Réseau d'Etudes Fonctionnelles sur les<br />
Organismes modèles<br />
1
1 st European Brachypodium Workshop<br />
19-21 October 2011<br />
Versailles-France<br />
Organizing committee<br />
Oumaya Bouchabké-Coussa (INRA, IJPB)<br />
Corine Enard (INRA, IJPB)<br />
Martine Gonneau (INRA, IJPB)<br />
Lise Jouanin (INRA, IJPB)<br />
Maria Jesus Lacruz (INRA, Versailles)<br />
Thierry Marcel (INRA, BIOGER-CPP)<br />
Jocelyne Picart (INRA, IJPB)<br />
Philippe Porée (INRA, Versailles)<br />
Stéphane Raude (INRA, Versailles)<br />
Richard Sibout (INRA, IJPB)<br />
Anne-Sophie Simon (INRA, Versailles)<br />
Maryvonne Thomas (INRA, Versailles)<br />
Scientific committee<br />
Dr. Herman Höfte (INRA, IJPB)<br />
Dr. Thierry C. Marcel (INRA, BIOGER-CPP)<br />
Dr. Jérôme Salse (INRA, GDEC)<br />
Dr. Arp Schnittger (CNRS, IBMP)<br />
Dr. Richard Sibout (INRA, IJPB)<br />
Dr. Philippe Vain (JIC, GB)<br />
Dr. John P. Vogel (USDA-ARS, USA)<br />
Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech<br />
INRA Centre de Versailles-Grignon<br />
Route de St-Cyr (RD10)<br />
78026 Versailles Cedex France<br />
tél : +33 (0)1 30 83 30 00 fax : +33 (0)1 30 83 33 19<br />
2
Contents<br />
Programme 4<br />
Oral presentations 9<br />
Session 1: Evolution and Natural Variation 10<br />
Session 2: Cell Wall, Biomass and Biofuels 16<br />
Session 3: Tools and Bioengineering 24<br />
Session 4: Biotic and Abiotic Stresses 31<br />
Session 5: Plant Development, Metabolism and Physiology 40<br />
Poster presentations 46<br />
Session 1: Evolution and Natural Variation 47<br />
Session 2: Cell Wall, Biomass and Biofuels 51<br />
Session 3: Tools and Bioengineering 60<br />
Session 4: Biotic and Abiotic Stresses 65<br />
Session 5: Plant Development, Metabolism and Physiology 68<br />
Authors Index 72<br />
List of participants 75<br />
3
PROGRAMME<br />
4
Wednesday 19 th October, 2011<br />
11:00 am to 7:00 pm Opening of Workshop registration<br />
Welcome and opening session<br />
1:00 pm Introductory Remarks<br />
Richard Sibout, INRA (IJPB), and Thierry Marcel, INRA (BIOGER-CPP), France<br />
1:10 Welcome Address<br />
David Bouchez, Director of IJPB, France<br />
1:30 Introductory talk: Dr. John Vogel, USDA-ARS, United-States<br />
Title: Brachypodium rises to a model.<br />
Evolution and Natural Variation<br />
Moderators: Jérome Salse and Richard Sibout<br />
2:00 Key Speaker: Dr. Jérôme Salse, INRA (UBP), France<br />
Title: What can model species offer cereal breeders?<br />
2:40 Pilar Catalan, University of Zaragoza, Spain<br />
Evolution and taxonomic split of the model grass Brachypodium distachyon (L.) P.<br />
Beauv.<br />
3:00 Antoine Peraldi, JIC, United Kingdom<br />
Using Brachypodium distachyon for model to crop translation of disease resistance in<br />
cereals<br />
3:20 pm Coffee Break<br />
4:00 Vibha Srivastava, University of Arkansas, United States<br />
Site-specific recombination systems for genetic modifications of cereals<br />
4:20 Stephanie Suer, Austrian Academy of Sciences, Austria<br />
Functional analysis of WOX4 in mono- and dicotyledonous plants and dissecting the<br />
evolution of vascular development<br />
4:40 David Pacheco-Villalobos, University of Lausanne, Switzerland<br />
Deciphering the function and evolution of BRX-like genes in Brachypodium distachyon<br />
Cell Wall, Biomass and Biofuels<br />
Moderators: Herman Höfte and John Vogel<br />
5:00 Christian A. Voigt, University of Hamburg, Germany<br />
Brachypodium distachyon as a model plant for lignocellulosic ethanol production<br />
5:20 Madeleine Bouvier d'Yvoire, INRA (IJPB), France<br />
Disrupting the cinnamyl alcohol dehydrogenase 1 (BdCAD1) gene leads to altered<br />
lignification and improved saccharification in Brachypodium distachyon<br />
5:40 Emiko Murozuka, University of Copenhagen, Danemark<br />
Identification of Brachypodium mutants defective in silicon uptake<br />
6:00 pm Poster Session<br />
Cocktail offered by Centre INRA Versailles-Grignon<br />
7:30 Departure by bus to centre of Versailles<br />
5
Thursday 20 th October, 2011<br />
Cell Wall, Biomass and Biofuels (followed)<br />
Moderators: Herman Höfte and John Vogel<br />
9:00 Key Speaker: Dr. John Vogel, USDA-ARS, United-States<br />
Title: Natural diversity from genomics to phenomics.<br />
9:40 Samuel Hazen, University of Massachusetts, United-States<br />
Functional characterization of candidate cell wall genes in B. distachyon<br />
10:00 Elisabeth Jamet, CNRS (LRSV), France<br />
Cell wall proteomics of Brachypodium distachyon<br />
10:20 Jozef Mravec, INRA (IJPB), France<br />
The role of pectins in Brachypodium development<br />
10:40 Poppy Marriott, University of York, United Kingdom<br />
Investigating biomass digestibility in Brachypodium<br />
11:00 pm Coffee and ‘viennoiseries’<br />
Tools and Bioengineering<br />
Moderators: Oumaya Bouchabke-Coussa and Philippe Vain<br />
11:40 Key Speaker: Dr. Philippe Vain, JIC, United Kingdom<br />
Title: T-DNA mutagenesis in Brachypodium distachyon.<br />
12:20 Dominika Idziak, University of Silesia, Poland<br />
Current status and future perspectives for molecular cytogenetic studies of<br />
Brachypodium<br />
12:40 Keiichi Mochida, RIKEN BMEP/PSC, Japan<br />
Functional annotation of a full-length Brachypodium cDNA resource<br />
1:00 pm Lunch at INRA Score Services<br />
Tools and Bioengineering (followed)<br />
Moderators: Oumaya Bouchabke-Coussa and Philippe Vain<br />
2:20 Marek Mutwil, Max Planck Institute of Molecular Plant Physiology, Germany<br />
BradiNet: co-expression platform and comparative transcriptomics for Brachypodium<br />
2:40 Richard Poiré, CSIRO Plant Industry, Australia<br />
Phenotyping the shoot and root diversity of Brachypodium distachyon to accelerate<br />
plant biofuel breeding<br />
3:00 Marion Dalmais, INRA (URGV), France<br />
TILLING in Brachypodium distachyon: development of a Sodium Azide mutant<br />
collection and screening for induced point mutations<br />
3:20 Jochen Kumlehn, IPK, Germany<br />
Novel tools for Brachypodium research: Genetic transformation using shoot segments<br />
and immature pollen-derived generation of instantly homozygous lines<br />
3:40 pm Coffee Break<br />
4:20 Round Tables: tba<br />
6:30 Departure by bus to restaurant ‘Maître Kanter’<br />
7:30 pm Dining cocktail at ‘Maître Kanter’ in Versailles<br />
6
Friday 21 st October, 2011<br />
Biotic and Abiotic Stresses<br />
Moderators: Thierry Marcel and Luis Mur<br />
9:00 am Key Speaker: Dr. Luis Mur, Aberystwyth University, United Kingdom<br />
Title: Exploiting Brachypodium distachyon biodiversity to establish sources of resistance<br />
to abiotic and biotic stress.<br />
9:40 Mica Erica, Scuola Superiore Sant’Anna, Italy<br />
Assessing the role of smallRNAs in leaf cell identity and growth reprogramming during<br />
drought stress in Brachypodium distachyon<br />
10 :00 Marie Dufresne, Université Paris-Sud 11, France<br />
Use of the pathosystem Brachypodium distachyon / Fusarium graminearum to<br />
investigate the detoxification mechanisms of mycotoxins by plants.<br />
10:20 Gerhard Adam, University of Natural Resources and Life Sciences, Austria<br />
Functional characterization of Brachypodium UDP-glucosyltransferases<br />
10:40 Aoife O’ Driscoll, University College Dublin, Ireland<br />
The Mycosphaerella graminicola-Brachypodium distachyon interaction: a new model<br />
pathosystem to study Septoria tritici blotch disease of wheat<br />
11:00 pm Coffee and ‘viennoiseries’<br />
11:40 Key Speaker: Dr. David Garvin, USDA-ARS, United-States<br />
Title: Brachypodium as a model for unraveling stem rust resistance.<br />
12:20 Thierry Marcel, INRA (BIOGER-CPP), France<br />
Brachypodium distachyon, a model grass to study plant-pathogen interactions<br />
12:40 Nicola Pecchioni, Università di Modena e Reggio Emilia, Italy<br />
QTLs for resistance to the leaf rust Puccinia brachypodii in the model grass<br />
Brachypodium distachyon<br />
1:00 pm Lunch at INRA Score Services<br />
2:20 Antje Bluemke, University of Hamburg, Germany<br />
Brachypodium distachyon: an excellent model for Fusarium graminearum infection in<br />
wheat<br />
Plant Development, Metabolism and Physiology<br />
Moderator: Arp Schnittger<br />
2:40 Fabienne Guillon, INRA (BIA), France<br />
Brachypodium distachyon grain: development-associated changes in morphology and<br />
storage accumulation<br />
3:00 Virginia González de la Calle, Universidad Politécnica de Madrid, Spain<br />
The transcription factor BdDOF24 interacts with BdGAMYB and regulates the cathepsin<br />
B-like gene during Brachypodium seed germination<br />
3:20 Pierre Delaplace, University of Liège, Belgium<br />
Rhizobacterial volatile organic compounds modulate biomass production and root<br />
architecture in Arabidopsis thaliana (L.) Heynh. and Brachypodium distachyon (L.) P.<br />
Beauv.<br />
3:40 John Doonan, Aberystwyth University, United Kingdom<br />
Using Natural And Induced Variation In Brachypodium To Study Grain Development<br />
4:00 pm Coffee Break<br />
7
4:20 Carl Ng, University College Dublin, Ireland<br />
Profiling the lipidome of Brachypodium distachyon<br />
4:40 Elene R. Valdivia, Universidad de Santiago de Compostela, Spain<br />
Inducible xylem differentiation in Brachypodium<br />
5:00 Closing Address<br />
8
ORAL COMMUNICATIONS<br />
9
Session 1 – Evolution and Natural Variation<br />
KEYNOTE LECTURE<br />
S1.1- What Can Model Species Offer Cereal Breeders<br />
Jerome Salse<br />
Laboratory ‘Plant Paleogenomics for Traits Improvement’, UMR INRA-UBP 1095, Domaine<br />
de Crouelle, 234, Avenue du Brézet, 63100 Clermont-Ferrand, FRANCE<br />
jsalse@clermont.inra.fr<br />
Abstract<br />
During the last decade, technological improvements led to the development of large<br />
sets of plant genomic resources including the Brachypodium genome release in 2010,<br />
permitting the emergence of high-resolution comparative genomic studies and<br />
translational genomics approaches to non-sequence cereal genomes. In an attempt to<br />
unravel the structure and evolution of the plant ancestor genome we have re-assed the<br />
synteny and duplications of Angiosperm genomes to identify and characterize shared<br />
duplications. We combined the data on the intra-genomic duplications with those on<br />
the colinear blocks and found duplicated segments that have been conserved at<br />
orthologous positions since the divergence of plants. By conducting detailed analysis of<br />
the length, composition, and divergence time of the conserved duplications, we<br />
identified common and lineage-specific patterns of conservation between the different<br />
genomes that allowed us to propose a model in which the plant genomes have evolved<br />
from a common ancestor with a basic number of five/seven chromosomes (90 MYA)<br />
through whole genome duplications (i.e. paleopolyploidization) and translocations<br />
followed by lineage specific segmental duplications, chromosome fusions and<br />
translocations (Abrouk et al. 2010; Murat et al. 2010; Salse et al. 2011).<br />
Based on these data an ‘inner circle’ comprising 5/7 ancestral chromosomes with 10000<br />
protogenes was defined providing a new reference for the plant chromosomes and new<br />
insights into their ancestral relationships that have led to arrange their chromosomes into<br />
concentric ‘crop circles’ of synteny blocks (Abrouk et al. 2010). The established plant<br />
ancestor genome structure, in term of chromosome structure and gene content, offer<br />
the opportunity to perform high resolution translational genomics in cereals, from model<br />
(i.e. sequenced) species to complex ones, through (i) marker development for<br />
physical/genetic mapping, (ii) gene or sequence annotation, (iii) trait dissection, that will<br />
be discussed in details in the current presentation (Quraishi et al 2011ab).<br />
References<br />
Abrouk M, Murat F, Pont C, et al. (2010) Paleogenomics of Plants: Modern Species<br />
Synteny-Based Modelling of Extinct Ancestors. Trends in Plant Science. 15(9):479-87.<br />
Murat F, Xu JH, Tannier E, et al. (2010) Ancestral Grass Karyotype Reconstrcution Unravels<br />
New Mechanisms of Genome Shuffling as a Source of Plant Evolution. Genome research.<br />
20(11):1545-1557<br />
Quraishi UM, Murat F, Abrouk M, et al. (2011a) Meta-Genomics Analysis of the Grain<br />
Dietary Fiber Content in Bread Wheat. Functional & Integrative Genomics. 11(1):71-83.<br />
Quraishi UM, Abrouk M, Murat F, et al. (2011b) Cross-Genome Map Based Dissection of a<br />
Nitrogen Use Efficiency Ortho-metaQTL in Bread Wheat Unravels Concerted Cereal<br />
Genome Evolution. Plant Journal. 65(5):745-56.<br />
Salse J, Feuillet C (2011) Palaeogenomics in cereals: modeling of ancestors for modern<br />
species improvement. C R Biol. 334(3):205-11.<br />
10
Session 1 – Evolution and Natural Variation<br />
S1.2- Evolution and taxonomic split of the model grass Brachypodium<br />
distachyon (L.) P. Beauv.<br />
Pilar Catalán 1, Jochen Müller 2, Robert Hasterok 3, Glyn Jenkins 4, Luis A. J. Mur 4, Tim<br />
Langdon 4, Alexander Betekhtin 3, Dorota Siwinska 3, Manuel Pimentel 5, Diana López-<br />
Alvarez 1<br />
1 Department of Agriculture (Botany), High Polytechnic School of Huesca, University of Zaragoza, Ctra. Cuarte km 1, 22071 Huesca,<br />
Spain<br />
2 Herbarium Haussknecht, Department of Systematic Botany, University of Jena, Fürstengraben 1, 07737 Jena, Germany<br />
3 Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28,<br />
40032 Katowice, Poland<br />
4 Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Plas Gogerddan, Aberystwyth SY23 3EB Wales, UK<br />
5 Department of Plant and Animal Biology and Ecology, Faculty of Biology, University of Coruña, Campus da Zapateira s. n., 15071 A<br />
Coruña, Spain<br />
pcatalan@unizar.es<br />
Abstract<br />
A multidisciplinary phenetic, cytogenetic and evolutionary study has been conducted<br />
on a representative sampling of the three known cytotypes of the model grass<br />
Brachypodium distachyon (2n = 10, 20, 30). Statistical analyses of 15 selected traits in<br />
greenhouse propagated individuals detected significant taxonomic differences<br />
between the three cytotypes that have been further validated through discriminant<br />
analysis of wild individuals, demonstrating stability of characters in natural populations.<br />
Cytogenetic analyses based on nuclear genome size estimation, fluorescence in situ<br />
hybridisation with genomic and multiple DNA sequences as probes and chromosome<br />
painting confirm that the 2n=10 and 2n=20 chromosome cytotypes correspond to two<br />
different diploid taxa, whereas the 2n=30 cytotype is a derived allotetraploid of the cross<br />
between them. Phylogenetic analysis based on two plastid (ndhF, trnLF) and five nuclear<br />
(ITS, ETS, CAL, DGAT, GI) genes have demonstrated that the 2n=20 and 2n=10 cytotypes<br />
were, respectively, the maternal and paternal genome donors of the 2n=30 cytotype.<br />
The substantial phenotypic, cytogenetic and molecular differences detected among<br />
the three B. distachyon s. l. cytotypes are indicative of major speciation processes within<br />
this complex that allow their taxonomic separation into three distinct species. We have<br />
kept the name B. distachyon for the 2n=10 cytotype, whose genome has been fully<br />
sequenced and which is being used as model grass for temperate cereals, and have<br />
consequently designated a new epitype for it; we have described two new species B.<br />
stacei and B. hybridum for, respectively, the 2n=20 and 2n=30 cytotypes. The completely<br />
sequenced B. distachyon genome and the future availability of fully sequenced B. stacei<br />
and B. hybridum genomes would provide an exceptional opportunity for thorough<br />
comparative phylogenomic analyses of these plants and other totally or partially<br />
sequenced grasses.<br />
References<br />
Catalán P, Müller J, Hasterok R, et al. (2011) Next generation systematics: evolution and taxonomic split of the model grass<br />
Brachypodium distachyon (L.) P. Beauv. (Poaceae). submitted.<br />
Hasterok R, Draper J, Jenkins G (2004) Laying the cytotaxonomic foundations of a new model grass, Brachypodium distachyon (L.)<br />
Beauv. Chromosome Research 12:397-403.<br />
Idziak D, Betekhtin A, Wolny E, et al. (2011) Painting the chromosomes of Brachypodium-current status and future prospects.<br />
Chromosoma: Epub ahead of print, doi 10.1007/s00412-00011-00326-00419<br />
Mur L-A, Allainguillaume J, Catalan P, et al. (2011) Exploiting the Brachypodium Tool Box in cereal and grass research. New Phytologist<br />
191: 334-347.<br />
Wolny E, Lesniewska K, Hasterok R, Langdon T (2011) Compact genomes and complex evolution in the genus Brachypodium.<br />
Chromosoma 120:199-212<br />
Keywords<br />
Brachypodium distachyon, Brachypodium stacei, Brachypodium hybridum, cytogenetics,<br />
evolutionary systematics<br />
11
Session 1 – Evolution and Natural Variation<br />
S1.3- Using Brachypodium distachyon for model to crop translation of disease<br />
resistance in cereals.<br />
Antoine Peraldi, Andrew Steed, Chris Burt, Paul Nicholson.<br />
Department of Disease and Stress and Stress Biology, John Innes Centre, Colney Lane,<br />
Norwich, NR4 7UH, UK.<br />
antoine.peraldi@bbsrc.ac.uk<br />
Abstract<br />
Cereal production needs to improve in both quality and yield during the twenty first<br />
century to cope with predicted global challenges such as climate change, rising biofuel<br />
demand and human demographic pressure. Cereal diseases such as Fusarium head<br />
blight (FHB) of wheat can reduce yield and also contaminate grain with mycotoxins<br />
harmful for both animal and human consumption. Despite numerous studies on the<br />
genetic components of resistance to Fusarium diseases, progress is impaired by the<br />
complexity of the wheat genome and lack of available genome sequence. Therefore<br />
research would benefit greatly from availability of a robust, tractable genetic model<br />
from which to undertake model to crop translation.<br />
Brachypodium distachyon (Bd) has numerous advantages both as a genetic model and<br />
in functional genetic studies because of the increasing availability of appropriate tools<br />
(Draper et al., 2001). We have demonstrated that Bd exhibits compatible interactions<br />
with the main FHB-causing species of Fusarium and mirrors the disease symptom<br />
development on wheat better that any other model proposed to date (Peraldi et al.,<br />
2011). This new pathosystem enables screening of Bd mutant populations (i.e.T-DNA, Vain<br />
et al., 2008) to identify genes involved in resistance to Fusarium. Once characterized,<br />
these gene candidates can be validated in the natural host using reverse genetics<br />
approaches (e.g TILLING).<br />
Using this approach, a Bd mutant was identified that conferred increased resistance to<br />
Fusarium infection in detached leaf and flower inoculation tests. Sequence alignments<br />
and dark-induced senescence tests were used to characterize this gene as a functional<br />
homologue of auxin response factor (ARF) 2. The potential role of ARF2 in the natural host<br />
was evaluated using virus-induced gene silencing (VIGS) of wheat ARF2 which resulted in<br />
an average reduction in FHB disease severity of 20%.<br />
References:<br />
Draper J, Mur LAJ, Jenkins G, et al. (2001) Brachypodium distachyon. A new model<br />
system for functional genomics in grasses. Plant Physiology 127:1539-1555.<br />
Peraldi A, Beccari G, Steed A, Nicholson P. (2011) Brachypodium distachyon: a new<br />
pathosystem to study Fusarium head blight and other Fusarium diseases of wheat. BMC<br />
Plant Biology 11:100.<br />
Vain P, Worland B, Thole V, et al. (2008) agrobacterium-mediated transformation of the<br />
temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional<br />
mutagenesis. Plant Biotechnology Journal 6:236-245.<br />
Keywords<br />
Brachypodium, Fusarium, wheat, pathogen resistance, model to crop translation.<br />
12
Session 1 – Evolution and Natural Variation<br />
S1.4- Site-specific recombination systems for genetic modifications of cereals<br />
Vibha Srivastava 1, Soumen Nandy, and M. Aydin Akbudak<br />
1 University of Arkansas, 115 Plant Science bldg, 72704 Fayetteville, USA<br />
vibhas@uark.edu<br />
Abstract<br />
Precise full-length integration of transgenes is desirable for ensuring optimum gene<br />
expression. Site-specific recombination systems are versatile tools for catalyzing DNA<br />
integration, excision or inversion. A number of site-specific recombination systems have<br />
been isolated and shown to excise specific DNA fragments from the transgene locus;<br />
however, their use for gene integration is rarely demonstrated. One such system is the<br />
yeast FLP-FRT system that has been widely used for DNA excision in plants, but not for<br />
gene integration. Here, we report the use of FLP-FRT system for efficient targeting of<br />
foreign genes into the engineered genomic sites in rice. The transgene vector<br />
containing a pair of directly oriented FRT sites was introduced by particle bombardment<br />
into cells containing the engineered target site. Transiently expressed FLP activity<br />
generated the backbone-free gene circle that integrated into the target site. The<br />
majority of the transgenic events contained the precise integration locus, expressed the<br />
transgene, and transmitted the stable site-specific integration locus to progeny. This<br />
robust transformation platform is useful for both biotechnology applications in crops, and<br />
functional genomics in model plant species such as Brachypodium.<br />
References<br />
Nandy S, Srivastava V (2011) Site-specific gene integration in rice genome mediated by<br />
the FLP-FRT recombination system. Plant Biotechnol J. 9: 713-721.<br />
Akbudak MA, Srivastava V (2011) Improved FLP recombinase, FLPe, efficiently removes<br />
marker gene from transgene locus developed by Cre-lox mediated site-specific gene<br />
integration in rice. Mol Biotechnol. 49: 82-89.<br />
Keywords<br />
Cereal transformation, transgene expression, site-specific recombination, FLP-FRT<br />
13
Session 1 – Evolution and Natural Variation<br />
S1.5- Functional analysis of WOX4 in mono- and dicotyledonous plants and<br />
dissecting the evolution of vascular development<br />
Stefanie Suer, Jasmin Bassler, Julia Riefler, Martina Schwarz, Thomas Greb<br />
Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr.<br />
Bohr-Gasse 3, 1030 Vienna, Austria<br />
Stefanie.suer@gmi.oeaw.ac.at<br />
Abstract<br />
Most dicotyledonous plants have the capacity to initiate secondary growth, i.e., the<br />
increase of the diameter of their shoots and roots by the activity of lateral meristems. The<br />
most prominent lateral meristem is the vascular cambium, producing secondary vascular<br />
tissues, thereby increasing long-distance transport capacities for nutrients and water, as<br />
well as the stability of the plant body. Recently, the cambium-specific activity of the<br />
homeobox-transcription factor WOX4 has been shown to be essential for cambium<br />
activity in dicotyledonous species such as Arabidopsis and Populus.<br />
In comparison, monocotyledonous plants do not undergo secondary growth.<br />
Interestingly however, monocot genomes encode a transcription factor highly similar to<br />
AtWOX4 raising the question as to the function of this factor in species without cambium<br />
activity and the presence of meristematic potential of monocot stems.<br />
In this study, we aim to elucidate the function of WOX4 in the monocot model<br />
Brachypodium distachyon and draw connections between mono- and dicotyledonous<br />
plants with regard to the regulation of vascular development. We show that BdWOX4 is<br />
expressed in stem nodes and in vascular bundles of young Brachypodium plants.<br />
Importantly, the BdWOX4 promoter recapitulates the activity of the AtWOX4 promoter<br />
when introduced into Arabidopsis and moreover, BdWOX4 is able to rescue the<br />
cambium defects of the Arabidopsis wox4 mutant. Taken together, our data<br />
demonstrate that, in spite of large anatomical differences, monocots and dicots utilize<br />
conserved molecular processes to regulate vascular developmental programs, and<br />
suggest that the cambium-specific regulatory network has been adapted to control<br />
different processes during the divergence of mono- and dicotyledonous species.<br />
References<br />
Alves SC, Worland B, Thole V, et al. (2009) A protocol for Agrobacterium-mediated<br />
transformation of Brachypodium distachyon community standard line Bd21. Nat. Protoc.<br />
4 (5): 638-649.<br />
Hirakawa Y, Kondo Y, Fukuda H (2010) TDIF peptide signaling regulates vascular stem cell<br />
proliferation via the WOX4 homeobox gene in Arabidopsis. Plant Cell 22: 2618-2629.<br />
Ji J, Strable J, Shimizu R, et al. (2009) WOX4 promotes procambial development. Plant<br />
Physiol. 152: 1346-1356.<br />
Vogel J, Hill T (2008) High-efficiency Agrobacterium-mediated transformation of<br />
Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep. 27: 471-478.<br />
Keywords<br />
Vascular development, WOX transcription factors<br />
14
Session 1 – Evolution and Natural Variation<br />
S1.6- Deciphering the function and evolution of BRX-like genes in<br />
Brachypodium distachyon<br />
David Pacheco-Villalobos, Martial Sankar & Christian S. Hardtke<br />
Department of Plant Molecular Biology, University of Lausanne, Biophore Building, CH-<br />
1015, Lausanne, Switwerland.<br />
David.PachecoVillalobos@unil.ch<br />
Abstract<br />
The BREVIS RADIX gene (BRX) was first identified by map-based cloning in Arabidopsis<br />
thaliana as a root growth QTL, for which both loss of function and hyperactive natural<br />
variants exist (1, 2). Arabidopsis brx mutants have short primary roots, because BRX is<br />
essential for root meristem growth in young seedlings (3). Although it is a transcriptional<br />
