pdf file

Acknowledgements:
Institut National de la Recherche
Agronomique – INRA
• Centre de Versailles-Grignon
• Département Santé des Plantes et
Environnement (SPE)
• Département de Biologie
Végétale (BV)
• Département Caractérisation et
Elaboration des Produits Issus de
l’Agriculture (CEPIA)
Région Ile-de-France
« action financée par la Région Ile-de-
France »
DIM ASTREA
« Agrosciences Territoires Ecologie
Alimentation »
IFR 87
« La plante et son environnement »
SFBV
Société Française de Biologie Végétale
EFOR
Réseau d'Etudes Fonctionnelles sur les
Organismes modèles
1

1 st European Brachypodium Workshop
19-21 October 2011
Versailles-France
Organizing committee
Oumaya Bouchabké-Coussa (INRA, IJPB)
Corine Enard (INRA, IJPB)
Martine Gonneau (INRA, IJPB)
Lise Jouanin (INRA, IJPB)
Maria Jesus Lacruz (INRA, Versailles)
Thierry Marcel (INRA, BIOGER-CPP)
Jocelyne Picart (INRA, IJPB)
Philippe Porée (INRA, Versailles)
Stéphane Raude (INRA, Versailles)
Richard Sibout (INRA, IJPB)
Anne-Sophie Simon (INRA, Versailles)
Maryvonne Thomas (INRA, Versailles)
Scientific committee
Dr. Herman Höfte (INRA, IJPB)
Dr. Thierry C. Marcel (INRA, BIOGER-CPP)
Dr. Jérôme Salse (INRA, GDEC)
Dr. Arp Schnittger (CNRS, IBMP)
Dr. Richard Sibout (INRA, IJPB)
Dr. Philippe Vain (JIC, GB)
Dr. John P. Vogel (USDA-ARS, USA)
Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech
INRA Centre de Versailles-Grignon
Route de St-Cyr (RD10)
78026 Versailles Cedex France
tél : +33 (0)1 30 83 30 00 fax : +33 (0)1 30 83 33 19
2

Contents
Programme 4
Oral presentations 9
Session 1: Evolution and Natural Variation 10
Session 2: Cell Wall, Biomass and Biofuels 16
Session 3: Tools and Bioengineering 24
Session 4: Biotic and Abiotic Stresses 31
Session 5: Plant Development, Metabolism and Physiology 40
Poster presentations 46
Session 1: Evolution and Natural Variation 47
Session 2: Cell Wall, Biomass and Biofuels 51
Session 3: Tools and Bioengineering 60
Session 4: Biotic and Abiotic Stresses 65
Session 5: Plant Development, Metabolism and Physiology 68
Authors Index 72
List of participants 75
3

PROGRAMME
4

Wednesday 19 th October, 2011
11:00 am to 7:00 pm Opening of Workshop registration
Welcome and opening session
1:00 pm Introductory Remarks
Richard Sibout, INRA (IJPB), and Thierry Marcel, INRA (BIOGER-CPP), France
1:10 Welcome Address
David Bouchez, Director of IJPB, France
1:30 Introductory talk: Dr. John Vogel, USDA-ARS, United-States
Title: Brachypodium rises to a model.
Evolution and Natural Variation
Moderators: Jérome Salse and Richard Sibout
2:00 Key Speaker: Dr. Jérôme Salse, INRA (UBP), France
Title: What can model species offer cereal breeders?
2:40 Pilar Catalan, University of Zaragoza, Spain
Evolution and taxonomic split of the model grass Brachypodium distachyon (L.) P.
Beauv.
3:00 Antoine Peraldi, JIC, United Kingdom
Using Brachypodium distachyon for model to crop translation of disease resistance in
cereals
3:20 pm Coffee Break
4:00 Vibha Srivastava, University of Arkansas, United States
Site-specific recombination systems for genetic modifications of cereals
4:20 Stephanie Suer, Austrian Academy of Sciences, Austria
Functional analysis of WOX4 in mono- and dicotyledonous plants and dissecting the
evolution of vascular development
4:40 David Pacheco-Villalobos, University of Lausanne, Switzerland
Deciphering the function and evolution of BRX-like genes in Brachypodium distachyon
Cell Wall, Biomass and Biofuels
Moderators: Herman Höfte and John Vogel
5:00 Christian A. Voigt, University of Hamburg, Germany
Brachypodium distachyon as a model plant for lignocellulosic ethanol production
5:20 Madeleine Bouvier d'Yvoire, INRA (IJPB), France
Disrupting the cinnamyl alcohol dehydrogenase 1 (BdCAD1) gene leads to altered
lignification and improved saccharification in Brachypodium distachyon
5:40 Emiko Murozuka, University of Copenhagen, Danemark
Identification of Brachypodium mutants defective in silicon uptake
6:00 pm Poster Session
Cocktail offered by Centre INRA Versailles-Grignon
7:30 Departure by bus to centre of Versailles
5

Thursday 20 th October, 2011
Cell Wall, Biomass and Biofuels (followed)
Moderators: Herman Höfte and John Vogel
9:00 Key Speaker: Dr. John Vogel, USDA-ARS, United-States
Title: Natural diversity from genomics to phenomics.
9:40 Samuel Hazen, University of Massachusetts, United-States
Functional characterization of candidate cell wall genes in B. distachyon
10:00 Elisabeth Jamet, CNRS (LRSV), France
Cell wall proteomics of Brachypodium distachyon
10:20 Jozef Mravec, INRA (IJPB), France
The role of pectins in Brachypodium development
10:40 Poppy Marriott, University of York, United Kingdom
Investigating biomass digestibility in Brachypodium
11:00 pm Coffee and ‘viennoiseries’
Tools and Bioengineering
Moderators: Oumaya Bouchabke-Coussa and Philippe Vain
11:40 Key Speaker: Dr. Philippe Vain, JIC, United Kingdom
Title: T-DNA mutagenesis in Brachypodium distachyon.
12:20 Dominika Idziak, University of Silesia, Poland
Current status and future perspectives for molecular cytogenetic studies of
Brachypodium
12:40 Keiichi Mochida, RIKEN BMEP/PSC, Japan
Functional annotation of a full-length Brachypodium cDNA resource
1:00 pm Lunch at INRA Score Services
Tools and Bioengineering (followed)
Moderators: Oumaya Bouchabke-Coussa and Philippe Vain
2:20 Marek Mutwil, Max Planck Institute of Molecular Plant Physiology, Germany
BradiNet: co-expression platform and comparative transcriptomics for Brachypodium
2:40 Richard Poiré, CSIRO Plant Industry, Australia
Phenotyping the shoot and root diversity of Brachypodium distachyon to accelerate
plant biofuel breeding
3:00 Marion Dalmais, INRA (URGV), France
TILLING in Brachypodium distachyon: development of a Sodium Azide mutant
collection and screening for induced point mutations
3:20 Jochen Kumlehn, IPK, Germany
Novel tools for Brachypodium research: Genetic transformation using shoot segments
and immature pollen-derived generation of instantly homozygous lines
3:40 pm Coffee Break
4:20 Round Tables: tba
6:30 Departure by bus to restaurant ‘Maître Kanter’
7:30 pm Dining cocktail at ‘Maître Kanter’ in Versailles
6

Friday 21 st October, 2011
Biotic and Abiotic Stresses
Moderators: Thierry Marcel and Luis Mur
9:00 am Key Speaker: Dr. Luis Mur, Aberystwyth University, United Kingdom
Title: Exploiting Brachypodium distachyon biodiversity to establish sources of resistance
to abiotic and biotic stress.
9:40 Mica Erica, Scuola Superiore Sant’Anna, Italy
Assessing the role of smallRNAs in leaf cell identity and growth reprogramming during
drought stress in Brachypodium distachyon
10 :00 Marie Dufresne, Université Paris-Sud 11, France
Use of the pathosystem Brachypodium distachyon / Fusarium graminearum to
investigate the detoxification mechanisms of mycotoxins by plants.
10:20 Gerhard Adam, University of Natural Resources and Life Sciences, Austria
Functional characterization of Brachypodium UDP-glucosyltransferases
10:40 Aoife O’ Driscoll, University College Dublin, Ireland
The Mycosphaerella graminicola-Brachypodium distachyon interaction: a new model
pathosystem to study Septoria tritici blotch disease of wheat
11:00 pm Coffee and ‘viennoiseries’
11:40 Key Speaker: Dr. David Garvin, USDA-ARS, United-States
Title: Brachypodium as a model for unraveling stem rust resistance.
12:20 Thierry Marcel, INRA (BIOGER-CPP), France
Brachypodium distachyon, a model grass to study plant-pathogen interactions
12:40 Nicola Pecchioni, Università di Modena e Reggio Emilia, Italy
QTLs for resistance to the leaf rust Puccinia brachypodii in the model grass
Brachypodium distachyon
1:00 pm Lunch at INRA Score Services
2:20 Antje Bluemke, University of Hamburg, Germany
Brachypodium distachyon: an excellent model for Fusarium graminearum infection in
wheat
Plant Development, Metabolism and Physiology
Moderator: Arp Schnittger
2:40 Fabienne Guillon, INRA (BIA), France
Brachypodium distachyon grain: development-associated changes in morphology and
storage accumulation
3:00 Virginia González de la Calle, Universidad Politécnica de Madrid, Spain
The transcription factor BdDOF24 interacts with BdGAMYB and regulates the cathepsin
B-like gene during Brachypodium seed germination
3:20 Pierre Delaplace, University of Liège, Belgium
Rhizobacterial volatile organic compounds modulate biomass production and root
architecture in Arabidopsis thaliana (L.) Heynh. and Brachypodium distachyon (L.) P.
Beauv.
3:40 John Doonan, Aberystwyth University, United Kingdom
Using Natural And Induced Variation In Brachypodium To Study Grain Development
4:00 pm Coffee Break
7

4:20 Carl Ng, University College Dublin, Ireland
Profiling the lipidome of Brachypodium distachyon
4:40 Elene R. Valdivia, Universidad de Santiago de Compostela, Spain
Inducible xylem differentiation in Brachypodium
5:00 Closing Address
8

ORAL COMMUNICATIONS
9

Session 1 – Evolution and Natural Variation
KEYNOTE LECTURE
S1.1- What Can Model Species Offer Cereal Breeders
Jerome Salse
Laboratory ‘Plant Paleogenomics for Traits Improvement’, UMR INRA-UBP 1095, Domaine
de Crouelle, 234, Avenue du Brézet, 63100 Clermont-Ferrand, FRANCE
jsalse@clermont.inra.fr
Abstract
During the last decade, technological improvements led to the development of large
sets of plant genomic resources including the Brachypodium genome release in 2010,
permitting the emergence of high-resolution comparative genomic studies and
translational genomics approaches to non-sequence cereal genomes. In an attempt to
unravel the structure and evolution of the plant ancestor genome we have re-assed the
synteny and duplications of Angiosperm genomes to identify and characterize shared
duplications. We combined the data on the intra-genomic duplications with those on
the colinear blocks and found duplicated segments that have been conserved at
orthologous positions since the divergence of plants. By conducting detailed analysis of
the length, composition, and divergence time of the conserved duplications, we
identified common and lineage-specific patterns of conservation between the different
genomes that allowed us to propose a model in which the plant genomes have evolved
from a common ancestor with a basic number of five/seven chromosomes (90 MYA)
through whole genome duplications (i.e. paleopolyploidization) and translocations
followed by lineage specific segmental duplications, chromosome fusions and
translocations (Abrouk et al. 2010; Murat et al. 2010; Salse et al. 2011).
Based on these data an ‘inner circle’ comprising 5/7 ancestral chromosomes with 10000
protogenes was defined providing a new reference for the plant chromosomes and new
insights into their ancestral relationships that have led to arrange their chromosomes into
concentric ‘crop circles’ of synteny blocks (Abrouk et al. 2010). The established plant
ancestor genome structure, in term of chromosome structure and gene content, offer
the opportunity to perform high resolution translational genomics in cereals, from model
(i.e. sequenced) species to complex ones, through (i) marker development for
physical/genetic mapping, (ii) gene or sequence annotation, (iii) trait dissection, that will
be discussed in details in the current presentation (Quraishi et al 2011ab).
References
Abrouk M, Murat F, Pont C, et al. (2010) Paleogenomics of Plants: Modern Species
Synteny-Based Modelling of Extinct Ancestors. Trends in Plant Science. 15(9):479-87.
Murat F, Xu JH, Tannier E, et al. (2010) Ancestral Grass Karyotype Reconstrcution Unravels
New Mechanisms of Genome Shuffling as a Source of Plant Evolution. Genome research.
20(11):1545-1557
Quraishi UM, Murat F, Abrouk M, et al. (2011a) Meta-Genomics Analysis of the Grain
Dietary Fiber Content in Bread Wheat. Functional & Integrative Genomics. 11(1):71-83.
Quraishi UM, Abrouk M, Murat F, et al. (2011b) Cross-Genome Map Based Dissection of a
Nitrogen Use Efficiency Ortho-metaQTL in Bread Wheat Unravels Concerted Cereal
Genome Evolution. Plant Journal. 65(5):745-56.
Salse J, Feuillet C (2011) Palaeogenomics in cereals: modeling of ancestors for modern
species improvement. C R Biol. 334(3):205-11.
10

Session 1 – Evolution and Natural Variation
S1.2- Evolution and taxonomic split of the model grass Brachypodium
distachyon (L.) P. Beauv.
Pilar Catalán 1, Jochen Müller 2, Robert Hasterok 3, Glyn Jenkins 4, Luis A. J. Mur 4, Tim
Langdon 4, Alexander Betekhtin 3, Dorota Siwinska 3, Manuel Pimentel 5, Diana López-
Alvarez 1
1 Department of Agriculture (Botany), High Polytechnic School of Huesca, University of Zaragoza, Ctra. Cuarte km 1, 22071 Huesca,
Spain
2 Herbarium Haussknecht, Department of Systematic Botany, University of Jena, Fürstengraben 1, 07737 Jena, Germany
3 Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28,
40032 Katowice, Poland
4 Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Plas Gogerddan, Aberystwyth SY23 3EB Wales, UK
5 Department of Plant and Animal Biology and Ecology, Faculty of Biology, University of Coruña, Campus da Zapateira s. n., 15071 A
Coruña, Spain
pcatalan@unizar.es
Abstract
A multidisciplinary phenetic, cytogenetic and evolutionary study has been conducted
on a representative sampling of the three known cytotypes of the model grass
Brachypodium distachyon (2n = 10, 20, 30). Statistical analyses of 15 selected traits in
greenhouse propagated individuals detected significant taxonomic differences
between the three cytotypes that have been further validated through discriminant
analysis of wild individuals, demonstrating stability of characters in natural populations.
Cytogenetic analyses based on nuclear genome size estimation, fluorescence in situ
hybridisation with genomic and multiple DNA sequences as probes and chromosome
painting confirm that the 2n=10 and 2n=20 chromosome cytotypes correspond to two
different diploid taxa, whereas the 2n=30 cytotype is a derived allotetraploid of the cross
between them. Phylogenetic analysis based on two plastid (ndhF, trnLF) and five nuclear
(ITS, ETS, CAL, DGAT, GI) genes have demonstrated that the 2n=20 and 2n=10 cytotypes
were, respectively, the maternal and paternal genome donors of the 2n=30 cytotype.
The substantial phenotypic, cytogenetic and molecular differences detected among
the three B. distachyon s. l. cytotypes are indicative of major speciation processes within
this complex that allow their taxonomic separation into three distinct species. We have
kept the name B. distachyon for the 2n=10 cytotype, whose genome has been fully
sequenced and which is being used as model grass for temperate cereals, and have
consequently designated a new epitype for it; we have described two new species B.
stacei and B. hybridum for, respectively, the 2n=20 and 2n=30 cytotypes. The completely
sequenced B. distachyon genome and the future availability of fully sequenced B. stacei
and B. hybridum genomes would provide an exceptional opportunity for thorough
comparative phylogenomic analyses of these plants and other totally or partially
sequenced grasses.
References
Catalán P, Müller J, Hasterok R, et al. (2011) Next generation systematics: evolution and taxonomic split of the model grass
Brachypodium distachyon (L.) P. Beauv. (Poaceae). submitted.
Hasterok R, Draper J, Jenkins G (2004) Laying the cytotaxonomic foundations of a new model grass, Brachypodium distachyon (L.)
Beauv. Chromosome Research 12:397-403.
Idziak D, Betekhtin A, Wolny E, et al. (2011) Painting the chromosomes of Brachypodium-current status and future prospects.
Chromosoma: Epub ahead of print, doi 10.1007/s00412-00011-00326-00419
Mur L-A, Allainguillaume J, Catalan P, et al. (2011) Exploiting the Brachypodium Tool Box in cereal and grass research. New Phytologist
191: 334-347.
Wolny E, Lesniewska K, Hasterok R, Langdon T (2011) Compact genomes and complex evolution in the genus Brachypodium.
Chromosoma 120:199-212
Keywords
Brachypodium distachyon, Brachypodium stacei, Brachypodium hybridum, cytogenetics,
evolutionary systematics
11

Session 1 – Evolution and Natural Variation
S1.3- Using Brachypodium distachyon for model to crop translation of disease
resistance in cereals.
Antoine Peraldi, Andrew Steed, Chris Burt, Paul Nicholson.
Department of Disease and Stress and Stress Biology, John Innes Centre, Colney Lane,
Norwich, NR4 7UH, UK.
antoine.peraldi@bbsrc.ac.uk
Abstract
Cereal production needs to improve in both quality and yield during the twenty first
century to cope with predicted global challenges such as climate change, rising biofuel
demand and human demographic pressure. Cereal diseases such as Fusarium head
blight (FHB) of wheat can reduce yield and also contaminate grain with mycotoxins
harmful for both animal and human consumption. Despite numerous studies on the
genetic components of resistance to Fusarium diseases, progress is impaired by the
complexity of the wheat genome and lack of available genome sequence. Therefore
research would benefit greatly from availability of a robust, tractable genetic model
from which to undertake model to crop translation.
Brachypodium distachyon (Bd) has numerous advantages both as a genetic model and
in functional genetic studies because of the increasing availability of appropriate tools
(Draper et al., 2001). We have demonstrated that Bd exhibits compatible interactions
with the main FHB-causing species of Fusarium and mirrors the disease symptom
development on wheat better that any other model proposed to date (Peraldi et al.,
2011). This new pathosystem enables screening of Bd mutant populations (i.e.T-DNA, Vain
et al., 2008) to identify genes involved in resistance to Fusarium. Once characterized,
these gene candidates can be validated in the natural host using reverse genetics
approaches (e.g TILLING).
Using this approach, a Bd mutant was identified that conferred increased resistance to
Fusarium infection in detached leaf and flower inoculation tests. Sequence alignments
and dark-induced senescence tests were used to characterize this gene as a functional
homologue of auxin response factor (ARF) 2. The potential role of ARF2 in the natural host
was evaluated using virus-induced gene silencing (VIGS) of wheat ARF2 which resulted in
an average reduction in FHB disease severity of 20%.
References:
Draper J, Mur LAJ, Jenkins G, et al. (2001) Brachypodium distachyon. A new model
system for functional genomics in grasses. Plant Physiology 127:1539-1555.
Peraldi A, Beccari G, Steed A, Nicholson P. (2011) Brachypodium distachyon: a new
pathosystem to study Fusarium head blight and other Fusarium diseases of wheat. BMC
Plant Biology 11:100.
Vain P, Worland B, Thole V, et al. (2008) agrobacterium-mediated transformation of the
temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional
mutagenesis. Plant Biotechnology Journal 6:236-245.
Keywords
Brachypodium, Fusarium, wheat, pathogen resistance, model to crop translation.
12

Session 1 – Evolution and Natural Variation
S1.4- Site-specific recombination systems for genetic modifications of cereals
Vibha Srivastava 1, Soumen Nandy, and M. Aydin Akbudak
1 University of Arkansas, 115 Plant Science bldg, 72704 Fayetteville, USA
vibhas@uark.edu
Abstract
Precise full-length integration of transgenes is desirable for ensuring optimum gene
expression. Site-specific recombination systems are versatile tools for catalyzing DNA
integration, excision or inversion. A number of site-specific recombination systems have
been isolated and shown to excise specific DNA fragments from the transgene locus;
however, their use for gene integration is rarely demonstrated. One such system is the
yeast FLP-FRT system that has been widely used for DNA excision in plants, but not for
gene integration. Here, we report the use of FLP-FRT system for efficient targeting of
foreign genes into the engineered genomic sites in rice. The transgene vector
containing a pair of directly oriented FRT sites was introduced by particle bombardment
into cells containing the engineered target site. Transiently expressed FLP activity
generated the backbone-free gene circle that integrated into the target site. The
majority of the transgenic events contained the precise integration locus, expressed the
transgene, and transmitted the stable site-specific integration locus to progeny. This
robust transformation platform is useful for both biotechnology applications in crops, and
functional genomics in model plant species such as Brachypodium.
References
Nandy S, Srivastava V (2011) Site-specific gene integration in rice genome mediated by
the FLP-FRT recombination system. Plant Biotechnol J. 9: 713-721.
Akbudak MA, Srivastava V (2011) Improved FLP recombinase, FLPe, efficiently removes
marker gene from transgene locus developed by Cre-lox mediated site-specific gene
integration in rice. Mol Biotechnol. 49: 82-89.
Keywords
Cereal transformation, transgene expression, site-specific recombination, FLP-FRT
13

Session 1 – Evolution and Natural Variation
S1.5- Functional analysis of WOX4 in mono- and dicotyledonous plants and
dissecting the evolution of vascular development
Stefanie Suer, Jasmin Bassler, Julia Riefler, Martina Schwarz, Thomas Greb
Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr.
Bohr-Gasse 3, 1030 Vienna, Austria
Stefanie.suer@gmi.oeaw.ac.at
Abstract
Most dicotyledonous plants have the capacity to initiate secondary growth, i.e., the
increase of the diameter of their shoots and roots by the activity of lateral meristems. The
most prominent lateral meristem is the vascular cambium, producing secondary vascular
tissues, thereby increasing long-distance transport capacities for nutrients and water, as
well as the stability of the plant body. Recently, the cambium-specific activity of the
homeobox-transcription factor WOX4 has been shown to be essential for cambium
activity in dicotyledonous species such as Arabidopsis and Populus.
In comparison, monocotyledonous plants do not undergo secondary growth.
Interestingly however, monocot genomes encode a transcription factor highly similar to
AtWOX4 raising the question as to the function of this factor in species without cambium
activity and the presence of meristematic potential of monocot stems.
In this study, we aim to elucidate the function of WOX4 in the monocot model
Brachypodium distachyon and draw connections between mono- and dicotyledonous
plants with regard to the regulation of vascular development. We show that BdWOX4 is
expressed in stem nodes and in vascular bundles of young Brachypodium plants.
Importantly, the BdWOX4 promoter recapitulates the activity of the AtWOX4 promoter
when introduced into Arabidopsis and moreover, BdWOX4 is able to rescue the
cambium defects of the Arabidopsis wox4 mutant. Taken together, our data
demonstrate that, in spite of large anatomical differences, monocots and dicots utilize
conserved molecular processes to regulate vascular developmental programs, and
suggest that the cambium-specific regulatory network has been adapted to control
different processes during the divergence of mono- and dicotyledonous species.
References
Alves SC, Worland B, Thole V, et al. (2009) A protocol for Agrobacterium-mediated
transformation of Brachypodium distachyon community standard line Bd21. Nat. Protoc.
4 (5): 638-649.
Hirakawa Y, Kondo Y, Fukuda H (2010) TDIF peptide signaling regulates vascular stem cell
proliferation via the WOX4 homeobox gene in Arabidopsis. Plant Cell 22: 2618-2629.
Ji J, Strable J, Shimizu R, et al. (2009) WOX4 promotes procambial development. Plant
Physiol. 152: 1346-1356.
Vogel J, Hill T (2008) High-efficiency Agrobacterium-mediated transformation of
Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep. 27: 471-478.
Keywords
Vascular development, WOX transcription factors
14

Session 1 – Evolution and Natural Variation
S1.6- Deciphering the function and evolution of BRX-like genes in
Brachypodium distachyon
David Pacheco-Villalobos, Martial Sankar & Christian S. Hardtke
Department of Plant Molecular Biology, University of Lausanne, Biophore Building, CH-
1015, Lausanne, Switwerland.
David.PachecoVillalobos@unil.ch
Abstract
The BREVIS RADIX gene (BRX) was first identified by map-based cloning in Arabidopsis
thaliana as a root growth QTL, for which both loss of function and hyperactive natural
variants exist (1, 2). Arabidopsis brx mutants have short primary roots, because BRX is
essential for root meristem growth in young seedlings (3). Although it is a transcriptional
co-regulator, BRX protein is primarily plasma membrane-associated, but can translocate
into the nucleus to regulate gene expression in an auxin-dependent manner to mediate
crosstalk between the auxin and brassinosteroid pathways (4, 5). BRX represented the first
member of a plant-specific gene family consisting of five paralogs in Arabidopsis (2). BRX
family genes have been also identified in poplar and Arabidopsis lyrata, as well as in
monocotyledons species, such as Brachypodium. Molecular and genetic analyses
revealed that dicotyledons BRX family genes display higher levels of functional
diversification than their monocotyledons counterparts (2). To examine their natural
genetic variation, Brachypodium BRX-like genes (BdBRXL-1 to 5) from 174 accessions
were sequenced in a pooled high throughput sequencing design and analysed to
determinate polymorphisms. A total of 79 variants (71 SNPs and 7 InDels) were found,
indicating high degree of conservation. Interestingly, a base substitution leading to a
STOP codon was found in BdBRXL2, the only BdBRXL gene which, if not mutated, could
not rescue the Arabidopsis brx phenotype. To further investigate the function of BRX-like
genes in monocotyledons we have generated Brachypodium transgenic lines carrying
various artificial microRNA (amiRNA) constructs. The amiRNAs were designed to
selectively knock-down the expression of each or several BRX-like genes (BdBRXL-1 to 5).
Primary root growth does not seem to be affected in the transgenic lines, possibly due to
functional redundancy or insufficient knock-down. However, a root system architecture
(branching) phenotype has been confirmed in certain lines and will be reported in detail.
References
1. Mouchel CF, Georgette B, Hardtke CS (2004) Natural genetic variation in Arabidopsis
identifies BREVIS RADIX, a novel regulator of cell proliferation and elongation in the root.
Genes and Development 18: 700-714.
2. Beuchat J, Li S, Ragni L, et al. (2010) A hyperactive quantitative trait locus allele of
Arabidopsis BRX contributes to natural variation in root growth vigor. Proccedings of the
National Academy of Sciences 107 : 8475-8480
3. Scacchi E, Salinas P, Gujas B, et al. (2010) Spatio-temporal sequence of crossregulatory
events in root meristem growth. Proccedings of the National Academy of
Sciences 107: 22734-22739.
4. Scacchi E, Osmont KS, Beuchat J, et al.. Dynamic, auxin-responsive plasma
membrane-to-nucleus movement of Arabidopsis BRX. Development 136: 2059-2067.
5. Mouchel CF, Osmont KS, Hardtke CS (2006) BRX mediates feedback between
brassinosteroid levels and auxin signalling in root growth. Nature 443: 458-461.
Keywords
Natural variation, root system architecture, amiRNA
15

Session 2 – Cell Wall, Biomass and Biofuels
S2.1- Brachypodium distachyon as a model plant for lignocellulosic ethanol
production
Till Meineke, Chithra Manisseri, Christian A. Voigt
Phytopathology & Biochemistry, Biocenter Klein Flottbek, University of Hamburg,
Ohnhorststr. 18, 22609 Hamburg, Germany
voigt@botanik.uni-hamburg.de
Abstract
The conversion of plant biomass to ethanol during fermentation is a strategy to mitigate
climate change by substituting fossil fuels, especially in transportation processes.
However, the conversion of biomass is mainly limited by the recalcitrant nature of the
plant cell wall. Apart from exploring cell wall degrading enzymes and pretreatment
methods for biomass, optimizing the plant cell wall itself for subsequent processing is a
promising approach. As an emerging model plant, B. distachyon is suitable to generate
cell wall mutants more easily than in designated biomass-delivering monocots. To test
putative B. distachyon cell wall mutants for altered ethanol production from biomass, we
wanted to know whether B. distachyon could be considered as a model plant in this
biotechnological application.
Therefore, we compared cell wall composition and ethanol production of leaf and stem
biomass from B. distachyon and the potential biomass-delivering monocots wheat,
maize, and Miscanthus x giganteus. Biomass was analyzed before and after different
steps of pretreatment and fermentation by pulsed amperometry (HPAEC-PAD) and
confocal laser-scanning microscopy. The accessible amount of glucose for fermentation
and the amount of produced ethanol was determined with refractive index detector in
a HPLC system. B. distachyon exhibited similar cell wall composition and fermentation
traits as the tested crops. Highest correlation in these data was not with the closelyrelated
wheat, but with maize and M. giganteus. Based on these results, we suggest B.
distachyon as model plant to study the impact of cell well alterations on lignocellulosic
ethanol production in energy crops.
Keywords
bio-ethanol, 2nd generation biofuels
16

Session 2 – Cell Wall, Biomass and Biofuels
S2.2- Disrupting the cinnamyl alcohol dehydrogenase 1 (BdCAD1) gene leads
to altered lignification and improved saccharification in Brachypodium
distachyon
Madeleine Bouvier d'Yvoire 1, Richard Sibout 1, Oumaya Bouchabke 1, Olivier Darracq 1,
Sebastien Antelme 1, Leonardo Gomez 2, Laurent Cezard 1, Catherine Lapierre 1, Lise
Jouanin 1.
1Institut Jean-Pierre Bourgin (IJPB), UMR1318 INRA-AgroParisTech, 78026 Versailles, FR
2Centre for Novel Agricultural Products, University of York, York Y010 5 YW, UK
Madeleine.Bouvier-Dyvoire@versailles.inra.fr
Abstract
Second generation biofuels using fermentable sugars from plant cell walls could provide
an answer to the augmentation of global energy needs with a limited impact on food
resources. As grasses show attractive features for this purpose, Brachypodium distachyon
was recently pointed as a valuable model plant for temperate grass species. Lignins are
major secondary cell wall polymers that derive from the polymerization of monolignols.
They confer rigidity and hydrophobicity to plant cell walls. However, they also constitute
an obstacle to most industrial processes targeting cellulose or hemicellulose, and they
need to be removed by costly, polluting and energy demanding processes. Plants with
altered lignification often display enhanced saccharification yields (enzymatic
conversion of cellulose into fermentable sugars).
In order to identify cell wall compositions more susceptible to saccharification, the
Sodium Azide and EMS mutageneized lines from the Versailles Brachypodium collection
were screened based on the colour of their stems. Indeed, orange, red or brown
vascular elements are often associated with altered lignification. The mutants so
identified, hereafter named « brown stem » (bs), were analyzed and several showed
improved saccharification. Among them, mutants in a CAD candidate gene (cinnamyl
alcohol dehydrogenase, involved in the last step of the monolignol biosynthesis
pathway) were found, that have the highest saccharification yields. These lines possess a
significative decrease in CAD activity and in their lignin contents. Their lignins are also
enriched in free phenolic groups, sinapaldehyde units and resistant bounds. Functional
complementation of Arabidopsis thaliana and Brachypodium cad mutants confirmed
the function of BdCAD1, and RNAi CAD lines are being selected in order to analyse the
effects of the silencing of several CAD genes.
References
Vanholme R, Moreel K, Ralph J, Boerjan W (2008) Lignin engineering. Current Opinion in
Plant Biology 11 :278-285
Marita JM, Vermerris W, Ralph J, Hatfield RD (2003) Variations in the cell wall composition
of Maize brown midrib mutants. Journal of Agricultural and Food Chemistry 51 : 1313-1321
Sibout R, Eudes A, Mouille G, et al. (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and
-D Are the Primary Genes Involved in Lignin Biosynthesis in the Floral Stem of Arabidopsis.
The Plant Cell 17 : 2059-2076
Youn B, Camacho R, Moinuddin SGA, et al. (2006) Crystal structures and catalytic
mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4.
Organic & Biomolecular Chemistry 4 : 1687-1697
Keywords
Brachypodium, lignin, cinnamyl alcohol dehydrogenase, saccharification
17

Session 2 – Cell Wall, Biomass and Biofuels
S2.3- Identification of Brachypodium mutants defective in silicon uptake
Emiko Murozuka, Pia Haugaard Nord-Larsen, Inge Skrumsager Møller, Thomas Paul Jahn
and Jan Kofod Schjoerring
Plant and Soil Science Laboratory, Department of Agriculture and Ecology, Faculty of Life
Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C
emikomu@life.ku.dk
Abstract
Silicon (Si) is a beneficial element for higher plants. The positive effect of Si is related to
the fact that Si increases the mechanical strength and rigidity of the cell walls, thereby
stimulating growth and contributing to the alleviation of biotic and abiotic stresses. On
the other hand, Si accumulation decreases the degradability of plant residues, which
may cause resistance to treatments for saccharification in cellulosic ethanol production
(Van Soest 2006). In addition, high content of Si in plant residues increases the amount of
ash during the combustion of biomass. There is accordingly a strong interest in reducing
Si accumulation in plant materials used for bioenergy purposes (Gressel 2008).
Si transport mechanisms have so far been studied in rice. Three Si transporters, Lsi1, Lsi2
and Lsi6 have been identified and characterized. Lsi1 and Lsi6 belong to aquaporins
while Lsi2 is a secondary active anion transporter (Ma and Yamaji 2008, Yamaji et al.,
2008). Lsi1 and Lsi2 are involved in uptake of Si, while Lsi6 functions in the distribution of Si
in the shoots. In our study, Brachypodium distachyon has been used to investigate the
mechanisms of uptake and distribution of Si in grasses. Phenotypic screening using
germanium (Ge) was carried out in a Brachypodium mutant population of about 1200
lines in order to identify mutants which were defective in Si uptake. After the screening,
about 30 lines were selected as candidates. So far one of the lines was found to be
defective in Lsi1. Characterization of the protein revealed that Brachypodium Lsi1
transports Si as well as other substances such as arsenite and boric acid, which
corresponds with the function of rice Lsi1. The mutant plant will be characterized after
backcrossing with the wild type focusing on degree of Si accumulation and the
incorporation of Si in the cell wall. Further screening and identification will be continued
in order to find more mutants in Si transporters.
References
Gressel J (2008) Transgenics are imperative for biofuel crops. Plant Sci 174: 246–263
Ma J.F, Yamaji N (2008) Functions and transport of silicon in plants. Cell Mol Life Sci 65:
3049-3057
Van Soest P.J (2006) Rice straw, the role of silica and treatments to improve quality. Anim.
Feed Sci. Tech. 130: 137–171
Yamaji N, Mitatni N, Ma J.F (2008) A transporter regulating silicon distribution in rice
shoots. Plant Cell 20:1381-1389
Keywords
silicon transporter, aquaporin, cell wall
18