co-regulator, BRX protein is primarily plasma membrane-associated, but can translocate<br />
into the nucleus to regulate gene expression in an auxin-dependent manner to mediate<br />
crosstalk between the auxin and brassinosteroid pathways (4, 5). BRX represented the first<br />
member of a plant-specific gene family consisting of five paralogs in Arabidopsis (2). BRX<br />
family genes have been also identified in poplar and Arabidopsis lyrata, as well as in<br />
monocotyledons species, such as Brachypodium. Molecular and genetic analyses<br />
revealed that dicotyledons BRX family genes display higher levels of functional<br />
diversification than their monocotyledons counterparts (2). To examine their natural<br />
genetic variation, Brachypodium BRX-like genes (BdBRXL-1 to 5) from 174 accessions<br />
were sequenced in a pooled high throughput sequencing design and analysed to<br />
determinate polymorphisms. A total of 79 variants (71 SNPs and 7 InDels) were found,<br />
indicating high degree of conservation. Interestingly, a base substitution leading to a<br />
STOP codon was found in BdBRXL2, the only BdBRXL gene which, if not mutated, could<br />
not rescue the Arabidopsis brx phenotype. To further investigate the function of BRX-like<br />
genes in monocotyledons we have generated Brachypodium transgenic lines carrying<br />
various artificial microRNA (amiRNA) constructs. The amiRNAs were designed to<br />
selectively knock-down the expression of each or several BRX-like genes (BdBRXL-1 to 5).<br />
Primary root growth does not seem to be affected in the transgenic lines, possibly due to<br />
functional redundancy or insufficient knock-down. However, a root system architecture<br />
(branching) phenotype has been confirmed in certain lines and will be reported in detail.<br />
References<br />
1. Mouchel CF, Georgette B, Hardtke CS (2004) Natural genetic variation in Arabidopsis<br />
identifies BREVIS RADIX, a novel regulator of cell proliferation and elongation in the root.<br />
Genes and Development 18: 700-714.<br />
2. Beuchat J, Li S, Ragni L, et al. (2010) A hyperactive quantitative trait locus allele of<br />
Arabidopsis BRX contributes to natural variation in root growth vigor. Proccedings of the<br />
National Academy of Sciences 107 : 8475-8480<br />
3. Scacchi E, Salinas P, Gujas B, et al. (2010) Spatio-temporal sequence of crossregulatory<br />
events in root meristem growth. Proccedings of the National Academy of<br />
Sciences 107: 22734-22739.<br />
4. Scacchi E, Osmont KS, Beuchat J, et al.. Dynamic, auxin-responsive plasma<br />
membrane-to-nucleus movement of Arabidopsis BRX. Development 136: 2059-2067.<br />
5. Mouchel CF, Osmont KS, Hardtke CS (2006) BRX mediates feedback between<br />
brassinosteroid levels and auxin signalling in root growth. Nature 443: 458-461.<br />
Keywords<br />
Natural variation, root system architecture, amiRNA<br />
15
Session 2 – Cell Wall, Biomass and Biofuels<br />
S2.1- Brachypodium distachyon as a model plant for lignocellulosic ethanol<br />
production<br />
Till Meineke, Chithra Manisseri, Christian A. Voigt<br />
Phytopathology & Biochemistry, Biocenter Klein Flottbek, University of Hamburg,<br />
Ohnhorststr. 18, 22609 Hamburg, Germany<br />
voigt@botanik.uni-hamburg.de<br />
Abstract<br />
The conversion of plant biomass to ethanol during fermentation is a strategy to mitigate<br />
climate change by substituting fossil fuels, especially in transportation processes.<br />
However, the conversion of biomass is mainly limited by the recalcitrant nature of the<br />
plant cell wall. Apart from exploring cell wall degrading enzymes and pretreatment<br />
methods for biomass, optimizing the plant cell wall itself for subsequent processing is a<br />
promising approach. As an emerging model plant, B. distachyon is suitable to generate<br />
cell wall mutants more easily than in designated biomass-delivering monocots. To test<br />
putative B. distachyon cell wall mutants for altered ethanol production from biomass, we<br />
wanted to know whether B. distachyon could be considered as a model plant in this<br />
biotechnological application.<br />
Therefore, we compared cell wall composition and ethanol production of leaf and stem<br />
biomass from B. distachyon and the potential biomass-delivering monocots wheat,<br />
maize, and Miscanthus x giganteus. Biomass was analyzed before and after different<br />
steps of pretreatment and fermentation by pulsed amperometry (HPAEC-PAD) and<br />
confocal laser-scanning microscopy. The accessible amount of glucose for fermentation<br />
and the amount of produced ethanol was determined with refractive index detector in<br />
a HPLC system. B. distachyon exhibited similar cell wall composition and fermentation<br />
traits as the tested crops. Highest correlation in these data was not with the closelyrelated<br />
wheat, but with maize and M. giganteus. Based on these results, we suggest B.<br />
distachyon as model plant to study the impact of cell well alterations on lignocellulosic<br />
ethanol production in energy crops.<br />
Keywords<br />
bio-ethanol, 2nd generation biofuels<br />
16
Session 2 – Cell Wall, Biomass and Biofuels<br />
S2.2- Disrupting the cinnamyl alcohol dehydrogenase 1 (BdCAD1) gene leads<br />
to altered lignification and improved saccharification in Brachypodium<br />
distachyon<br />
Madeleine Bouvier d'Yvoire 1, Richard Sibout 1, Oumaya Bouchabke 1, Olivier Darracq 1,<br />
Sebastien Antelme 1, Leonardo Gomez 2, Laurent Cezard 1, Catherine Lapierre 1, Lise<br />
Jouanin 1.<br />
1Institut Jean-Pierre Bourgin (IJPB), UMR1318 INRA-AgroParisTech, 78026 Versailles, FR<br />
2Centre for Novel Agricultural Products, University of York, York Y010 5 YW, UK<br />
Madeleine.Bouvier-Dyvoire@versailles.inra.fr<br />
Abstract<br />
Second generation biofuels using fermentable sugars from plant cell walls could provide<br />
an answer to the augmentation of global energy needs with a limited impact on food<br />
resources. As grasses show attractive features for this purpose, Brachypodium distachyon<br />
was recently pointed as a valuable model plant for temperate grass species. Lignins are<br />
major secondary cell wall polymers that derive from the polymerization of monolignols.<br />
They confer rigidity and hydrophobicity to plant cell walls. However, they also constitute<br />
an obstacle to most industrial processes targeting cellulose or hemicellulose, and they<br />
need to be removed by costly, polluting and energy demanding processes. Plants with<br />
altered lignification often display enhanced saccharification yields (enzymatic<br />
conversion of cellulose into fermentable sugars).<br />
In order to identify cell wall compositions more susceptible to saccharification, the<br />
Sodium Azide and EMS mutageneized lines from the Versailles Brachypodium collection<br />
were screened based on the colour of their stems. Indeed, orange, red or brown<br />
vascular elements are often associated with altered lignification. The mutants so<br />
identified, hereafter named « brown stem » (bs), were analyzed and several showed<br />
improved saccharification. Among them, mutants in a CAD candidate gene (cinnamyl<br />
alcohol dehydrogenase, involved in the last step of the monolignol biosynthesis<br />
pathway) were found, that have the highest saccharification yields. These lines possess a<br />
significative decrease in CAD activity and in their lignin contents. Their lignins are also<br />
enriched in free phenolic groups, sinapaldehyde units and resistant bounds. Functional<br />
complementation of Arabidopsis thaliana and Brachypodium cad mutants confirmed<br />
the function of BdCAD1, and RNAi CAD lines are being selected in order to analyse the<br />
effects of the silencing of several CAD genes.<br />
References<br />
Vanholme R, Moreel K, Ralph J, Boerjan W (2008) Lignin engineering. Current Opinion in<br />
Plant Biology 11 :278-285<br />
Marita JM, Vermerris W, Ralph J, Hatfield RD (2003) Variations in the cell wall composition<br />
of Maize brown midrib mutants. Journal of Agricultural and Food Chemistry 51 : 1313-1321<br />
Sibout R, Eudes A, Mouille G, et al. (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and<br />
-D Are the Primary Genes Involved in Lignin Biosynthesis in the Floral Stem of Arabidopsis.<br />
The Plant Cell 17 : 2059-2076<br />
Youn B, Camacho R, Moinuddin SGA, et al. (2006) Crystal structures and catalytic<br />
mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4.<br />
Organic & Biomolecular Chemistry 4 : 1687-1697<br />
Keywords<br />
Brachypodium, lignin, cinnamyl alcohol dehydrogenase, saccharification<br />
17
Session 2 – Cell Wall, Biomass and Biofuels<br />
S2.3- Identification of Brachypodium mutants defective in silicon uptake<br />
Emiko Murozuka, Pia Haugaard Nord-Larsen, Inge Skrumsager Møller, Thomas Paul Jahn<br />
and Jan Kofod Schjoerring<br />
Plant and Soil Science Laboratory, Department of Agriculture and Ecology, Faculty of Life<br />
Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C<br />
emikomu@life.ku.dk<br />
Abstract<br />
Silicon (Si) is a beneficial element for higher plants. The positive effect of Si is related to<br />
the fact that Si increases the mechanical strength and rigidity of the cell walls, thereby<br />
stimulating growth and contributing to the alleviation of biotic and abiotic stresses. On<br />
the other hand, Si accumulation decreases the degradability of plant residues, which<br />
may cause resistance to treatments for saccharification in cellulosic ethanol production<br />
(Van Soest 2006). In addition, high content of Si in plant residues increases the amount of<br />
ash during the combustion of biomass. There is accordingly a strong interest in reducing<br />
Si accumulation in plant materials used for bioenergy purposes (Gressel 2008).<br />
Si transport mechanisms have so far been studied in rice. Three Si transporters, Lsi1, Lsi2<br />
and Lsi6 have been identified and characterized. Lsi1 and Lsi6 belong to aquaporins<br />
while Lsi2 is a secondary active anion transporter (Ma and Yamaji 2008, Yamaji et al.,<br />
2008). Lsi1 and Lsi2 are involved in uptake of Si, while Lsi6 functions in the distribution of Si<br />
in the shoots. In our study, Brachypodium distachyon has been used to investigate the<br />
mechanisms of uptake and distribution of Si in grasses. Phenotypic screening using<br />
germanium (Ge) was carried out in a Brachypodium mutant population of about 1200<br />
lines in order to identify mutants which were defective in Si uptake. After the screening,<br />
about 30 lines were selected as candidates. So far one of the lines was found to be<br />
defective in Lsi1. Characterization of the protein revealed that Brachypodium Lsi1<br />
transports Si as well as other substances such as arsenite and boric acid, which<br />
corresponds with the function of rice Lsi1. The mutant plant will be characterized after<br />
backcrossing with the wild type focusing on degree of Si accumulation and the<br />
incorporation of Si in the cell wall. Further screening and identification will be continued<br />
in order to find more mutants in Si transporters.<br />
References<br />
Gressel J (2008) Transgenics are imperative for biofuel crops. Plant Sci 174: 246–263<br />
Ma J.F, Yamaji N (2008) Functions and transport of silicon in plants. Cell Mol Life Sci 65:<br />
3049-3057<br />
Van Soest P.J (2006) Rice straw, the role of silica and treatments to improve quality. Anim.<br />
Feed Sci. Tech. 130: 137–171<br />
Yamaji N, Mitatni N, Ma J.F (2008) A transporter regulating silicon distribution in rice<br />
shoots. Plant Cell 20:1381-1389<br />
Keywords<br />
silicon transporter, aquaporin, cell wall<br />
18
Session 2 – Cell Wall, Biomass and Biofuels<br />
KEYNOTE LECTURE<br />
S2.4- Natural diversity from genomics to phenomics<br />
John P. Vogel<br />
USDA-ARS Western Regional Research Center, Albany, CA, 94710 USA<br />
john.vogel@ars.usda.gov<br />
Abstract<br />
Natural variation in genomic sequence and the resultant phenotypic variation are<br />
valuable tools for determining gene function. To develop resources to allow researchers<br />
to use natural variation as a research tool my laboratory together with an international<br />
group of collaborators has initiated several projects focused on natural diversity in<br />
Brachypodium distachyon. To provide a genomic foundation for these studies we are<br />
resequencing 56 natural accessions through the US Department of Energy Joint Genome<br />
Institute. Analysis of the first six accessions is nearly complete and shows tremendous<br />
diversity with SNP frequencies ranging from 200-600 base pairs per SNP. To survey the<br />
phenotypic diversity in a large collection of natural accessions we are collaborating with<br />
the High Resolution Plant Phenomics Facility in Canberra, Australia. In addition, we are<br />
creating homozygous T-DNA lines for phenotypic analysis. Another area of interest is the<br />
use of the related, but perennial, species B. sylvaticum as a tool to understand the<br />
genetic basis of perenniality. Initial steps in this direction include the development of an<br />
efficient transformation system and surveying genetic diversity in a small collection of B.<br />
sylvaticum accessions.<br />
Keywords<br />
natural diversity, resequencing, phenotype, Brachypodium sylvaticum, transformation<br />
19
Session 2 – Cell Wall, Biomass and Biofuels<br />
S2.5- Functional characterization of candidate cell wall genes in B. distachyon<br />
Pubudu Handakumbura, Gina M. Trabucco, Michael V. Veling, Dominick A. Matos, Karen<br />
S. Osmont, Scott J. Lee, Samuel P. Hazen<br />
Biology Department, University of Massachusetts, 221 Morrill Science Center III, Amherst,<br />
MA, 01003, USA<br />
hazen@bio.umass.edu<br />
Abstract<br />
The cell wall is a complex composite of polysaccharides, proteins, and lignin, with lignin<br />
and cellulose representing two of the most abundant bio-organic compounds on Earth.<br />
Much of what is currently understood about the transcriptional regulation of cell wall<br />
biosynthesis is from the study of Arabidopsis thaliana xylem vessels and fibers, yet this<br />
understanding may not be generalizable across land plants. The cell walls of grasses,<br />
including domesticated cereals that provide the majority of human calories and the<br />
perennials under development as biofuel energy crops, differ significantly in morphology<br />
and composition from the eudicot A. thaliana. We are taking a reverse genetics<br />
approach to understand the transcriptional regulation of secondary cell wall biosynthesis<br />
using the grass model system Brachypodium distachyon. In order to confirm transcription<br />
factor targets, we have silenced and confirmed candidate cellulose and lignin<br />
associated genes using artificial microRNAs. Using our well establish yeast one-hybrid<br />
assay, we are testing for activity between cis-regulatory regions of those genes and a<br />
collection of transcription factor proteins. In doing so, we seek to resolve the regulatory<br />
networks leading to cellulose and lignin synthesis.<br />
Keywords<br />
Transcription factor, secondary cell wall, gene expression<br />
20
Session 2 – Cell Wall, Biomass and Biofuels<br />
S2.6- Cell wall proteomics of Brachypodium distachyon<br />
Elisabeth JAMET, Thibaut DOUCHE, David ROUJOL, Hélène San Clemente, Benoît VALOT,<br />
Michel ZIVY, Rafael PONT-LEZICA<br />
LRSV; UMR5546 UPS/CNRS; BP42617, F-31326 Castanet Tolosan, France<br />
PAPPSO; UMR0320/UMR8120 INRA/CNRS/Université Paris X Génétique végétale; F-91190<br />
Gif sur Yvette<br />
jamet@lrsv.ups-tlse.fr<br />
Abstract<br />
Plant biomass is one of the greatest untapped reserves on the planet and is mostly<br />
composed of cell walls. Energy-rich polysaccharides make up about 75% of plant cell<br />
walls. These polymers can be broken down to produce sugar substrates through the<br />
saccharification process. A whole range of products can be obtained from these sugars<br />
including bioethanol. However, the complex structure of cell walls, consisting in a<br />
network of various polysaccharides and glycoproteins, encrusted by phenolic polymers<br />
in secondary walls, makes them very resistant to degradation. Improving the ease and<br />
yield of saccharification represents the major technological hurdle that must be<br />
overcome before the full vision of the plant-fuelled biorefinery can be realized.<br />
Brachypodium distachyon has many qualities that make it a model for study of<br />
dedicated bioethanol crops such as Schwitchgrass or Miscanthus. Since the full<br />
sequencing of its genome in 2008, it is possible to perform accurate transcriptomics and<br />
proteomics studies. The aim of this project (ANR KBBE CELLWALL) was to identify cell wall<br />
proteins playing roles in cell wall extension and in secondary wall formation. Recent cell<br />
wall proteomics studies mainly performed in Arabidopsis thaliana led to the identification<br />
of about 500, i.e. approximately one fourth of the expected, cell wall proteins falling into<br />
nine functional classes (Jamet et al. 2008; San Clemente et al. 2009). With regard to the<br />
objective of the project, two classes were of special interest, i.e. proteins acting on<br />
polysaccharides and oxido-reductases. The present work focused on cell wall<br />
transcriptomics and proteomics of growing vs mature leaves and internodes. About 360<br />
cell wall proteins could be identified by mass spectrometry and bioinformatics among<br />
which some were more abundant in young or mature organs. Relative quantification<br />
could be done for one third of them, thus allowing to propose candidates of interest for<br />
further functional studies.<br />
References<br />
ANR KBBE CELLWALL (2009-2012). Coordinator: Persson S (MPI Golm, Germany). Partner 3:<br />
Jamet E (LRSV, Toulouse, France).<br />
Feiz L, Irshad M, Pont-Lezica R, et al. (2006) Evaluation of cell wall preparations for<br />
proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls. Plant<br />
Methods 2: 10.<br />
Jamet E, Albenne C, Boudart G, et al. (2008) Recent advances in plant cell wall<br />
proteomics. Proteomics 8: 893-908.<br />
San Clemente H, Pont-Lezica R, Jamet E (2009) Bioinformatics as a tool for assessing the<br />
quality of sub-cellular proteomic strategies and inferring functions of proteins : plant cell<br />
wall proteomics as a test case. Bioinform Biol Insights 3 : 15-28<br />
Keywords<br />
biomass, cell wall, mass spectrometry, proteomics, quantification<br />
21
Session 2 – Cell Wall, Biomass and Biofuels<br />
S2.7- The role of pectins in Brachypodium development<br />
Jozef Mravec, Oumaya Bouchabké-Coussa, Yves Chupeau, Grégory Mouille, Richard<br />
Sibout, Herman Höfte<br />
Institut Jean-Pierre Bourgin (IJPB), INRA, route de Saint Cyr, 78026 Versailles, France<br />
jmravec@versailles.inra.fr<br />
Abstract<br />
One of the most remarkable differences between the primary cell walls of dicots (type I)<br />
and commelinoid monocots (type II) is the abundance of pectin polymers. Type II cell<br />
walls are generally considered to be poor in pectin content. The biological relevance of<br />
this divergence has never been elaborated. We took the advantage of Brachypodium<br />
distachyon as an emerging plant model to study the synthesis, distribution and the<br />
downstream modification of pectins in Poales. We generated and phenotyped the RNAi<br />
mutant in BdQUA1 (Bradi3g43810) -- the closest ortholog of Arabidopsis QUASIMODO1<br />
gene which encodes for a glycosyltransferase involved in pectin synthesis. We also<br />
determined the expresion of BdQUA1 by in situ hybridization and by a promoter-GUS<br />
construct. The spatial distribution of various pectins in the root meristem was mapped by<br />
immunolocalizations using various pectin-specific antibodies. Our results suggest, that like<br />
in dicots, pectins play an irreplaceable role in grass development and that the pectin<br />
metabolism, especially in the meristematic tissues, is tightly regulated. Currently we also<br />
study the role of pectins in the secondary cell wall deposition and the pectin influence<br />
on the saccharification yield.<br />
References<br />
Keywords<br />
monocot, cell wall, pectins, development, QUASIMODO 1<br />
22
Session 2 – Cell Wall, Biomass and Biofuels<br />
S2.8- Investigating biomass digestibility in Brachypodium distachyon<br />
Poppy E. Marriott, Leonardo D. Gomez, Caragh Whitehead and Simon J. McQueen-<br />
Mason<br />
CNAP, Department of Biology, University of York, York. YO10 5DD. UK<br />
pm518@york.ac.uk<br />
Abstract<br />
Lignocellulosic biomass is largely composed of polysaccharides which can be broken<br />
down to produce sugars for the production of ethanol through fermentation. This<br />
bioethanol could provide a sustainable replacement for fossil fuel derived transportation<br />
fuels. However, lignocellulose is extremely resistant to digestion and converting it to<br />
fermentable sugars requires energetic pretreatment and expensive enzyme applications.<br />
To make second generation bioethanol a commercial reality we therefore need to<br />
improve the conversion efficiency. One way to achieve this is by producing plants with<br />
more digestible lignocellulose. This project involves a forward genetic screen with the<br />
model grass, Brachypodium distachyon, to identify plants with an alteration in digestibility<br />
from large, randomly mutated populations. Such a large scale screen requires a high<br />
throughput approach and at the University of York an analytical platform has been<br />
developed that can perform saccharification analysis in a 96-well plate format. The<br />
system can reliably detect differences in the saccharification of plant tissues and is able<br />
to rapidly process large numbers of samples with a minimum amount of human<br />
intervention.<br />
Two chemically mutagenised populations were screened (from INRA and the USDA) and<br />
a relatively large amount of variation in sugar release was identified (up to +70% and -<br />
50% compared to WT). Mutants with a significant difference in sugar release in<br />
comparison to WT were isolated and saccharification of the offspring was determined to<br />
test for heritability of the trait. Plants with heritable mutations will be taken forward for in<br />
depth cell wall analysis to understand the effect of the mutation on the structure and<br />
digestibility of the cell wall. Mapping of the mutations will also be undertaken in order to<br />
identify genes involved in the phenotypic variation. This will provide novel genetic<br />
markers to aid in selection of the best genotypes for industrial production of bioethanol.<br />
References<br />
Gomez LD, Whitehead C, Barakate A, et al. (2010) Automated saccharification assay for<br />
determination of digestibility in plant materials. Biotechnology for Biofuels 3: 23.<br />
Gomez LD, Bristow JK, Statham ER, McQueen-Mason SJ (2008) Analysis of<br />
saccharification in Brachypodium distachyon stems under mild conditions of hydrolysis.<br />
Biotechnology for Biofuels 178: 61-72.<br />
Abramson M, Shoseyov O, Shani Z (2010) Plant cell wall reconstruction toward improved<br />
lignocellulosic production and processability. Plant Science 178: 61-72<br />
Garvin DF, Gu YQ, Hasterok R, et al. (2008) Development of genetic and genomic<br />
research resources for Brachypodium distachyon, a new model system for grass crop<br />
research. Crop Science 48: S69-S84<br />
Keywords<br />
Brachypodium, Biofuels, lignocellulose, saccharification, genetic screen<br />
23
Session 3 – Tools and Bioengineering<br />
KEYNOTE LECTURE<br />
S3.1- T-DNA mutagenesis in Brachypodium distachyon<br />
Vera Thole and Philippe Vain<br />
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich NR4<br />
7UH, United Kingdom.<br />
philippe.vain@jic.ac.uk<br />
Abstract<br />
Brachypodium distachyon is emerging as a new experimental and genomics model for<br />
temperate cereal crops and biomass grasses. Recently, the International Brachypodium<br />
Tagging Consortium (IBTC), which includes eight laboratories from five countries, has<br />
been established to scale-up the production and characterisation of T-DNA mutants. The<br />
BrachyTAG program is part of the IBTC and has generated a collection of 5,000 fertile<br />
plant lines (genotype Bd21) transformed with the binary vector pVec8-GFP.<br />
Characterisation of an initial population of 741 plant lines (Thole et al., 2010) has shown<br />
that analysing the regions flanking both borders of the T-DNA inserts nearly doubled the<br />
recovery of Brachypodium flanking sequence tags (FSTs) available to describe the<br />
different T-DNA insertions. Overall, more than 2,200 flanking regions were identified with<br />
44% corresponding to Brachypodium FSTs. Approximately 50% of the T-DNA insertions<br />
interrupted a predicted gene in the Bd21 annotated genome sequence. The distribution<br />
and density of T-DNA inserts will be presented as well as examples of T-DNA mutant<br />
genotyping, phenotyping and complementation. Novel binary vectors (pBrachyTAG)<br />
have also been developed to improve T-DNA tagging and trapping in Brachypodium.<br />
The T-DNA lines generated by the BrachyTAG program are available as a community<br />
resource and have been distributed internationally since 2008 via the BrachyTAG.org<br />
website.<br />
References<br />
Thole V, Worland B, Wright J, Bevan MW, Vain P (2010) Distribution and characterization<br />
of more than 1000 T-DNA tags in the genome of Brachypodium distachyon community<br />
standard line Bd21. Plant Biotechnology Journal 8:734–747.<br />
Keywords<br />
mutagenesis, T-DNA tagging, reverse genetics, functional genomics, flanking sequence<br />
tags (FST).<br />
24
Session 3 – Tools and Bioengineering<br />
S3.2- Current status and future perspectives for molecular cytogenetic studies<br />
of Brachypodium<br />