Session 2 – Cell Wall, Biomass and Biofuels
KEYNOTE LECTURE
S2.4- Natural diversity from genomics to phenomics
John P. Vogel
USDA-ARS Western Regional Research Center, Albany, CA, 94710 USA
john.vogel@ars.usda.gov
Abstract
Natural variation in genomic sequence and the resultant phenotypic variation are
valuable tools for determining gene function. To develop resources to allow researchers
to use natural variation as a research tool my laboratory together with an international
group of collaborators has initiated several projects focused on natural diversity in
Brachypodium distachyon. To provide a genomic foundation for these studies we are
resequencing 56 natural accessions through the US Department of Energy Joint Genome
Institute. Analysis of the first six accessions is nearly complete and shows tremendous
diversity with SNP frequencies ranging from 200-600 base pairs per SNP. To survey the
phenotypic diversity in a large collection of natural accessions we are collaborating with
the High Resolution Plant Phenomics Facility in Canberra, Australia. In addition, we are
creating homozygous T-DNA lines for phenotypic analysis. Another area of interest is the
use of the related, but perennial, species B. sylvaticum as a tool to understand the
genetic basis of perenniality. Initial steps in this direction include the development of an
efficient transformation system and surveying genetic diversity in a small collection of B.
sylvaticum accessions.
Keywords
natural diversity, resequencing, phenotype, Brachypodium sylvaticum, transformation
19

Session 2 – Cell Wall, Biomass and Biofuels
S2.5- Functional characterization of candidate cell wall genes in B. distachyon
Pubudu Handakumbura, Gina M. Trabucco, Michael V. Veling, Dominick A. Matos, Karen
S. Osmont, Scott J. Lee, Samuel P. Hazen
Biology Department, University of Massachusetts, 221 Morrill Science Center III, Amherst,
MA, 01003, USA
hazen@bio.umass.edu
Abstract
The cell wall is a complex composite of polysaccharides, proteins, and lignin, with lignin
and cellulose representing two of the most abundant bio-organic compounds on Earth.
Much of what is currently understood about the transcriptional regulation of cell wall
biosynthesis is from the study of Arabidopsis thaliana xylem vessels and fibers, yet this
understanding may not be generalizable across land plants. The cell walls of grasses,
including domesticated cereals that provide the majority of human calories and the
perennials under development as biofuel energy crops, differ significantly in morphology
and composition from the eudicot A. thaliana. We are taking a reverse genetics
approach to understand the transcriptional regulation of secondary cell wall biosynthesis
using the grass model system Brachypodium distachyon. In order to confirm transcription
factor targets, we have silenced and confirmed candidate cellulose and lignin
associated genes using artificial microRNAs. Using our well establish yeast one-hybrid
assay, we are testing for activity between cis-regulatory regions of those genes and a
collection of transcription factor proteins. In doing so, we seek to resolve the regulatory
networks leading to cellulose and lignin synthesis.
Keywords
Transcription factor, secondary cell wall, gene expression
20

Session 2 – Cell Wall, Biomass and Biofuels
S2.6- Cell wall proteomics of Brachypodium distachyon
Elisabeth JAMET, Thibaut DOUCHE, David ROUJOL, Hélène San Clemente, Benoît VALOT,
Michel ZIVY, Rafael PONT-LEZICA
LRSV; UMR5546 UPS/CNRS; BP42617, F-31326 Castanet Tolosan, France
PAPPSO; UMR0320/UMR8120 INRA/CNRS/Université Paris X Génétique végétale; F-91190
Gif sur Yvette
jamet@lrsv.ups-tlse.fr
Abstract
Plant biomass is one of the greatest untapped reserves on the planet and is mostly
composed of cell walls. Energy-rich polysaccharides make up about 75% of plant cell
walls. These polymers can be broken down to produce sugar substrates through the
saccharification process. A whole range of products can be obtained from these sugars
including bioethanol. However, the complex structure of cell walls, consisting in a
network of various polysaccharides and glycoproteins, encrusted by phenolic polymers
in secondary walls, makes them very resistant to degradation. Improving the ease and
yield of saccharification represents the major technological hurdle that must be
overcome before the full vision of the plant-fuelled biorefinery can be realized.
Brachypodium distachyon has many qualities that make it a model for study of
dedicated bioethanol crops such as Schwitchgrass or Miscanthus. Since the full
sequencing of its genome in 2008, it is possible to perform accurate transcriptomics and
proteomics studies. The aim of this project (ANR KBBE CELLWALL) was to identify cell wall
proteins playing roles in cell wall extension and in secondary wall formation. Recent cell
wall proteomics studies mainly performed in Arabidopsis thaliana led to the identification
of about 500, i.e. approximately one fourth of the expected, cell wall proteins falling into
nine functional classes (Jamet et al. 2008; San Clemente et al. 2009). With regard to the
objective of the project, two classes were of special interest, i.e. proteins acting on
polysaccharides and oxido-reductases. The present work focused on cell wall
transcriptomics and proteomics of growing vs mature leaves and internodes. About 360
cell wall proteins could be identified by mass spectrometry and bioinformatics among
which some were more abundant in young or mature organs. Relative quantification
could be done for one third of them, thus allowing to propose candidates of interest for
further functional studies.
References
ANR KBBE CELLWALL (2009-2012). Coordinator: Persson S (MPI Golm, Germany). Partner 3:
Jamet E (LRSV, Toulouse, France).
Feiz L, Irshad M, Pont-Lezica R, et al. (2006) Evaluation of cell wall preparations for
proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls. Plant
Methods 2: 10.
Jamet E, Albenne C, Boudart G, et al. (2008) Recent advances in plant cell wall
proteomics. Proteomics 8: 893-908.
San Clemente H, Pont-Lezica R, Jamet E (2009) Bioinformatics as a tool for assessing the
quality of sub-cellular proteomic strategies and inferring functions of proteins : plant cell
wall proteomics as a test case. Bioinform Biol Insights 3 : 15-28
Keywords
biomass, cell wall, mass spectrometry, proteomics, quantification
21

Session 2 – Cell Wall, Biomass and Biofuels
S2.7- The role of pectins in Brachypodium development
Jozef Mravec, Oumaya Bouchabké-Coussa, Yves Chupeau, Grégory Mouille, Richard
Sibout, Herman Höfte
Institut Jean-Pierre Bourgin (IJPB), INRA, route de Saint Cyr, 78026 Versailles, France
jmravec@versailles.inra.fr
Abstract
One of the most remarkable differences between the primary cell walls of dicots (type I)
and commelinoid monocots (type II) is the abundance of pectin polymers. Type II cell
walls are generally considered to be poor in pectin content. The biological relevance of
this divergence has never been elaborated. We took the advantage of Brachypodium
distachyon as an emerging plant model to study the synthesis, distribution and the
downstream modification of pectins in Poales. We generated and phenotyped the RNAi
mutant in BdQUA1 (Bradi3g43810) -- the closest ortholog of Arabidopsis QUASIMODO1
gene which encodes for a glycosyltransferase involved in pectin synthesis. We also
determined the expresion of BdQUA1 by in situ hybridization and by a promoter-GUS
construct. The spatial distribution of various pectins in the root meristem was mapped by
immunolocalizations using various pectin-specific antibodies. Our results suggest, that like
in dicots, pectins play an irreplaceable role in grass development and that the pectin
metabolism, especially in the meristematic tissues, is tightly regulated. Currently we also
study the role of pectins in the secondary cell wall deposition and the pectin influence
on the saccharification yield.
References
Keywords
monocot, cell wall, pectins, development, QUASIMODO 1
22

Session 2 – Cell Wall, Biomass and Biofuels
S2.8- Investigating biomass digestibility in Brachypodium distachyon
Poppy E. Marriott, Leonardo D. Gomez, Caragh Whitehead and Simon J. McQueen-
Mason
CNAP, Department of Biology, University of York, York. YO10 5DD. UK
pm518@york.ac.uk
Abstract
Lignocellulosic biomass is largely composed of polysaccharides which can be broken
down to produce sugars for the production of ethanol through fermentation. This
bioethanol could provide a sustainable replacement for fossil fuel derived transportation
fuels. However, lignocellulose is extremely resistant to digestion and converting it to
fermentable sugars requires energetic pretreatment and expensive enzyme applications.
To make second generation bioethanol a commercial reality we therefore need to
improve the conversion efficiency. One way to achieve this is by producing plants with
more digestible lignocellulose. This project involves a forward genetic screen with the
model grass, Brachypodium distachyon, to identify plants with an alteration in digestibility
from large, randomly mutated populations. Such a large scale screen requires a high
throughput approach and at the University of York an analytical platform has been
developed that can perform saccharification analysis in a 96-well plate format. The
system can reliably detect differences in the saccharification of plant tissues and is able
to rapidly process large numbers of samples with a minimum amount of human
intervention.
Two chemically mutagenised populations were screened (from INRA and the USDA) and
a relatively large amount of variation in sugar release was identified (up to +70% and -
50% compared to WT). Mutants with a significant difference in sugar release in
comparison to WT were isolated and saccharification of the offspring was determined to
test for heritability of the trait. Plants with heritable mutations will be taken forward for in
depth cell wall analysis to understand the effect of the mutation on the structure and
digestibility of the cell wall. Mapping of the mutations will also be undertaken in order to
identify genes involved in the phenotypic variation. This will provide novel genetic
markers to aid in selection of the best genotypes for industrial production of bioethanol.
References
Gomez LD, Whitehead C, Barakate A, et al. (2010) Automated saccharification assay for
determination of digestibility in plant materials. Biotechnology for Biofuels 3: 23.
Gomez LD, Bristow JK, Statham ER, McQueen-Mason SJ (2008) Analysis of
saccharification in Brachypodium distachyon stems under mild conditions of hydrolysis.
Biotechnology for Biofuels 178: 61-72.
Abramson M, Shoseyov O, Shani Z (2010) Plant cell wall reconstruction toward improved
lignocellulosic production and processability. Plant Science 178: 61-72
Garvin DF, Gu YQ, Hasterok R, et al. (2008) Development of genetic and genomic
research resources for Brachypodium distachyon, a new model system for grass crop
research. Crop Science 48: S69-S84
Keywords
Brachypodium, Biofuels, lignocellulose, saccharification, genetic screen
23

Session 3 – Tools and Bioengineering
KEYNOTE LECTURE
S3.1- T-DNA mutagenesis in Brachypodium distachyon
Vera Thole and Philippe Vain
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich NR4
7UH, United Kingdom.
philippe.vain@jic.ac.uk
Abstract
Brachypodium distachyon is emerging as a new experimental and genomics model for
temperate cereal crops and biomass grasses. Recently, the International Brachypodium
Tagging Consortium (IBTC), which includes eight laboratories from five countries, has
been established to scale-up the production and characterisation of T-DNA mutants. The
BrachyTAG program is part of the IBTC and has generated a collection of 5,000 fertile
plant lines (genotype Bd21) transformed with the binary vector pVec8-GFP.
Characterisation of an initial population of 741 plant lines (Thole et al., 2010) has shown
that analysing the regions flanking both borders of the T-DNA inserts nearly doubled the
recovery of Brachypodium flanking sequence tags (FSTs) available to describe the
different T-DNA insertions. Overall, more than 2,200 flanking regions were identified with
44% corresponding to Brachypodium FSTs. Approximately 50% of the T-DNA insertions
interrupted a predicted gene in the Bd21 annotated genome sequence. The distribution
and density of T-DNA inserts will be presented as well as examples of T-DNA mutant
genotyping, phenotyping and complementation. Novel binary vectors (pBrachyTAG)
have also been developed to improve T-DNA tagging and trapping in Brachypodium.
The T-DNA lines generated by the BrachyTAG program are available as a community
resource and have been distributed internationally since 2008 via the BrachyTAG.org
website.
References
Thole V, Worland B, Wright J, Bevan MW, Vain P (2010) Distribution and characterization
of more than 1000 T-DNA tags in the genome of Brachypodium distachyon community
standard line Bd21. Plant Biotechnology Journal 8:734–747.
Keywords
mutagenesis, T-DNA tagging, reverse genetics, functional genomics, flanking sequence
tags (FST).
24

Session 3 – Tools and Bioengineering
S3.2- Current status and future perspectives for molecular cytogenetic studies
of Brachypodium
Dominika Idziak, Elzbieta Wolny, Karolina Lesniewska, Alexander Betekhtin, Natalia
Borowska, Ewa Breda, Maja Jankowska, Robert Hasterok
Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental
Protection, University of Silesia, Jagiellonska 28 St, 40‑032 Katowice, Poland
didziak@us.edu.pl
Abstract
Molecular cytogenetics contributes to better understanding of the structure, function
and evolution of genomes by bridging the DNA sequence data and information about
chromosome biology at the cellular, tissue and organismal level. The key method of
modern molecular cytogenetics is fluorescence in situ hybridisation (FISH) which allows
direct microscopic observation of the physical position and abundance of a
fluorescently labelled DNA sequence in mitotic and meiotic chromosomes, interphase
nuclei or DNA fibers. The primary uses of FISH in plant biology studies are identification of
chromosomes or chromosome segments and physical mapping of plant genomes, for
structural genomics and phylogenetic analyses. In B. distachyon sequencing project FISH
using BAC clones from Bd21 genomic libraries provided a valuable complementary
approach in ordering genome sequence assemblies and detecting false joints in the
sequence scaffolds as well as aligning the sequence assemblies integrated with physical
and genetic maps to individual B. distachyon chromosomes (Febrer et al., 2010; IBI,
2010). In the presented work we summarise research projects currently carried out in our
laboratory that are based on molecular cytogenetics methods. The on‑going
investigation comprises studies of karyotype evolution using chromosome painting and
single-BAC FISH, analysis of nucleus architecture and studies of chromosome behaviour
during meiosis. FISH coupled with immunoassays is used for cytological analysis of
chromatin features which affect gene expression.
References
Febrer M, Goicoechea J L, Wright J, et al. (2010) An integrated physical, genetic and
cytogenetic map of Brachypodium distachyon, a model system for grass research.
PloS ONE 5: e13461.
International Brachypodium Initiative (2010) Genome Sequencing and Analysis of the
model grass Brachypodium distachyon. Nature 463: 763–768.
Keywords
molecular cytogenetics, physical mapping, chromosome biology
25

Session 3 – Tools and Bioengineering
S3.3- Functional annotation of a full-length Brachypodium cDNA resource
Keiichi Mochida, Yukiko Uehara, Fuminori Takahashi, Takuhiro Yoshida, Tetsuya Sakurai,
Kazuo Shinozaki
RIKEN BMEP/PSC
mochida@psc.riken.jp
Abstract
Full-length cDNA libraries and large-scale sequence data sets of clones are invaluable
resources for life science projects concerning the study of various species [1-3].
Collection of full-length cDNA is an essential genomic resource for the correct
annotation of genomic sequences as well as for the functional analysis of genes and
their products. We constructed a mixed full-length cDNA library of various tissues of
Brachypodium distachyon Bd21 strain by using the biotinylated CAP trapper method [4].
Then, we obtained 79,097 expressed sequence tags (ESTs), which were sequenced from
both ends. We mapped the ESTs to the B. distachyon genome sequence together with
the available gene annotation. As a result of this mapping, we allocated 66,140 ESTs of
33,070 clones to the representative 8,461 genomic positions of the currently annotated
genes. We also allocated 3,018 ESTs of 1,509 clones to the 670 non-redundant genomic
positions without any annotated genes. Furthermore, through this mapping analysis, we
also identified transcripts putatively derived from the trans-splicing event in
Brachypodium. To update the orthologous ORFeome resources in Pooideae, we
performed comparative analyses of the full-length cDNA dataset of Brachypodium and
those of wheat and barley. To integrate our full-length cDNA information with other
related significant information resources, we constructed an integrated database, RIKEN
BrahcyFLcDNA DB, which is an information gateway to access the resource. The
database provides full-length cDNA-based gene structural annotation, predicted gene
functions, and information on corresponding putative orthologs in grass species for
comparative analysis. Therefore, this full-length cDNA resource should be beneficial in
accelerating the discovery and functional analysis of genes in Brachypodium.
References
1. Seki M, Narusaka M, Kamiya A, Ishida J, Satou M, et al. (2002) Functional annotation of
a full-length Arabidopsis cDNA collection. Science 296: 141-145.
2. Seki M, Shinozaki K (2009) Functional genomics using RIKEN Arabidopsis thaliana fulllength
cDNAs. J Plant Res. 122: 355-366
3. Mochida K, Shinozaki K (2010) Genomics and bioinformatics resources for crop
improvement. Plant Cell Physiol 51: 497-523.
4. Carninci P, Kvam C, Kitamura A, Ohsumi T, Okazaki Y, et al. (1996) High-efficiency fulllength
cDNA cloning by biotinylated CAP trapper. Genomics 37: 327-336.
Keywords
Full-length cDNA, database, gene annotation
26