Dominika Idziak, Elzbieta Wolny, Karolina Lesniewska, Alexander Betekhtin, Natalia<br />
Borowska, Ewa Breda, Maja Jankowska, Robert Hasterok<br />
Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental<br />
Protection, University of Silesia, Jagiellonska 28 St, 40‑032 Katowice, Poland<br />
didziak@us.edu.pl<br />
Abstract<br />
Molecular cytogenetics contributes to better understanding of the structure, function<br />
and evolution of genomes by bridging the DNA sequence data and information about<br />
chromosome biology at the cellular, tissue and organismal level. The key method of<br />
modern molecular cytogenetics is fluorescence in situ hybridisation (FISH) which allows<br />
direct microscopic observation of the physical position and abundance of a<br />
fluorescently labelled DNA sequence in mitotic and meiotic chromosomes, interphase<br />
nuclei or DNA fibers. The primary uses of FISH in plant biology studies are identification of<br />
chromosomes or chromosome segments and physical mapping of plant genomes, for<br />
structural genomics and phylogenetic analyses. In B. distachyon sequencing project FISH<br />
using BAC clones from Bd21 genomic libraries provided a valuable complementary<br />
approach in ordering genome sequence assemblies and detecting false joints in the<br />
sequence scaffolds as well as aligning the sequence assemblies integrated with physical<br />
and genetic maps to individual B. distachyon chromosomes (Febrer et al., 2010; IBI,<br />
2010). In the presented work we summarise research projects currently carried out in our<br />
laboratory that are based on molecular cytogenetics methods. The on‑going<br />
investigation comprises studies of karyotype evolution using chromosome painting and<br />
single-BAC FISH, analysis of nucleus architecture and studies of chromosome behaviour<br />
during meiosis. FISH coupled with immunoassays is used for cytological analysis of<br />
chromatin features which affect gene expression.<br />
References<br />
Febrer M, Goicoechea J L, Wright J, et al. (2010) An integrated physical, genetic and<br />
cytogenetic map of Brachypodium distachyon, a model system for grass research.<br />
PloS ONE 5: e13461.<br />
International Brachypodium Initiative (2010) Genome Sequencing and Analysis of the<br />
model grass Brachypodium distachyon. Nature 463: 763–768.<br />
Keywords<br />
molecular cytogenetics, physical mapping, chromosome biology<br />
25
Session 3 – Tools and Bioengineering<br />
S3.3- Functional annotation of a full-length Brachypodium cDNA resource<br />
Keiichi Mochida, Yukiko Uehara, Fuminori Takahashi, Takuhiro Yoshida, Tetsuya Sakurai,<br />
Kazuo Shinozaki<br />
RIKEN BMEP/PSC<br />
mochida@psc.riken.jp<br />
Abstract<br />
Full-length cDNA libraries and large-scale sequence data sets of clones are invaluable<br />
resources for life science projects concerning the study of various species [1-3].<br />
Collection of full-length cDNA is an essential genomic resource for the correct<br />
annotation of genomic sequences as well as for the functional analysis of genes and<br />
their products. We constructed a mixed full-length cDNA library of various tissues of<br />
Brachypodium distachyon Bd21 strain by using the biotinylated CAP trapper method [4].<br />
Then, we obtained 79,097 expressed sequence tags (ESTs), which were sequenced from<br />
both ends. We mapped the ESTs to the B. distachyon genome sequence together with<br />
the available gene annotation. As a result of this mapping, we allocated 66,140 ESTs of<br />
33,070 clones to the representative 8,461 genomic positions of the currently annotated<br />
genes. We also allocated 3,018 ESTs of 1,509 clones to the 670 non-redundant genomic<br />
positions without any annotated genes. Furthermore, through this mapping analysis, we<br />
also identified transcripts putatively derived from the trans-splicing event in<br />
Brachypodium. To update the orthologous ORFeome resources in Pooideae, we<br />
performed comparative analyses of the full-length cDNA dataset of Brachypodium and<br />
those of wheat and barley. To integrate our full-length cDNA information with other<br />
related significant information resources, we constructed an integrated database, RIKEN<br />
BrahcyFLcDNA DB, which is an information gateway to access the resource. The<br />
database provides full-length cDNA-based gene structural annotation, predicted gene<br />
functions, and information on corresponding putative orthologs in grass species for<br />
comparative analysis. Therefore, this full-length cDNA resource should be beneficial in<br />
accelerating the discovery and functional analysis of genes in Brachypodium.<br />
References<br />
1. Seki M, Narusaka M, Kamiya A, Ishida J, Satou M, et al. (2002) Functional annotation of<br />
a full-length Arabidopsis cDNA collection. Science 296: 141-145.<br />
2. Seki M, Shinozaki K (2009) Functional genomics using RIKEN Arabidopsis thaliana fulllength<br />
cDNAs. J Plant Res. 122: 355-366<br />
3. Mochida K, Shinozaki K (2010) Genomics and bioinformatics resources for crop<br />
improvement. Plant Cell Physiol 51: 497-523.<br />
4. Carninci P, Kvam C, Kitamura A, Ohsumi T, Okazaki Y, et al. (1996) High-efficiency fulllength<br />
cDNA cloning by biotinylated CAP trapper. Genomics 37: 327-336.<br />
Keywords<br />
Full-length cDNA, database, gene annotation<br />
26
Session 3 – Tools and Bioengineering<br />
S3.4- BradiNet: co-expression platform and comparative transcriptomics for<br />
Brachypodium<br />
Marek Mutwil 1, Richard Sibout 2, Herman Höfte 2 and Staffan Persson 1<br />
1 Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam –<br />
Golm, Germany<br />
2 Institut Jean-Pierre Bourgin, UMR1318-INRA-AgroParisTech, Centre de Versailles-Grignon,<br />
Route de St-Cyr, 78026 Versailles Cedex-France<br />
mutwil@mpimp-golm.mpg.de<br />
Abstract<br />
It is well documented that transcriptionally coordinated genes tend to be functionally<br />
related and that such relationships may be conserved across different species and even<br />
kingdoms. To exploit such relationships, we have performed microarray analysis of all<br />
major tissues during different developmental stages of Brachypodium distachyon. The<br />
data was used to create co-expression network for Brachypodium and included into<br />
PlaNet database (http://aranet.mpimp-golm.mpg.de/bradinet). One major problem<br />
with transferring knowledge from a model organism is that since plants harbor large gene<br />
families, standard phylogenetic methods might not be sufficient to identify functional<br />
homologs. To remedy this, we implemented a comparative network algorithm that<br />
estimates similarities between network structures. Thus, the platform can be used to swiftly<br />
infer similar coexpressed network vicinities within and across species and can predict the<br />
identity of functional homologs. We exemplify this with comparative analysis of<br />
Brachypodium cellulose synthase gene networks to find corresponding functional<br />
homologs between Arabidopsis, Brachypodium, Medicago, poplar, rice, soybean and<br />
wheat. The data support the contention that this platform will considerably improve<br />
transfer of knowledge between model organisms Arabidopsis and Brachypodium and<br />
crop species such as rice and wheat.<br />
27
Session 3 – Tools and Bioengineering<br />
S3.5- Phenotyping the shoot and root diversity of Brachypodium distachyon to<br />
accelerate plant biofuel breeding<br />
1Richard Poiré, 2Vincent Chochois, 1Solène Callarec, 1Xavier Sirault, 3John Vogel,<br />
2Michelle Watt, 1Robert Furbank<br />
1High Resolution Plant Phenomics Centre, CSIRO Plant Industry, Canberra, ACT 2601,<br />
Australia<br />
2CSIRO Plant Industry, Canberra, ACT 2601, Australia<br />
3USDA, ARS, WRRC Albany, California 94710, USA<br />
richard.poire@csiro.au<br />
Abstract<br />
The High Resolution Plant Phenomics Centre (HRPPC) is the Canberra node of the<br />
Australian Plant Phenomics Facility. The HRPPC focuses on deep phenotyping and<br />
reverse phenomics through development of next generation tools to measure<br />
performance of plants ranging from model species to major crops.<br />
A major aim of the HRPPC model plant module is to find genes of agricultural<br />
importance using high resolution phenotyping of model plant species. One such project<br />
is an international collaboration using the model species Brachypodium distachyon to<br />
speed up the breeding of next generation biofuel crops and discover genes responsible<br />
for important traits in wheat. Brachypodium, unlike switchgrass or miscanthus, has all the<br />
attributes of a model species (small and fully sequenced genome, self-fertile, short life<br />
cycle, easily transformed and crossed) allowing high throughput screening and<br />
characterisation of traits relevant to biofuel research.<br />
We have developed a variety of non-invasive and destructive assays for growth,<br />
biomass, photosynthesis and root growth and architecture in Brachypodium. Large<br />
phenotypic variability of biomass accumulation was observed in a set of 160 natural<br />
accessions of Brachypodium [1]. Root growth and architecture was characterised in<br />
these ecotypes and show significant variation in total root length, root type distribution<br />
(nodal versus seminal root systems) or shoot/root ratio.<br />
Contrasting ecotypes are currently under investigation using high resolution imaging<br />
analysis techniques to identify the optimal combination of these phenotypic traits under<br />
limiting water and nutrients and to determine the underlying genomic regions<br />
responsible. Since Brachypodium is a typical grass and shares a high degree of genetic<br />
similarity and markers with commercial crops, it would be possible to extend the<br />
knowledge gained from this project to improve plants species with a much more<br />
complex and large genome such as switchgrass and wheat.<br />
References<br />
1. Vogel J, Tuna M, Budak H, et al. (2009) Development of SSR markers and analysis of<br />
diversity in Turkish populations of Brachypodium distachyon. BMC Plant Biology 9: 88.<br />
Keywords<br />
Phenotyping, shoot, root architecture, biofuel, diversity<br />
28
Session 3 – Tools and Bioengineering<br />
S3.6- TILLING in Brachypodium distachyon: development of a Sodium Azide<br />
mutant collection and screening for induced point mutations<br />
Dalmais M. 1, Darracq O. 2, Oria N. 2, Antelme S. 2, Troadec C. 1, Bouvier d’Yvoire M. 2,<br />
Jouanin 2, Lapierre C. 2 Höfte H. 2, Sibout R. 2, Bendahmane A. 1<br />
1 Unité de recherche en génomique végétale (URGV), 2 rue Gaston Crémieux, 91057<br />
Evry FR<br />
2 Institut Jean-Pierre Bourgin (IJPB), UMR1318 INRA-AgroParisTech, 78026 Versailles, FR<br />
dalmais@evry.inra.fr<br />
Abstract<br />
Brachypodium distachyon is an established model plant for grasses. With the completion<br />
of the genome sequencing project in 2010, a major challenge is to determine gene<br />
functions. In plants, the most common techniques to produce altered or loss of function<br />
mutations are T-DNA or transposon insertional mutagenesis and RNA interference.<br />
However, unless a highthroughput transformation protocols become available for<br />
Brachypodium, functional analysis of Brachypodium genes with the tagging approaches<br />
remain expensive and time consuming. On the other hand, chemical mutagenesis is a<br />
straightforward and cost-effective way to saturate a genome with mutations. TILLING<br />
(Targeting Induced Local Lesions IN Genomes) uses chemical mutagenesis coupled with<br />
gene-specific detection of single-nucleotide mutations. This strategy generates allelic<br />
series of the targeted genes which makes it possible to dissect the function of the protein<br />
as well as to investigate the role of essential genes that are otherwise not likely to be<br />
recovered in genetic screens based on insertional mutagenesis. To investigate the<br />
capacity of TILLING as a powerful tool of reverse genetics in Brachypodium we have set<br />
up a TILLING platform and performed a screen for mutations in genes from the lignin<br />
biosynthesis pathway. First we have constructed a reference sodium azide mutant<br />
population of 5530 M2 families in Bd21-3 genetic background and developed a<br />
database, UTILLdb (http://urgv.evry.inra.fr/UTILLdb), that contains phenotypic as well as<br />
sequence information on mutant genes. UTILLdb can be searched online for TILLING<br />
alleles, through the BLAST tool, or for phenotypic information about mutants by keywords.<br />
To setup the TILLING platform, genomic DNA was prepared from the mutant lines and<br />
organized in pools for bulked screening using the mismatch specific endonuclease,<br />
ENDO1. To assess the quality of Brachypodium mutant collection and to estimate the<br />
mutation density, we screened for induced mutations in 6 genes related lignin<br />
biosynthesis pathway. In total, we identified and confirmed by sequencing 92 induced<br />
mutations. We also estimated the overall mutation rate as one mutation every 525 kb in<br />
our Bd21-3 mutant collection. This mutation frequency is similar to the rate of one<br />
mutation per 500 Kb reported both for sorghum and barley (respectively Xin et al., 2008<br />
and Gottwald et al., 2009). A much more saturated mutation density has been observed<br />
in polyploid grasses such as tetraploid wheat, with one mutation per 40 kb, and<br />
hexaploid wheat with one mutation per 24 kb, which withstand much higher doses of<br />
EMS without obvious impact on plant survival. In all together, we routinely identify about<br />
15 alleles per tilled genes which are sufficient to genetically characterize the function of<br />
a gene using the TILLING approach. Several phenotypes will be displayed in this<br />
presentation.<br />
29
Session 3 – Tools and Bioengineering<br />
S3.7- Novel tools for Brachypodium research: Genetic transformation using<br />
shoot segments and immature pollen-derived generation of instantly<br />
homozygous lines<br />
Bianca Melzer, Tilo Guse, Christine Kastner, Katarzyna Plasun, Carolin Berger, Eszter<br />
Kapusi, Götz Hensel, Cornelia Marthe, Petra Hoffmeister and Jochen Kumlehn<br />
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Plant<br />
Reproductive Biology, Corrensstraße 3, 06466 Gatersleben, Germany<br />
kumlehn@ipk-gatersleben.de<br />
Abstract<br />
Brachypodium distachyon is a valuable experimental model species for the<br />
economically important temperate cereals of the tribe Triticeae. To comprehensively<br />
validate gene functions, a viable transformation system is required. Current<br />
transformation methods rest on the use of immature embryos or immature embryoderived<br />
callus as gene transfer recipient tissues. However, the tiny immature embryos are<br />
difficult to dissect in large scale and callus propagated over longer periods of time is<br />
anticipated to undergo significant variation of the genetic and epigenetic background.<br />
In this study, we have demonstrated that efficient plant regeneration can be achieved<br />
from shoot segments of B. distachyon seedlings. A major advantage of this method is<br />
that the explants can be readily produced in large scale from mature grains, i.e. there is<br />
no routine cultivation of donor plants required. Taking advantage of this regeneration<br />
method, callus derived from shoot segments was inoculated using hypervirulent<br />
Agrobacterium carrying the hpt gene and the gfp and gus-intron genes as plant<br />
selectable marker and reporter genes, respectively. This approach resulted in more than<br />
a hundred independent primary transgenic lines. In these plants and their progeny,<br />
reporter gene expression was observed and stable integration of T-DNAs confirmed by<br />
Southern blot, which provided evidence of generative transmission of the transgenes. In<br />
a second approach, a method of immature pollen-derived embryogenesis and plant<br />
regeneration was established in B. distachyon. Since pollen constitutes the haploid<br />
outcome of meiotic recombination and pollen embryogenesis frequently includes<br />
identical genome duplication, this method may facilitate the production of entirely<br />
homozygous mutant or transgenic lines in just one step, which is especially useful when<br />
multiple transgenes or insertion-derived mutations have to be combined.<br />
Keywords<br />
doubled haploid, embryogenesis, homozygous, pollen, transgenic<br />
30
Session 4 – Biotic and Abiotic Stresses<br />
KEYNOTE LECTURE<br />
S4.1- Exploiting Brachypodium distachyon biodiversity to establish sources of<br />
resistance to abiotic and biotic stress.<br />
Luis A. J. Mur 1, Joel Allaingullianme 1, Jessica Gough 1, Rasim Uman 1, Ianto Thomas 1, J.<br />
William Allwood 1, Joel V. Smith, 1 Greg Shelley 1, Andrew P. M. Routledge 1, Adina<br />
Breiman 2, Elena Benavente 3 and Pilar Calatan 4.<br />
1 Institute of Biological, Environmental and Rural Science, Penglais Campus, Aberystwyth University<br />
2 Dept of Plant Sciences Tel Aviv University, Tel Aviv Israel 69978<br />
3 Department of Biotechnology, Polytechnic University of Madrid, 28040-Madrid, Spain<br />
4 Department of Agriculture (Botany), High Polytechnic School of Huesca, University of Zaragoza, Ctra. Cuarte km 1, 22071 Huesca,<br />
Spain.<br />
lum@aber.ac.uk<br />
Abstract<br />
Brachypodium distachyon is an undomesticated model grass species which offers the<br />
possibly of elucidating the genetic bases of multigenic traits associated with abiotic stress<br />
tolerance and durable disease resistance (Mur et al. 2011). To exploit this potential we have<br />
established collections of Brachypodium distachyon and Brachypodium hybridum (formerly<br />
the 2n =30 cytotype of B. distachyon; see presentation by Catalan et al.) germplasm from<br />
Northern Spain and also via, collaboration, incorporated collections from Southern Spain,<br />
Morocco, Israel as well as accessions from Italy, France and Bulgaria. Genetic diversity within<br />
Brachypodium germplasm was assessed through polymorphism at microsatellite loci.<br />
Accession genetic diversity was related to geographical origin with distinct polymorphisms<br />
between Western and Eastern European accession and also between Brachypodium<br />
species. Biochemical variation amongst the accessions was investigated by metabolite<br />
fingerprinting using Fourier Transform Infra-Red (FT-IR) spectroscopy. Using the supervised<br />
multivariate approach – Discriminant Function Analysis (DFA) - variation in metabolites in the<br />
first emerged leaf of Brachypodium genotypes could be linked to geographical origin.<br />
Our assessments of drought tolerance focused on inbred lines developed from our collection<br />
and also the ABR1 through 7 series, USDA lines Bd21, Bd2-3, Bd3-1 as well as from the Turkish<br />
collection established by Vogel et al. (2009). A total of 118 Brachypodium genotypes have<br />
been assessed for drought tolerance and, based on estimates of relative water content and<br />
cytoplasmic membrane stability, Bd2-3 proved to be most drought susceptible and ABR5 the<br />
most tolerant. Metabolomic approaches were employed to further investigate the basis of<br />
the variation in tolerance, based on electrospray ionisation Mass Spectrometry (ESI-MS) of<br />
polar and non-polar extracts from drought and non-droughted plants. Multivariate data<br />
mining indicated that polyamine, arginine and antioxidant metabolites were all elevated in<br />
ABR5 and not Bd2-3. We are now seeking to develop mapping populations to deduce<br />
genetic traits linked to drought tolerance.<br />
Our work on responses to pathogens has focused on responses to Magnaporthe grisea - the<br />
causal agent of rice blast - and rust pathogens, primarily crown rust (Puccina coronata) and<br />
the emerging grassland pathogen, Dreschlera spp. Leafspot in the ABR1 through 7<br />
genotypes. Events associated with local and systemic resistance to pathogens have been<br />
elucidated. Resistance to pathogens was correlated with an elevation in jasmonic acid but<br />
not salicylic acid. Inhibition of jasmonic acid biosynthesis using ursolic acid resulted in<br />
reduced local and systemic resistance. Lipoxygenase (LOX) is a key biosynthetic enzyme in<br />
the jasmonate biosynthetic pathway. Two LOX Bd21 T-DNA mutants are available within the<br />
JIC (UK) T-DNA population (BdAAA466, BdAAA615) and both exhibited reduced jasmonate<br />
accumulation on infection and compromised resistance to rice blast. These data suggest the<br />
importance of jasmonate signalling in Brachypodium and most likely, temperate cereals.<br />
References<br />
Mur LA, Allainguillaume J, Catalán P, et al. (2011) Exploiting the Brachypodium Tool Box in cereal and grass research. New Phytol. 2011<br />
91, 334-47.<br />
Vogel JP, Tuna M, Budak H, et al. (2009). Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium<br />
distachyon. BMC Plant Biology: 13:9:88<br />
31
Session 4 – Biotic and Abiotic Stresses<br />
S4.2- Assessing the role of smallRNAs in leaf cell identity and growth<br />
reprogramming during drought stress in Brachypodium distachyon<br />
Mica, Erica 1 ; Bertolini, Edoardo 1,2; Verelst, Wim 2; Piccolo, Viviana 3; Inzé, Dirk 2; Horner,<br />
David 3 ; Pè, Mario Enrico 1<br />
1 Scuola Superiore Sant’Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy<br />
2 Department of Molecular Genetics, Ghent University, Technologiepark 927, 9052 Ghent,<br />
Belgium<br />
3 Department of Biomolecular Sciences and Biotechnology, University of Milan, via<br />
Celoria 26, 20133 Milan, Italy<br />
erica.mica@sssup.it<br />
Abstract<br />
Food production is limited primarily by environmental stresses, of which drought is one of<br />
the most common. Water deficiency in fact affects both growth and development of<br />
crop plants through alterations in metabolism and gene expression.<br />
As a drought tolerant grass, Brachypodium may possess specific adaptations and<br />
tolerance mechanisms whose characterization could be useful to address drought<br />
tolerance in related crops, such as wheat and barley.<br />
The drought response is a complex trait regulated by a plethora of genetic and<br />
epigenetic networks. Here we address the role of smallRNA molecules, in particular<br />
miRNAs, in fine tuning gene expression in developing leaves under drought stress.<br />
In our soil-based assay, we subjected the reference accession Bd21 to growth-limiting,<br />
non-lethal drought stress. In this condition, we observed that drought stress has no effect<br />
on cell division rates but affects cell expansion resulting in a reduced leaf size. From two<br />
biological replicates we produced four smallRNA libraries obtained from expanding and<br />
proliferating leaf zones subjected either to drought stress or control conditions. Using nextgeneration<br />
sequencing techniques the libraries were sequenced, producing nearly 30<br />
million reads. The bioinformatics pipeline that we developed identified conserved and<br />
species-specific miRNAs, some of which were detected for the first time in<br />
Brachypodium. Statistical analyses were applied to detect miRNAs whose expression is<br />
modulated under stress or in different developmental conditions. Our data suggest that<br />
a higher proportion of miRNAs are involved in developmental programming, while only<br />
few miRNAs are regulated under drought stress in Brachypodium.<br />
We are currently analyzing putative miRNAs targets investigating their possible<br />
involvement in leaf development both in normal and stressed conditions. Combining<br />
miRNAs and miRNA target genes analyses we expect to obtain a more complete picture<br />
of the biological function of these molecules.<br />
Keywords<br />
miRNA, smallRNA, drought, growth-reprogramming, leaf<br />
32
Session 4 – Biotic and Abiotic Stresses<br />
S4.3- Use of the pathosystem Brachypodium distachyon / Fusarium<br />
graminearum to investigate the detoxification mechanisms of mycotoxins by<br />
plants.<br />
Jean-Claude Pasquet, Catherine Macadré, Xavier Deguercy, Patrick Saindrenan and<br />
Marie Dufresne<br />
Institut de Biologie des Plantes, Université Paris-Sud 11, 91405 Orsay, France<br />
marie.dufresne@u-psud.fr<br />
Abstract<br />
Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is one of the<br />
most damaging diseases on small-grain cereals, including wheat (Goswami and Kistler,<br />
2004). During its development in the host plant, this pathogen produces trichothecene<br />
mycotoxins (including deoxynivalenol or DON) that inhibit eukaryotic protein translation,<br />
are harmful to humans, animals (Rocha et al. 2005) and also phytotoxic (Desmond et al.<br />
2008). No gene-for-gene resistance has been reported towards F. graminearum but<br />
quantitative trait loci (QTL) were identified in bread wheat. Genetic, metabolic or<br />
transcriptional analyses have correlated some of these QTLs with functions involved in<br />
DON detoxification, including UDP-glycosyltransferases (UGTs). Our team recently<br />
developed a new pathosystem, the intereaction between the small cereal species<br />
Brachypodium distachyon and F. graminearum, and uses it to study the mechanisms of<br />
DON detoxification. Based on phylogenetic relationships with UGTs identified in<br />
Arabidopsis (Poppenberger et al. 2003) or barley (Gardiner et al. 2010), we have<br />
identified four candidate UGTs from B. distachyon potentially involved in this process and<br />
first investigated how DON or infection by F. graminearum can regulate the expression of<br />
the corresponding genes, using quantitative RT-PCR analyses. We have shown that the<br />
expression of all four candidate genes is strongly induced between 72 and 96 hours after<br />
inoculation by F. graminearum and as soon as 3 hours after application of DON. By<br />
monitoring the expression of the Tri5 gene (gene encoding the first enzyme of the<br />