Session 3 – Tools and Bioengineering
S3.4- BradiNet: co-expression platform and comparative transcriptomics for
Brachypodium
Marek Mutwil 1, Richard Sibout 2, Herman Höfte 2 and Staffan Persson 1
1 Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam –
Golm, Germany
2 Institut Jean-Pierre Bourgin, UMR1318-INRA-AgroParisTech, Centre de Versailles-Grignon,
Route de St-Cyr, 78026 Versailles Cedex-France
mutwil@mpimp-golm.mpg.de
Abstract
It is well documented that transcriptionally coordinated genes tend to be functionally
related and that such relationships may be conserved across different species and even
kingdoms. To exploit such relationships, we have performed microarray analysis of all
major tissues during different developmental stages of Brachypodium distachyon. The
data was used to create co-expression network for Brachypodium and included into
PlaNet database (http://aranet.mpimp-golm.mpg.de/bradinet). One major problem
with transferring knowledge from a model organism is that since plants harbor large gene
families, standard phylogenetic methods might not be sufficient to identify functional
homologs. To remedy this, we implemented a comparative network algorithm that
estimates similarities between network structures. Thus, the platform can be used to swiftly
infer similar coexpressed network vicinities within and across species and can predict the
identity of functional homologs. We exemplify this with comparative analysis of
Brachypodium cellulose synthase gene networks to find corresponding functional
homologs between Arabidopsis, Brachypodium, Medicago, poplar, rice, soybean and
wheat. The data support the contention that this platform will considerably improve
transfer of knowledge between model organisms Arabidopsis and Brachypodium and
crop species such as rice and wheat.
27

Session 3 – Tools and Bioengineering
S3.5- Phenotyping the shoot and root diversity of Brachypodium distachyon to
accelerate plant biofuel breeding
1Richard Poiré, 2Vincent Chochois, 1Solène Callarec, 1Xavier Sirault, 3John Vogel,
2Michelle Watt, 1Robert Furbank
1High Resolution Plant Phenomics Centre, CSIRO Plant Industry, Canberra, ACT 2601,
Australia
2CSIRO Plant Industry, Canberra, ACT 2601, Australia
3USDA, ARS, WRRC Albany, California 94710, USA
richard.poire@csiro.au
Abstract
The High Resolution Plant Phenomics Centre (HRPPC) is the Canberra node of the
Australian Plant Phenomics Facility. The HRPPC focuses on deep phenotyping and
reverse phenomics through development of next generation tools to measure
performance of plants ranging from model species to major crops.
A major aim of the HRPPC model plant module is to find genes of agricultural
importance using high resolution phenotyping of model plant species. One such project
is an international collaboration using the model species Brachypodium distachyon to
speed up the breeding of next generation biofuel crops and discover genes responsible
for important traits in wheat. Brachypodium, unlike switchgrass or miscanthus, has all the
attributes of a model species (small and fully sequenced genome, self-fertile, short life
cycle, easily transformed and crossed) allowing high throughput screening and
characterisation of traits relevant to biofuel research.
We have developed a variety of non-invasive and destructive assays for growth,
biomass, photosynthesis and root growth and architecture in Brachypodium. Large
phenotypic variability of biomass accumulation was observed in a set of 160 natural
accessions of Brachypodium [1]. Root growth and architecture was characterised in
these ecotypes and show significant variation in total root length, root type distribution
(nodal versus seminal root systems) or shoot/root ratio.
Contrasting ecotypes are currently under investigation using high resolution imaging
analysis techniques to identify the optimal combination of these phenotypic traits under
limiting water and nutrients and to determine the underlying genomic regions
responsible. Since Brachypodium is a typical grass and shares a high degree of genetic
similarity and markers with commercial crops, it would be possible to extend the
knowledge gained from this project to improve plants species with a much more
complex and large genome such as switchgrass and wheat.
References
1. Vogel J, Tuna M, Budak H, et al. (2009) Development of SSR markers and analysis of
diversity in Turkish populations of Brachypodium distachyon. BMC Plant Biology 9: 88.
Keywords
Phenotyping, shoot, root architecture, biofuel, diversity
28

Session 3 – Tools and Bioengineering
S3.6- TILLING in Brachypodium distachyon: development of a Sodium Azide
mutant collection and screening for induced point mutations
Dalmais M. 1, Darracq O. 2, Oria N. 2, Antelme S. 2, Troadec C. 1, Bouvier d’Yvoire M. 2,
Jouanin 2, Lapierre C. 2 Höfte H. 2, Sibout R. 2, Bendahmane A. 1
1 Unité de recherche en génomique végétale (URGV), 2 rue Gaston Crémieux, 91057
Evry FR
2 Institut Jean-Pierre Bourgin (IJPB), UMR1318 INRA-AgroParisTech, 78026 Versailles, FR
dalmais@evry.inra.fr
Abstract
Brachypodium distachyon is an established model plant for grasses. With the completion
of the genome sequencing project in 2010, a major challenge is to determine gene
functions. In plants, the most common techniques to produce altered or loss of function
mutations are T-DNA or transposon insertional mutagenesis and RNA interference.
However, unless a highthroughput transformation protocols become available for
Brachypodium, functional analysis of Brachypodium genes with the tagging approaches
remain expensive and time consuming. On the other hand, chemical mutagenesis is a
straightforward and cost-effective way to saturate a genome with mutations. TILLING
(Targeting Induced Local Lesions IN Genomes) uses chemical mutagenesis coupled with
gene-specific detection of single-nucleotide mutations. This strategy generates allelic
series of the targeted genes which makes it possible to dissect the function of the protein
as well as to investigate the role of essential genes that are otherwise not likely to be
recovered in genetic screens based on insertional mutagenesis. To investigate the
capacity of TILLING as a powerful tool of reverse genetics in Brachypodium we have set
up a TILLING platform and performed a screen for mutations in genes from the lignin
biosynthesis pathway. First we have constructed a reference sodium azide mutant
population of 5530 M2 families in Bd21-3 genetic background and developed a
database, UTILLdb (http://urgv.evry.inra.fr/UTILLdb), that contains phenotypic as well as
sequence information on mutant genes. UTILLdb can be searched online for TILLING
alleles, through the BLAST tool, or for phenotypic information about mutants by keywords.
To setup the TILLING platform, genomic DNA was prepared from the mutant lines and
organized in pools for bulked screening using the mismatch specific endonuclease,
ENDO1. To assess the quality of Brachypodium mutant collection and to estimate the
mutation density, we screened for induced mutations in 6 genes related lignin
biosynthesis pathway. In total, we identified and confirmed by sequencing 92 induced
mutations. We also estimated the overall mutation rate as one mutation every 525 kb in
our Bd21-3 mutant collection. This mutation frequency is similar to the rate of one
mutation per 500 Kb reported both for sorghum and barley (respectively Xin et al., 2008
and Gottwald et al., 2009). A much more saturated mutation density has been observed
in polyploid grasses such as tetraploid wheat, with one mutation per 40 kb, and
hexaploid wheat with one mutation per 24 kb, which withstand much higher doses of
EMS without obvious impact on plant survival. In all together, we routinely identify about
15 alleles per tilled genes which are sufficient to genetically characterize the function of
a gene using the TILLING approach. Several phenotypes will be displayed in this
presentation.
29

Session 3 – Tools and Bioengineering
S3.7- Novel tools for Brachypodium research: Genetic transformation using
shoot segments and immature pollen-derived generation of instantly
homozygous lines
Bianca Melzer, Tilo Guse, Christine Kastner, Katarzyna Plasun, Carolin Berger, Eszter
Kapusi, Götz Hensel, Cornelia Marthe, Petra Hoffmeister and Jochen Kumlehn
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Plant
Reproductive Biology, Corrensstraße 3, 06466 Gatersleben, Germany
kumlehn@ipk-gatersleben.de
Abstract
Brachypodium distachyon is a valuable experimental model species for the
economically important temperate cereals of the tribe Triticeae. To comprehensively
validate gene functions, a viable transformation system is required. Current
transformation methods rest on the use of immature embryos or immature embryoderived
callus as gene transfer recipient tissues. However, the tiny immature embryos are
difficult to dissect in large scale and callus propagated over longer periods of time is
anticipated to undergo significant variation of the genetic and epigenetic background.
In this study, we have demonstrated that efficient plant regeneration can be achieved
from shoot segments of B. distachyon seedlings. A major advantage of this method is
that the explants can be readily produced in large scale from mature grains, i.e. there is
no routine cultivation of donor plants required. Taking advantage of this regeneration
method, callus derived from shoot segments was inoculated using hypervirulent
Agrobacterium carrying the hpt gene and the gfp and gus-intron genes as plant
selectable marker and reporter genes, respectively. This approach resulted in more than
a hundred independent primary transgenic lines. In these plants and their progeny,
reporter gene expression was observed and stable integration of T-DNAs confirmed by
Southern blot, which provided evidence of generative transmission of the transgenes. In
a second approach, a method of immature pollen-derived embryogenesis and plant
regeneration was established in B. distachyon. Since pollen constitutes the haploid
outcome of meiotic recombination and pollen embryogenesis frequently includes
identical genome duplication, this method may facilitate the production of entirely
homozygous mutant or transgenic lines in just one step, which is especially useful when
multiple transgenes or insertion-derived mutations have to be combined.
Keywords
doubled haploid, embryogenesis, homozygous, pollen, transgenic
30

Session 4 – Biotic and Abiotic Stresses
KEYNOTE LECTURE
S4.1- Exploiting Brachypodium distachyon biodiversity to establish sources of
resistance to abiotic and biotic stress.
Luis A. J. Mur 1, Joel Allaingullianme 1, Jessica Gough 1, Rasim Uman 1, Ianto Thomas 1, J.
William Allwood 1, Joel V. Smith, 1 Greg Shelley 1, Andrew P. M. Routledge 1, Adina
Breiman 2, Elena Benavente 3 and Pilar Calatan 4.
1 Institute of Biological, Environmental and Rural Science, Penglais Campus, Aberystwyth University
2 Dept of Plant Sciences Tel Aviv University, Tel Aviv Israel 69978
3 Department of Biotechnology, Polytechnic University of Madrid, 28040-Madrid, Spain
4 Department of Agriculture (Botany), High Polytechnic School of Huesca, University of Zaragoza, Ctra. Cuarte km 1, 22071 Huesca,
Spain.
lum@aber.ac.uk
Abstract
Brachypodium distachyon is an undomesticated model grass species which offers the
possibly of elucidating the genetic bases of multigenic traits associated with abiotic stress
tolerance and durable disease resistance (Mur et al. 2011). To exploit this potential we have
established collections of Brachypodium distachyon and Brachypodium hybridum (formerly
the 2n =30 cytotype of B. distachyon; see presentation by Catalan et al.) germplasm from
Northern Spain and also via, collaboration, incorporated collections from Southern Spain,
Morocco, Israel as well as accessions from Italy, France and Bulgaria. Genetic diversity within
Brachypodium germplasm was assessed through polymorphism at microsatellite loci.
Accession genetic diversity was related to geographical origin with distinct polymorphisms
between Western and Eastern European accession and also between Brachypodium
species. Biochemical variation amongst the accessions was investigated by metabolite
fingerprinting using Fourier Transform Infra-Red (FT-IR) spectroscopy. Using the supervised
multivariate approach – Discriminant Function Analysis (DFA) - variation in metabolites in the
first emerged leaf of Brachypodium genotypes could be linked to geographical origin.
Our assessments of drought tolerance focused on inbred lines developed from our collection
and also the ABR1 through 7 series, USDA lines Bd21, Bd2-3, Bd3-1 as well as from the Turkish
collection established by Vogel et al. (2009). A total of 118 Brachypodium genotypes have
been assessed for drought tolerance and, based on estimates of relative water content and
cytoplasmic membrane stability, Bd2-3 proved to be most drought susceptible and ABR5 the
most tolerant. Metabolomic approaches were employed to further investigate the basis of
the variation in tolerance, based on electrospray ionisation Mass Spectrometry (ESI-MS) of
polar and non-polar extracts from drought and non-droughted plants. Multivariate data
mining indicated that polyamine, arginine and antioxidant metabolites were all elevated in
ABR5 and not Bd2-3. We are now seeking to develop mapping populations to deduce
genetic traits linked to drought tolerance.
Our work on responses to pathogens has focused on responses to Magnaporthe grisea - the
causal agent of rice blast - and rust pathogens, primarily crown rust (Puccina coronata) and
the emerging grassland pathogen, Dreschlera spp. Leafspot in the ABR1 through 7
genotypes. Events associated with local and systemic resistance to pathogens have been
elucidated. Resistance to pathogens was correlated with an elevation in jasmonic acid but
not salicylic acid. Inhibition of jasmonic acid biosynthesis using ursolic acid resulted in
reduced local and systemic resistance. Lipoxygenase (LOX) is a key biosynthetic enzyme in
the jasmonate biosynthetic pathway. Two LOX Bd21 T-DNA mutants are available within the
JIC (UK) T-DNA population (BdAAA466, BdAAA615) and both exhibited reduced jasmonate
accumulation on infection and compromised resistance to rice blast. These data suggest the
importance of jasmonate signalling in Brachypodium and most likely, temperate cereals.
References
Mur LA, Allainguillaume J, Catalán P, et al. (2011) Exploiting the Brachypodium Tool Box in cereal and grass research. New Phytol. 2011
91, 334-47.
Vogel JP, Tuna M, Budak H, et al. (2009). Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium
distachyon. BMC Plant Biology: 13:9:88
31

Session 4 – Biotic and Abiotic Stresses
S4.2- Assessing the role of smallRNAs in leaf cell identity and growth
reprogramming during drought stress in Brachypodium distachyon
Mica, Erica 1 ; Bertolini, Edoardo 1,2; Verelst, Wim 2; Piccolo, Viviana 3; Inzé, Dirk 2; Horner,
David 3 ; Pè, Mario Enrico 1
1 Scuola Superiore Sant’Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy
2 Department of Molecular Genetics, Ghent University, Technologiepark 927, 9052 Ghent,
Belgium
3 Department of Biomolecular Sciences and Biotechnology, University of Milan, via
Celoria 26, 20133 Milan, Italy
erica.mica@sssup.it
Abstract
Food production is limited primarily by environmental stresses, of which drought is one of
the most common. Water deficiency in fact affects both growth and development of
crop plants through alterations in metabolism and gene expression.
As a drought tolerant grass, Brachypodium may possess specific adaptations and
tolerance mechanisms whose characterization could be useful to address drought
tolerance in related crops, such as wheat and barley.
The drought response is a complex trait regulated by a plethora of genetic and
epigenetic networks. Here we address the role of smallRNA molecules, in particular
miRNAs, in fine tuning gene expression in developing leaves under drought stress.
In our soil-based assay, we subjected the reference accession Bd21 to growth-limiting,
non-lethal drought stress. In this condition, we observed that drought stress has no effect
on cell division rates but affects cell expansion resulting in a reduced leaf size. From two
biological replicates we produced four smallRNA libraries obtained from expanding and
proliferating leaf zones subjected either to drought stress or control conditions. Using nextgeneration
sequencing techniques the libraries were sequenced, producing nearly 30
million reads. The bioinformatics pipeline that we developed identified conserved and
species-specific miRNAs, some of which were detected for the first time in
Brachypodium. Statistical analyses were applied to detect miRNAs whose expression is
modulated under stress or in different developmental conditions. Our data suggest that
a higher proportion of miRNAs are involved in developmental programming, while only
few miRNAs are regulated under drought stress in Brachypodium.
We are currently analyzing putative miRNAs targets investigating their possible
involvement in leaf development both in normal and stressed conditions. Combining
miRNAs and miRNA target genes analyses we expect to obtain a more complete picture
of the biological function of these molecules.
Keywords
miRNA, smallRNA, drought, growth-reprogramming, leaf
32

Session 4 – Biotic and Abiotic Stresses
S4.3- Use of the pathosystem Brachypodium distachyon / Fusarium
graminearum to investigate the detoxification mechanisms of mycotoxins by
plants.
Jean-Claude Pasquet, Catherine Macadré, Xavier Deguercy, Patrick Saindrenan and
Marie Dufresne
Institut de Biologie des Plantes, Université Paris-Sud 11, 91405 Orsay, France
marie.dufresne@u-psud.fr
Abstract
Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is one of the
most damaging diseases on small-grain cereals, including wheat (Goswami and Kistler,
2004). During its development in the host plant, this pathogen produces trichothecene
mycotoxins (including deoxynivalenol or DON) that inhibit eukaryotic protein translation,
are harmful to humans, animals (Rocha et al. 2005) and also phytotoxic (Desmond et al.
2008). No gene-for-gene resistance has been reported towards F. graminearum but
quantitative trait loci (QTL) were identified in bread wheat. Genetic, metabolic or
transcriptional analyses have correlated some of these QTLs with functions involved in
DON detoxification, including UDP-glycosyltransferases (UGTs). Our team recently
developed a new pathosystem, the intereaction between the small cereal species
Brachypodium distachyon and F. graminearum, and uses it to study the mechanisms of
DON detoxification. Based on phylogenetic relationships with UGTs identified in
Arabidopsis (Poppenberger et al. 2003) or barley (Gardiner et al. 2010), we have
identified four candidate UGTs from B. distachyon potentially involved in this process and
first investigated how DON or infection by F. graminearum can regulate the expression of
the corresponding genes, using quantitative RT-PCR analyses. We have shown that the
expression of all four candidate genes is strongly induced between 72 and 96 hours after
inoculation by F. graminearum and as soon as 3 hours after application of DON. By
monitoring the expression of the Tri5 gene (gene encoding the first enzyme of the
trichothecene biosynthetic pathway, Foroud and Eudes, 2009), we have further
demonstrated that DON is likely produced at stages where the candidate genes are
induced. Functional analyses of these genes are underway in B. distachyon to decipher
more precisely their involvement in DON detoxification and in resistance towards FHB.
References
Foroud NA, Eudes F (2009) Trichothecenes in cereals grains. International Journal of
Molecular Sciences 10: 147-173
Gardiner SA, Boddu J, Berthiller F, et al. (2010) Transcriptome analysis of the barleydeoxynivalenol
interaction: evidence for a role of glutathione in deoxynivalenol
detoxification. Molecular Plant-Microbe Interactions 23: 962-976
Goswami RS, Kistler HC (2004) Heading for disaster: Fusarium graminearum on cereal
crops. Molecular Plant Pathology 5: 515-525
Poppenberger B, Berthiller F, Lucyshyn D, et al. (2003) Detoxification of the Fusarium
mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana. The
Journal of Biological Chemistry 48: 47905-47914
Rocha O, Ansari K, Dooham FM (2005) Effects of trichothecens mycotoxins on eukaryotic
cells: A review. Food additives and contaminants 22: 369-378
Keywords
Fusarium graminearum, deoxynivalenol, detoxification, UDP-glycosyltransferases
33