trichothecene biosynthetic pathway, Foroud and Eudes, 2009), we have further<br />
demonstrated that DON is likely produced at stages where the candidate genes are<br />
induced. Functional analyses of these genes are underway in B. distachyon to decipher<br />
more precisely their involvement in DON detoxification and in resistance towards FHB.<br />
References<br />
Foroud NA, Eudes F (2009) Trichothecenes in cereals grains. International Journal of<br />
Molecular Sciences 10: 147-173<br />
Gardiner SA, Boddu J, Berthiller F, et al. (2010) Transcriptome analysis of the barleydeoxynivalenol<br />
interaction: evidence for a role of glutathione in deoxynivalenol<br />
detoxification. Molecular Plant-Microbe Interactions 23: 962-976<br />
Goswami RS, Kistler HC (2004) Heading for disaster: Fusarium graminearum on cereal<br />
crops. Molecular Plant Pathology 5: 515-525<br />
Poppenberger B, Berthiller F, Lucyshyn D, et al. (2003) Detoxification of the Fusarium<br />
mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana. The<br />
Journal of Biological Chemistry 48: 47905-47914<br />
Rocha O, Ansari K, Dooham FM (2005) Effects of trichothecens mycotoxins on eukaryotic<br />
cells: A review. Food additives and contaminants 22: 369-378<br />
Keywords<br />
Fusarium graminearum, deoxynivalenol, detoxification, UDP-glycosyltransferases<br />
33
Session 4 – Biotic and Abiotic Stresses<br />
S4.4- Functional characterization of Brachypodium UDP-glucosyltransferases<br />
1Wolfgang Schweiger, 1Maria Paula Kovalski, 1Juan Antonio Torres Acosta, 2Marc<br />
Lemmens, 3Hikmet Budak, 3Franz Berthiller, 4Thomas Nussbaumer, 4Klaus Mayer, 1Gerhard<br />
Adam<br />
1 Department of Applied Genetics and Cell Biology, University of Natural Resources and<br />
Life Sciences (BOKU), Konrad Lorenz Str. 25, A-3430 Tulln, Austria<br />
2 Department IFA Tulln (BOKU), Konrad Lorenz Str. 20, A-3430 Tulln, Austria<br />
3 Sabanci University Faculty of Engineering and Natural Sciences, Orhanli, Tuzla-Istanbul,<br />
Turkey<br />
4 Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München,<br />
Neuherberg, Germany<br />
gerhard.adam@boku.ac.at<br />
Abstract<br />
Fusarium graminearum is an important fungal pathogen of small grain cereals and<br />
maize. It produces the protein biosynthesis inhibitor and mycotoxin deoxynivalenol, which<br />
is a virulence factor supporting fungal spread in infected ears. Resistance to Fusarium is<br />
at least partly determined by the ability to convert DON into the non-toxic conjugate<br />
DON-3-O-glucoside (Poppenberger et al. 2003). Also Brachypodium can be infected<br />
with Fusarium, and different accessions can form the detoxification product to a variable<br />
extent.<br />
The gene family of Bd UDP-glucosyltransferases (UGTs) consists of 177 predicted genes.<br />
We characterized the cluster of 6 Bd genes which have the highest sequence similarity<br />
with a barley gene HvUGT13248 conferring toxin resistance in yeast (Schweiger et al.<br />
2010). Only two out of the six Bd homologs confered DON resistance. We conclude that<br />
the UGTs, which frequently occur in clusters containing a variable number of genes<br />
compared to rice and sorghum, seem to evolve very rapidly. It is therefore nontrivial to<br />
identify the true orthologs in crop plants. Validation of the presumed function of<br />
candidate genes by heterologus expression and functional testing in yeast is warranted.<br />
References<br />
References<br />
Poppenberger B, Berthiller F, Lucyshyn D, Sieberer T, Schuhmacher R, Krska R, Kuchler K,<br />
Glössl J, Luschnig C, Adam G (2003) Detoxification of the Fusarium mycotoxin<br />
deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana. J. Biol. Chem.<br />
278: 47905-47914.<br />
Schweiger W, Boddu J, Shin S, Poppenberger B, Berthiller F, Lemmens M, Muehlbauer GJ,<br />
Adam G (2010) Validation of a candidate deoxynivalenol-inactivating UDPglucosyltransferase<br />
from barley by heterologous expression in yeast. Mol. Plant Microbe<br />
Interact. 23: 962-76.<br />
Keywords<br />
Fusarium, mycotoxin, detoxification, resistance<br />
34
Session 4 – Biotic and Abiotic Stresses<br />
S4.5- The Mycosphaerella graminicola-Brachypodium distachyon interaction:<br />
a new model pathosystem to study Septoria tritici blotch disease of wheat.<br />
Aoife O’ Driscoll 1, 2, Fiona Doohan 2 and Ewen Mullins 1<br />
1 Dept. Crop Science, Teagasc Crops, Environment and Land Use Programme, Oak Park,<br />
Carlow.<br />
2 Molecular Plant-Microbe Interactions Laboratory, University College Dublin, Ireland.<br />
aoife.odriscoll@teagasc.ie<br />
Abstract<br />
Mycosphaerella graminicola is the causal agent of Septoria tritici blotch (STB) disease of<br />
wheat, the most economically important foliar pathogen of wheat in Europe. Arising from<br />
a lack of durable resistance in current wheat varieties, wheat producers rely solely on<br />
fungicides to preserve yields. However, the emergence of fungicide resistance and<br />
insensitivity among M. graminicola populations means that the need for improved<br />
varietal resistance is now more imperative than ever before. Quantitative trait loci<br />
associated with STB resistance have been mapped to the chromosomes of the<br />
hexaploid wheat genome but no Stb resistance gene has yet been cloned; neither has a<br />
M. graminicola avirulence gene. Studies on STB are complicated by the complex wheat<br />
genome, long generation time, demanding growth requirements and the limited number<br />
of mutant lines available. The use of model plant species has proven beneficial for the<br />
elucidation of complex host-pathogen systems. Arabidopsis has been used extensively in<br />
such studies but its potential efficacy is limited for STB disease as it is not a natural host of<br />
M. graminicola.<br />
In response to this challenge, we have focussed on the utility of Brachypodium<br />
distachyon as a viable model host species for M. graminicola. The research presented<br />
here demonstrates that Brachypodium is a natural host for M. graminicola and is readily<br />
amenable to infection by multiple isolates of M. graminicola. Displayed disease<br />
symptoms are comparable to those observed in wheat and a screen of multiple<br />
ecotypes has identified a differential response to STB disease within the Brachypodium<br />
gene pool. In addition, we have recorded disease symptoms on all aerial parts of<br />
Brachypodium post inoculation. This report is the first of its kind, and lays the foundations<br />
for the first model pathosystem with which to investigate the mechanisms underlying STB<br />
resistance in a genetically characterised monocotyledonous model species.<br />
Keywords<br />
Septoria, Brachypodium, wheat, model species<br />
35
Session 4 – Biotic and Abiotic Stresses<br />
KEYNOTE LECTURE<br />
S4.6- Brachypodium as a model for unraveling stem rust resistance<br />
David F. Garvin<br />
USDA-ARS Plant Science Research Unit, St. Paul, MN 55108 USA<br />
David.Garvin@ars.usda.gov<br />
Abstract<br />
Members belonging to the fungal genus Puccinia are highly destructive to cool season<br />
cereal crops. Of particular concern is, P. graminis, the causal agent of stem rust, which<br />
poses a threat to wheat production globally due to the emergence of new races that<br />
defeat previously effective resistance genes. Unlike Arabidopsis, Brachypodium<br />
distachyon (Brachypodium) is reported to serve as a host to Puccinia species, and thus it<br />
may be a valuable surrogate for exploring Brachypodium-rust pathosystems. We have<br />
found that Brachypodium can be colonized by different formae specialis of P. graminis,<br />
and that natural variation for resistance resides in Brachypodium germplasm. Genetic<br />
analysis of resistance to timothy stem rust, P. graminis phlei-pratensis, in one recombinant<br />
inbred population suggests major gene control, and efforts are underway to isolate this<br />
gene. Similarly, there is significant variation for resistance to wheat stem rust (P. graminis<br />
tritici). However, unlike timothy stem rust reactions, the disease phenotypes here are<br />
highly diverse, suggesting that the resistance may be quantitative in nature. We<br />
hypothesize that some of this broad variation may reflect minor gene variation<br />
associated with non-host resistance. Screens of mutant populations have identified<br />
validated genotypes both with increased susceptibility and with enhanced resistance,<br />
and genetic studies are ongoing to further explore their biological basis.<br />
Keywords<br />
Plant disease, natural variation, molecular genetics, gene cloning, resistance genes<br />
36
Session 4 – Biotic and Abiotic Stresses<br />
S4.7- Brachypodium distachyon, a model grass to study plant-pathogen<br />
interactions<br />
Thierry C Marcel 1, Rients E Niks 2, June Chun Wang 2, Sébastien Antelme 3, Jean-Benoit<br />
Morel 4, Marie Dufresne 5, Thierry Langin 6, Diana Fernandez 7, Stéphane Bellafiore 7, Jia<br />
Chen 7, Christophe Brugidou 8, Richard Sibout 3<br />
1 INRA-AgroParisTech, UR1290 BIOGER-CPP, Avenue Lucien Brétignières BP01, 78850 Thiverval-Grignon, France<br />
2 Laboratory of Plant Breeding, Wageningen University, Droevendaalsesteeg 1, 6708 Wageningen, the Netherlands<br />
3 INRA-AgroParisTech, UMR1318 Institut Jean-Pierre Bourgin, Route de St-Cyr (RD10), 78026 Versailles Cedex, France<br />
4 INRA Campus International de Baillarguet, UMR BGPI, INRA TA A-54/K, 34398 Montpellier, France<br />
5 Institut de Biologie des Plantes, Université Paris-Sud 11, 91405 Orsay, France<br />
6 UMR INRA-UBP 1095, GDEC, 234 avenue des Landais, 63100 Clermont-Ferrand, France.<br />
7 Centre IRD, UMR 186 IRD-UM2-Cirad Résistance des Plantes aux Bioagresseurs, 911 avenue Agropolis, 34394 Montpellier Cedex 5,<br />
France<br />
8 Centre IRD, UMR 5096 UP-IRD-CNRS, 911 avenue Agropolis, 34394 Montpellier Cedex 5, France<br />
thierry.marcel@versailles.inra.fr<br />
Abstract<br />
Brachypodium (alias Brachypodium distachyon) serves as a model plant species for grass<br />
genomics. There is notably a growing interest in developing Brachypodium as a model<br />
plant species to study economically significant pathogens of cereals. The discovery of<br />
key genes determining the outcome of the interaction between Brachypodium and<br />
pathogens will permit the detection of possible orthologues in temperate cereals that<br />
could be exploited as source of genetic resistance to these pathogens. We explored two<br />
approaches aimed at using the natural diversity of Brachypodium to unravel such genes<br />
involved in plant-pathogen interactions. First, Brachypodium is a host species for some<br />
pathogens of cereals and any type of resistance found in Brachypodium accessions may<br />
be exploited. Brachypodium indeed is a host to the fungal pathogens Magnaporthe<br />
oryzae (Routledge et al. 2004; Wang et al. 2011) and Fusarium graminearum (Peraldi et<br />
al. 2011) that normally infect rice, wheat and barley, respectively. Second,<br />
Brachypodium is a non-host (all accessions are resistant to all isolates of the pathogen) to<br />
other cereal pathogens. Then, related pathogens may be collected from Brachypodium<br />
in nature and used for further research. This was recently proposed by Barbieri et al.<br />
(2011) who showed that Brachypodium has susceptible and resistant accessions to<br />
isolates of the leaf rust Puccinia brachypodii. We established a core collection of diploid<br />
Brachypodium inbred lines by selecting 40 lines available to the research community that<br />
maximize the phenotypic and genotypic diversity in the species. We used this core<br />
collection to determine the host range and response of Brachypodium to the fungal<br />
pathogens Magnaporthe oryzae, Fusarium graminearum, Puccinia graminis, P. striiformis,<br />
P. brachypodii, and to the rice root-knot nematode Meloidogyne graminicola and rice<br />
yellow mosaic virus. Our effort is providing a general overview of the possibility to use<br />
Brachypodium as a model system to study important grass and cereal pathogens, and<br />
will reveal key lines and interactions to perform further genetic and functional studies.<br />
References<br />
Barbieri M, Marcel TCM, Niks RE (2011) Host status of false brome grass (Brachypodium) to the leaf rust fungus Puccinia brachypodii<br />
and the stripe rust fungus P. striiformis. Plant Disease: In Press.<br />
Peraldi A, Beccari G, Steed A, Nicholson P (2011) Brachypodium distachyon: a new pathosystem to study Fusarium head blight and<br />
other Fusarium diseases of wheat. BMC Plant Biology: In Press.<br />
Routledge APM, Shelley G, Smith JV, et al. (2004) Magnaporthe grisea interactions with the model grass Brachypodium distachyon<br />
closely resemble those with rice (Oryza sativa). Molecular Plant Pathology 5: 253-265.<br />
Wang X-Y, Wang J-Y, Jiang H, et al. (2011) Pathogenicity of rice blast fungus Magnaporthe oryzae on Brachypodium distachyon.<br />
Chinese Journal of Rice Science 25: 314-320.<br />
Keywords<br />
Pathogen, Natural variation, Host status, Core collection<br />
37
Session 4 – Biotic and Abiotic Stresses<br />
S4.8- QTLs for resistance to the leaf rust Puccinia brachypodii in the model<br />
grass Brachypodium distachyon<br />
Mirko Barbieri*, Thierry C. Marcel** , ***, Rients E. Niks***, Enrico Francia*, Marianna<br />
Pasquariello*, Valentina Mazzamurro*, David F. Garvin****, Nicola Pecchioni*<br />
*Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio Emilia,<br />
Via Amendola 2, 42122 Reggio Emilia, Italy<br />
**INRA-AgroParisTech, UR1290 BIOGER-CPP, Avenue Lucien Brétignières BP01, 78850<br />
Thierval-Grignon, France<br />
***Laboratory of Plant Breeding, Wageningen University, Droevendaalsesteeg 1 6708<br />
Wageningen, The Netherlands<br />
****USDA-ARS Plant Science Research Unit, 411 Borlaug Hall, University of Minnesota, 1991<br />
Upper Buford Circle, St. Paul, MN, 55108, USA<br />
nicola.pecchioni@unimore.it<br />
Abstract<br />
The wild grass Brachypodium distachyon is a useful model for temperate cereals (Draper<br />
et al. 2001; Garvin et al. 2008), but its potential to study the interactions with pathogens<br />
remains underexploited. Leaf rust is one of the major fungal diseases affecting cereals,<br />
and recently the host status of Brachypodium to Puccinia rusts was investigated (Barbieri<br />
et al. 2011).We aimed to identify genomic regions associated with quantitative<br />
resistance to leaf rust in Brachypodium. Two inbred lines, Bd3-1 and Bd1-1, with<br />
quantitative differences in their level of resistance to Puccinia brachypodii, were crossed<br />
to develop an F2 population of 110 plants, that were evaluated for reaction to a P.<br />
brachypodii isolate at both seedling and advanced growth stages, by means of the<br />
AUDPC (Area Under Disease Progress Curve) score. Results from the F2 plants were<br />
validated in F2-derived F3 families. The mapping population showed quantitative and<br />
transgressive segregation for leaf rust resistance. We applied AFLP, SNP and SSR markers<br />
to develop a new Bachypodium linkage map of 203 loci spanning 811.8 cM, and<br />
anchored to its genome sequence.<br />
Three leaf rust resistance QTLs (Rpbq1, Rpbq2 and Rpbq3) were identified on<br />
chromosomes 2, 3 and 4. The resistant alleles of Rpbq1 and Rpbq2 were contributed by<br />
the resistant parent Bd3-1, while for Rpbq3 the resistant allele came from Bd1-1. This study<br />
was, to our knowledge, the first quantitative analysis of any trait in Brachypodium<br />
(Barbieri et al., in preparation). To begin the process of isolating the QTLs, we have<br />
chosen the 8 candidate genes closest to the peaks of Rpbq2 and Rpbq3, based on<br />
genome sequence information. The genes were resequenced in the parents, and<br />
polymorphisms identified for mapping efforts. The validation of gene sequences for a leaf<br />
rust resistance QTL will lead to a deeper understanding of mechanisms of quantitative<br />
resistance to this and to other rust pathogens that infect members of the Triticeae.<br />
References<br />
Draper J., Mur L.A., Jenkins G., et al. (2001) Brachypodium distachyon. A new model system for functional genomics in grasses. Plant<br />
Physiol. 127:1539-1555<br />
Garvin D.F., Gu Y.Q., Hasterok R., et al. (2008) Development of genetic and genomic research resources for Brachypodium<br />
distachyon, a new model system for grass crop research. Crop Sci 48:69-84<br />
Barbieri M., Marcel T.C., Niks R.E. (2011) Host status of false brome grass (Brachypodium) to the leaf rust fungus Puccinia brachypodii<br />
and the stripe rust fungus P. striiformis. Plant Disease, Volume 0, Number ja (doi: 10.1094/PDIS-11-10-0825)<br />
Barbieri M., Garvin D. F., Marcel T.C., Niks R.E., Francia E., Pasquariello M., Mazzamurro V., Pecchioni N. QTLs for resistance to the leaf<br />
rust Puccinia brachypodii in the model plant Brachypodium distachyon. In preparation<br />
Keywords<br />
Puccinia brachypodii, genetic map, leaf rust, quantitative resistance, QTL<br />
38
Session 4 – Biotic and Abiotic Stresses<br />
S4.9- Brachypodium distachyon: an excellent model for Fusarium<br />
graminearum infection in wheat<br />
Antje Bluemke, Christian A. Voigt<br />
Molecular Phytopathology and Genetics, Biocenter Klein Flottbek, University of Hamburg,<br />
22609 Hamburg, Germany.<br />
antje.bluemke@botanik.uni-hamburg.de<br />
Abstract<br />
Fusarium head blight (FHB) is one of the most important cereal diseases. It leads to severe<br />
mycotoxin contamination of grain and extensive yield losses. The plant pathogen<br />
Fusarium graminearum is the most common agent of FHB. Investigating the interaction<br />
between F. graminearum and cereals, esp. wheat, was always limited by the genetic<br />
inaccessibility of these plants. During the last years Brachypodium distachyon has been<br />
established as a model system for the temperate grasses, including wheat.<br />
In a first step, we established the F. graminearum infection of B. distachyon. Disease<br />
symptoms resembled that in wheat. To investigate the established pathosystem on a<br />
cellular level, F. graminearum wild-type and defined mutants with reduced virulence on<br />
wheat ( fgl1, secreted lipase [1] tri5, trichodiene synthase [2], gpmk1, mitogenactivated<br />
protein kinase [3]) were used for infection. All F. graminearum strains expressed<br />
GFP constitutively to follow infection in planta. A comprehensive view on the<br />
pathosystem was gained by combining data of mycotoxin (deoxynivalenol (DON))<br />
production and pathogen-induced alteration of the cell wall composition with the in<br />
planta infection progress.<br />
Regarding DON accumulation in plant tissue, all data from B. distachyon resembled<br />
those known from wheat. A strong increase in DON occurred during F. graminearum<br />
wild-type and fgl1 infection, whereas for the mutant strains tri5 and gpmk1 DON was<br />
not detectable. Similar alterations of the cell wall composition were observed in B.<br />
distachyon as well as in wheat after infection with F. graminearum. Also the infection<br />
progress of all tested F. graminearum strains was comparable to the described progress<br />
in wheat.<br />
Our results show that B. distachyon responds to F. graminearum infection on a visual and<br />
cellular level like wheat. Therefore, Brachypodium serves as an excellent model to study<br />
FHB in small grain cereals, especially wheat.<br />
References<br />
1. Voigt CA, Schäfer W, Salomon S (2005) A secreted lipase of Fusarium graminearum is a<br />
virulence factor required for infection of cereals. The Plant Journal 42: 364-375.2.<br />
2. Jansen C, von Wettstein D, Schafer W, et al. (2005) Infection patterns in barley and<br />
wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted<br />
Fusarium graminearum. Proc Natl Acad Sci U S A 102: 16892-16897.<br />
3. Jenczmionka NJ, Maier FJ, Lösch AP, Schäfer W (2003) Mating, conidiation and<br />
pathogenicity of Fusarium graminearum, the main causal agent of the head-blight<br />
disease of wheat, are regulated by the MAP kinase gpmk1. Current Genetics 43: 87-95.<br />
Keywords<br />
Brachypodium, Fusarium<br />
39
Session 5 – Plant Development, Metabolism and Physiology<br />
S5.1- Brachypodium distachyon grain: development-associated changes in<br />
morphology and storage accumulation<br />
Colette Larré 1, Anne-Laure Chateigner-Boutin 1, Brigitte Bouchet 1, Fabrice Petipas 2,<br />
Hélène Rogniaux 1, Luc Saulnier 1, Bertrand Dubreucq 2 , Fabienne Guillon 1<br />
1 UR1268, INRA BIA, Rue de la Géraudière, BP71627, 44316 Nantes, France<br />
2 Unité de Biologie des Semences, INRA, RD 10, 78026 Versailles cedex, France<br />
Colette.Larre@nantes.inra.fr<br />
Abstract<br />
Brachypodium distachyon is a promising new model for temperate cereals.<br />
In an effort to characterize this model, we recently analysed the composition of<br />
Brachypodium mature grain. Brachypodium grain is notable for its low starch content<br />
(less than 10% of the endosperm compared to 60-70% in other cereals), and its high cell<br />
wall polysaccharide content (>55%) compared to 2–7% in other cereals and consisting<br />
especially of (1-3)(1-4) D-glucans (48% grain dry weight) and arabinoxylans. Protein<br />
content is in the upper range reported for other cereals (17%). The major grain<br />
components, which serve as reservoir for carbon, nitrogen and sulphur for postgerminative<br />
growth, appear therefore to be -glucans and proteins (globulins and<br />
glutelins).<br />
Here we present the characterization of Brachypodium grain development. Grain<br />
morphology as well as polysaccharides and protein accumulation were studied by<br />
microscopy. Proteins were identified by mass spectrometry. The grain development<br />
spans 54 days after flowering (DAF). Maximum size (about 6 mm) is reached at 14 DAF<br />
when the grain fresh weight is about 7 mg. From 14 to 40 DAF, storage compounds<br />
accumulate to reach a weight of about 8.5 mg. At the end of dehydration grain weight<br />
is about 5 mg.<br />
Although starch is not detected in the mature grain outer layers, it accumulates in the<br />
early stages of development. In the endosperm, starch is detected from 14 DAF and<br />
during grain development starch granules increase in size. Nevertheless, their final<br />
number and size are limited. -glucans accumulate in endosperm cell wall from 7 DAF<br />
during cellularisation.<br />
Storage proteins accumulate from 15 DAF. At this stage the major types of storage<br />
proteins are detected. Protein analysis during early development, reveals enzymes<br />
involved in many metabolic pathways. From 15 DAF, the accumulation of storage<br />
proteins hinders the detection of other proteins. In mature grain, storage proteins are<br />
stored in small and large « vesicles ».<br />
References<br />
Guillon F, Bouchet B, Jamme F, Robert P, Quémener B, Barron C, Larré C, Dumas P,<br />
Saulnier L (2011) Brachypodium distachyon grain : characterization of endosperm cell<br />
walls. Journal of Experimental Botany 62 : 1001-1015.<br />
Larré C, Penninck S, Bouchet B, Lollier V, Denery-Papini S, Guillon F, Rogniaux H (2010).<br />
Brachypodium distachyon grain : identification and subcellular localization of storage<br />
proteins. Journal of Experimental Botany 61 : 1771-1783.<br />
Keywords<br />
Grain development, grain composition (proteins and carbohydrates)<br />
40
Session 5 – Plant Development, Metabolism and Physiology<br />
S5.2- The transcription factor BdDOF24 interacts with BdGAMYB and regulates<br />
the cathepsin B-like gene during Brachypodium seed germination<br />
Virginia González-de la Calle, Sara Hernando-Amado, Raquel Iglesias-Fernández, Cristina<br />
Barrero-Sicilia and Pilar Carbonero<br />
CBGP (Centro de Biotecnología y Genómica de Plantas) ETSI Agrónomos. Universidad<br />
Politécnica de Madrid. Campus de Montegancedo 28223 Pozuelo de Alarcón (Madrid)<br />
virginia.gonzalez@upm.es<br />
Abstract<br />
Transcription factors (TFs) are important regulators of gene expression. According to the<br />
specificity of the DNA-binding domain, TFs can be classified into different families. The<br />
DOF (DNA binding with One Finger) transcription factor family belongs to the class of zinc<br />
finger domains and it is characterized by a conserved region of 52 amino acid residues<br />
that is structured as a Cys2/Cys2 Zn 2+ finger that binds specifically the CREs containing<br />
the common core 5’-T/AAAAG-3’. DOF transcription factors have been associated with<br />
regulation during seed maturation and germination in cereals. We have annotated 27<br />
putative BdDof genes of Brachypodium distachyon; their phylogenetic relationship with<br />
closely related DOF proteins from rice and barley have been established and their<br />
global expression levels in different tissues, as well as the expression pro<strong>file</strong> of the most<br />
abundant BdDof genes during seed germination have been determined. One such a<br />
gene, BdDof24 was predominantly expressed in germinating seeds.<br />
Promoters of hydrolase genes, induced upon germination, such are those encoding -<br />
amylase, EII(1-3, 1-4)- -glucanase and thiol-proteases (cathepsin B-like) contain, in<br />
addition to the 5’-T/AAAAG-3’ motif, a GA responsive element (GARE-5’-TAACAAA-3’).<br />
Two TFs binding to these two motives have been identified: BdDOF24 and BdGAMYB. We<br />
have also annotated the putative ortholog of the barley CathB-like gene (Al21):<br />
BdCathB, we established its expression pro<strong>file</strong> in different organs and throughout seed<br />
germination. The expression pattern of genes encoding BdGAMYB and BdDOF24<br />
suggests a possible transcriptional control of the CathB gene by these TFs during seed<br />
germination. This hypothesis has been further validated by EMSA, by molecular<br />
interactions (Y2Hassays) between both TFs and by transient assays in planta.<br />
References<br />
Gubler F, Raventos D, Keys M, et al. (1999) Target genes and regulatory domains of the<br />
GAMYB transcriptional activator in cereal aleurone. Plant J 17(1): 1-9.<br />
Mena M, Cejudo FJ, Isabel-Lamoneda I, Carbonero P (2002) A role for the DOF<br />
transcription factor BPBF in the regulation of gibberellin-responsive genes in barley<br />
aleurone. Plant Physiol 130: 111-119.<br />
Moreno-Risueno MA, Martínez M, Vicente-Carbajosa J, Carbonero P (2007) The family of<br />
DOF transcription factors: from green unicellular algae to vascular plants. Mol Genet<br />
Genomics 277:379-390.<br />
Keywords<br />
Transcriptional regulation, Germination, DOF transcription factors, Hydrolases<br />
41
Session 5 – Plant Development, Metabolism and Physiology<br />
S5.3- Rhizobacterial volatile organic compounds modulate biomass<br />
production and root architecture in Arabidopsis thaliana (L.) Heynh. and<br />
Brachypodium distachyon (L.) P. Beauv.<br />
Delaplace Pierre 1*, Varin Sébastien 1, Ormeño-Lafuente Elena 2, Spaepen Stijn 3, Saunier de<br />
Cazenave Magdalena 1, Blondiaux-Hendrick Adeline 1, Wathelet Jean-Paul 4, du Jardin<br />
Patrick 1<br />
1 University of Liège, Gembloux Agro-Bio Tech, Plant Biology Unit, Gembloux, Belgium<br />
2 Current address : Centre National de la Recherche Scientifique, Institut Ecologie et<br />
Environnement, Institut Méditerranéen d'Ecologie et Paléoécologie, UMR CNRS 6116,<br />
Laboratoire de Diversité Fonctionnelle des Communautés Végétales, Marseille, France<br />
3 Centre of Microbial and Plant Genetics, K.U.Leuven, Bio-incubator, Heverlee, Belgium<br />
4 University of Liège, Gembloux Agro-Bio Tech, General and Organic Chemistry Unit,<br />
Gembloux, Belgium<br />
* Pierre.delaplace@ulg.ac.be<br />
Abstract<br />
The ability of plants to take up water and mineral nutrients from the soil depends on their<br />
capacity to develop an extensive root system and on the interactions of roots with their<br />
soil environment, the rhizosphere. Studies of the mechanisms involved in the<br />
communication between plant growth-promoting soil micro-organisms and the root<br />
system is expected to lead to improved fertility management strategies. Up to now, the<br />
characterization of such interactions has been mainly focused on liquid diffusates but it<br />
has been recently reported that volatile organic compounds (VOC) also play a role as<br />
chemical messengers in positive interactions occurring in the rhizosphere and involving<br />
plants, bacteria, fungi and insects [1-2].<br />
In this context, this project aims to better understand the ecophysiology of the<br />
rhizosphere of Arabidopsis thaliana and Brachypodium distachyon. For this purpose, a<br />
collection of 18 rhizobacterial strains belonging to nine genera were selected for their<br />
potential growth promotion ability. The VOC-emission capacity of each strain was<br />
measured after 24 and 48 hours of growth on agar media [3] using solid-phase<br />
microextraction followed by gas chromatography coupled to a mass spectrometer.<br />
In parallel, an in vitro screening system was set up to assess VOC-mediated plant-growth<br />
promotion ability of the selected strains. This system separates physically the plant and<br />
the bacteria while allowing interactions through VOC for 10 days within a shared<br />
atmosphere. For Arabidopsis, two inoculum sizes were considered: 2*10 5 and 2*10 6 cfu,<br />
using biological triplicates. At the end of the process, leaf and root biomasses, leaf area,<br />
primary root length, lateral root number and lateral root length were measured to assess<br />
the effects of bacterial VOC on plant growth. Experiments are in progress to characterize<br />
such parameters on Brachypodium in order to compare the response of both model<br />
species to rhizobacterial volatile perception.<br />
Acknowledgements: this work was supported by the Belgian Fonds de la Recherche<br />
Scientifique (FRFC project 2.4.591.10. and postdoctoral grant 1808458).<br />
References<br />
1. Ryu CM, Farag MA, Hu CH, et al. (2003) Bacterial volatiles promote growth in Arabidopsis. Proc Natl Acad Sci USA 8: 4927-4932.<br />
2. Kai M, Effmert U, Berg G, Piechulla B (2007) Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen<br />
Rhizoctonia solani. Arch Microbiol 187: 351-360.<br />
3. Farag MA, Ryu CM, Sumner LW, Paré PW (2006) GC–MS SPME profiling of rhizobacterial volatiles reveals prospective inducers of<br />
growth promotion and induced systemic resistance in plants. Phytochemistry 67: 2262-2268.<br />
Keywords<br />
root architecture, plant growth-promoting rhizobacteria, volatile organic compounds<br />
42
Session 5 – Plant Development, Metabolism and Physiology<br />
S5.4- Using Natural And Induced Variation In Brachypodium To Study Grain<br />
Development<br />
John H Doonan 1, Magdalena Opanowicz 2, Max Bush 2 , Kay Trafford 2 , Vera Thole 2 ,<br />
Sinead Drea 3 , Phillip Hands 3, Philippe Vain 2<br />
1 Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,<br />
Ceredigion, UK<br />
2 John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK<br />
3 University of Leicester, University Road, Leicester, LE1 7RH, UK<br />
john.doonan@aber.ac.uk<br />
Abstract<br />
Grains provide the vast majority of calories consumed by humans and projected yields<br />
must dramatically and rapidly increase if global food supply is to keep up with demand.<br />
While it is clear that our major grain crops have much potential to deliver such increases<br />
in yield, it is imperative that the rate of yield increase should itself be increased. This will<br />
require the exploration of different complementary approaches, including conventional<br />
breeding, through transgenics and improved agronomy. A thorough understanding of<br />
grain development will underpin such efforts and we have begun to compare grain<br />
development in different species to understand the molecular and cellular basis of the<br />
diversity in grain development and composition (Opanowicz et al. 2011).<br />
Brachypodium distachyon is an attractive fast cycling small grass being developed as a<br />
model for grass and cereal biology and also is of interest as a widely distributed wild grass<br />
that is adapted to a range of habitats (Opanowicz et al. 2008). Grain development, for<br />
example, is largely similar to that of wheat and other temperate cereals, but displays<br />
some important differences (Opanowicz et al. 2011). Natural and induced variation in<br />
Brachypodium grain development can be exploited to understand the regulation of<br />
grain development. Natural accessions collected from diverse locations display<br />
variation in grain size and shattering as well as in plant morphology, offering the possibility<br />
of understanding the genetic control of grain traits in an undomesticated species.<br />
Selected T-DNA tagged lines, where specific genes have been disrupted, also display<br />
defects in plant and grain development, providing the opportunity to understand in<br />
detail the genetic regulation of grain development and growth (Vain et al. 2011).<br />
References<br />
Opanowicz M, Vain P, Draper J, Parker D, Doonan JH (2008) Brachypodium distachyon:<br />
making hay with a wild grass. Trends Plant Sci 13: 172-177.<br />
Opanowicz M, Hands P, Betts D, Parker ML, Toole GA et al. (2011) Endosperm<br />
development in Brachypodium distachyon. J Exp Bot 62: 735-748.<br />
Vain P, Thole V, Worland B, Opanowicz M, Bush MS et al. (2011) A T-DNA mutation in the<br />
RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium<br />
distachyon. Plant J 66: 929–940.<br />
43
Session 5 – Plant Development, Metabolism and Physiology<br />
S5.5- Profiling the lipidome of Brachypodium distachyon<br />
M. Nurul Islam, John P. Chambers, Carl K.-Y. Ng<br />
School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4,<br />
Ireland<br />
carl.ng@ucd.ie<br />
Abstract<br />
Lipids are essential metabolites in cells and they fulfil a variety of functions, including<br />
structural components of cellular membranes, energy storage, cell signalling, and<br />
membrane trafficking. In plants, changes in lipid composition have been observed in<br />
diverse responses ranging from abiotic and biotic stress to organogenesis. Knowledge of<br />
the lipid composition is an important first step towards understanding the function of lipids<br />
in any given biological system. As Brachypodium distachyon is emerging as the model<br />
species for temperate grass research, it is therefore fundamentally important to gain<br />
insights of its lipid composition. We used HPLC-coupled with tandem mass spectrometry<br />
to pro<strong>file</strong> and quantify levels of sphingolipids and glycerophospholipids in B. distachyon<br />
plants. A total of 123 lipids belonging to 10 classes were identified and quantified.<br />
Additionally, we showed that 4-sphingenine (d18:1 4) is the main unsaturated dihydroxy-<br />
LCB in B. distachyon, and we were unable to detect d18:1 8, which is the main<br />
unsaturated dihydroxy-LCB in the model dicotyledonous species, Arabidopsis thaliana.<br />
This work serves as the first step towards a comprehensive characterisation of the B.<br />
distachyon lipidome that will complement future biochemical studies.<br />
Keywords<br />
Brachypodium distachyon, lipidome, glycerophospholipids, sphingolipids, LC-MS/MS<br />
44
Session 5 – Plant Development, Metabolism and Physiology<br />
S5.6- Inducible xylem differentiation in Brachypodium<br />
Elene R. Valdivia, María Teresa Herrera, Cristina Gianzo, Gloria Revilla, Ignacio Zarra y<br />
Javier Sampedro<br />
Departamento de Fisiología Vegetal, Facultad de Biología, Universidad de Santiago de<br />
Compostela<br />
elenevaldivia@gmail.com<br />
Abstract<br />
The manipulation of secondary cell wall composition in biomass crops is one of the most<br />
promising strategies to reduce the processing cost of lignocellulosic materials, which<br />
could then become an abundant source of cheap biofuels. Grasses are good<br />
candidates for this approach, but very little is known about the regulation of grass cell<br />
wall synthesis.<br />
In the dicot Arabidopsis several of the transcription factors (TFs) involved in secondary<br />
cell wall synthesis have been characterized in detail. In particular, VND genes seem to<br />
act at very early stages of xylem differentiation and the expression of some of the VNDs is<br />
sufficient to start the process of secondary cell wall deposition. We have identified and<br />
cloned Brachypodium orthologs of the Arabidopsis VNDs. When three of these genes<br />
(VND2, VND4 and VND5) are overexpressed transiently in tobacco leaves, they are<br />
capable of inducing transdifferentiation into tracheary elements suggesting a<br />
conservation of function.<br />
Brachypodium can be transformed with high efficiency through cocultivation of<br />
embryogenic calli with Agrobacterium. We have adapted the estradiol inducible system<br />
for use in Brachypodium and have transformed this species with inducible overexpression<br />
constructs for VND2, VND4 and VND5, as well as chimeric respressors obtained from the<br />
same genes by addition of a repressor domain. Inducible overexpression of VND5 results<br />
in extensive transdifferentiation of different cell types into tracheary element. Through RT-<br />
PCR we have detected an increase expression of cellulose synthase, as well as some TFs<br />
that could work downstream of the VND genes. We are currently characterizing this<br />
plants which could provide a first draft of the regulatory network of secondary cell wall<br />
synthesis in grasses. The repressor lines will be characterized soon and could allow us to<br />
identify feedback mechanisms that detect secondary cell wall alterations.<br />
Keywords<br />
Brachypodium, Secondary cell wall synthesis, Transcription Factors<br />
45
POSTER PRESENTATIONS<br />
46
Session 1 – Evolution and Natural Variation<br />
P1.1- Characterization of the diploid and tetraploid Brachypodium<br />
distachyonpopulations in Israel<br />
Assaf Distelfeld, Smadar Ezrati, Tamar Eilam, Hanan Sela, Raz Avni, and Adina Breiman<br />
Dept. Molecular Biology and Ecology of Plants, The Institute for Cereal Crops<br />
Improvement, Tel Aviv University, Israel.<br />
adinaB@tauex.tau.ac.il<br />
Abstract<br />
Brachypodium distachyon that has emerged as a powerful model system to study the<br />
unique aspects of the grasses is very abundant in Israel. It grows in diverse habitats from<br />
the dry desert areas in the south to the relatively rainy Mediterranean areas in the north<br />
and on different soil types. The Institute for Cereal Crops Improvement in Tel-Aviv<br />
University holds a collection of crops wild relatives, including B. distachyon (BD) from<br />
Israel. The BD collection includes accessions representing the diverse habitats. In a<br />
preliminary study, to determine their ploidy level, 272 plants from the BD collection were<br />
evaluated for their DNA content. Ninety three plants from 23 sites were found to have<br />
similar DNA content as the diploid sequenced line BD21 while the rest had double<br />
amount of DNA content and most likely are allopolyploids. The plants were grown under<br />
controlled greenhouse conditions and showed considerable morphologic and<br />
phenotypic variability in plant architecture, leaf size, leaf color, flowering time, spike<br />
morphology and biomass. Our aim is to associate this phenotypic data with<br />
transcriptomic changes among the diploid genotypes during the vegetative stage in<br />
order to identify genes that control biomass production. The identification of novel genes<br />
conferring increase biomass production will have a significant practical relevance. In<br />
addition, we plan to genotype the collection with SSR markers to study the population<br />
structure and gene flow.<br />
Keywords<br />
Brachypodium distachyon collection, phenotypic variation, biomass, diverse habitats.<br />
47
Session 1 – Evolution and Natural Variation<br />
P1.2- Diversity analysis in Spanish populations of Brachypodium distachyon<br />
Patricia Giraldo, Gaspar Soria, Iciar Sevilla, Marta Rodríguez-Quijano, Jose F Vázquez,<br />
Jose M Carrillo, Elena Benavente<br />
Departamento de Biotecnología (Unidad de Genética). E.T.S. Ingenieros Agrónomos.<br />
Universidad Politécnica de Madrid<br />
patricia.giraldo@upm.es<br />
Abstract<br />
The objective of this work has been to analyze the variability of a germplasm collection<br />
held at ETSI Agrónomos (Universidad Politécnica de Madrid). It is composed by 64 natural<br />
populations morphologically ascribable to Brachypodium distachyon that were<br />
collected by our research group in a wide area of the continental Spain. Climate and<br />
geographical parameters from the collection sites were recorded in order to explore<br />
their correlation with genetic variability data.<br />
The cytological analysis has shown that most of the accessions belong to the diploid (2n=<br />
10) and allotetraploid (2n=30) cytotypes, but a few populations with 2n= 20 individuals<br />
have also been found. We have tried to distinguish between the diploid and<br />
allotetraploid cytotypes by electrophoretic analysis of endosperm proteins, but no<br />
differential marker has been found. The protein pattern of allotetraploid accessions has<br />
revealed a higher degree of polymorphism compared to diploid accessions.<br />
One hundred and ninety-three individuals from 32 diploid (2n=10) populations have been<br />
genotyped with 15 microsatellites (SSRs) selected based on their genomic distribution<br />
and pro<strong>file</strong> quality. The total number of amplified alleles was equal to 94. The number of<br />
alleles per locus varied from 1 to 24, with a mean of 6.26 alleles per locus. Diversity<br />
analysis reflected a comparable diversity both between and within wild populations<br />
(Total gene diversity Ht= 0.464; gene differentiation between populations Gst= 0.514). No<br />
correlation has been found between genetic and ecoclimatic variables.<br />
Four microsatellites have shown a different allelic pattern in diploid versus allotetraploid<br />
individuals, thus providing a useful tool for to discriminate between both cytotypes.<br />
Inbred lines and RILs, developed from selected individuals, will be available for the<br />
scientific community.<br />
Keywords<br />
genetic variability, wild populations, molecular markers<br />
48
Session 1 – Evolution and Natural Variation<br />
P1.3- Morpho-phenologic diversity among Tunisian populations of<br />
Brachypodium Distachyon<br />
Mohamed Neji, Sondes Rahmouni, Warda Saoudi, Malek Smida, Wael Tamaalli<br />
Mounawer Badri, Chedly Abdelly and Gandour Mhemmed<br />
Center of Biotechnology of Borj-Cédria, BP 901 hammam lif 2050 Tunisia<br />
mnmedneji@gmail.com<br />
Abstract<br />
Brachypodium distachyon, commonly called ‘purple false brome’, belongs to Poacea<br />
grass subfamily. This species is mainly distributed in South Europe, North Africa, Southwest<br />
Asia and many other places. It is frequently diploid and has a small genome size<br />
(300Mb). B. distachyon has a close genetic relationship with temperate cereal crops. In<br />
order to bring a better knowledge on the genetic diversity of this species, 9 populations<br />
of B. distachyon representative of all the eco-climatic stages of Tunisia were<br />
characterized on the basis of 18 morpho-phenologic features. Data obtained for the 180<br />
evaluated lines were subjected to various statistical analyses. It arises from the statistical<br />
analyses significant variations for all studied features.<br />
Correlations analysis shows that the majority of morphometric traits were negatively and<br />
significantly correlated between them. The relative importance of the components inter<br />
and intra-population of the phenotypical variation was different according to the<br />
evaluated feature.<br />
Relative importance of intra and inter-populations components variation for phenotypic<br />
variation varied between traits.<br />
Variation of spikelet emergence date, means number of enternode per stem, mean<br />
length of leafs and shape number were dominated by the inter-populations component<br />
but the remaining traits were dominated by the intra-populations component.<br />
The similarities between populations were analyzed by a hierarchical classification. The<br />
dendrogram obtained showed the presence of two principal groups, which reflect a<br />
marked geographical structuring. The first grouped populations originated from (Fayadh<br />
and Zagouan) and the second one grouped the remaining populations.<br />
Keywords<br />
B. distachyon; morphological diversity; geographical structuration<br />
49
Session 1 – Evolution and Natural Variation<br />
P1.4- Genetic Characterization of New Brachypodium distachyon Populations<br />
from Diverse Geographic Regions in Turkey<br />
M. Tuna 1, . Nizam 1, S. Öney 2, G. Sava 1 and J. Vogel 3<br />
1Namik Kemal University, Faculty of Agriculture, Department of Field Crops, 59100,<br />
Tekirdag, Turkey<br />
2Suleyman Demirel University, Faculty of Art and Science, Department of Biology, 32260,<br />
Isparta, Turkey.<br />
3USDA-ARS Western Regional Research Center, Genomics and Gene Discovery Unit,<br />
Albany, CA 94710, USA<br />
metintuna66@yahoo.com<br />
Abstract<br />
The small annual grass Brachypodium distachyon is rapidly emerging as a powerful<br />
model system for temperate grasses due to its biological, physical and genomic<br />
characteristics. The natural range of B. distachyon largely overlaps the ancestral range of<br />
cultivated small grains and Turkey lies in the center of that geographical area thus<br />
granting a great amount of genetic variability.<br />
The objective of this study was to establish and genetically characterize a large B.<br />
distachyon germplasm collection originating from diverse geographical regions in Turkey.<br />
Seeds from approximately 20 individual plants were collected from each of the 118<br />
locations throughout Turkey. Flow cytometry was used to determine the ploidy of<br />
representatives from each location. Seventy five of the populations contained only<br />
diploid plants, 35 of the populations contained only polyploid plants and 8 populations<br />
contained a mix of diploid and polyploid plants. To examine the genetic diversity of this<br />
new collection, a representative subset of 185 diploid individuals was genotyped using<br />
SSR markers. Our preliminary results show considerable genetic diversity both between<br />
and within populations. We also compared the SSR pro<strong>file</strong>s of the new lines to previously<br />
examined lines. Significantly, some of the new accessions were contained in new clades<br />
indicating that we have not sampled Turkish B. distachyon diversity to saturation. Future<br />
plans for this collection include phenotypic characterization with an emphasis on biotic<br />
and abiotic stress. We also plan to develop recombinant inbred lines from divergent lines.<br />
50
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.1- Brachypodium mutants, new tools to validate candidate genes<br />
underlying lignin content and digestibility QTL in maize?<br />
Audrey Courtial 1,2, Matthieu Reymond 3, Valérie Méchin 3, Jacqueline Grima-Pettenati 2<br />
and Yves Barrière 1.<br />
1 INRA, UGAPF, BP80006, 86600 Lusignan, France<br />
2 UMR5546 CNRS - UPS, 31326 Castanet-Tolosan, France<br />
3 INRA, Institut Jean-Pierre Bourgin, 78026 Versailles, France<br />
courtial@lrsv.ups-tlse.fr<br />
Abstract<br />
Deciphering the genetic and genomic determinants involved in the maize cell wall<br />
biosynthesis and assembly is a strategic issue for both ruminant feeding and biofuels<br />
production. The identification of candidate genes underlying QTL for high cell wall<br />
degradability is a relevant strategy for further marker-assisted selection.<br />
In the F288 x F271 early maize RIL progeny, several QTL were mapped, of which those<br />
located on bin 6.06 explain nearly 50% of the phenotypic variation for both lignin content<br />
and cell wall digestibility (Roussel et al., 2002; Courtial et al., in preparation). In order to<br />
improve the resolution mapping of QTL, new markers were genotyped using HRM (High<br />
Resolution Melting) technology and QTL physical positions were then estimated based on<br />
the publicly available maize genomic sequence (line B73). All the genes located within<br />
the QTL support interval are potential candidate genes explaining the lignin and<br />
digestibility variability. Among them those involved in cell wall biosynthesis and assembly,<br />
and/or regulation of these processes are positional and functional candidate genes.<br />
Complementary strategies are underway for the validation of these candidate genes.<br />
Expression studies will be performed using contrasting lines and allele sequencing of<br />
candidate genes will be considered for both parental lines to reduce the number of<br />
candidate genes. As a first functional validation, we could use the new emerging<br />
monocot model Brachypodium distachyon which offers mutants well suited to study the<br />
composition and degradability of monocot walls.<br />
References<br />
Roussel V, Gibelin C, Fontaine A-S, Barrière Y (2002) Genetic analysis in recombinant<br />
inbred lines of early dent forage maize. II - QTL mapping for cell wall constituents and cell<br />
wall digestibility from per se value and top cross experiments. Maydica 47: 9-20.<br />
Keywords<br />
Maize, cell wall digestibility, lignin, candidate genes, brachypodium<br />
51
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.2- Characterization of secondary wall deposition in Brachypodium stems<br />
towards the goal of fiber cell engineering for biomass feedstocks<br />
Michael Harrington, Nicolas Noria, Richard Sibout, Gregory Mouille, Catherine Lapierre,<br />
Notburger Gierlinger*, Herman Höfte<br />
Institut Jean Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la<br />
Recherche Agronomique-AgroParisTech, 78026 Versailles, France.<br />
*Faculty 5/Biomimetics, Biological Materials, University of Applied Sciences Bremen,<br />
28199, Bremen, Germany.<br />
Michael.Harrington@versailles.inra.fr<br />
Abstract<br />
Worldwide the demand for renewable energy made from non-food plant biomass is<br />
increasing. Plant biomass (or ‘lignocellulose’) found in plant cell walls serves as an<br />
attractive alternative to the current biomass feedstocks, i.e., corn and sugar cane. Cell<br />
walls of most plants are composed of energy-rich polysaccharide polymers that can be<br />
broken down (‘saccharification’) to produce many bio-based products (e.g., bioplastics,<br />
fibers for textile) and bioethanol. Previous studies have provided valuable information in<br />
our understanding of cell wall biosynthesis, particularly towards our understanding of the<br />
molecules involved in this process, however little is known about the cellular behaviors<br />
associated with wall deposition and how they are regulated by these genes.<br />
The goal of this work is to address both the cellular and molecular mechanisms<br />
underlying secondary wall synthesis during sclerenchyma cell develpment using<br />
Brachypodium distachyon, a model for biomass feedstocks. To date we have begun to<br />
characterize sclerenchyma cell formation using a variety of molecular and 3D<br />
recontruction tools, to gain a better understanding of the timing of secondary wall<br />
deposition. In addition, we are now focusing our attention to the molecular regulators<br />
(i.e., transcription factors or/and processing enzymes) of sclerenchyma cell<br />
differentiation and maturation by applying several physical and chemical approaches,<br />
including laser microdissection, transcriptomics, to cell wall composition analysis assays<br />