Session 4 – Biotic and Abiotic Stresses
S4.4- Functional characterization of Brachypodium UDP-glucosyltransferases
1Wolfgang Schweiger, 1Maria Paula Kovalski, 1Juan Antonio Torres Acosta, 2Marc
Lemmens, 3Hikmet Budak, 3Franz Berthiller, 4Thomas Nussbaumer, 4Klaus Mayer, 1Gerhard
Adam
1 Department of Applied Genetics and Cell Biology, University of Natural Resources and
Life Sciences (BOKU), Konrad Lorenz Str. 25, A-3430 Tulln, Austria
2 Department IFA Tulln (BOKU), Konrad Lorenz Str. 20, A-3430 Tulln, Austria
3 Sabanci University Faculty of Engineering and Natural Sciences, Orhanli, Tuzla-Istanbul,
Turkey
4 Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München,
Neuherberg, Germany
gerhard.adam@boku.ac.at
Abstract
Fusarium graminearum is an important fungal pathogen of small grain cereals and
maize. It produces the protein biosynthesis inhibitor and mycotoxin deoxynivalenol, which
is a virulence factor supporting fungal spread in infected ears. Resistance to Fusarium is
at least partly determined by the ability to convert DON into the non-toxic conjugate
DON-3-O-glucoside (Poppenberger et al. 2003). Also Brachypodium can be infected
with Fusarium, and different accessions can form the detoxification product to a variable
extent.
The gene family of Bd UDP-glucosyltransferases (UGTs) consists of 177 predicted genes.
We characterized the cluster of 6 Bd genes which have the highest sequence similarity
with a barley gene HvUGT13248 conferring toxin resistance in yeast (Schweiger et al.
2010). Only two out of the six Bd homologs confered DON resistance. We conclude that
the UGTs, which frequently occur in clusters containing a variable number of genes
compared to rice and sorghum, seem to evolve very rapidly. It is therefore nontrivial to
identify the true orthologs in crop plants. Validation of the presumed function of
candidate genes by heterologus expression and functional testing in yeast is warranted.
References
References
Poppenberger B, Berthiller F, Lucyshyn D, Sieberer T, Schuhmacher R, Krska R, Kuchler K,
Glössl J, Luschnig C, Adam G (2003) Detoxification of the Fusarium mycotoxin
deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana. J. Biol. Chem.
278: 47905-47914.
Schweiger W, Boddu J, Shin S, Poppenberger B, Berthiller F, Lemmens M, Muehlbauer GJ,
Adam G (2010) Validation of a candidate deoxynivalenol-inactivating UDPglucosyltransferase
from barley by heterologous expression in yeast. Mol. Plant Microbe
Interact. 23: 962-76.
Keywords
Fusarium, mycotoxin, detoxification, resistance
34

Session 4 – Biotic and Abiotic Stresses
S4.5- The Mycosphaerella graminicola-Brachypodium distachyon interaction:
a new model pathosystem to study Septoria tritici blotch disease of wheat.
Aoife O’ Driscoll 1, 2, Fiona Doohan 2 and Ewen Mullins 1
1 Dept. Crop Science, Teagasc Crops, Environment and Land Use Programme, Oak Park,
Carlow.
2 Molecular Plant-Microbe Interactions Laboratory, University College Dublin, Ireland.
aoife.odriscoll@teagasc.ie
Abstract
Mycosphaerella graminicola is the causal agent of Septoria tritici blotch (STB) disease of
wheat, the most economically important foliar pathogen of wheat in Europe. Arising from
a lack of durable resistance in current wheat varieties, wheat producers rely solely on
fungicides to preserve yields. However, the emergence of fungicide resistance and
insensitivity among M. graminicola populations means that the need for improved
varietal resistance is now more imperative than ever before. Quantitative trait loci
associated with STB resistance have been mapped to the chromosomes of the
hexaploid wheat genome but no Stb resistance gene has yet been cloned; neither has a
M. graminicola avirulence gene. Studies on STB are complicated by the complex wheat
genome, long generation time, demanding growth requirements and the limited number
of mutant lines available. The use of model plant species has proven beneficial for the
elucidation of complex host-pathogen systems. Arabidopsis has been used extensively in
such studies but its potential efficacy is limited for STB disease as it is not a natural host of
M. graminicola.
In response to this challenge, we have focussed on the utility of Brachypodium
distachyon as a viable model host species for M. graminicola. The research presented
here demonstrates that Brachypodium is a natural host for M. graminicola and is readily
amenable to infection by multiple isolates of M. graminicola. Displayed disease
symptoms are comparable to those observed in wheat and a screen of multiple
ecotypes has identified a differential response to STB disease within the Brachypodium
gene pool. In addition, we have recorded disease symptoms on all aerial parts of
Brachypodium post inoculation. This report is the first of its kind, and lays the foundations
for the first model pathosystem with which to investigate the mechanisms underlying STB
resistance in a genetically characterised monocotyledonous model species.
Keywords
Septoria, Brachypodium, wheat, model species
35

Session 4 – Biotic and Abiotic Stresses
KEYNOTE LECTURE
S4.6- Brachypodium as a model for unraveling stem rust resistance
David F. Garvin
USDA-ARS Plant Science Research Unit, St. Paul, MN 55108 USA
David.Garvin@ars.usda.gov
Abstract
Members belonging to the fungal genus Puccinia are highly destructive to cool season
cereal crops. Of particular concern is, P. graminis, the causal agent of stem rust, which
poses a threat to wheat production globally due to the emergence of new races that
defeat previously effective resistance genes. Unlike Arabidopsis, Brachypodium
distachyon (Brachypodium) is reported to serve as a host to Puccinia species, and thus it
may be a valuable surrogate for exploring Brachypodium-rust pathosystems. We have
found that Brachypodium can be colonized by different formae specialis of P. graminis,
and that natural variation for resistance resides in Brachypodium germplasm. Genetic
analysis of resistance to timothy stem rust, P. graminis phlei-pratensis, in one recombinant
inbred population suggests major gene control, and efforts are underway to isolate this
gene. Similarly, there is significant variation for resistance to wheat stem rust (P. graminis
tritici). However, unlike timothy stem rust reactions, the disease phenotypes here are
highly diverse, suggesting that the resistance may be quantitative in nature. We
hypothesize that some of this broad variation may reflect minor gene variation
associated with non-host resistance. Screens of mutant populations have identified
validated genotypes both with increased susceptibility and with enhanced resistance,
and genetic studies are ongoing to further explore their biological basis.
Keywords
Plant disease, natural variation, molecular genetics, gene cloning, resistance genes
36

Session 4 – Biotic and Abiotic Stresses
S4.7- Brachypodium distachyon, a model grass to study plant-pathogen
interactions
Thierry C Marcel 1, Rients E Niks 2, June Chun Wang 2, Sébastien Antelme 3, Jean-Benoit
Morel 4, Marie Dufresne 5, Thierry Langin 6, Diana Fernandez 7, Stéphane Bellafiore 7, Jia
Chen 7, Christophe Brugidou 8, Richard Sibout 3
1 INRA-AgroParisTech, UR1290 BIOGER-CPP, Avenue Lucien Brétignières BP01, 78850 Thiverval-Grignon, France
2 Laboratory of Plant Breeding, Wageningen University, Droevendaalsesteeg 1, 6708 Wageningen, the Netherlands
3 INRA-AgroParisTech, UMR1318 Institut Jean-Pierre Bourgin, Route de St-Cyr (RD10), 78026 Versailles Cedex, France
4 INRA Campus International de Baillarguet, UMR BGPI, INRA TA A-54/K, 34398 Montpellier, France
5 Institut de Biologie des Plantes, Université Paris-Sud 11, 91405 Orsay, France
6 UMR INRA-UBP 1095, GDEC, 234 avenue des Landais, 63100 Clermont-Ferrand, France.
7 Centre IRD, UMR 186 IRD-UM2-Cirad Résistance des Plantes aux Bioagresseurs, 911 avenue Agropolis, 34394 Montpellier Cedex 5,
France
8 Centre IRD, UMR 5096 UP-IRD-CNRS, 911 avenue Agropolis, 34394 Montpellier Cedex 5, France
thierry.marcel@versailles.inra.fr
Abstract
Brachypodium (alias Brachypodium distachyon) serves as a model plant species for grass
genomics. There is notably a growing interest in developing Brachypodium as a model
plant species to study economically significant pathogens of cereals. The discovery of
key genes determining the outcome of the interaction between Brachypodium and
pathogens will permit the detection of possible orthologues in temperate cereals that
could be exploited as source of genetic resistance to these pathogens. We explored two
approaches aimed at using the natural diversity of Brachypodium to unravel such genes
involved in plant-pathogen interactions. First, Brachypodium is a host species for some
pathogens of cereals and any type of resistance found in Brachypodium accessions may
be exploited. Brachypodium indeed is a host to the fungal pathogens Magnaporthe
oryzae (Routledge et al. 2004; Wang et al. 2011) and Fusarium graminearum (Peraldi et
al. 2011) that normally infect rice, wheat and barley, respectively. Second,
Brachypodium is a non-host (all accessions are resistant to all isolates of the pathogen) to
other cereal pathogens. Then, related pathogens may be collected from Brachypodium
in nature and used for further research. This was recently proposed by Barbieri et al.
(2011) who showed that Brachypodium has susceptible and resistant accessions to
isolates of the leaf rust Puccinia brachypodii. We established a core collection of diploid
Brachypodium inbred lines by selecting 40 lines available to the research community that
maximize the phenotypic and genotypic diversity in the species. We used this core
collection to determine the host range and response of Brachypodium to the fungal
pathogens Magnaporthe oryzae, Fusarium graminearum, Puccinia graminis, P. striiformis,
P. brachypodii, and to the rice root-knot nematode Meloidogyne graminicola and rice
yellow mosaic virus. Our effort is providing a general overview of the possibility to use
Brachypodium as a model system to study important grass and cereal pathogens, and
will reveal key lines and interactions to perform further genetic and functional studies.
References
Barbieri M, Marcel TCM, Niks RE (2011) Host status of false brome grass (Brachypodium) to the leaf rust fungus Puccinia brachypodii
and the stripe rust fungus P. striiformis. Plant Disease: In Press.
Peraldi A, Beccari G, Steed A, Nicholson P (2011) Brachypodium distachyon: a new pathosystem to study Fusarium head blight and
other Fusarium diseases of wheat. BMC Plant Biology: In Press.
Routledge APM, Shelley G, Smith JV, et al. (2004) Magnaporthe grisea interactions with the model grass Brachypodium distachyon
closely resemble those with rice (Oryza sativa). Molecular Plant Pathology 5: 253-265.
Wang X-Y, Wang J-Y, Jiang H, et al. (2011) Pathogenicity of rice blast fungus Magnaporthe oryzae on Brachypodium distachyon.
Chinese Journal of Rice Science 25: 314-320.
Keywords
Pathogen, Natural variation, Host status, Core collection
37

Session 4 – Biotic and Abiotic Stresses
S4.8- QTLs for resistance to the leaf rust Puccinia brachypodii in the model
grass Brachypodium distachyon
Mirko Barbieri*, Thierry C. Marcel** , ***, Rients E. Niks***, Enrico Francia*, Marianna
Pasquariello*, Valentina Mazzamurro*, David F. Garvin****, Nicola Pecchioni*
*Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio Emilia,
Via Amendola 2, 42122 Reggio Emilia, Italy
**INRA-AgroParisTech, UR1290 BIOGER-CPP, Avenue Lucien Brétignières BP01, 78850
Thierval-Grignon, France
***Laboratory of Plant Breeding, Wageningen University, Droevendaalsesteeg 1 6708
Wageningen, The Netherlands
****USDA-ARS Plant Science Research Unit, 411 Borlaug Hall, University of Minnesota, 1991
Upper Buford Circle, St. Paul, MN, 55108, USA
nicola.pecchioni@unimore.it
Abstract
The wild grass Brachypodium distachyon is a useful model for temperate cereals (Draper
et al. 2001; Garvin et al. 2008), but its potential to study the interactions with pathogens
remains underexploited. Leaf rust is one of the major fungal diseases affecting cereals,
and recently the host status of Brachypodium to Puccinia rusts was investigated (Barbieri
et al. 2011).We aimed to identify genomic regions associated with quantitative
resistance to leaf rust in Brachypodium. Two inbred lines, Bd3-1 and Bd1-1, with
quantitative differences in their level of resistance to Puccinia brachypodii, were crossed
to develop an F2 population of 110 plants, that were evaluated for reaction to a P.
brachypodii isolate at both seedling and advanced growth stages, by means of the
AUDPC (Area Under Disease Progress Curve) score. Results from the F2 plants were
validated in F2-derived F3 families. The mapping population showed quantitative and
transgressive segregation for leaf rust resistance. We applied AFLP, SNP and SSR markers
to develop a new Bachypodium linkage map of 203 loci spanning 811.8 cM, and
anchored to its genome sequence.
Three leaf rust resistance QTLs (Rpbq1, Rpbq2 and Rpbq3) were identified on
chromosomes 2, 3 and 4. The resistant alleles of Rpbq1 and Rpbq2 were contributed by
the resistant parent Bd3-1, while for Rpbq3 the resistant allele came from Bd1-1. This study
was, to our knowledge, the first quantitative analysis of any trait in Brachypodium
(Barbieri et al., in preparation). To begin the process of isolating the QTLs, we have
chosen the 8 candidate genes closest to the peaks of Rpbq2 and Rpbq3, based on
genome sequence information. The genes were resequenced in the parents, and
polymorphisms identified for mapping efforts. The validation of gene sequences for a leaf
rust resistance QTL will lead to a deeper understanding of mechanisms of quantitative
resistance to this and to other rust pathogens that infect members of the Triticeae.
References
Draper J., Mur L.A., Jenkins G., et al. (2001) Brachypodium distachyon. A new model system for functional genomics in grasses. Plant
Physiol. 127:1539-1555
Garvin D.F., Gu Y.Q., Hasterok R., et al. (2008) Development of genetic and genomic research resources for Brachypodium
distachyon, a new model system for grass crop research. Crop Sci 48:69-84
Barbieri M., Marcel T.C., Niks R.E. (2011) Host status of false brome grass (Brachypodium) to the leaf rust fungus Puccinia brachypodii
and the stripe rust fungus P. striiformis. Plant Disease, Volume 0, Number ja (doi: 10.1094/PDIS-11-10-0825)
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
rust Puccinia brachypodii in the model plant Brachypodium distachyon. In preparation
Keywords
Puccinia brachypodii, genetic map, leaf rust, quantitative resistance, QTL
38

Session 4 – Biotic and Abiotic Stresses
S4.9- Brachypodium distachyon: an excellent model for Fusarium
graminearum infection in wheat
Antje Bluemke, Christian A. Voigt
Molecular Phytopathology and Genetics, Biocenter Klein Flottbek, University of Hamburg,
22609 Hamburg, Germany.
antje.bluemke@botanik.uni-hamburg.de
Abstract
Fusarium head blight (FHB) is one of the most important cereal diseases. It leads to severe
mycotoxin contamination of grain and extensive yield losses. The plant pathogen
Fusarium graminearum is the most common agent of FHB. Investigating the interaction
between F. graminearum and cereals, esp. wheat, was always limited by the genetic
inaccessibility of these plants. During the last years Brachypodium distachyon has been
established as a model system for the temperate grasses, including wheat.
In a first step, we established the F. graminearum infection of B. distachyon. Disease
symptoms resembled that in wheat. To investigate the established pathosystem on a
cellular level, F. graminearum wild-type and defined mutants with reduced virulence on
wheat ( fgl1, secreted lipase [1] tri5, trichodiene synthase [2], gpmk1, mitogenactivated
protein kinase [3]) were used for infection. All F. graminearum strains expressed
GFP constitutively to follow infection in planta. A comprehensive view on the
pathosystem was gained by combining data of mycotoxin (deoxynivalenol (DON))
production and pathogen-induced alteration of the cell wall composition with the in
planta infection progress.
Regarding DON accumulation in plant tissue, all data from B. distachyon resembled
those known from wheat. A strong increase in DON occurred during F. graminearum
wild-type and fgl1 infection, whereas for the mutant strains tri5 and gpmk1 DON was
not detectable. Similar alterations of the cell wall composition were observed in B.
distachyon as well as in wheat after infection with F. graminearum. Also the infection
progress of all tested F. graminearum strains was comparable to the described progress
in wheat.
Our results show that B. distachyon responds to F. graminearum infection on a visual and
cellular level like wheat. Therefore, Brachypodium serves as an excellent model to study
FHB in small grain cereals, especially wheat.
References
1. Voigt CA, Schäfer W, Salomon S (2005) A secreted lipase of Fusarium graminearum is a
virulence factor required for infection of cereals. The Plant Journal 42: 364-375.2.
2. Jansen C, von Wettstein D, Schafer W, et al. (2005) Infection patterns in barley and
wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted
Fusarium graminearum. Proc Natl Acad Sci U S A 102: 16892-16897.
3. Jenczmionka NJ, Maier FJ, Lösch AP, Schäfer W (2003) Mating, conidiation and
pathogenicity of Fusarium graminearum, the main causal agent of the head-blight
disease of wheat, are regulated by the MAP kinase gpmk1. Current Genetics 43: 87-95.
Keywords
Brachypodium, Fusarium
39