towards these goals.<br />
Ultimately, this work is improving our knowledge of secondary cell wall synthesis while<br />
identifying the regulators of this process to improve biomass saccharification. Conversely,<br />
this work addresses concerns for sustainable sources of energy, fibers, and chemicals by<br />
creating a dedicated biomass crop.<br />
Keywords<br />
seconday wall, sclerenchyma, lignin, micrsodissection<br />
52
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.3- Phenotyping of yield components and saccharification efficiency in<br />
Bd21, Bd21-3 and 4 wild accessions of diploid Brachypodium distachyon<br />
Van Hulle Steven, Roldán-Ruiz Isabel, Muylle Hilde<br />
Institute of Agricultural and Fisheries Research, Plant Sciences Unit, Caritasstraat 21, 9090<br />
Melle, Belgium<br />
Hilde.Muylle@ilvo.vlaanderen.be<br />
Abstract<br />
Brachypodium distachyon is a temperate wild grass species and is a powerful model<br />
system for other Poaceae. Exploring the natural variation in the yield characteristics and<br />
saccharification potential of Brachypodium provides an important basis for genetic<br />
dissection of these traits. Bd21, Bd21-3 and 4 wild accessions (pi185133, pi185134,<br />
pi245730 and pi254867) were phenotyped. Significant differences were found in yield<br />
traits such as total number of branches, dry matter yield and seed yield. Using a small<br />
scale enzymatic hydrolysis assay, significant differences were found between the studied<br />
accessions for saccharification potential, ranging between 132.75 mg glucose/g biomass<br />
(pi185133) till 187,23 mg glucose/g biomass (pi254867). Bd21 and Bd21-3 showed<br />
intermediate sugar release (151,27 and 155.56 mg glucose / g biomass respectively). The<br />
identified phenotypic variation of Brachypodium in yield traits and saccharification<br />
potential can be used to identify genes and alleles important for the complex trait as<br />
yield and cell wall accessibility.<br />
Keywords<br />
Biomass, Bioenergy, Yield, Saccharification, Cell Wall<br />
53
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.4- Impact of cell wall variability on saccharification yield in natural<br />
Brachypodium distachyon accessions<br />
Legay S. 1, Oudihat M. 1, Darracq O. 1, Antelme S. 1, Whitehead C. 2, Gomez L. 2, Mouille G.<br />
1, Lapierre C. 1, Jouanin L. 1, Sibout R 1.<br />
1Institut Jean-Pierre Bourgin (IJPB), UMR1318 INRA-AgroParisTech, 78026 Versailles, FR<br />
2Centre for Novel Agricultural Products, University of York, York Y010 5 YW, UK<br />
sylvain.legay@versailles.inra.fr<br />
Abstract<br />
Lignocellulosic derived biomass represents one of the most abundant renewable sources<br />
of carbohydrate for the production of bioenergy. Amongst the available biomass,<br />
emerging bioenergy crops such as grasses show great potential for the production of<br />
lignocellulose. In the last few years Brachypodium distachyon has come forth as a model<br />
for temperate grass species due to numerous advantages: small stature, low growth<br />
requirements, genomic resources … Still relatively little is known about the secondary cell<br />
wall composition in Brachypodium. To gain insight on the variability of the secondary cell<br />
wall composition of Brachypodium, we selected a collection of natural accessions of<br />
Brachypodium in order to conduct a detailed biochemical analysis of the different<br />
secondary cell wall polymers. A total of 22 natural accessions of Brachypodium were<br />
selected based on differences of ploïdy, genotypes and phenotypes. Total biomass<br />
production, as well as NIRS and FTIR spectral analysis of stems enabled the classification<br />
of the accessions in 3 groups. Detailed analysis of lignin and polysaccharide composition<br />
revealed a large variation amongst the different accessions. Histological analysis of the<br />
stems of the most extreme lines was achieved to understand if the observed differences<br />
were linked to anatomical variability. Finally the potential impact of the cell wall<br />
composition variability on the production of bioethanol was assessed through<br />
saccharification assays. The results from this study will enable the selection of accessions<br />
with contrasted cell wall and saccharification properties and in the near future lead to<br />
the identification of QTLs for these corresponding traits.<br />
Keywords<br />
biofuel, cell wall, saccharification, natural accessions<br />
54
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.5- Cellulose biosynthesis and growth anisotropy in Brachypodium<br />
distachyon<br />
Karen Sanguinet Osmont, Samuel Hazen, Tobias I. Baskin<br />
Department of Biology, 611 North Pleasant St. UMass-Amherst, Amherst, MA 01003<br />
ksosmont@bio.umass.edu<br />
Abstract<br />
Cellulose is Earth's most abundant polymer and humanity's most useful building material,<br />
comprising upwards of 30% total plant biomass. Cellulose regulates the direction of plant<br />
growth. In a growing organ, cells expand at different rates in different directions, that is,<br />
anisotropically. Cellular growth anisotropy must be controlled precisely to build organs of<br />
specific, heritable shapes. Anisotropic expansion is enabled by cellulose microfibrils,<br />
which thereby help determine the shape and size of the mature plant. Our aim is to study<br />
cellulose synthesis and the control of growth anisotropy in the emerging model plant,<br />
Brachypodium distachyon. Our understanding of cell wall biosynthesis and CESA<br />
functionality has stemmed from mutant analyses in plants such as arabidopsis, but little<br />
functional analysis has been performed in the grasses. We are using both forward and<br />
reverse genetic approaches to help understand cellulose biosynthesis and formation of<br />
the primary cell wall. As in arabidopsis, brachypodium contains ten cellulose biosynthesis<br />
A (CESA) genes, whose expression patterns were determined in roots using quantitative<br />
RT-PCR and microarray analysis. We found BdCESA1, BdCESA3, BdCESA6, and BdCESA9<br />
were most highly expressed in roots. Therefore, we generated a series of artificial<br />
microRNA (amiR) constructs to knock down expression of these genes. We report the<br />
initial characterization of some of these lines. Because knockouts in key primary cell wall<br />
synthesis genes are often lethal, we screened EMS-mutagenized families for temperaturesensitive<br />
root swelling. We are currently screening 4,600 M2 families for conditional,<br />
recessive alleles. We have identified several segregating families with putative short or<br />
swollen root phenotypes, which are being examined further. Overall, our hope is to shed<br />
light on and identify new players in cellulose biosynthesis and anisotropic growth in the<br />
grasses.<br />
Keywords<br />
Cellulose, Anisotropic growth, CESA, Root development<br />
55
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.6- Towards the identification of new cell wall-related genes in<br />
Brachypodium distachyon<br />
Timpano H, Darracq O, Badel E, Pollet B, Lapierre C, Höfte H, Sibout R, Vernhettes S, and<br />
Gonneau M.<br />
Institut Jean-Pierre Bourgin, UMR1318-INRA-AgroParisTech, Centre de Versailles-Grignon,<br />
Route de St-Cyr, 78026 Versailles Cedex-France<br />
helene.timpano@versailles.inra.fr<br />
Abstract<br />
The production of second-generation biofuels based on the transformation of plant<br />
biomass is a pressing issue. Biomass is represented by cell walls of the plant cells consisting<br />
of a network of cellulose microfibrils and matrix polysaccharides encrusted by lignin. To<br />
enhance the potential of plant biomass, we first need to provide insights on the<br />
mechanisms of the biosynthesis of cell wall polymers and in particular that of cellulose<br />
microfibrils.<br />
The saccharification yield of the cellulose is the major trait we can improve to use the<br />
biomass as a competitive alternative of fossil energy and Brachypodium is an essential<br />
model species to facilitate research on monocotyledonous crops dedicated to biofuel<br />
production.<br />
From the mutagenized population obtained at the IJPB we selected by visual screening<br />
a particular mutant called spa1. This mutant shares characteristics of the brittle culm<br />
mutants of rice and barley, such as brittleness, irregular xylem, and a cellulose content<br />
deficiency especially in stems, with 50% of the amount found in the wild type.<br />
Interestingly, this mutant is also floppy unlike others brittle culm mutants which remain<br />
upright. Lignin assays indicate a higher amount of lignin and the mechanical strength<br />
defects of spa1 is illustrated by a Young’s modulus three times lower than that of WT.<br />
Complementary approaches are in progress to identify the SPA1 gene : sequencing of<br />
candidate genes related to cell wall synthesis or co-expressed with secondary cell wall<br />
cellulose synthases and a classical mapping strategy combined with NGS methods.<br />
Moreover within the framework of the European RENEWALL and KBBE CellWall projects<br />
and thanks to the co-expression network tool BradiNet (M. Mutwill, KBBE project), RNAi<br />
strategies are in progress to inactivate few genes selected according to specific<br />
expression criteria and potentially involved in cell wall synthesis.<br />
References<br />
Brkljacic J GE, Scholl R, Mockler T, et al. (2011) Brachypodium as a model for the grasses:<br />
Today and the future. Plant Physiol 157: 3-13.<br />
Zhang B, Zhou Y (2011) Rice Brittleness Mutants : A way to open the black box of<br />
monocot cell wall biosynthesis. JIPB 53 : 136-142.<br />
Keywords<br />
Cell wall, cellulose, biofuels, mechanical properties<br />
56
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.7- Enhancement of biomass production and cell wall degradability by<br />
AtGA20ox1 overexpression and Bd4CL1 downregulation<br />
Voorend Wannes (a)(b), Sibout Richard (c), Bendahmane Abdelahafid (d), Oria Nicolas (c),<br />
Roldán-Ruiz Isabel (a), Muylle Hilde (a), Inzé Dirk (b)<br />
(a) Institute for Agricultural and Fisheries Research (ILVO), Plant Science Unit, Caritasstraat<br />
21, 9090 Melle, Belgium<br />
(b) VIB, Department of Plant Systems Biology, Ghent University, Technologiepark 927, 9052<br />
Gent, Belgium<br />
(c) IJPB-INRA Centre de Versailles-Grignon, Route de St-Cyr (RD10), 78026 Versailles<br />
Cedex, France<br />
(d) URGV-INRACNRS, 2 rue Gaston Crémieux, CP5708 91057 Evry cedex, France<br />
Wannes.Voorend@ilvo.vlaanderen.be<br />
Abstract<br />
Food and feed have always originated mainly from plants, but they can now also supply<br />
energy in the form of biofuel. The supply will be under great strain, however, from the<br />
combination of a rapidly increasing population, higher living standards in developing<br />
countries, and environmental changes. Additionally, future agriculture will have to meet<br />
the needs of a low-carbon economy. For all of these reasons, the next generation of<br />
crops will need to exhibit increased yield and quality. When using whole plant as energy<br />
source, such as for animal feed and bio-ethanol feedstock, more biomass and a higher<br />
glucose release from cell walls will also be required.<br />
This presentation describes a proof of concept to improve cell wall degradability or<br />
digestibility as well as biomass production through transformation and mutagenesis in<br />
Brachypodium Bd21-3 plants.<br />
Our focus lies on altering the lignin content, as this is often related to improved cell wall<br />
degradability. The Brachypodium Bd21-3 TILLING population of INRA Versailles was<br />
screened for mutants in the 4-coumarate ligase (4CL) gene. Fourteen mutations were<br />
identified and plants will be scored for glucose release upon enzymatic hydrolysis.<br />
Lignin mutants can show improved degradability, but these lines often exhibit a lower<br />
yield. An increase in biomass production can be achieved by overexpressing genes (e.g.<br />
GA20ox1 in Arabidopsis) that are known to enhance organ size (leaf and stem). We<br />
currently have 18 UBIL::AtGA20ox1 lines in the Bd21-3 inbred line. Expression levels are<br />
now being analyzed. Promising lines will be phenotyped for enhanced biomass<br />
production.<br />
Ultimately, we will seek to combine the improved properties of cell wall degradability or<br />
digestibility and biomass production by either transforming the plants in a mutant<br />
background or crossing transformed lines.<br />
Keywords<br />
Degradability, biomass, transformation, mutagenesis<br />
57
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.8- Relationships between maize morphological and biochemical traits with<br />
in vitro cell wall degradability<br />
Yu Zhang 1 Tanya Culhaoglu 1, Brigitte Pollet 1, Corine Melin 2, Dominique Denoue 2, Yves<br />
Barrière 2, Stéphanie Baumberger 1, Valérie Mechin 1<br />
1Institut Jean-Pierre Bourgin, UMR 1318 INRA/AgroParisTech, Pôle SCSM, 78000 Versailles,<br />
France<br />
2Unité de Recherche Pluridisciplinaire Prairies et Plantes Fourragères, 86600 Lusignan,<br />
France<br />
Yu.Zhang@versailles.inra.fr<br />
Abstract<br />
Grasses (maize, rice, wheat, oats, rye…) dominate cultivated cropland, supplying most of<br />
the dietary energy needs of many classes of livestock. Nowadays, the bioenergy’ boom<br />
is an international preoccupation and justifies all the more an investment on<br />
graminaceaous lignocellulosic biomass. Grass cell walls are complex molecular<br />
assemblies involving polysaccharidic (cellulose and hemicelluloses) and phenolic (lignins<br />
and p-hydroxycinnamic acids) components. Lignin association with other cell wall<br />
constituents greatly modifies cell wall properties, including enzymatic degradability of<br />
structural polysaccharides.<br />
In this study 8 maize recombinant inbred lines were selected for their similar lignin content<br />
and variable cell wall degradability to assess both the impact of lignin structure and<br />
organization, and the impact of cell wall reticulation by p-hydroxycinnamic acids on cell<br />
wall degradability independently of the main “lignin content” factor. These recombinant<br />
lines and there parents were analyzed for cell wall residue content, esterified and<br />
etherified ferulic and p-coumaric acids content, lignin content and structure and in vitro<br />
degradability. Amongst these biochemical parameters, the proportion of uncondensed<br />
lignin and the esterified p-coumaric acid content showed high significant correlation<br />
with in vitro cell wall degradability (r = -0.82** and r = -0.72* respectively). We also<br />
investigated the relationships between maize morphological traits and biochemical<br />
factors., The 10 genotypes were thus measured for their plant height (Hplant), the height<br />
and the diameter of the node of the principal ears (Hears, Dears) and the line yield (Yield)<br />
at the ensiling stage..The esterified p-coumaric acid content showed high positive<br />
correlation coefficient with all of the morphological traits (r = 0.63* versus Hplant, r = 0.68*<br />
versus Hears, r = 0.64* versus Dears and r = 0.72* versus Yield). Moreover, morphological traits<br />
were found to be negatively correlated with in vitro cell wall degradability (r = -0.70*<br />
versus Hplant, r = -0.65* versus Hears,). This study revealed that plants with higher in vitro<br />
degradability were also the ones with the more reduced agronomic value.<br />
Keywords<br />
cell wall; in vitro cell wall degradability; esterified p-coumaric acid content; agronomic<br />
value<br />
58
Session 2 – Cell Wall, Biomass and Biofuels<br />
P2.9- Brachypodium as a model for studying traits relevant for the<br />
development of bioenergy grasses<br />
Maurice Bosch 1, Luned Roberts 1, Sue Dalton 1, Lorraine Fisher 1, Paul Robson 1, Iain S<br />
Donnison 1, Thomas Didion 2, Klaus Nielsen 2, Luis A J Mur 1<br />
1Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,<br />
Plas Gogerddan, Aberystwyth SY23 3EB, UK<br />
2 DLF-TRIFOLIUM A/S, Højerupvej 31, 4660 Store Heddinge, Denmark<br />
mub@aber.ac.uk<br />
Abstract<br />
Several perennial grasses, including Miscanthus and switchgrass, are currently being<br />
developed as a renewable source of lignocellulosic biomass derived bioenergy. One of<br />
the main bottlenecks for the commercial exploitation of these feedstocks is the<br />
recalcitrance of the cell wall biomass to conversion into fermentable sugars. A detailed<br />
understanding of the regulatory networks controlling cell wall biogenesis is required to<br />
enable the genetic engineering and molecular breeding of energy crops with improved<br />
conversion characteristics. Brachypodium represents an excellent model for functional<br />
genomic studies that can underpin and accelerate the establishment of energy grasses<br />
as a sustainable replacement of fossil fuels. Several candidate genes identified in a<br />
gene-expression profiling experiment in maize (Bosch et al., 2011) are currently being<br />
tested in Brachypodium for involvement in cell wall biogenesis. Subsequently this<br />
information can be translated to genetically more recalcitrant and undomesticated<br />
energy grasses.<br />
The plant cell wall not only represents a major energy store but is also involved in<br />
defence against environmental stresses, including drought. There is a requirement to<br />
develop drought-tolerant crops that produce significant yields with reduced amounts of<br />
water. In collaboration with DLF-TRIFOLIUM A/S, a leading grass biotechnology company,<br />
we are instigating a project in which we will assess drought responses in the<br />
Brachypodium germplasm collection available at IBERS (Mur et al., 2011) and correlate<br />
changes in expression levels of cell wall related genes with changes in secondary<br />
metabolite pro<strong>file</strong>s and cell wall polysaccharides during drought. We are also initiating a<br />
programme of work that will exploit available genetic resources for Brachypodium to<br />
study the genotypic variation in other important traits that affect yield and composition<br />
in bioenergy crops.<br />
References<br />
Bosch M, Mayer CD, Cookson A, Donnison IS (2011) Identification of genes involved in<br />
cell wall biogenesis in grasses by differential gene expression profiling of elongating and<br />
non-elongating maize internodes. Journal of Experimental Botany 62: 3545-3561.<br />
Mur LAJ, Allainguillaume J, Catalán P, Hasterok R, Jenkins G, Lesniewska K, Thomas I,<br />
Vogel J (2011) Exploiting the Brachypodium Tool Box in cereal and grass research. New<br />
Phytologist 91: 334-347<br />
Keywords<br />
Cell wall, drought, biofuel, yield, Miscanthus<br />
59
Session 3 – Tools and Bioengineering<br />
P3.1- Cold treatment, maltose and modified MS medium improve<br />
embryogenesis in Brachypodium BD21<br />
Sue Dalton, Maurice Bosch, Iain S Donnison<br />
Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,<br />
Plas Gogerddan, Aberystwyth SY23 3EB, UK<br />
snd@aber.ac.uk<br />
Abstract<br />
Brachypodium distachyon accession BD21 identified by Vogel and Hill (2008) is<br />
commonly used for genetic transformation. The media developed for immature embryo<br />
callus induction include Linsmaier and Skoog (LS) medium with 3% sucrose, 2.5mgl -1 2,4-D,<br />
0.6 mgl -1 CuSO4 and no vitamins except 1mgl -1 thiamine and a similar Murashige and<br />
Skoog (MS) medium (Vain et al, 2008) with cysteine and ‘M5’ vitamins (0.5mgl -1 thiamine).<br />
A ‘Modified’ MS medium (MODMS, Duchefa) contains x10 thiamine (1mgl -1) and<br />
improves embryogenesis in Poa and Miscanthus. Maltose also produces softer, faster<br />
growing embryogenic callus than sucrose in some grass species. BD21 embryogenic<br />
callus induction was compared on LS and MODMS media with sucrose and maltose.<br />
Cold treatment and CuSO4 concentration were also examined.<br />
Whole inflorescences were surface sterilised and immature embryos excised and<br />
cultured on eight media comprising MODMS or LS medium, maltose or sucrose and 0.3 or<br />
0.6mgl -1 CuSO4. Embryos were cultured fresh or from inflorescences stored overnight at<br />
4 oC. Embryo age and size varied within each inflorescence, but all were cultured (150-<br />
200 embryos per treatment).<br />
Embryos with embryogenic proliferation were counted after 4 weeks culture at 25 oC in<br />
darkness. With sucrose more cultures were embryogenic on MODMS than on LS based<br />
media. However maltose was generally superior to sucrose and there was an interaction<br />
between cold treatment and medium. With maltose more freshly cultured embryos were<br />
embryogenic on LS based media, but after cold treatment many more were<br />
embryogenic on MODMS media, which contained more vitamins. Copper concentration<br />
made little difference to embryogenesis on MODMS media, although the highest<br />
percentage (42%) was achieved with 0.3mgl -1 CuSO4. After cold treatment,<br />
embryogenesis on LS media was much improved by 0.3 compared with 0.6mgl -1 CuSO4.<br />
Overall the best treatment was overnight cold and culture on MODMS medium with 3%<br />
maltose, 3mgl -1 2,4-D and 0.3 mg/l -1 CuSO4.<br />
References<br />
Vogel J and Hill T. (2008). High-efficiency Agrobacterium-mediated transformation of<br />
Brachypodium distachyon inbred line Bd21-3. Plant Cell Reports, 27: 471-478.<br />
Vain P, Worland B, Thole V, et al. (2008). Agrobacterium-mediated transformation of the<br />
temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional<br />
mutagenesis. Plant Biotechnology Journal, 6: 236–245<br />
Keywords<br />
Embryogenesis, cold treatment, maltose, thiamine, vitamins<br />
60
Session 3 – Tools and Bioengineering<br />
P3.2- Neomycin Phosphotransferase Gene (nptII) as a selectable marker and<br />
enhanced rice ubiquitin promoter as a constitutive promoter for<br />
Brachypodium BD21<br />
Sue Dalton, Maurice Bosch, Paul Robson, Iain S Donnison<br />
Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,<br />
Plas Gogerddan, Aberystwyth SY23 3EB, UK<br />
snd@aber.ac.uk<br />
Abstract<br />
Brachypodium distachyon BD21 is an embryogenic diploid accession and is the most<br />
commonly used material for genetic transformation. Unfortunately hygromycin selection<br />
can be unclear in this and other embryogenic Brachypodium accessions and the<br />
established protocol (Alves et al, 2009) uses GFP as an additional selection. Without GFP<br />
this protocol allows many escapes as selection is reduced from 40 to 30 to 20 mgl -1<br />
hygromycin as calli grow and regenerate plants. This author found only calli that grow<br />
readily and regenerate on continuous selection of 40mgl -1 hygromycin to be definitely<br />
transformed. Experiments with the hygromycin gene under the CaMV promoter (pROB5,<br />
Bilang et al, 1991) have yielded few such easily identifiable transformants and<br />
hygromycin under the actin promoter (pAct1HPT, Cho et al, 1998) is preferred. This<br />
selection gene was compared with the bar gene (pUBA, Toki et al, 1992) and nptII gene<br />
(pBKS ubi-nptII, Paul Robson, IBERS) under the maize ubiquitin promoter and cobombarded<br />
with gus under an enhanced rubi3 rice ubiquitin promoter (pRESQ48,<br />
Sivamani and Qu, 2006).<br />
Calli bombarded with pUBA and selected on bialophos became friable and were not<br />
continued. In this experiment calli bombarded with pAct1HPT produced no obvious<br />
transformants, although weak plants grew. These were not GUS positive however. Calli<br />
bombarded with pUbi-nptII and selected on 100-150 mgl -1 paromomycin were obviously<br />
transformed or not and plants were regenerated under selection. One plant<br />
constitutively expressed GUS and is the first grass to do so with the pRESQ48 construct,<br />
although the plant may be infertile. The nptII gene appears to be a useful and easily<br />
selectable gene for Brachypodium, and the enhanced rice ubiquitin promoter appears<br />
to be constitutive and to not co-suppress the maize ubiquitin promoter.<br />
References<br />
Alves SC, Worland B, Thole V, Snape JW, Bevan MW and Vain P. (2009) A protocol for<br />
Agrobacterium-mediated transformation of Brachypodium distachyon community<br />
standard line Bd21. Nature Protocols 4: 638-649<br />
Bilang R, Iida S, Peterhans A, Potrykus I, Paszkowski J. (1991) The 3’-terminal region of the<br />
hygromycin-B-resistance gene is important for its activity in Eschiricia coli and Nicotiana<br />
tabacum. Gene 100: 247-250<br />
Cho M-J,Jiang W, Lemaux PG. (1998) Transformation of recalcitrant barley cultivars<br />
through improvement of regenerability and decreased albinism. Plant Sci. 138:229-244.<br />
Sivamani E and Qu R. (2006) Expression enhancement of a rice polyubiquitin gene<br />
promoter. Plant Molecular Biology 60:225-239<br />
Toki S, Takamatsu S, Nojiri C, Ooba S, Anzai H, Iwata M, Christensen A, Quail PH, Uchimiya<br />
H. (1992) Expression of maize ubiquitin gene promoter-bar chimeric gene in transgenic<br />
rice. Plant Physiol 100:1503-1507<br />
Keywords<br />
Selection, promoters, enhanced rubi3, nptII, gus<br />
61
Session 3 – Tools and Bioengineering<br />
P3.3- Tools and methods for functional gene analysis in Brachypodium<br />
distachyon. Examples of genes involved in cell wall synthesis.<br />
Oumaya Bouchabké-Coussa*, Camille Soulhat, Madeleine Bouvier d’Yvoire, Sebastien<br />
Antelme, Philippe Lebris and Richard Sibout<br />
INRA, Institut Jean-Pierre Bourgin, 78026 Versailles, France<br />
Oumaya.Bouchabke@versailles.inra.fr<br />
Abstract<br />
We are involved in developing many resources for Brachypodium: TILLING collection,<br />
natural accessions collection, phenotyping and gene transfer via Agrobacterium<br />
tumefaciens. We developed a genetic transformation platform for efficient gene<br />
functional analysis. A set of binary vectors specific for monocots is available in our lab as<br />
well as an efficient method using different selective agents. We use Brachypodium as a<br />
model system for studying how to modify grass crops for increased biofuel production<br />
made from lignocellulosic cell walls. Some lignin mutants from the monolignol<br />
biosynthesis pathway were recently identified by phenotyping and TILLING, their<br />
functional analysis is underway notably through complementation by overexpression of<br />
concerned gene in mutant background. Other interesting candidate genes involved in<br />
cell wall biosynthesis were identified by sequence similarity between Brachypodium and<br />
other plant species. When mutants were not available, corresponding sequences were<br />
cloned from Brachypodium genome and their validation is in progress through RNAi,<br />
amiRNA or over-expression strategies. Preliminary characterizations of transgenics plants<br />
are shown.<br />
Keywords<br />
Brachypodium distachyon, gene transfer, Agrobacterium tumefaciens, cell wall.<br />
62
Session 3 – Tools and Bioengineering<br />
P3.4- Construction of Activation T-DNA-Tagging Mutant Pool in Brachypodium<br />
distachyon<br />
Shin-Young Hong, Jae-Hoon Jung, Pil Joon Seo, Chung-Mo Park<br />
Department of Chemistry, Seoul National University, Seoul 151-741, Korea<br />
cmpark@snu.ac.kr<br />
Abstract<br />
Activation tagging is a powerful tool for gene discovery and functional genomics in<br />
plants. A pool of mutant can be generated by randomly inserting a T-DNA enhancer<br />
element into the genome of target plants. Consequently, a gene can be<br />
transcriptionally activated by the nearby insertion of the enhancer element, resulting in a<br />
gain-of function phenotype. The activation tagging mutagenesis has been employed<br />
successfully in both dicot and monocot plants species, such as Arabidopsis and rice, and<br />
contributed to functional characterization of numerous genes.<br />
Here, we report an activation tagging system that was successfully employed in a<br />
model grass Brachypodium distachyon, in which a multimeric transcriptional enhancer<br />
element of the Cauliflower Mosaic Virus (CaMV) 35S promoter was randomly integrated<br />
into the genome of an inbred line Bd21-3. The activation tagging T-DNA vector pGA2715<br />
contains a promoterless b-glucuronidase reporter gene (GUS) with an intron and multiple<br />
splicing donor and acceptor sequences immediately next to the right border. The<br />
multimeric transcriptional enhancer of the CaMV 35S promoter is located adjacent to<br />
the left border.<br />
So far, we obtained more than 1,200 transgenic lines. The genomic sequence<br />
regions flanking the T-DNA insertion sites were determined by inverse PCR and DNA<br />
sequencing. More than 472 transgenic lines were characterized, and 315 independent T-<br />
DNA loci, which represents 67% of the sequence transgenic lines, were assigned to<br />
different genes. We are aiming to obtain at least 1000 independent tagging lines by the<br />
end of 2011 and carry out gene functional studies.<br />
References<br />
Ayliffe MA, Pryor AJ (2011) Activation tagging and insertional mutagenesis in barley.<br />
Methods in Molecular Biology 678: 107-128.<br />
Wan S, Wu J, Zhang Z, et al. (2009) Activation tagging, an efficient tool for functional<br />
analysis of the rice genome. Plant Molecular Biology 69: 69-80.<br />
Pogorelko GV, Fursova OV, Ogarkova OA, Tarasov VA (2008) A new technique for<br />
activation tagging in Arabidopsis. Gene 414: 67-75.<br />
Jeong DH, An S, Park S, et al. (2006) Generation of a flanking sequence-tag database for<br />
activation-tagging lines in japonica rice. Plant Journal 45: 123-132.<br />
Jeong DH, An S, Kang HG, et al. (2002) T-DNA insertional mutagenesis for activation<br />
tagging in rice. Plant Physiology 130: 1636-1644.<br />
Keywords<br />
Activation tagging, T-DNA tagging, Brachypodium distachyon<br />
63
Session 3 – Tools and Bioengineering<br />
P3.5- pBrachyTAG: A novel vector system for T-DNA tagging and trapping in<br />
grasses<br />
Vera Thole, Barbara Worland and Philippe Vain<br />
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich NR4<br />
7UH, United Kingdom.<br />
vera.thole@bbsrc.ac.uk<br />
Abstract<br />
Over the past decade, Brachypodium distachyon has been established as a new<br />
experimental and genomics model system to bridge research into temperate cereals<br />
and to promote research in biomass crops. Recently, we have developed highly efficient<br />
systems to produce T-DNA insertion lines (Alves et al., 2009, Vain et al., 2008) and to<br />
retrieve flanking sequence tags (FSTs) (Thole et al., 2009) from mutant lines. Here, we<br />
report the construction and testing of improved transformation vectors, pBrachyTAG, for<br />
T-DNA tagging and promoter trapping in grasses. The pBrachyTAG vectors are based on<br />
the pCLEAN dual binary vector system (Thole et al., 2007) and are designed to address<br />
current limitations in characterising flanking plant genomic sequences and creating<br />
functional gene fusions in trapping strategies. These new vectors contain features known<br />
(i) to reduce vector backbone transfer, (ii) to limit the introduction of superfluous DNA<br />
sequences, (iii) to improve transformation efficiency, and (iv) to enable more than one<br />
FST retrieval strategy. The performance of the pBrachyTAG vectors for insertional<br />
mutagenesis and promoter trapping will be presented, discussed and compared to<br />
generic binary vectors such as pVec8-GFP previously used in our studies (Thole et al.,<br />
2010).<br />
References<br />
Alves SC, Worland B, Thole V, et al. (2009) A protocol for Agrobacterium-mediated<br />
transformation of Brachypodium distachyon community standard line Bd21. Nature<br />
Protocols 4: 638-649.<br />
Thole V, Alves SC, Worland B, et al. (2009) A protocol for efficiently retrieving and<br />
characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA<br />
insertional mutants. Nature Protocols 4: 650-661.<br />
Thole V, Worland B, Snape JW, Vain P (2007) The pCLEAN dual binary vector system for<br />
Agrobacterium-mediated plant transformation. Plant Physiology 145: 1211-1219.<br />
Thole V, Worland B, Wright J, et al. (2010) Distribution and characterization of more than<br />
1000 T-DNA tags in the genome of Brachypodium distachyon community standard line<br />
Bd21. Plant Biotechnology Journal 8:734-747.<br />
Vain P, Worland B, Thole V, et al. (2008) Agrobacterium-mediated transformation of the<br />
temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional<br />
mutagenesis. Plant Biotechnology Journal 6: 236-245.<br />
Keywords<br />
Binary vector, Agrobacterium, T-DNA tagging, T-DNA trapping, Flanking sequence tag<br />
64
Session 4 – Biotic and Abiotic Stresses<br />
P4.1- Genetics of Rice (Oryza sativa L.) under Normal and Water Stress<br />
Conditions<br />
Muhammad Ashfaq and Abdus Salam Khan<br />
Department of Plant Breeding and Genetics, University of Agriculture Faisalabad,<br />
Pakistan<br />
ashfaq_qs@yahoo.com, muhammad.ashfaq@mail.mcgill.ca,<br />
Abstract<br />
The experiment was conducted in Department of Plant Breeding and Genetics, University<br />
of Agriculture Faisalabad to study the genetic basis of drought tolerance in rice (Oryza<br />
sativa). Forty diverse rice genotypes were studied under field condition for various<br />
morphological traits in year 2008. These genotypes were evaluated for drought tolerance<br />
on the basis of physiomorphological traits and some seed traits of rice grain. After this<br />
from these forty, twenty genotypes were grown in polythene bags to study the root shoot<br />
traits at seedling stage under normal and water stress condition for the selection of<br />
diverse parents. A total 28 SSR markers were also used to see genetic diversity among the<br />
twenty genotypes of rice. More diversity was observed between improved basmati rice<br />
varieties and advance breeding lines. Less diversity was observed in basmati varieties in<br />
comparison with the rice advance lines. All the 28 SSR markers showed greatest<br />
polymorphism among the rice genotypes. But some SSR markers showed highest<br />
polymorphism among the rice genotypes. Some highest polymorphic markers showed<br />
more variation among the genotypes namely RM-421, RM-254, RM-235, RM-544, RM-257,<br />
RM-224, RM-248 and RM-590. Eight parents were selected on the basis of phenotypic and<br />
genotypic screening for the development of F1 hybrids by using diallel mating design to<br />
see the gene action among the parents and their F1 hybrids. All the possible<br />
combinations were made between the parents excluding reciprocals. These experiments<br />
were conducted in the green house and various morphological traits were studied under<br />
both normal and stress conditions. All the possible combinations were made between<br />
the parents excluding reciprocals. These experiments were conducted in the green<br />
house and various morphological traits were studied under both normal and stress<br />
conditions. Stress was given at the reproductive stage. Data were analyzed by using<br />
Hayman graphical approach and Griffing approach to study the genetic variance and<br />
combining ability analysis among the parents and their F1 hybrids. Griffing analysis<br />
genotypes CB-17, CB-32 and Basmati-198 were found good general combiners for<br />
productive tillers per plant, primary branches per panicle and yield per plant, especially<br />
under water stress condition. Maximum specific combining ability was found in Basmati-<br />
198 × CB-17 for Productive tillers per plant, Basmati-198 × CB-42 for Primary branches per<br />
panicle and CB-32 × CB-14 for Yield per plant.<br />
References<br />
Hayman, B.I. 1954. The theory and analysis of diallel crosses. Genetics 30:789-809.<br />
Griffing, B. 1956. Concept of general and specific combining ability in relation to<br />
diallelmating systems. Australian J. Biol. Sci. 9:463-493.<br />
Keywords<br />
Rice, Genetic diversity, Correlation, SSR marker, Morphological traits<br />
65
Session 4 – Biotic and Abiotic Stresses<br />
P4.2- Metabolic reprogramming in pre-symptomatic phases of Magnaporthe<br />
grisea infection of susceptible and resistant Brachypodium distachyon plants<br />
Hassan Zubair, Manfred Beckmann, Kathleen Tailliart and John Draper<br />
Institute of Biological, Environmental and Rural Sciences, Aberystwyth University,<br />
Ceredigion, UK<br />
hhz@aber.ac.uk<br />
Abstract<br />
Metabolite fingerprinting using flow infusion electrospray MS in combination with<br />
machine learning data mining provided a primary discovery tool to investigate the reprogramming<br />
of plant’s early defence metabolism in pre-symptomatic leaf tissues during<br />
Magnaporthe grisea (M. grisea) infection of Brachypodium distachyon (B. distachyon).<br />
Two B. distachyon ecotypes, one susceptible (ABR1) and other resistant (ABR5) to blast<br />
infection were used to elucidate pathogen-induced changes. Explanatory m/z signals<br />
were annotated using ultra-high accurate mass analysis by Fourier-transform ion<br />
cyclotron resonance (FT-ICR) MS and the accurate mass database MZedDB searched at<br />
< 1ppm mass accuracy.<br />
Susceptible interactions of M. grisea with ABR1 plants showed an early metabolic shift<br />
during penetration of the fungal pathogen into epidermal cells 24-28 h after inoculation.<br />
A disruption of the TCA cycle and energy metabolism, as well as amino acid biosynthesis<br />
was evident during this phase. Effects on trisaccharide and flavonoid synthesis were also<br />
evident. However, most of these changes were not observed in the infection of resistant<br />
ABR5 plants.<br />
A metabolic response to M. grisea infection in ABR5 started < 8 h after inoculation. By 24<br />
h (pre-penetration stage) infected ABR5 plants had already accumulated specific<br />
antifungal compounds (e.g. sphingofungin E) at significantly high levels. A range of other<br />
compounds accumulated in ABR5 plants by 24 h included phospholipids such as<br />
PC(18:2), PC(18:3) and 2-16:1-lysoPG, along with pantothenate and melibionate.<br />
These metabolome alterations in pathogen-challenged leaf tissues (both ABR1 and ABR5<br />
plants) prior to penetration of first epidermal cell, and during penetration itself were<br />
striking and suggests that M. grisea is able to manipulate host’s defence metabolism in<br />
susceptible plants in a very early phase of the infection process. Host enzyme-activities,<br />
potentially modulated by the invading pathogen, are currently being explored.<br />
Keywords<br />
Metabolic re-programming, Magnaporthe grisea, Brachypodium distachyon, presymptomatic<br />
infection, susceptible and resistance responses<br />
66
Session 4 – Biotic and Abiotic Stresses<br />
P4.3- Brachypodium distachyon diversity and drought response: establishing<br />
a reference experiment for automated phenotyping<br />
John Draper, Alan Gay, Hassan Zubair, Gordon Allison, Manfred Beckmann, Kathleen<br />
Tailiart and John Doonan<br />
Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,<br />
Ceredigion, UK<br />
hhz@aber.ac.uk<br />
Abstract<br />
Automated, comprehensive phenotyping of plant genotype collections is expected to<br />
offer new insight into genetic diversity and functional genomics. The UK National Plant<br />
Phenomics Centre based at IBERS will be working with partners internationally to refine<br />
protocols, calibrate, and where possible standardise cross-platform phenotyping<br />
approaches suitable for future data integration. The phenotyping approach in<br />
Aberystwyth is based on a LemnaTec conveyor system that offers opportunity to<br />
measure non-invasively dynamic aspects of growth architecture and development (both<br />
aerial and underground) as well as monitor aspects of water physiology, temperature<br />
control and stress responses. Specific sampling stations allow for destructive sampling of<br />
plant tissue for further analysis , such as transcriptomics and metabolomics.<br />
We describe the functional aspects of the National Plant Phenomics Centre and outline<br />
plans for characterising the growth, development and responses to drought stress in a<br />
small collection of diverse B. distachyon ecotypes. Specifically we intend to image a<br />
selection of highly diverse B. distachyon genotypes collected from a range of habitats<br />
differing in altitude, average temperature and rainfall.<br />
One aspect of EU FP7 plans to use automated phenotyping in systems level biology is to<br />
integrate data with that derived from comprehensive ‘chemical phenotyping’. With this<br />
in mind metabolite fingerprinting using flow infusion electrospray MS in combination with<br />
machine learning data mining will provide a primary discovery tool to investigate the reprogramming<br />
of plant metabolism in response to drought.<br />
Keywords<br />
Automated phenotyping, Imaging, Metabolic re-programming, Brachypodium<br />
distachyon, drought stress<br />
67
Session 5 – Plant Development, Metabolism and Physiology<br />
P5.1- Describing and modelling root and shoot growth and development in<br />
Brachypodium distachyon (L.) Beauv.<br />
Delory Benjamin*, Delaplace Pierre, Gfeller Aurélie, Fauconnier Marie-Laure, du Jardin<br />
Patrick<br />
University of Liège, Gembloux Agro-Bio Tech, Plant Biology Unit, Gembloux, Belgium<br />
* Benjamin.Delory@student.ulg.ac.be<br />
Abstract<br />
Due to its small size, its short developmental cycle and its close phylogenetic relationship<br />
with the Triticeae tribe, Brachypodium distachyon (L.) Beauv. has been proposed as a<br />
model species for temperate cereals [1-3]. In this context, this work aims to describe and<br />
model root and shoot growth and development of B. distachyon (Bd21-1) grown under<br />
controlled environmental conditions [22°C, 65% RH, 20h light, 95 µmol.m -2.s -1 (PAR, LED<br />
lighting)]. For this purpose, vernalized caryopses were sown in a substrate consisting of<br />
vermiculite and compost (80/20, v/v). Growth and development of the above and<br />
belowground parts were monitored for 70 days. Dry and fresh masses of plant organs<br />
were measured every seven days from sowing. Biomasses of adventitious and seminal<br />
roots were measured separately. The number of spikelets on the main stem and on tillers<br />
was also counted on plants aged of 70 days.<br />
The modelling of root and shoot growth was achieved by calibrating sigmoidal growth<br />
models to the mean biomass values measured at each day of analysis. For each plant<br />
organ, the growth model selected was the one with the lowest residual variance. Finally,<br />
developmental stages identified for B. distachyon were compared with those defined for<br />
cereal crops by Zadoks et al. (1974) [4].<br />
Maximum rates of fresh and dry shoot biomass production were 29,5 and 14,1 mg.day -1<br />
respectively. Based on modelling, these values seem to be reached 49 and 72 days after<br />
sowing. Results also show that the fresh mass of adventitious roots at day 42 is significantly<br />
higher than that of seminal roots. Maximum rates of fresh and dry root biomass<br />
production were 6,9 and 0,8 mg.day -1 respectively, and were reached after 37 and 43<br />
days.<br />
References<br />
1. Draper J, Mur LAJ, Jenkins G, et al. (2001) Brachypodium distachyon. A new model<br />
system for functional genomics in grasses. Plant Physiology 127: 1539-1555.<br />
2. Garvin DF (2007) Brachypodium: a new monocot model plant system emerges.<br />
Journal of the Science of Food and Agriculture 87: 1177-1179.<br />
3. Watt M, Schneebeli K, Dong P, Wilson IW (2009) The shoot and root growth of<br />
Brachypodium and its potential as a model for wheat and other cereal crops. Functional<br />
Plant Biology 36: 960-969.<br />
4. Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the growth stages of<br />
cereals. Weed Research 14: 415-421.<br />
Keywords<br />
root growth, shoot growth, Brachypodium distachyon, growth modelling<br />
68
Session 5 – Plant Development, Metabolism and Physiology<br />
P5.2- Endosperm development in Brachypodium<br />
Philip Hands 1, Magdalena Opanowicz 3, Donna Betts 1, Mary L. Parker 2, Geraldine A.<br />
Toole 2, E. N. Claire Mills 2, John H. Doonan 3, Sinéad Drea 1<br />
1 Department of Biology, University of Leicester, University Rd, Leicester LE1 7RH, UK.<br />
2 Institute of Food Research, Norwich, NR4 7UA, UK<br />
3 The Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth<br />
University, Penglais, Aberystwyth, Ceredigion, SY23 3DA<br />
psh14@le.ac.uk<br />
Abstract<br />
Brachypodium distachyon has become an important model system for the temperate<br />
grasses. It is the first member of the economically important Pooideae group and the first<br />
wild grass species to be sequenced. This comparative analysis of grain structure and<br />
development in B. distachyon to the cultivated cereal grains provides insight into the<br />
nature of temperate cereal grain development and to the effects of the domestication<br />
process. Analyses reveal differences in endosperm tissue organisation, cell structure and<br />
grain storage reserves between Brachypodium and wheat. Molecular marking of<br />
domains within the developing Brachypodium grain using mRNA in situ hybridisation<br />
reveals distinct differences in endosperm differentiation. Most notably, the aleurone<br />
layer is not as distinct or regionally differentiated as in wheat and a modified aleurone<br />
region is absent. Cell walls in the central endosperm and nucellar epidermis are<br />
unusually prominent and the large nucellar epidermis layer is persistent throughout grain<br />
development, suggesting a role in grain filling mechanisms. These significant grain<br />
structural differences may reflect Brachypodium’s phylogenetic position and its position<br />
intermediate between the triticeae and rice.<br />
References<br />
Opanowicz M, Hands P, Betts D, Parker ML, Toole GA, Mills ENC, Doonan JH, Drea S (2011)<br />
Endosperm development in Brachypodium distachyon. J Exp Bot 62: 735–748<br />
Keywords<br />
Grain development, endosperm, aleurone<br />
69
Session 5 – Plant Development, Metabolism and Physiology<br />
P5.3- Unraveling the functional diversity of the CYP73 family in Brachypodium:<br />
is there more than 4-hydroxylation of cinnamic acid?<br />
Hugues Renault 1, Jean-Etienne Bassard 1, Richard Sibout 2, Martine Schmitt 3, François<br />
André 4, Danièle Werck-Reichhart 1<br />
1Institut de Biologie Moléculaire des Plantes – CNRS UPR2357, Université de Strasbourg, 28<br />
rue Goethe, 67083 Strasbourg, France<br />
2Institut Jean-Pierre Bourgin – UMR1318 INRA-AgroParisTech, Route de Saint-Cyr (RD10),<br />
78026 Versailles, France<br />
3Laboratoire d’Innovation Thérapeutique (LIT) – CNRS, Université de Strasbourg, Faculté<br />
de Pharmacie, 74 route du rhin, 67400 Illkirch, France<br />
4Service de Bioénergétique, Biologie Structurale et Mécanismes (SB 2SM) – CEA, CNRS<br />
URA2096, Gif-sur-Yvette, France<br />
hugues.renault@ibmp-cnrs.unistra.fr<br />
Abstract<br />
Plant phenolic compounds contribute to the agronomic, industrial and nutritional<br />
performances of agricultural and forest resources (Vogt, 2010). Genes in the phenolic<br />
metabolism are usually present in single or low copy number. Duplications are conserved<br />
across taxa, which is indicative of gene specialization and branching in the pathway.<br />
Cytochromes P450 of the CYP73 family catalyze the 4-hydroxylation of cinnamic acid<br />
(C4H activity), an early and obligatory step in the biosynthesis of most phenolic<br />
compounds such as lignins monomers, flavonoids, coumarins, stilbenes, lignans and<br />
tannins. Growing evidence, based on protein alignment and phylogenetic analysis,<br />
suggest that two classes of CYP73 occur in angiosperms (Ehlting et al., 2006). Class I<br />
CYP73s are mainly expressed in lignified tissues and are meant to play function in the<br />
general phenylpropanoid pathway. Class II CYP73s are atypical in that they have a<br />
longer N-terminal membrane anchor and preferential expression in flower and roots. In<br />
Brachypodium genome, we identified three CYP73 genes. Two paralogs encode class I<br />
C4Hs while the third one encodes a class II protein. Orthologs of the three genes are<br />
found in all sequenced monocot genomes. The PHENOWALL ANR project is aimed at<br />
clarifying the specific functions of the three C4Hs. Each protein will be expressed in yeast<br />
for testing catalytic properties with a large set of natural and synthetic substrates.<br />
Resulting data will help elaborating substrate pharmacophores to be docked in 3D<br />
model of each protein. Enzyme functions in planta will be addressed by investigating<br />
phenotype of TILLING mutants and silenced/overexpressor lines. Likewise, promoter-GUS<br />
lines are being produced for fine description of the expression pattern of each CYP73.<br />
References<br />
Ehlting J, Hamberger B, Million-Rousseau R, Werck-Reichhart D (2006) Cytochromes P450<br />
in phenolic metabolism. Phytochemistry 5: 239-270<br />
Vogt T (2010) Phenylpropanoid biosynthesis. Molecular Plant 3: 2-20<br />
Keywords<br />
Phenolic metabolism, lignin, cell wall, cytochrome P450, gene duplication<br />
70
Session 5 – Plant Development, Metabolism and Physiology<br />
P5.4- Starch bioengineering in Brachypodium distachyon<br />
Vanja Tanackovic 1, Jan T. Svensson 1, Mikkel A. Glaring 2, Massimiliano Carciofi 3, Susanne<br />
Langgård Jensen 1, Andreas Blennow 1<br />
1 VKR Research Centre Pro-Active Plants, Department of Plant Biology and<br />
Biotechnology, Faculty of Life Sciences, University of Copenhagen, DK-1871,<br />
Frederiksberg C, Denmark<br />
2 Department of Agriculture and Ecology, Faculty of Life Sciences, University of<br />
Copenhagen, DK-1871, Frederiksberg C, Denmark<br />
3 Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus<br />
University, DK-4200 Slagelse, Denmark<br />
vtana@life.ku.dk<br />
Abstract<br />
Brachypodium distachyon was recently introduced as a model plant for temperate<br />
cereals (Opanowicz et al., 2008; IBI 2010). In order to explore pre-domesticated and<br />
novel features of cereal starch metabolism we aim to establish Brachypodium as a<br />
model. Bioinformatics analysis identified starch biosynthesis genes including seven soluble<br />
starch synthases (SS), two granule bound starch syntheses (GBSS), four starch branching<br />
enzymes (SBE), two glucan- and one phosphoglucan- water dikinases (GWD, PWD).<br />
Transit peptides and putative carbohydrate-binding modules (CBMs) of the families<br />
CBM20, CBM45, CBM48 and CBM53 were identified. The gene setup for starch<br />
biosynthesis is very similar to barley and a phylogenetic analysis based on the SS genes<br />
provided evidence for a close relation to barley and wheat.<br />
To investigate the starch structural features, grain starch from two lines, Bd21 and Bd21-3<br />
were characterized. Microscopic, chemical and structural data including amylopectin<br />
chain length distribution, phosphate content, amylose content of the starch granules<br />
provided evidence for a close structural relationship to temperate cereals even though<br />
kernel starch content and starch granule size were considerably lower and -glucan<br />
content was much higher than that for barley (Hordeum vulgare). Small-angle and wideangle<br />
X-ray scattering (SAXS and WAXS) show low crystallinity of Brachypodium starch<br />
granules as compared to barley. These data were confirmed by differential scanning<br />
calorimetry (DSC) data.<br />
Further steps include Agrobacterium-mediated and biolistic transformations and mutant<br />
analyses of starch biosynthesis genes of interest to provide evidence for their specific<br />
action in this grass as compared to domesticated cereals.<br />