Session 5 – Plant Development, Metabolism and Physiology
S5.1- Brachypodium distachyon grain: development-associated changes in
morphology and storage accumulation
Colette Larré 1, Anne-Laure Chateigner-Boutin 1, Brigitte Bouchet 1, Fabrice Petipas 2,
Hélène Rogniaux 1, Luc Saulnier 1, Bertrand Dubreucq 2 , Fabienne Guillon 1
1 UR1268, INRA BIA, Rue de la Géraudière, BP71627, 44316 Nantes, France
2 Unité de Biologie des Semences, INRA, RD 10, 78026 Versailles cedex, France
Colette.Larre@nantes.inra.fr
Abstract
Brachypodium distachyon is a promising new model for temperate cereals.
In an effort to characterize this model, we recently analysed the composition of
Brachypodium mature grain. Brachypodium grain is notable for its low starch content
(less than 10% of the endosperm compared to 60-70% in other cereals), and its high cell
wall polysaccharide content (>55%) compared to 2–7% in other cereals and consisting
especially of (1-3)(1-4) D-glucans (48% grain dry weight) and arabinoxylans. Protein
content is in the upper range reported for other cereals (17%). The major grain
components, which serve as reservoir for carbon, nitrogen and sulphur for postgerminative
growth, appear therefore to be -glucans and proteins (globulins and
glutelins).
Here we present the characterization of Brachypodium grain development. Grain
morphology as well as polysaccharides and protein accumulation were studied by
microscopy. Proteins were identified by mass spectrometry. The grain development
spans 54 days after flowering (DAF). Maximum size (about 6 mm) is reached at 14 DAF
when the grain fresh weight is about 7 mg. From 14 to 40 DAF, storage compounds
accumulate to reach a weight of about 8.5 mg. At the end of dehydration grain weight
is about 5 mg.
Although starch is not detected in the mature grain outer layers, it accumulates in the
early stages of development. In the endosperm, starch is detected from 14 DAF and
during grain development starch granules increase in size. Nevertheless, their final
number and size are limited. -glucans accumulate in endosperm cell wall from 7 DAF
during cellularisation.
Storage proteins accumulate from 15 DAF. At this stage the major types of storage
proteins are detected. Protein analysis during early development, reveals enzymes
involved in many metabolic pathways. From 15 DAF, the accumulation of storage
proteins hinders the detection of other proteins. In mature grain, storage proteins are
stored in small and large « vesicles ».
References
Guillon F, Bouchet B, Jamme F, Robert P, Quémener B, Barron C, Larré C, Dumas P,
Saulnier L (2011) Brachypodium distachyon grain : characterization of endosperm cell
walls. Journal of Experimental Botany 62 : 1001-1015.
Larré C, Penninck S, Bouchet B, Lollier V, Denery-Papini S, Guillon F, Rogniaux H (2010).
Brachypodium distachyon grain : identification and subcellular localization of storage
proteins. Journal of Experimental Botany 61 : 1771-1783.
Keywords
Grain development, grain composition (proteins and carbohydrates)
40

Session 5 – Plant Development, Metabolism and Physiology
S5.2- The transcription factor BdDOF24 interacts with BdGAMYB and regulates
the cathepsin B-like gene during Brachypodium seed germination
Virginia González-de la Calle, Sara Hernando-Amado, Raquel Iglesias-Fernández, Cristina
Barrero-Sicilia and Pilar Carbonero
CBGP (Centro de Biotecnología y Genómica de Plantas) ETSI Agrónomos. Universidad
Politécnica de Madrid. Campus de Montegancedo 28223 Pozuelo de Alarcón (Madrid)
virginia.gonzalez@upm.es
Abstract
Transcription factors (TFs) are important regulators of gene expression. According to the
specificity of the DNA-binding domain, TFs can be classified into different families. The
DOF (DNA binding with One Finger) transcription factor family belongs to the class of zinc
finger domains and it is characterized by a conserved region of 52 amino acid residues
that is structured as a Cys2/Cys2 Zn 2+ finger that binds specifically the CREs containing
the common core 5’-T/AAAAG-3’. DOF transcription factors have been associated with
regulation during seed maturation and germination in cereals. We have annotated 27
putative BdDof genes of Brachypodium distachyon; their phylogenetic relationship with
closely related DOF proteins from rice and barley have been established and their
global expression levels in different tissues, as well as the expression profile of the most
abundant BdDof genes during seed germination have been determined. One such a
gene, BdDof24 was predominantly expressed in germinating seeds.
Promoters of hydrolase genes, induced upon germination, such are those encoding -
amylase, EII(1-3, 1-4)- -glucanase and thiol-proteases (cathepsin B-like) contain, in
addition to the 5’-T/AAAAG-3’ motif, a GA responsive element (GARE-5’-TAACAAA-3’).
Two TFs binding to these two motives have been identified: BdDOF24 and BdGAMYB. We
have also annotated the putative ortholog of the barley CathB-like gene (Al21):
BdCathB, we established its expression profile in different organs and throughout seed
germination. The expression pattern of genes encoding BdGAMYB and BdDOF24
suggests a possible transcriptional control of the CathB gene by these TFs during seed
germination. This hypothesis has been further validated by EMSA, by molecular
interactions (Y2Hassays) between both TFs and by transient assays in planta.
References
Gubler F, Raventos D, Keys M, et al. (1999) Target genes and regulatory domains of the
GAMYB transcriptional activator in cereal aleurone. Plant J 17(1): 1-9.
Mena M, Cejudo FJ, Isabel-Lamoneda I, Carbonero P (2002) A role for the DOF
transcription factor BPBF in the regulation of gibberellin-responsive genes in barley
aleurone. Plant Physiol 130: 111-119.
Moreno-Risueno MA, Martínez M, Vicente-Carbajosa J, Carbonero P (2007) The family of
DOF transcription factors: from green unicellular algae to vascular plants. Mol Genet
Genomics 277:379-390.
Keywords
Transcriptional regulation, Germination, DOF transcription factors, Hydrolases
41

Session 5 – Plant Development, Metabolism and Physiology
S5.3- Rhizobacterial volatile organic compounds modulate biomass
production and root architecture in Arabidopsis thaliana (L.) Heynh. and
Brachypodium distachyon (L.) P. Beauv.
Delaplace Pierre 1*, Varin Sébastien 1, Ormeño-Lafuente Elena 2, Spaepen Stijn 3, Saunier de
Cazenave Magdalena 1, Blondiaux-Hendrick Adeline 1, Wathelet Jean-Paul 4, du Jardin
Patrick 1
1 University of Liège, Gembloux Agro-Bio Tech, Plant Biology Unit, Gembloux, Belgium
2 Current address : Centre National de la Recherche Scientifique, Institut Ecologie et
Environnement, Institut Méditerranéen d'Ecologie et Paléoécologie, UMR CNRS 6116,
Laboratoire de Diversité Fonctionnelle des Communautés Végétales, Marseille, France
3 Centre of Microbial and Plant Genetics, K.U.Leuven, Bio-incubator, Heverlee, Belgium
4 University of Liège, Gembloux Agro-Bio Tech, General and Organic Chemistry Unit,
Gembloux, Belgium
* Pierre.delaplace@ulg.ac.be
Abstract
The ability of plants to take up water and mineral nutrients from the soil depends on their
capacity to develop an extensive root system and on the interactions of roots with their
soil environment, the rhizosphere. Studies of the mechanisms involved in the
communication between plant growth-promoting soil micro-organisms and the root
system is expected to lead to improved fertility management strategies. Up to now, the
characterization of such interactions has been mainly focused on liquid diffusates but it
has been recently reported that volatile organic compounds (VOC) also play a role as
chemical messengers in positive interactions occurring in the rhizosphere and involving
plants, bacteria, fungi and insects [1-2].
In this context, this project aims to better understand the ecophysiology of the
rhizosphere of Arabidopsis thaliana and Brachypodium distachyon. For this purpose, a
collection of 18 rhizobacterial strains belonging to nine genera were selected for their
potential growth promotion ability. The VOC-emission capacity of each strain was
measured after 24 and 48 hours of growth on agar media [3] using solid-phase
microextraction followed by gas chromatography coupled to a mass spectrometer.
In parallel, an in vitro screening system was set up to assess VOC-mediated plant-growth
promotion ability of the selected strains. This system separates physically the plant and
the bacteria while allowing interactions through VOC for 10 days within a shared
atmosphere. For Arabidopsis, two inoculum sizes were considered: 2*10 5 and 2*10 6 cfu,
using biological triplicates. At the end of the process, leaf and root biomasses, leaf area,
primary root length, lateral root number and lateral root length were measured to assess
the effects of bacterial VOC on plant growth. Experiments are in progress to characterize
such parameters on Brachypodium in order to compare the response of both model
species to rhizobacterial volatile perception.
Acknowledgements: this work was supported by the Belgian Fonds de la Recherche
Scientifique (FRFC project 2.4.591.10. and postdoctoral grant 1808458).
References
1. Ryu CM, Farag MA, Hu CH, et al. (2003) Bacterial volatiles promote growth in Arabidopsis. Proc Natl Acad Sci USA 8: 4927-4932.
2. Kai M, Effmert U, Berg G, Piechulla B (2007) Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen
Rhizoctonia solani. Arch Microbiol 187: 351-360.
3. Farag MA, Ryu CM, Sumner LW, Paré PW (2006) GC–MS SPME profiling of rhizobacterial volatiles reveals prospective inducers of
growth promotion and induced systemic resistance in plants. Phytochemistry 67: 2262-2268.
Keywords
root architecture, plant growth-promoting rhizobacteria, volatile organic compounds
42

Session 5 – Plant Development, Metabolism and Physiology
S5.4- Using Natural And Induced Variation In Brachypodium To Study Grain
Development
John H Doonan 1, Magdalena Opanowicz 2, Max Bush 2 , Kay Trafford 2 , Vera Thole 2 ,
Sinead Drea 3 , Phillip Hands 3, Philippe Vain 2
1 Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,
Ceredigion, UK
2 John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK
3 University of Leicester, University Road, Leicester, LE1 7RH, UK
john.doonan@aber.ac.uk
Abstract
Grains provide the vast majority of calories consumed by humans and projected yields
must dramatically and rapidly increase if global food supply is to keep up with demand.
While it is clear that our major grain crops have much potential to deliver such increases
in yield, it is imperative that the rate of yield increase should itself be increased. This will
require the exploration of different complementary approaches, including conventional
breeding, through transgenics and improved agronomy. A thorough understanding of
grain development will underpin such efforts and we have begun to compare grain
development in different species to understand the molecular and cellular basis of the
diversity in grain development and composition (Opanowicz et al. 2011).
Brachypodium distachyon is an attractive fast cycling small grass being developed as a
model for grass and cereal biology and also is of interest as a widely distributed wild grass
that is adapted to a range of habitats (Opanowicz et al. 2008). Grain development, for
example, is largely similar to that of wheat and other temperate cereals, but displays
some important differences (Opanowicz et al. 2011). Natural and induced variation in
Brachypodium grain development can be exploited to understand the regulation of
grain development. Natural accessions collected from diverse locations display
variation in grain size and shattering as well as in plant morphology, offering the possibility
of understanding the genetic control of grain traits in an undomesticated species.
Selected T-DNA tagged lines, where specific genes have been disrupted, also display
defects in plant and grain development, providing the opportunity to understand in
detail the genetic regulation of grain development and growth (Vain et al. 2011).
References
Opanowicz M, Vain P, Draper J, Parker D, Doonan JH (2008) Brachypodium distachyon:
making hay with a wild grass. Trends Plant Sci 13: 172-177.
Opanowicz M, Hands P, Betts D, Parker ML, Toole GA et al. (2011) Endosperm
development in Brachypodium distachyon. J Exp Bot 62: 735-748.
Vain P, Thole V, Worland B, Opanowicz M, Bush MS et al. (2011) A T-DNA mutation in the
RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium
distachyon. Plant J 66: 929–940.
43

Session 5 – Plant Development, Metabolism and Physiology
S5.5- Profiling the lipidome of Brachypodium distachyon
M. Nurul Islam, John P. Chambers, Carl K.-Y. Ng
School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4,
Ireland
carl.ng@ucd.ie
Abstract
Lipids are essential metabolites in cells and they fulfil a variety of functions, including
structural components of cellular membranes, energy storage, cell signalling, and
membrane trafficking. In plants, changes in lipid composition have been observed in
diverse responses ranging from abiotic and biotic stress to organogenesis. Knowledge of
the lipid composition is an important first step towards understanding the function of lipids
in any given biological system. As Brachypodium distachyon is emerging as the model
species for temperate grass research, it is therefore fundamentally important to gain
insights of its lipid composition. We used HPLC-coupled with tandem mass spectrometry
to profile and quantify levels of sphingolipids and glycerophospholipids in B. distachyon
plants. A total of 123 lipids belonging to 10 classes were identified and quantified.
Additionally, we showed that 4-sphingenine (d18:1 4) is the main unsaturated dihydroxy-
LCB in B. distachyon, and we were unable to detect d18:1 8, which is the main
unsaturated dihydroxy-LCB in the model dicotyledonous species, Arabidopsis thaliana.
This work serves as the first step towards a comprehensive characterisation of the B.
distachyon lipidome that will complement future biochemical studies.
Keywords
Brachypodium distachyon, lipidome, glycerophospholipids, sphingolipids, LC-MS/MS
44

Session 5 – Plant Development, Metabolism and Physiology
S5.6- Inducible xylem differentiation in Brachypodium
Elene R. Valdivia, María Teresa Herrera, Cristina Gianzo, Gloria Revilla, Ignacio Zarra y
Javier Sampedro
Departamento de Fisiología Vegetal, Facultad de Biología, Universidad de Santiago de
Compostela
elenevaldivia@gmail.com
Abstract
The manipulation of secondary cell wall composition in biomass crops is one of the most
promising strategies to reduce the processing cost of lignocellulosic materials, which
could then become an abundant source of cheap biofuels. Grasses are good
candidates for this approach, but very little is known about the regulation of grass cell
wall synthesis.
In the dicot Arabidopsis several of the transcription factors (TFs) involved in secondary
cell wall synthesis have been characterized in detail. In particular, VND genes seem to
act at very early stages of xylem differentiation and the expression of some of the VNDs is
sufficient to start the process of secondary cell wall deposition. We have identified and
cloned Brachypodium orthologs of the Arabidopsis VNDs. When three of these genes
(VND2, VND4 and VND5) are overexpressed transiently in tobacco leaves, they are
capable of inducing transdifferentiation into tracheary elements suggesting a
conservation of function.
Brachypodium can be transformed with high efficiency through cocultivation of
embryogenic calli with Agrobacterium. We have adapted the estradiol inducible system
for use in Brachypodium and have transformed this species with inducible overexpression
constructs for VND2, VND4 and VND5, as well as chimeric respressors obtained from the
same genes by addition of a repressor domain. Inducible overexpression of VND5 results
in extensive transdifferentiation of different cell types into tracheary element. Through RT-
PCR we have detected an increase expression of cellulose synthase, as well as some TFs
that could work downstream of the VND genes. We are currently characterizing this
plants which could provide a first draft of the regulatory network of secondary cell wall
synthesis in grasses. The repressor lines will be characterized soon and could allow us to
identify feedback mechanisms that detect secondary cell wall alterations.
Keywords
Brachypodium, Secondary cell wall synthesis, Transcription Factors
45

POSTER PRESENTATIONS
46

Session 1 – Evolution and Natural Variation
P1.1- Characterization of the diploid and tetraploid Brachypodium
distachyonpopulations in Israel
Assaf Distelfeld, Smadar Ezrati, Tamar Eilam, Hanan Sela, Raz Avni, and Adina Breiman
Dept. Molecular Biology and Ecology of Plants, The Institute for Cereal Crops
Improvement, Tel Aviv University, Israel.
adinaB@tauex.tau.ac.il
Abstract
Brachypodium distachyon that has emerged as a powerful model system to study the
unique aspects of the grasses is very abundant in Israel. It grows in diverse habitats from
the dry desert areas in the south to the relatively rainy Mediterranean areas in the north
and on different soil types. The Institute for Cereal Crops Improvement in Tel-Aviv
University holds a collection of crops wild relatives, including B. distachyon (BD) from
Israel. The BD collection includes accessions representing the diverse habitats. In a
preliminary study, to determine their ploidy level, 272 plants from the BD collection were
evaluated for their DNA content. Ninety three plants from 23 sites were found to have
similar DNA content as the diploid sequenced line BD21 while the rest had double
amount of DNA content and most likely are allopolyploids. The plants were grown under
controlled greenhouse conditions and showed considerable morphologic and
phenotypic variability in plant architecture, leaf size, leaf color, flowering time, spike
morphology and biomass. Our aim is to associate this phenotypic data with
transcriptomic changes among the diploid genotypes during the vegetative stage in
order to identify genes that control biomass production. The identification of novel genes
conferring increase biomass production will have a significant practical relevance. In
addition, we plan to genotype the collection with SSR markers to study the population
structure and gene flow.
Keywords
Brachypodium distachyon collection, phenotypic variation, biomass, diverse habitats.
47

Session 1 – Evolution and Natural Variation
P1.2- Diversity analysis in Spanish populations of Brachypodium distachyon
Patricia Giraldo, Gaspar Soria, Iciar Sevilla, Marta Rodríguez-Quijano, Jose F Vázquez,
Jose M Carrillo, Elena Benavente
Departamento de Biotecnología (Unidad de Genética). E.T.S. Ingenieros Agrónomos.
Universidad Politécnica de Madrid
patricia.giraldo@upm.es
Abstract
The objective of this work has been to analyze the variability of a germplasm collection
held at ETSI Agrónomos (Universidad Politécnica de Madrid). It is composed by 64 natural
populations morphologically ascribable to Brachypodium distachyon that were
collected by our research group in a wide area of the continental Spain. Climate and
geographical parameters from the collection sites were recorded in order to explore
their correlation with genetic variability data.
The cytological analysis has shown that most of the accessions belong to the diploid (2n=
10) and allotetraploid (2n=30) cytotypes, but a few populations with 2n= 20 individuals
have also been found. We have tried to distinguish between the diploid and
allotetraploid cytotypes by electrophoretic analysis of endosperm proteins, but no
differential marker has been found. The protein pattern of allotetraploid accessions has
revealed a higher degree of polymorphism compared to diploid accessions.
One hundred and ninety-three individuals from 32 diploid (2n=10) populations have been
genotyped with 15 microsatellites (SSRs) selected based on their genomic distribution
and profile quality. The total number of amplified alleles was equal to 94. The number of
alleles per locus varied from 1 to 24, with a mean of 6.26 alleles per locus. Diversity
analysis reflected a comparable diversity both between and within wild populations
(Total gene diversity Ht= 0.464; gene differentiation between populations Gst= 0.514). No
correlation has been found between genetic and ecoclimatic variables.
Four microsatellites have shown a different allelic pattern in diploid versus allotetraploid
individuals, thus providing a useful tool for to discriminate between both cytotypes.
Inbred lines and RILs, developed from selected individuals, will be available for the
scientific community.
Keywords
genetic variability, wild populations, molecular markers
48

Session 1 – Evolution and Natural Variation
P1.3- Morpho-phenologic diversity among Tunisian populations of
Brachypodium Distachyon
Mohamed Neji, Sondes Rahmouni, Warda Saoudi, Malek Smida, Wael Tamaalli
Mounawer Badri, Chedly Abdelly and Gandour Mhemmed
Center of Biotechnology of Borj-Cédria, BP 901 hammam lif 2050 Tunisia
mnmedneji@gmail.com
Abstract
Brachypodium distachyon, commonly called ‘purple false brome’, belongs to Poacea
grass subfamily. This species is mainly distributed in South Europe, North Africa, Southwest
Asia and many other places. It is frequently diploid and has a small genome size
(300Mb). B. distachyon has a close genetic relationship with temperate cereal crops. In
order to bring a better knowledge on the genetic diversity of this species, 9 populations
of B. distachyon representative of all the eco-climatic stages of Tunisia were
characterized on the basis of 18 morpho-phenologic features. Data obtained for the 180
evaluated lines were subjected to various statistical analyses. It arises from the statistical
analyses significant variations for all studied features.
Correlations analysis shows that the majority of morphometric traits were negatively and
significantly correlated between them. The relative importance of the components inter
and intra-population of the phenotypical variation was different according to the
evaluated feature.
Relative importance of intra and inter-populations components variation for phenotypic
variation varied between traits.
Variation of spikelet emergence date, means number of enternode per stem, mean
length of leafs and shape number were dominated by the inter-populations component
but the remaining traits were dominated by the intra-populations component.
The similarities between populations were analyzed by a hierarchical classification. The
dendrogram obtained showed the presence of two principal groups, which reflect a
marked geographical structuring. The first grouped populations originated from (Fayadh
and Zagouan) and the second one grouped the remaining populations.
Keywords
B. distachyon; morphological diversity; geographical structuration
49

Session 1 – Evolution and Natural Variation
P1.4- Genetic Characterization of New Brachypodium distachyon Populations
from Diverse Geographic Regions in Turkey
M. Tuna 1, . Nizam 1, S. Öney 2, G. Sava 1 and J. Vogel 3
1Namik Kemal University, Faculty of Agriculture, Department of Field Crops, 59100,
Tekirdag, Turkey
2Suleyman Demirel University, Faculty of Art and Science, Department of Biology, 32260,
Isparta, Turkey.
3USDA-ARS Western Regional Research Center, Genomics and Gene Discovery Unit,
Albany, CA 94710, USA
metintuna66@yahoo.com
Abstract
The small annual grass Brachypodium distachyon is rapidly emerging as a powerful
model system for temperate grasses due to its biological, physical and genomic
characteristics. The natural range of B. distachyon largely overlaps the ancestral range of
cultivated small grains and Turkey lies in the center of that geographical area thus
granting a great amount of genetic variability.
The objective of this study was to establish and genetically characterize a large B.
distachyon germplasm collection originating from diverse geographical regions in Turkey.
Seeds from approximately 20 individual plants were collected from each of the 118
locations throughout Turkey. Flow cytometry was used to determine the ploidy of
representatives from each location. Seventy five of the populations contained only
diploid plants, 35 of the populations contained only polyploid plants and 8 populations
contained a mix of diploid and polyploid plants. To examine the genetic diversity of this
new collection, a representative subset of 185 diploid individuals was genotyped using
SSR markers. Our preliminary results show considerable genetic diversity both between
and within populations. We also compared the SSR profiles of the new lines to previously
examined lines. Significantly, some of the new accessions were contained in new clades
indicating that we have not sampled Turkish B. distachyon diversity to saturation. Future
plans for this collection include phenotypic characterization with an emphasis on biotic
and abiotic stress. We also plan to develop recombinant inbred lines from divergent lines.
50

Session 2 – Cell Wall, Biomass and Biofuels
P2.1- Brachypodium mutants, new tools to validate candidate genes
underlying lignin content and digestibility QTL in maize?
Audrey Courtial 1,2, Matthieu Reymond 3, Valérie Méchin 3, Jacqueline Grima-Pettenati 2
and Yves Barrière 1.
1 INRA, UGAPF, BP80006, 86600 Lusignan, France
2 UMR5546 CNRS - UPS, 31326 Castanet-Tolosan, France
3 INRA, Institut Jean-Pierre Bourgin, 78026 Versailles, France
courtial@lrsv.ups-tlse.fr
Abstract
Deciphering the genetic and genomic determinants involved in the maize cell wall
biosynthesis and assembly is a strategic issue for both ruminant feeding and biofuels
production. The identification of candidate genes underlying QTL for high cell wall
degradability is a relevant strategy for further marker-assisted selection.
In the F288 x F271 early maize RIL progeny, several QTL were mapped, of which those
located on bin 6.06 explain nearly 50% of the phenotypic variation for both lignin content
and cell wall digestibility (Roussel et al., 2002; Courtial et al., in preparation). In order to
improve the resolution mapping of QTL, new markers were genotyped using HRM (High
Resolution Melting) technology and QTL physical positions were then estimated based on
the publicly available maize genomic sequence (line B73). All the genes located within
the QTL support interval are potential candidate genes explaining the lignin and
digestibility variability. Among them those involved in cell wall biosynthesis and assembly,
and/or regulation of these processes are positional and functional candidate genes.
Complementary strategies are underway for the validation of these candidate genes.
Expression studies will be performed using contrasting lines and allele sequencing of
candidate genes will be considered for both parental lines to reduce the number of
candidate genes. As a first functional validation, we could use the new emerging
monocot model Brachypodium distachyon which offers mutants well suited to study the
composition and degradability of monocot walls.
References
Roussel V, Gibelin C, Fontaine A-S, Barrière Y (2002) Genetic analysis in recombinant
inbred lines of early dent forage maize. II - QTL mapping for cell wall constituents and cell
wall digestibility from per se value and top cross experiments. Maydica 47: 9-20.
Keywords
Maize, cell wall digestibility, lignin, candidate genes, brachypodium
51

Session 2 – Cell Wall, Biomass and Biofuels
P2.2- Characterization of secondary wall deposition in Brachypodium stems
towards the goal of fiber cell engineering for biomass feedstocks
Michael Harrington, Nicolas Noria, Richard Sibout, Gregory Mouille, Catherine Lapierre,
Notburger Gierlinger*, Herman Höfte
Institut Jean Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la
Recherche Agronomique-AgroParisTech, 78026 Versailles, France.
*Faculty 5/Biomimetics, Biological Materials, University of Applied Sciences Bremen,
28199, Bremen, Germany.
Michael.Harrington@versailles.inra.fr
Abstract
Worldwide the demand for renewable energy made from non-food plant biomass is
increasing. Plant biomass (or ‘lignocellulose’) found in plant cell walls serves as an
attractive alternative to the current biomass feedstocks, i.e., corn and sugar cane. Cell
walls of most plants are composed of energy-rich polysaccharide polymers that can be
broken down (‘saccharification’) to produce many bio-based products (e.g., bioplastics,
fibers for textile) and bioethanol. Previous studies have provided valuable information in
our understanding of cell wall biosynthesis, particularly towards our understanding of the
molecules involved in this process, however little is known about the cellular behaviors
associated with wall deposition and how they are regulated by these genes.
The goal of this work is to address both the cellular and molecular mechanisms
underlying secondary wall synthesis during sclerenchyma cell develpment using
Brachypodium distachyon, a model for biomass feedstocks. To date we have begun to
characterize sclerenchyma cell formation using a variety of molecular and 3D
recontruction tools, to gain a better understanding of the timing of secondary wall
deposition. In addition, we are now focusing our attention to the molecular regulators
(i.e., transcription factors or/and processing enzymes) of sclerenchyma cell
differentiation and maturation by applying several physical and chemical approaches,
including laser microdissection, transcriptomics, to cell wall composition analysis assays
towards these goals.
Ultimately, this work is improving our knowledge of secondary cell wall synthesis while
identifying the regulators of this process to improve biomass saccharification. Conversely,
this work addresses concerns for sustainable sources of energy, fibers, and chemicals by
creating a dedicated biomass crop.
Keywords
seconday wall, sclerenchyma, lignin, micrsodissection
52

Session 2 – Cell Wall, Biomass and Biofuels
P2.3- Phenotyping of yield components and saccharification efficiency in
Bd21, Bd21-3 and 4 wild accessions of diploid Brachypodium distachyon
Van Hulle Steven, Roldán-Ruiz Isabel, Muylle Hilde
Institute of Agricultural and Fisheries Research, Plant Sciences Unit, Caritasstraat 21, 9090
Melle, Belgium
Hilde.Muylle@ilvo.vlaanderen.be
Abstract
Brachypodium distachyon is a temperate wild grass species and is a powerful model
system for other Poaceae. Exploring the natural variation in the yield characteristics and
saccharification potential of Brachypodium provides an important basis for genetic
dissection of these traits. Bd21, Bd21-3 and 4 wild accessions (pi185133, pi185134,
pi245730 and pi254867) were phenotyped. Significant differences were found in yield
traits such as total number of branches, dry matter yield and seed yield. Using a small
scale enzymatic hydrolysis assay, significant differences were found between the studied
accessions for saccharification potential, ranging between 132.75 mg glucose/g biomass
(pi185133) till 187,23 mg glucose/g biomass (pi254867). Bd21 and Bd21-3 showed
intermediate sugar release (151,27 and 155.56 mg glucose / g biomass respectively). The
identified phenotypic variation of Brachypodium in yield traits and saccharification
potential can be used to identify genes and alleles important for the complex trait as
yield and cell wall accessibility.
Keywords
Biomass, Bioenergy, Yield, Saccharification, Cell Wall
53

Session 2 – Cell Wall, Biomass and Biofuels
P2.4- Impact of cell wall variability on saccharification yield in natural
Brachypodium distachyon accessions
Legay S. 1, Oudihat M. 1, Darracq O. 1, Antelme S. 1, Whitehead C. 2, Gomez L. 2, Mouille G.
1, Lapierre C. 1, Jouanin L. 1, Sibout R 1.
1Institut Jean-Pierre Bourgin (IJPB), UMR1318 INRA-AgroParisTech, 78026 Versailles, FR
2Centre for Novel Agricultural Products, University of York, York Y010 5 YW, UK
sylvain.legay@versailles.inra.fr
Abstract
Lignocellulosic derived biomass represents one of the most abundant renewable sources
of carbohydrate for the production of bioenergy. Amongst the available biomass,
emerging bioenergy crops such as grasses show great potential for the production of
lignocellulose. In the last few years Brachypodium distachyon has come forth as a model
for temperate grass species due to numerous advantages: small stature, low growth
requirements, genomic resources … Still relatively little is known about the secondary cell
wall composition in Brachypodium. To gain insight on the variability of the secondary cell
wall composition of Brachypodium, we selected a collection of natural accessions of
Brachypodium in order to conduct a detailed biochemical analysis of the different
secondary cell wall polymers. A total of 22 natural accessions of Brachypodium were
selected based on differences of ploïdy, genotypes and phenotypes. Total biomass
production, as well as NIRS and FTIR spectral analysis of stems enabled the classification
of the accessions in 3 groups. Detailed analysis of lignin and polysaccharide composition
revealed a large variation amongst the different accessions. Histological analysis of the
stems of the most extreme lines was achieved to understand if the observed differences
were linked to anatomical variability. Finally the potential impact of the cell wall
composition variability on the production of bioethanol was assessed through
saccharification assays. The results from this study will enable the selection of accessions
with contrasted cell wall and saccharification properties and in the near future lead to
the identification of QTLs for these corresponding traits.
Keywords
biofuel, cell wall, saccharification, natural accessions
54

Session 2 – Cell Wall, Biomass and Biofuels
P2.5- Cellulose biosynthesis and growth anisotropy in Brachypodium
distachyon
Karen Sanguinet Osmont, Samuel Hazen, Tobias I. Baskin
Department of Biology, 611 North Pleasant St. UMass-Amherst, Amherst, MA 01003
ksosmont@bio.umass.edu
Abstract
Cellulose is Earth's most abundant polymer and humanity's most useful building material,
comprising upwards of 30% total plant biomass. Cellulose regulates the direction of plant
growth. In a growing organ, cells expand at different rates in different directions, that is,
anisotropically. Cellular growth anisotropy must be controlled precisely to build organs of
specific, heritable shapes. Anisotropic expansion is enabled by cellulose microfibrils,
which thereby help determine the shape and size of the mature plant. Our aim is to study
cellulose synthesis and the control of growth anisotropy in the emerging model plant,
Brachypodium distachyon. Our understanding of cell wall biosynthesis and CESA
functionality has stemmed from mutant analyses in plants such as arabidopsis, but little
functional analysis has been performed in the grasses. We are using both forward and
reverse genetic approaches to help understand cellulose biosynthesis and formation of
the primary cell wall. As in arabidopsis, brachypodium contains ten cellulose biosynthesis
A (CESA) genes, whose expression patterns were determined in roots using quantitative
RT-PCR and microarray analysis. We found BdCESA1, BdCESA3, BdCESA6, and BdCESA9
were most highly expressed in roots. Therefore, we generated a series of artificial
microRNA (amiR) constructs to knock down expression of these genes. We report the
initial characterization of some of these lines. Because knockouts in key primary cell wall
synthesis genes are often lethal, we screened EMS-mutagenized families for temperaturesensitive
root swelling. We are currently screening 4,600 M2 families for conditional,
recessive alleles. We have identified several segregating families with putative short or
swollen root phenotypes, which are being examined further. Overall, our hope is to shed
light on and identify new players in cellulose biosynthesis and anisotropic growth in the
grasses.
Keywords
Cellulose, Anisotropic growth, CESA, Root development
55

Session 2 – Cell Wall, Biomass and Biofuels
P2.6- Towards the identification of new cell wall-related genes in
Brachypodium distachyon
Timpano H, Darracq O, Badel E, Pollet B, Lapierre C, Höfte H, Sibout R, Vernhettes S, and
Gonneau M.
Institut Jean-Pierre Bourgin, UMR1318-INRA-AgroParisTech, Centre de Versailles-Grignon,
Route de St-Cyr, 78026 Versailles Cedex-France
helene.timpano@versailles.inra.fr
Abstract
The production of second-generation biofuels based on the transformation of plant
biomass is a pressing issue. Biomass is represented by cell walls of the plant cells consisting
of a network of cellulose microfibrils and matrix polysaccharides encrusted by lignin. To
enhance the potential of plant biomass, we first need to provide insights on the
mechanisms of the biosynthesis of cell wall polymers and in particular that of cellulose
microfibrils.
The saccharification yield of the cellulose is the major trait we can improve to use the
biomass as a competitive alternative of fossil energy and Brachypodium is an essential
model species to facilitate research on monocotyledonous crops dedicated to biofuel
production.
From the mutagenized population obtained at the IJPB we selected by visual screening
a particular mutant called spa1. This mutant shares characteristics of the brittle culm
mutants of rice and barley, such as brittleness, irregular xylem, and a cellulose content
deficiency especially in stems, with 50% of the amount found in the wild type.
Interestingly, this mutant is also floppy unlike others brittle culm mutants which remain
upright. Lignin assays indicate a higher amount of lignin and the mechanical strength
defects of spa1 is illustrated by a Young’s modulus three times lower than that of WT.
Complementary approaches are in progress to identify the SPA1 gene : sequencing of
candidate genes related to cell wall synthesis or co-expressed with secondary cell wall
cellulose synthases and a classical mapping strategy combined with NGS methods.
Moreover within the framework of the European RENEWALL and KBBE CellWall projects
and thanks to the co-expression network tool BradiNet (M. Mutwill, KBBE project), RNAi
strategies are in progress to inactivate few genes selected according to specific
expression criteria and potentially involved in cell wall synthesis.
References
Brkljacic J GE, Scholl R, Mockler T, et al. (2011) Brachypodium as a model for the grasses:
Today and the future. Plant Physiol 157: 3-13.
Zhang B, Zhou Y (2011) Rice Brittleness Mutants : A way to open the black box of
monocot cell wall biosynthesis. JIPB 53 : 136-142.
Keywords
Cell wall, cellulose, biofuels, mechanical properties
56