Our data show that Brachypodium distachyon can provide a valuable and efficient<br />
model for starch bioengineering in temperate cereals.<br />
References<br />
Opanowicz, M, Vain, P, Draper, J, Parker D and Doonan, JH (2008) Brachypodium<br />
distachyon: making hay with a wild grass, Trends Plant Sci. 13, 172-177.<br />
The International Brachypodium Initiative (2010) Genome sequencing and analysis of the<br />
model grass Brachypodium distachyon, Nature 463, 763–768.<br />
Keywords<br />
Brachypodium distachyon, model plant, grain starch, starch biosynthesis genes, starch<br />
bioengineering<br />
71
AUTHORS INDEX<br />
72
S: Speakers’ abstracts P: Posters’ abstracts<br />
ADAM G. S4.4 GAO C. -<br />
ANTELME S. S2.2, S3.6, GARVIN D.F. S4.6, S4.8<br />
S4.7, P2.4, GAYMARD F. -<br />
P3.3 GIRALDO P. P1.2<br />
ASHFAQ M. P4.1 GONNEAU M. P2.6<br />
BAKAN B. - GONZALEZ DE LA CALLE V. H. S5.2<br />
BARATINY D. - GONZALEZ GARCIA M. de la C. -<br />
BARRIERE Y. P2.1, P2.8 GOUJON T. -<br />
BELCRAM H. - GUBAEVA E. -<br />
BENAVENTE E. S4.1, P1.2 HANDS P. S5.4, P5.2<br />
BERGER A. - HARDTKE C. S1.6<br />
BERQUIN P. - HARRINGTON M. P2.2<br />
BERTOLINI E. S4.2 HAZEN S. S2.5, P2.5<br />
BHALLA P. - HIMURO Y. -<br />
BILU A. - HOFTE H. S2.7, S3.4,<br />
BLONDET E. - S3.6, P2.2,<br />
BLUEMKE A. S4.9 P2.6<br />
BOSCH M. P2.9, P3.1, HO-YUE-KUANG M.S. -<br />
P3.2 IDZIAK D. S3.2<br />
BOUCHABKE-COUSSA O. S2.7, P3.3 JAMET E. S2.6<br />
BOUCHEZ D. - JØRGENSEN B. -<br />
BOUVIER D'YVOIRE M. S2.2, S3.6, JOUANIN L. S2.2, S3.6,<br />
P3.3 P2.4<br />
BOUYER D. - JUNG J.-H. P3.4<br />
BREIMAN A. S4.1, P1.1 JURANIEC M. -<br />
CATALAN P. S1.2 KRAPP A. -<br />
CAZALIS R. - KUMLEHN J. S3.7<br />
CHATEIGNER-BOUTIN A.-L. S5.1 LAPIERRE C. S2.2, S3.6,<br />
CHUPEAU M.-C. - P2.2, P2.4,<br />
CHUPEAU Y. S2.7 P2.6<br />
CLAUSEN S.S. - LARRE C. S5.1<br />
COURTIAL A. P2.1 LE BRIS P. -<br />
DALMAIS M. S3.6 LECOMTE P. -<br />
DARRACQ O. S2.2, S3.6, LEGAY S. P2.4<br />
P2.4, P2.6 LEPINIEC L. -<br />
DAVID L. - LEVI BAR SHALOM A. -<br />
DELAPLACE P. S5.3, P5.1 LOUDET O. -<br />
DELL'ACQUA M. - MACADRE C. S4.3<br />
DELORY B. P5.1 MAILLET F. -<br />
DINH THI V. H. - MARCEL T.C. S4.7, S4.8<br />
DOOHAN F. S4.5 MARION-POLL A. -<br />
DOONAN J. S5.4, P4.3, MARRIOTT P. S2.8<br />
P5.2 MARTIN M. -<br />
DOUCHE T. S2.6 MASCLAUX-DAUBRESSE C. -<br />
DUBREUCQ B. S5.1 MAZEL J. -<br />
DUFRESNE M. S4.3, S4.7 MAZZAMURRO V. S4.8<br />
DURAND-TARDIF M. - MECHIN V. P2.8<br />
EL GUEDDARI N.E. - MICA E. S4.2<br />
ENARD C. - MOCHIDA K. S3.3<br />
FERRARIO-MERY S. - MORIN H. -<br />
GANDOUR M. P1.3 MOSCOU M. -<br />
73
MOUILLE G. S2.7, S2.2, VALDIVIA E. S5.6<br />
S2.4 VARIN S. S5.3<br />
MRAVEC J. S2.7 VEDELE F. -<br />
MULLINS E. S4.5 VERBRUGGEN N. -<br />
MUR L.A.J. S1.2, S4.1, VERNHETTES S. P2.6<br />
P2.9<br />
MUROZUKA E. S4.5 VOGEL J.P. S2.4, S3.5,<br />
MUTWIL M. S3.4 P1.4<br />
MUYLLE H. P2.3, P2.7 VOIGT C. S4.9, S2.1<br />
NEJI M. P1.3 VOOREND W. P2.7<br />
NG C. S5.5 VOZABOVA T. -<br />
NICHOLSON P. S1.3 WANG Y. -<br />
O'DRISCOLL A. S4.5 WARD E. -<br />
ORIA N. S3.6, P2.7 WIESENBERGER G. -<br />
PACHECO VILLALOBOS D. S1.6 WORLAND B. P3.5<br />
PANNETIER C. - WULFF B. -<br />
PARK C.-M. P3.4 ZHANG Y. P2.8<br />
PASQUET J.-C. S4.3 ZUBAIR H. P4.2, P4.3<br />
PECCHIONI N. S4.8<br />
PELLETIER G. -<br />
PERALDI A. S1.3<br />
PERSSON S. S3.4<br />
POIRE R. S3.5<br />
PUENTE P. -<br />
RASMUSSEN S.K. -<br />
RENAULT H. P5.3<br />
REYMOND M. P2.1<br />
RIZZOLATTI C. -<br />
SALSE J. S1.1<br />
SANGUINET OSMONT K. S2.5, P2.5<br />
SAUNIER DE CAZENAVE M. S5.3<br />
SAVAS G. -<br />
SCAGNELLI A. -<br />
SCHNITTGER A. -<br />
SCHULMAN A. -<br />
SEO P.J. P3.4<br />
SIBOUT R. S2.2, S2.7,<br />
S3.4, S3.6,<br />
S4.7, P2.2,<br />
P2.4, P2.6,<br />
P2.7, P3.3,<br />
P5.3<br />
SOULHAT C. P3.3<br />
SRIVASTAVA V. S1.4<br />
SUER S. S1.5<br />
TANACKOVIC V. P5.4<br />
THEVENIN J. -<br />
THOLE V. S3.1, S5.4,<br />
P3.5<br />
TIMPANO H. P2.6<br />
TORRES-ACOSTA J.A. S4.4<br />
TUNA M. P1.4<br />
VAIN P. S3.1, S5.4,<br />
P3.5<br />
74
LIST OF PARTICIPANTS<br />
75
ADAM Gerhard<br />
Univ. of Natural Res. & Life Sci. (BOKU),<br />
Konrad Lorenz Str. 24,<br />
3430 Tulln , Austria<br />
gerhard.adam@boku.ac.at<br />
ANTELME Sébastien<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr<br />
78000 Versailles, France<br />
sebastien.antelme@versailles.inra.fr<br />
ASHFAQ Muhammad<br />
Department of Plant Breeding and<br />
Genetics, University of Agriculture,<br />
Room No 6-D Z.A, Hashmi Hall,<br />
38000 Faisalabad, Pakistan<br />
muhammad.ashfaq@mail.mcgill.ca<br />
BAKAN Bénédicte<br />
INRA BIA, Rue de la Géraudière, BP71627,<br />
44316 Nantes, France<br />
bakan@nantes.inra.fr<br />
BARATINY Davy<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
davy.baratiny@versailles.inra.fr<br />
BARRIERE Yves<br />
INRA Lusignan, RD 150,<br />
86600 Lusignan, France<br />
yves.barriere@lusignan.inra.fr<br />
BELCRAM Harry<br />
INRA URGV - Génomique Végétale,<br />
2 rue Gaston Crémieux,<br />
91000 Evry, France<br />
belcram@evry.inra.fr<br />
BENAVENTE Elena<br />
Universidad Politecnica de Madrid, Dpto.<br />
Biotecnoloía, ETSIA<br />
28040 Madrid, Spain<br />
e.benavente@upm.es<br />
BERGER Adeline, INRA IJPB Institut Jean-<br />
Pierre Bourgin - umr 1318, Rd 10 - Route de<br />
Saint-Cyr,<br />
78000 Versailles, France<br />
adeline.berger@versailles.inra.fr<br />
BERQUIN Patrick<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr<br />
78000, Versailles, France<br />
patrick.berquin@versailles.inra.fr<br />
BERTOLINI Edoardo<br />
Scuola Superiore Sant'Anna of Pisa, c/o<br />
ENEA RC Casaccia V. Angiullarese 301,<br />
123 Rome, Italy<br />
e.bertolini@sssup.it<br />
BHALLA Prem<br />
The University of Melbourne, Grattan<br />
Street,<br />
VIC 3010 Parkville, Australia<br />
premlb@unimelb.edu.au<br />
BILU Alon<br />
Evogene Ltd., Gad Fainstiain 13,<br />
Rehovot, Israel<br />
alonb@evogene.com<br />
BLONDET Eddy<br />
INRA URGV - Génomique Végétale, 2 rue<br />
Gaston Crémieux,<br />
91000 Evry, France<br />
blondet@evry.inra.fr<br />
BLUEMKE Antje<br />
University of Hamburg, Biocenter Klein<br />
Flottbek, Ohnhorststr. 18,<br />
22609 Hamburg, Germany<br />
antje.bluemke@botanik.uni-hamburg.de<br />
BOSCH Maurice<br />
Institute of Biological, Environmental &<br />
Rural Sciences (IBERS), Aberystwyth<br />
University, Gogerddan, Aberystwyth,<br />
SY23 3EB, Aberystwyth, United Kingdom<br />
bosch.maurice@gmail.com<br />
76
BOUCHABKE-COUSSA Oumaya<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Oumaya.Bouchabke-Coussa@versailles.inra.fr<br />
BOUCHEZ David<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
david.bouchez@versailles.inra.fr<br />
BOUVIER D'YVOIRE Madeleine<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
madeleine.bouvier-dyvoire@versailles.inra.fr<br />
BOUYER Daniel<br />
CNRS - IBMP Institut de Biologie Moléculaire<br />
des Plantes, 12, Rue General Zimmer,<br />
67084 Strasbourg, France<br />
daniel.bouyer@ibmp-cnrs.unistra.fr<br />
BREIMAN Adina<br />
Tel Aviv University, Dept of Plant Sciences,<br />
69978 Tel Aviv, Israel<br />
adina@post.tau.ac.il<br />
CATALAN Pilar<br />
High Polytechnic School of Huesca,<br />
University of Zaragoza, Ctra. Cuarte km 1,<br />
22071 Huesca, Spain<br />
pilar.catalan09@gmail.com<br />
CAZALIS Roland<br />
URBV, University of Namur,<br />
61 rue de Bruxelles,<br />
5000 Namur, Belgium<br />
roland.cazalis@fundp.ac.be<br />
CHATEIGNER-BOUTIN Anne-Laure<br />
INRA BIA, Rue de la Géraudière, BP71627,<br />
44316 Nantes, France<br />
anne-laure.chateigner-boutin@nantes.inra.fr<br />
CHUPEAU Marie-Christine<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
marie-christine.chupeau@versailles.inra.fr<br />
CHUPEAU Yves<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
yves.chupeau@versailles.inra.fr<br />
CLAUSEN Signe Sandbech<br />
Risø National Laboratory for Sustainable<br />
Energy, BIO-309, P.O. Box 49,<br />
Frederiksborgvej 399,<br />
4000 Roskilde, Denmark<br />
sicl@risoe.dtu.dk<br />
COURTIAL Audrey<br />
UPS LRSV UMR5546 UPS/CNRS,<br />
Pôle de Biotechnologies Végétales 24,<br />
Chemin de Borde Rouge, BP42617,<br />
31326 Castanet-Tolosan, France<br />
courtial@lrsv.ups-tlse.fr<br />
DALMAIS Marion<br />
INRA URGV - Génomique Végétale,<br />
2 rue Gaston Cremieux,<br />
91000 Evry, France<br />
m.dalmais@evry.inra.fr<br />
DARRACQ Olivier<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
o_darracq@hotmail.com<br />
DAVID Laure<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Laure.David@versailles.inra.fr<br />
DELAPLACE Pierre<br />
University of Liège,<br />
Gembloux Agro-Bio Tech,<br />
2, Passage des Deportes,<br />
5030 Gembloux, Belgium<br />
pierre.delaplace@ulg.ac.be<br />
77
DELL'ACQUA Matteo<br />
Scuola Superiore Sant'Anna of Pisa,<br />
Piazza martiri della Libertà, 33 ,<br />
56127 Pisa, Italy<br />
m.dellacqua@sssup.it<br />
DELORY Benjamin<br />
University of Liège, Gembloux Agro-Bio<br />
Tech, 2, Passage des Deportes,<br />
5030 Gembloux, Belgium<br />
Benjamin.Delory@student.ulg.ac.be<br />
DINH THI Vinh Ha<br />
INRA URGV - Génomique Végétale,<br />
2 rue gaston crémieux,<br />
91057 Evry, France<br />
dinhthi@evry.inra.fr<br />
DOOHAN Fiona<br />
University College Dublin,<br />
Belfield, Dublin 4,<br />
Dublin, Ireland<br />
fiona.doohan@ucd.ie<br />
DOONAN John<br />
National Plant Phenomics Centre,<br />
Gogerddan campus, Aberystwyth<br />
University,<br />
SY23 3EB Aberystwyth, United Kingdom<br />
john.doonan@aber.ac.uk<br />
DOUCHE Thibaut<br />
UPS LRSV UMR5546 UPS/CNRS, Pôle de<br />
Biotechnologies Végétales,<br />
24, chemin de Borde Rouge, BP42617,<br />
31326 Castanet-Tolosan, France<br />
douche@lrsv.ups-tlse.fr<br />
DUBREUCQ Bertrand<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
bertrand.dubreucq@versailles.inra.fr<br />
DUFRESNE Marie<br />
Institut de Biologie des Plantes,<br />
rue Noetzlin,<br />
91405 Orsay cedex, France<br />
marie.dufresne@u-psud.fr<br />
DURAND-TARDIF Mylène<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Mylene.Durand-Tardif@versailles.inra.fr<br />
EL GUEDDARI Nour Eddine<br />
IBBP, Hindenburgplatz 55,<br />
48143 Münster, Germany<br />
guedari@uni-muenster.de<br />
ENARD Corine<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Corine.Enard@versailles.inra.fr<br />
FERRARIO-MERY Sylvie<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Sylvie.Ferrario@versailles.inra.fr<br />
GANDOUR Mhemmed<br />
Center of Biotechnology of Borj-Cédria,<br />
BP 901 hammam lif,<br />
2050 Hammam-lif, Tunisia<br />
gandourmed@yahoo.fr<br />
GAO Caixia<br />
Institute of Genetics and Developmental<br />
Biology, Da Tun Lu Rd,<br />
Chaoyang District,<br />
100101 Beijing, P.R.China<br />
cxgao@genetics.ac.cn<br />
GARVIN David F.<br />
USDA-ARS Plant Science Research Unit,<br />
St. Paul, MN 55108, Unit. States of America<br />
David.Garvin@ars.usda.gov<br />
GAYMARD Frédéric<br />
INRA Dept BV, Bat 7, 2 Place Viala,<br />
34060 Montpellier, France<br />
bv@supagro.inra.fr<br />
78
GIRALDO Patricia<br />
E.T.S.I. Agronomos. Universidad Politecnica<br />
de Madrid,<br />
Av. Complutense s/n. ,<br />
28040 Madrid, Spain<br />
patricia.giraldo@upm.es<br />
GONNEAU Martine<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Martine.Gonneau@versailles.inra.fr<br />
GONZALEZ DE LA CALLE Virginia<br />
Centro de Biotecnología y Genómica de<br />
Plantas, ETSI Agrónomos,<br />
Universidad Politécnica de Madrid,<br />
Campus de Montegancedo,<br />
28223 Pozuelo de Alarcón, Spain<br />
virginia.gonzalez@upm.es<br />
GONZALEZ GARCIA María de la Cruz<br />
Instituto de Bioquímica Vegetal y<br />
Fotosíntesis, Avda. Americo Vespucio 49,<br />
41092 Seville, Spain<br />
maricruz@ibvf.csic.es<br />
GOUJON Thomas<br />
INRA Dept BV, 46 Allée d'Italie,<br />
69364 Lyon cedex 07, France<br />
thomas.goujon@ens-lyon.fr<br />
GUBAEVA Ekaterina<br />
Westfalische Wilhelms University,<br />
Hindenburgplatz 55,<br />
48143 Muenster, Germany<br />
i_guba01@uni-muenster.de<br />
HANDS Philip<br />
University of Leicester, University Rd,<br />
LE1 7RH, Leicester, United Kingdom<br />
psh14@le.ac.uk<br />
HARDTKE Christian<br />
Dept. Plant Mol. Biol., Biophore Bldg.,<br />
DBMV,<br />
CH-1015 Lausanne, Switzerland<br />
christian.hardtke@unil.ch<br />
HARRINGTON Michael<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
mjhphd@gmail.com<br />
HAZEN Samuel<br />
University of Massachusetts, 221 Morrill<br />
Science Center,<br />
1003 Amherst, United States of America<br />
hazen@bio.umass.edu<br />
HIMURO Yasuyo<br />
RIKEN Tsukuba Institute,<br />
3-1-1 Koyadai,<br />
Tsukuba-shi, Ibaraki,<br />
305-0074 Tsukuba, Japan<br />
yasuyo.himuro@riken.jp<br />
HOFTE Herman<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
herman.hofte@versailles.inra.fr<br />
HO-YUE-KUANG Marie Séverine<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
severine.hyk@gmail.com<br />
IDZIAK Dominika<br />
University of Silesia,<br />
ul. Bankowa 12,<br />
Katowice, Poland<br />
didziak@us.edu.pl<br />
JAMET Elisabeth<br />
UPS LRSV UMR5546 UPS/CNRS, Pôle de<br />
Biotechnologies Végétales<br />
24, chemin de Borde Rouge, BP42617,<br />
31326 Castanet-Tolosan, France<br />
jamet@lrsv.ups-tlse.fr<br />
JØRGENSEN Bodil<br />
Department of Agriculture and Ecology,<br />
Thorvaldsensvej 40,<br />
1871 Frederiksberg, Denmark<br />
boj@life.ku.dk<br />
79
JOUANIN Lise<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
lise.jouanin@versailles.inra.fr<br />
JUNG Jae-Hoon<br />
Séoul University,<br />
Gwanak _ 599 Gwanak-ro, Gwanak-gu,<br />
151-742 Seoul, Korea<br />
hanbari22@korea.com<br />
JURANIEC Michal<br />
Laboratoire de Physiologie et de<br />
Génétique Moléculaire des Plantes,<br />
Université Libre de Bruxelles,<br />
Campus Plaine- CP242,<br />
1050 Brussels, Belgium<br />
juraniec@univ.rzeszow.pl<br />
KRAPP Anne<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
anne.krapp@versailles.inra.fr<br />
KUMLEHN Jochen<br />
IPK Gatersleben,<br />
Corrensstrasse 3,<br />
6466 Gatersleben, Germany<br />
kumlehn@ipk-gatersleben.de<br />
LAPIERRE Catherine<br />
AgroParisTech, UMR1318,<br />
Route de St Cyr,<br />
78026 Versailles, France<br />
catherine.lapierre@versailles.inra.fr<br />
LARRE Colette<br />
INRA BIA, Rue de la Géraudière,<br />
BP71627,<br />
44316 Nantes, France<br />
larre@nantes.inra.fr<br />
LE BRIS Philippe<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France, philippe.l<br />
bris@versailles.inra.fr<br />
LECOMTE Philippe<br />
INRA - UMR GDEC,<br />
234, avenue du Brézet,<br />
63100 Clermont-Ferrand, France<br />
philippe.lecomte@clermont.inra.fr<br />
LEGAY Sylvain<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
sylvain.legay@versailles.inra.fr<br />
LEPINIEC Loïc<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
loic.lepiniec@versailles.inra.fr<br />
LEVI BAR SHALOM Avishag<br />
Evogene Ltd.,<br />
13 Gad Feinstein street,<br />
76121 Rehovot, Israel<br />
avishaglb@evogene.com<br />
LOUDET Olivier<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Olivier.Loudet@versailles.inra.fr<br />
MACADRE Catherine<br />
Institut de Biologie des Plantes,<br />
rue Noetzlin,<br />
91405 Orsay cedex, France<br />
catherine.macadre@u-psud.fr<br />
MAILLET Fabienne<br />
INRA LIPM,<br />
Chemin de Borde Rouge, BP 52627,<br />
31326 Castanet-Tolosan, France<br />
maillet@toulouse.inra.fr<br />
MARCEL Thierry<br />
INRA BIOGER, UR1290 BIOGER-CPP,<br />
Avenue Lucien Brétignières, BP01,<br />
78850 Thiverval-Grignon, France<br />
Thierry.Marcel@versailles.inra.fr<br />
80
MARION-POLL Annie<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
annie.marion-poll@versailles.inra.fr<br />
MARRIOTT Poppy<br />
Univeristy of York, CNAP, Department of<br />
Biology, University of York,<br />
YO10 5DD York, United Kingdom<br />
pm518@york.ac.uk<br />
MARTIN Marjolaine<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
marjolaine.martin@hotmail.fr<br />
MASCLAUX-DAUBRESSE Céline<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
masclaux@versailles.inra.fr<br />
MAZEL Julien<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
julien.mazel@versailles.inra.fr<br />
MAZZAMURRO Valentina<br />
Dipartimento di Scienze Agrarie e degli<br />
Alimenti, Università di Modena e Reggio<br />
Emilia, Via Amendola, 2 - Padiglione Besta,<br />
42122 Reggio Emilia, Italy<br />
valentina.mazzamurro@unimore.it<br />
MECHIN Valérie<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
vmechin@versailles.inra.fr<br />
MICA Erica<br />
Scuola Superiore Sant'Anna of Pisa, Piazza<br />
martiri della Libertà, 33 ,<br />
56127 Pisa, Italy<br />
erica.mica@sssup.it<br />
MOCHIDA Keiichi,<br />
RIKEN Biomass Engineering Program, 1-7-<br />
22, Suehiro-cho, Tsurumi-ku,<br />
230-0045 Yokohama, Japan<br />
mochida@psc.riken.jp<br />
MORIN Halima<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
halima.morin@versailles.inra.fr<br />
MOSCOU Matthew<br />
The Sainsbury Laboratory,<br />
Norwich Research Park,<br />
NR4 7UH Norwich, United Kingdom<br />
matthew.moscou@tsl.ac.uk<br />
MOUILLE Grégory<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
gregory.mouille@versailles.inra.fr<br />
MRAVEC Jozef<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
jmravec@versailles.inra.fr<br />
MULLINS Ewen<br />
Teagasc, Dept. Crop Science,<br />
Oak Park,<br />
Carlow, Ireland<br />
ewen.mullins@teagasc.ie<br />
MUR Luis A. J.<br />
Aberystwyth University,<br />
"IBERS" - Institute of Biological,<br />
Environmental and Rural Sciences.<br />
Edward Llwyd Building,<br />
Aberystwyth SY23 3DA, Wales, United<br />
Kingdom<br />
lum@aber.ac.uk<br />
MUROZUKA Emiko<br />
Faculty of Life Sciences, University of<br />
Copenhagen,<br />
Thorvaldsensvej 40, opg. 8, 3rd floor,<br />
DK-1871 Copenhagen, Denmark<br />
emikomu@life.ku.dk<br />
81
MUTWIL Marek<br />
Max-Planck-Institute for Molecular Plant<br />
Physiology, Am Mühlenberg 1,<br />
14476 Potsdam, Germany<br />
mutwil@mpimp-golm.mpg.de<br />
MUYLLE Hilde<br />
ILVO, Caritasstraat 21<br />
9090 Melle, Belgium<br />
Hilde.Muylle@ilvo.vlaanderen.be<br />
NEJI Mohamed<br />
Center of Biotechnology of Borj-Cédria,<br />
Borj cedria Hammam Lif,<br />
2050 Tunis, Tunisia<br />
mnmedneji@gmail.com<br />
NG Carl<br />
University College Dublin, School of Biology<br />
and Environmental Science, Dublin 4,<br />
Dublin, Ireland<br />
carl.ng@ucd.ie<br />
NICHOLSON Paul<br />
John Innes Centre, Norwich Research Park,<br />
NR4 7UH Norwich, United Kingdom<br />
paul.nicholson@bbsrc.ac.uk<br />
O'DRISCOLL Aoife<br />
Teagasc, Dept. Crop Science,<br />
Oak Park,<br />
Carlow, Ireland<br />
aoife.odriscoll@teagasc.ie<br />
ORIA Nicolas<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
nicolas.oria@versailles.inra.fr<br />
PACHECO VILLALOBOS David<br />
Department of Plant Molecular Biology,<br />
University of Lausanne, Biophore Building,<br />
CH-1015 Lausanne, Switzerland<br />
David.PachecoVillalobos@unil.ch<br />
PANNETIER Catherine<br />
INRA-CIRAD, IJPB UMR1318, Bât 2,<br />
Route de St-Cyr (RD10),<br />
78026 Versailles, France<br />
catherine.pannetier@versailles.inra.fr<br />
PARK Chung-Mo<br />
Séoul University, Gwanak _ 599 Gwanak-ro,<br />
Gwanak-gu,<br />
151-742 Seoul, Korea<br />
cmpark@snu.ac.kr<br />
PASQUET Jean-Claude<br />
IBP, rue Noetzlin,<br />
91405 Orsay cedex, France<br />
jean-claude.pasquet@u-psud.fr<br />
PECCHIONI Nicola<br />
Dipartimento di Scienze Agrarie e degli<br />
Alimenti, Università di Modena e Reggio<br />
Emilia, Via Amendola, 2 - Padiglione Besta<br />
42122 Reggio Emilia, Italy<br />
nicola.pecchioni@unimore.it<br />
PELLETIER Georges<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
georges.pelletier@versailles.inra.fr<br />
PERALDI Antoine<br />
John Innes Centre, Norwich Research Park,<br />
NR4 7UH Norwich, United Kingdom<br />
antoine.peraldi@bbsrc.ac.uk<br />
PERSSON Staffan<br />
Max-Planck-Institute for Molecular Plant<br />
Physiology, Am Mühlenberg 1,<br />
14476 Potsdam, Germany<br />
persson@mpimp-golm.mpg.de<br />
POIRE Richard<br />
CSIRO Plant Industry Black Mountain<br />
Laboratories, Clunies Ross Street,<br />
2601 Canberra, Australia<br />
richard.poire@csiro.au<br />
PUENTE Pilar<br />
BASF SE, Speyerer St 2,<br />
67117 Limburgerhof, Germany<br />
pilar.puente@basf.com<br />
RASMUSSEN Søren K.<br />
University of Copenhagen,<br />
Thorvaldsensvej 40,<br />
1871 Frederiksberg, Denmark<br />
skr@life.ku.dk<br />
82
RENAULT Hugues<br />
CNRS - IBMP Institut de Biologie Moléculaire<br />
des Plantes, 28 rue Goethe,<br />
67083 Strasbourg, France<br />
hugues.renault@ibmp-cnrs.unistra.fr<br />
REYMOND Matthieu<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
mreymond@versailles.inra.fr<br />
RIZZOLATTI Carine<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Carine.Rizzolatti@versailles.inra.fr<br />
SALSE Jérôme<br />
Laboratory ‘Plant Paleogenomics for Traits<br />
Improvement’, UMR INRA-UBP 1095,<br />
Domaine de Crouelle,<br />
234 Avenue du Brézet,<br />
63100 Clermont-Ferrand, France<br />
jsalse@clermont.inra.fr<br />
SANGUINET OSMONT Karen<br />
UMass-Amhert, 611 N. Pleasant St.,<br />
1002 Amherst, United States of America<br />
ksosmont@bio.umass.edu<br />
SAUNIER DE CAZENAVE Magdalena<br />
University of Liège, Gembloux Agro-Bio<br />
Tech, 2 Passage des Deportes,<br />
5030 Gembloux, Belgium<br />
m.saunierdecazenave@ulg.ac.be<br />
SAVAS Gulsemin<br />
Namik Kemal University - Faculty of<br />
Agriculture - Department of Field Crops,<br />
De irmenaltı Campus,<br />
59030 Tekirdag, Turkey<br />
SCAGNELLI Aurélie<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
aurelie.scagnelli@versailles.inra.fr<br />
SCHNITTGER Arp<br />
CNRS - IBMP Institut de Biologie Moléculaire<br />
des Plantes, 28 rue Goethe<br />
67083 Strasbourg, France<br />
Arp.Schnittger@ibmp-cnrs.unistra.fr<br />
SCHULMAN Alan<br />
MTT & University of Helsinki, PO Box 65,<br />
FIN-00014 Helsinki, Finland<br />
alan.schulman@helsinki.fi<br />
SEO Pil Joon<br />
Séoul University, Gwanak _ 599 Gwanak-ro,<br />
Gwanak-gu,<br />
151-742, Seoul, Korea<br />
dualnt83@snu.ac.kr<br />
SIBOUT Richard<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Richard.Sibout@versailles.inra.fr<br />
SOULHAT Camille<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Camille.Soulhat@versailles.inra.fr<br />
SRIVASTAVA Vibha<br />
University of Arkansas,<br />
115 Plant Science bldg,<br />
72704 Fayetteville, Unit. States of America<br />
vibhas@uark.edu<br />
SUER Stefanie<br />
Gregor Mendel Institute of Molecular Plant<br />
Biology, Dr. Bohr-Gasse 3,<br />
1030 Vienna, Austria<br />
stefanie.suer@gmi.oeaw.ac.at<br />
TANACKOVIC Vanja<br />
Faculty of Life Sciences, Department of<br />
Plant Biology and Biotechnology,<br />
Thorvaldsensvej 40, opg. 10, 1,<br />
1871 Copenhagen, Denmark<br />
vtana@life.ku.dk<br />
83
THEVENIN Johanne<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Johanne.Thevenin@versailles.inra.fr<br />
THOLE Vera<br />
John Innes Centre, Norwich Research Park,<br />
NR4 7UH Norwich, United Kingdom<br />
vera.thole@bbsrc.ac.uk<br />
TIMPANO Hélène<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
helene.timpano@versailles.inra.fr<br />
TORRES-ACOSTA Juan Antonio<br />
Universität für Bodenkultur Department für<br />
Angewandte Genetik und Zellbiologie,<br />
Konrad Lorenzstrasse 24,<br />
3430 Tulln , Austria<br />
juan-antonio.torres-acosta@boku.ac.at<br />
TUNA Metin<br />
Namik Kemal University - Faculty of<br />
Agriculture - Department of Field Crops,<br />
De irmenaltı Campus,<br />
59030 Tekirdag, Turkey<br />
metintuna66@yahoo.com<br />
VAIN Philippe<br />
Department of Crop Genetics, John Innes<br />
Centre, Norwich Research Park,<br />
Norwich NR4 7UH, United Kingdom<br />
philippe.vain@jic.ac.uk<br />
VALDIVIA Elene<br />
Univ Santiago de Compostela ,<br />
Departamento de Fisiología Vegetal ,<br />
15782 Santiago de Compostela , Spain<br />
elenevaldivia@gmail.com<br />
VARIN Sébastien<br />
University of Liège, Gembloux Agro-Bio<br />
Tech, 2 Passage des Deportes,<br />
5030 Gembloux , Belgium<br />
seb.varin@orange.fr<br />
VEDELE Françoise<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
francoise.vedele@versailles.inra.fr<br />
VERBRUGGEN Nathalie<br />
Laboratoire de Physiologie et de<br />
Génétique Moléculaire des Plantes,<br />
Université Libre de Bruxelles, Campus<br />
Plaine- CP242,<br />
1050 Brussels, Belgium<br />
nverbru@ulb.ac.be<br />
VERNHETTES Samantha<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
samantha.vernhettes@versailles.inra.fr<br />
VOGEL John P.<br />
USDA-ARS Western Regional Research<br />
Center,<br />
Albany, CA, 94710, Unit. States of America<br />
john.vogel@ars.usda.gov<br />
VOIGT Christian<br />
Biocenter Klein Flottbek,<br />
Ohnhorststr. 18,<br />
22609 Hamburg, Germany<br />
voigt@botanik.uni-hamburg.de<br />
VOOREND Wannes,<br />
ILVO,<br />
Caritasstraat 21,<br />
9090 Melle, Belgium<br />
wannes.voorend@ilvo.vlaanderen.be<br />
VOZABOVA Tereza<br />
Laboratory of Plant Breeding, PO box 386,<br />
Droevendaalsesteeg,<br />
6708PB Wageningen, The Netherlands<br />
T.Vozabova@seznam.cz<br />
WANG Yin<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
yin.wang@versailles.inra.fr<br />
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WARD Eric<br />
Two Blades Foundation, P.O. Box 51291,<br />
27717 Durham, United States of America<br />
erw@2blades.org<br />
WIESENBERGER Gerlinde<br />
Univ. of Natural Res. & Life Sci. (BOKU),<br />
Konrad Lorenz Str. 24,<br />
3430 Tulln, Austria<br />
gerlinde.wiesenberger@boku.ac.at<br />
WORLAND Barbara<br />
John Innes Centre,<br />
Norwich Research Park,<br />
NR4 7UH Norwich, United Kingdom<br />
barbara.worland@bbsrc.ac.uk<br />
WULFF Brande<br />
The Sainsbury Laboratory,<br />
Norwich Research Park,<br />
NR4 7UH Norwich, United Kingdom<br />
brande.wulff@sainsbury-laboratory.ac.uk<br />
ZHANG Yu<br />
INRA IJPB Institut Jean-Pierre Bourgin - umr<br />
1318, Rd 10 - Route de Saint-Cyr,<br />
78000 Versailles, France<br />
Yu.Zhang@versaillesinra.fr<br />
ZUBAIR Hassan<br />
IBERS, Aberystwyth University, Edward<br />
Llwyd Building,<br />
SY23 3DA Aberystwyth, United Kingdom<br />
hhz@aber.ac.uk<br />
85