Session 2 – Cell Wall, Biomass and Biofuels
P2.7- Enhancement of biomass production and cell wall degradability by
AtGA20ox1 overexpression and Bd4CL1 downregulation
Voorend Wannes (a)(b), Sibout Richard (c), Bendahmane Abdelahafid (d), Oria Nicolas (c),
Roldán-Ruiz Isabel (a), Muylle Hilde (a), Inzé Dirk (b)
(a) Institute for Agricultural and Fisheries Research (ILVO), Plant Science Unit, Caritasstraat
21, 9090 Melle, Belgium
(b) VIB, Department of Plant Systems Biology, Ghent University, Technologiepark 927, 9052
Gent, Belgium
(c) IJPB-INRA Centre de Versailles-Grignon, Route de St-Cyr (RD10), 78026 Versailles
Cedex, France
(d) URGV-INRACNRS, 2 rue Gaston Crémieux, CP5708 91057 Evry cedex, France
Wannes.Voorend@ilvo.vlaanderen.be
Abstract
Food and feed have always originated mainly from plants, but they can now also supply
energy in the form of biofuel. The supply will be under great strain, however, from the
combination of a rapidly increasing population, higher living standards in developing
countries, and environmental changes. Additionally, future agriculture will have to meet
the needs of a low-carbon economy. For all of these reasons, the next generation of
crops will need to exhibit increased yield and quality. When using whole plant as energy
source, such as for animal feed and bio-ethanol feedstock, more biomass and a higher
glucose release from cell walls will also be required.
This presentation describes a proof of concept to improve cell wall degradability or
digestibility as well as biomass production through transformation and mutagenesis in
Brachypodium Bd21-3 plants.
Our focus lies on altering the lignin content, as this is often related to improved cell wall
degradability. The Brachypodium Bd21-3 TILLING population of INRA Versailles was
screened for mutants in the 4-coumarate ligase (4CL) gene. Fourteen mutations were
identified and plants will be scored for glucose release upon enzymatic hydrolysis.
Lignin mutants can show improved degradability, but these lines often exhibit a lower
yield. An increase in biomass production can be achieved by overexpressing genes (e.g.
GA20ox1 in Arabidopsis) that are known to enhance organ size (leaf and stem). We
currently have 18 UBIL::AtGA20ox1 lines in the Bd21-3 inbred line. Expression levels are
now being analyzed. Promising lines will be phenotyped for enhanced biomass
production.
Ultimately, we will seek to combine the improved properties of cell wall degradability or
digestibility and biomass production by either transforming the plants in a mutant
background or crossing transformed lines.
Keywords
Degradability, biomass, transformation, mutagenesis
57

Session 2 – Cell Wall, Biomass and Biofuels
P2.8- Relationships between maize morphological and biochemical traits with
in vitro cell wall degradability
Yu Zhang 1 Tanya Culhaoglu 1, Brigitte Pollet 1, Corine Melin 2, Dominique Denoue 2, Yves
Barrière 2, Stéphanie Baumberger 1, Valérie Mechin 1
1Institut Jean-Pierre Bourgin, UMR 1318 INRA/AgroParisTech, Pôle SCSM, 78000 Versailles,
France
2Unité de Recherche Pluridisciplinaire Prairies et Plantes Fourragères, 86600 Lusignan,
France
Yu.Zhang@versailles.inra.fr
Abstract
Grasses (maize, rice, wheat, oats, rye…) dominate cultivated cropland, supplying most of
the dietary energy needs of many classes of livestock. Nowadays, the bioenergy’ boom
is an international preoccupation and justifies all the more an investment on
graminaceaous lignocellulosic biomass. Grass cell walls are complex molecular
assemblies involving polysaccharidic (cellulose and hemicelluloses) and phenolic (lignins
and p-hydroxycinnamic acids) components. Lignin association with other cell wall
constituents greatly modifies cell wall properties, including enzymatic degradability of
structural polysaccharides.
In this study 8 maize recombinant inbred lines were selected for their similar lignin content
and variable cell wall degradability to assess both the impact of lignin structure and
organization, and the impact of cell wall reticulation by p-hydroxycinnamic acids on cell
wall degradability independently of the main “lignin content” factor. These recombinant
lines and there parents were analyzed for cell wall residue content, esterified and
etherified ferulic and p-coumaric acids content, lignin content and structure and in vitro
degradability. Amongst these biochemical parameters, the proportion of uncondensed
lignin and the esterified p-coumaric acid content showed high significant correlation
with in vitro cell wall degradability (r = -0.82** and r = -0.72* respectively). We also
investigated the relationships between maize morphological traits and biochemical
factors., The 10 genotypes were thus measured for their plant height (Hplant), the height
and the diameter of the node of the principal ears (Hears, Dears) and the line yield (Yield)
at the ensiling stage..The esterified p-coumaric acid content showed high positive
correlation coefficient with all of the morphological traits (r = 0.63* versus Hplant, r = 0.68*
versus Hears, r = 0.64* versus Dears and r = 0.72* versus Yield). Moreover, morphological traits
were found to be negatively correlated with in vitro cell wall degradability (r = -0.70*
versus Hplant, r = -0.65* versus Hears,). This study revealed that plants with higher in vitro
degradability were also the ones with the more reduced agronomic value.
Keywords
cell wall; in vitro cell wall degradability; esterified p-coumaric acid content; agronomic
value
58

Session 2 – Cell Wall, Biomass and Biofuels
P2.9- Brachypodium as a model for studying traits relevant for the
development of bioenergy grasses
Maurice Bosch 1, Luned Roberts 1, Sue Dalton 1, Lorraine Fisher 1, Paul Robson 1, Iain S
Donnison 1, Thomas Didion 2, Klaus Nielsen 2, Luis A J Mur 1
1Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,
Plas Gogerddan, Aberystwyth SY23 3EB, UK
2 DLF-TRIFOLIUM A/S, Højerupvej 31, 4660 Store Heddinge, Denmark
mub@aber.ac.uk
Abstract
Several perennial grasses, including Miscanthus and switchgrass, are currently being
developed as a renewable source of lignocellulosic biomass derived bioenergy. One of
the main bottlenecks for the commercial exploitation of these feedstocks is the
recalcitrance of the cell wall biomass to conversion into fermentable sugars. A detailed
understanding of the regulatory networks controlling cell wall biogenesis is required to
enable the genetic engineering and molecular breeding of energy crops with improved
conversion characteristics. Brachypodium represents an excellent model for functional
genomic studies that can underpin and accelerate the establishment of energy grasses
as a sustainable replacement of fossil fuels. Several candidate genes identified in a
gene-expression profiling experiment in maize (Bosch et al., 2011) are currently being
tested in Brachypodium for involvement in cell wall biogenesis. Subsequently this
information can be translated to genetically more recalcitrant and undomesticated
energy grasses.
The plant cell wall not only represents a major energy store but is also involved in
defence against environmental stresses, including drought. There is a requirement to
develop drought-tolerant crops that produce significant yields with reduced amounts of
water. In collaboration with DLF-TRIFOLIUM A/S, a leading grass biotechnology company,
we are instigating a project in which we will assess drought responses in the
Brachypodium germplasm collection available at IBERS (Mur et al., 2011) and correlate
changes in expression levels of cell wall related genes with changes in secondary
metabolite profiles and cell wall polysaccharides during drought. We are also initiating a
programme of work that will exploit available genetic resources for Brachypodium to
study the genotypic variation in other important traits that affect yield and composition
in bioenergy crops.
References
Bosch M, Mayer CD, Cookson A, Donnison IS (2011) Identification of genes involved in
cell wall biogenesis in grasses by differential gene expression profiling of elongating and
non-elongating maize internodes. Journal of Experimental Botany 62: 3545-3561.
Mur LAJ, Allainguillaume J, Catalán P, Hasterok R, Jenkins G, Lesniewska K, Thomas I,
Vogel J (2011) Exploiting the Brachypodium Tool Box in cereal and grass research. New
Phytologist 91: 334-347
Keywords
Cell wall, drought, biofuel, yield, Miscanthus
59

Session 3 – Tools and Bioengineering
P3.1- Cold treatment, maltose and modified MS medium improve
embryogenesis in Brachypodium BD21
Sue Dalton, Maurice Bosch, Iain S Donnison
Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,
Plas Gogerddan, Aberystwyth SY23 3EB, UK
snd@aber.ac.uk
Abstract
Brachypodium distachyon accession BD21 identified by Vogel and Hill (2008) is
commonly used for genetic transformation. The media developed for immature embryo
callus induction include Linsmaier and Skoog (LS) medium with 3% sucrose, 2.5mgl -1 2,4-D,
0.6 mgl -1 CuSO4 and no vitamins except 1mgl -1 thiamine and a similar Murashige and
Skoog (MS) medium (Vain et al, 2008) with cysteine and ‘M5’ vitamins (0.5mgl -1 thiamine).
A ‘Modified’ MS medium (MODMS, Duchefa) contains x10 thiamine (1mgl -1) and
improves embryogenesis in Poa and Miscanthus. Maltose also produces softer, faster
growing embryogenic callus than sucrose in some grass species. BD21 embryogenic
callus induction was compared on LS and MODMS media with sucrose and maltose.
Cold treatment and CuSO4 concentration were also examined.
Whole inflorescences were surface sterilised and immature embryos excised and
cultured on eight media comprising MODMS or LS medium, maltose or sucrose and 0.3 or
0.6mgl -1 CuSO4. Embryos were cultured fresh or from inflorescences stored overnight at
4 oC. Embryo age and size varied within each inflorescence, but all were cultured (150-
200 embryos per treatment).
Embryos with embryogenic proliferation were counted after 4 weeks culture at 25 oC in
darkness. With sucrose more cultures were embryogenic on MODMS than on LS based
media. However maltose was generally superior to sucrose and there was an interaction
between cold treatment and medium. With maltose more freshly cultured embryos were
embryogenic on LS based media, but after cold treatment many more were
embryogenic on MODMS media, which contained more vitamins. Copper concentration
made little difference to embryogenesis on MODMS media, although the highest
percentage (42%) was achieved with 0.3mgl -1 CuSO4. After cold treatment,
embryogenesis on LS media was much improved by 0.3 compared with 0.6mgl -1 CuSO4.
Overall the best treatment was overnight cold and culture on MODMS medium with 3%
maltose, 3mgl -1 2,4-D and 0.3 mg/l -1 CuSO4.
References
Vogel J and Hill T. (2008). High-efficiency Agrobacterium-mediated transformation of
Brachypodium distachyon inbred line Bd21-3. Plant Cell Reports, 27: 471-478.
Vain P, Worland B, Thole V, et al. (2008). Agrobacterium-mediated transformation of the
temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional
mutagenesis. Plant Biotechnology Journal, 6: 236–245
Keywords
Embryogenesis, cold treatment, maltose, thiamine, vitamins
60

Session 3 – Tools and Bioengineering
P3.2- Neomycin Phosphotransferase Gene (nptII) as a selectable marker and
enhanced rice ubiquitin promoter as a constitutive promoter for
Brachypodium BD21
Sue Dalton, Maurice Bosch, Paul Robson, Iain S Donnison
Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,
Plas Gogerddan, Aberystwyth SY23 3EB, UK
snd@aber.ac.uk
Abstract
Brachypodium distachyon BD21 is an embryogenic diploid accession and is the most
commonly used material for genetic transformation. Unfortunately hygromycin selection
can be unclear in this and other embryogenic Brachypodium accessions and the
established protocol (Alves et al, 2009) uses GFP as an additional selection. Without GFP
this protocol allows many escapes as selection is reduced from 40 to 30 to 20 mgl -1
hygromycin as calli grow and regenerate plants. This author found only calli that grow
readily and regenerate on continuous selection of 40mgl -1 hygromycin to be definitely
transformed. Experiments with the hygromycin gene under the CaMV promoter (pROB5,
Bilang et al, 1991) have yielded few such easily identifiable transformants and
hygromycin under the actin promoter (pAct1HPT, Cho et al, 1998) is preferred. This
selection gene was compared with the bar gene (pUBA, Toki et al, 1992) and nptII gene
(pBKS ubi-nptII, Paul Robson, IBERS) under the maize ubiquitin promoter and cobombarded
with gus under an enhanced rubi3 rice ubiquitin promoter (pRESQ48,
Sivamani and Qu, 2006).
Calli bombarded with pUBA and selected on bialophos became friable and were not
continued. In this experiment calli bombarded with pAct1HPT produced no obvious
transformants, although weak plants grew. These were not GUS positive however. Calli
bombarded with pUbi-nptII and selected on 100-150 mgl -1 paromomycin were obviously
transformed or not and plants were regenerated under selection. One plant
constitutively expressed GUS and is the first grass to do so with the pRESQ48 construct,
although the plant may be infertile. The nptII gene appears to be a useful and easily
selectable gene for Brachypodium, and the enhanced rice ubiquitin promoter appears
to be constitutive and to not co-suppress the maize ubiquitin promoter.
References
Alves SC, Worland B, Thole V, Snape JW, Bevan MW and Vain P. (2009) A protocol for
Agrobacterium-mediated transformation of Brachypodium distachyon community
standard line Bd21. Nature Protocols 4: 638-649
Bilang R, Iida S, Peterhans A, Potrykus I, Paszkowski J. (1991) The 3’-terminal region of the
hygromycin-B-resistance gene is important for its activity in Eschiricia coli and Nicotiana
tabacum. Gene 100: 247-250
Cho M-J,Jiang W, Lemaux PG. (1998) Transformation of recalcitrant barley cultivars
through improvement of regenerability and decreased albinism. Plant Sci. 138:229-244.
Sivamani E and Qu R. (2006) Expression enhancement of a rice polyubiquitin gene
promoter. Plant Molecular Biology 60:225-239
Toki S, Takamatsu S, Nojiri C, Ooba S, Anzai H, Iwata M, Christensen A, Quail PH, Uchimiya
H. (1992) Expression of maize ubiquitin gene promoter-bar chimeric gene in transgenic
rice. Plant Physiol 100:1503-1507
Keywords
Selection, promoters, enhanced rubi3, nptII, gus
61

Session 3 – Tools and Bioengineering
P3.3- Tools and methods for functional gene analysis in Brachypodium
distachyon. Examples of genes involved in cell wall synthesis.
Oumaya Bouchabké-Coussa*, Camille Soulhat, Madeleine Bouvier d’Yvoire, Sebastien
Antelme, Philippe Lebris and Richard Sibout
INRA, Institut Jean-Pierre Bourgin, 78026 Versailles, France
Oumaya.Bouchabke@versailles.inra.fr
Abstract
We are involved in developing many resources for Brachypodium: TILLING collection,
natural accessions collection, phenotyping and gene transfer via Agrobacterium
tumefaciens. We developed a genetic transformation platform for efficient gene
functional analysis. A set of binary vectors specific for monocots is available in our lab as
well as an efficient method using different selective agents. We use Brachypodium as a
model system for studying how to modify grass crops for increased biofuel production
made from lignocellulosic cell walls. Some lignin mutants from the monolignol
biosynthesis pathway were recently identified by phenotyping and TILLING, their
functional analysis is underway notably through complementation by overexpression of
concerned gene in mutant background. Other interesting candidate genes involved in
cell wall biosynthesis were identified by sequence similarity between Brachypodium and
other plant species. When mutants were not available, corresponding sequences were
cloned from Brachypodium genome and their validation is in progress through RNAi,
amiRNA or over-expression strategies. Preliminary characterizations of transgenics plants
are shown.
Keywords
Brachypodium distachyon, gene transfer, Agrobacterium tumefaciens, cell wall.
62

Session 3 – Tools and Bioengineering
P3.4- Construction of Activation T-DNA-Tagging Mutant Pool in Brachypodium
distachyon
Shin-Young Hong, Jae-Hoon Jung, Pil Joon Seo, Chung-Mo Park
Department of Chemistry, Seoul National University, Seoul 151-741, Korea
cmpark@snu.ac.kr
Abstract
Activation tagging is a powerful tool for gene discovery and functional genomics in
plants. A pool of mutant can be generated by randomly inserting a T-DNA enhancer
element into the genome of target plants. Consequently, a gene can be
transcriptionally activated by the nearby insertion of the enhancer element, resulting in a
gain-of function phenotype. The activation tagging mutagenesis has been employed
successfully in both dicot and monocot plants species, such as Arabidopsis and rice, and
contributed to functional characterization of numerous genes.
Here, we report an activation tagging system that was successfully employed in a
model grass Brachypodium distachyon, in which a multimeric transcriptional enhancer
element of the Cauliflower Mosaic Virus (CaMV) 35S promoter was randomly integrated
into the genome of an inbred line Bd21-3. The activation tagging T-DNA vector pGA2715
contains a promoterless b-glucuronidase reporter gene (GUS) with an intron and multiple
splicing donor and acceptor sequences immediately next to the right border. The
multimeric transcriptional enhancer of the CaMV 35S promoter is located adjacent to
the left border.
So far, we obtained more than 1,200 transgenic lines. The genomic sequence
regions flanking the T-DNA insertion sites were determined by inverse PCR and DNA
sequencing. More than 472 transgenic lines were characterized, and 315 independent T-
DNA loci, which represents 67% of the sequence transgenic lines, were assigned to
different genes. We are aiming to obtain at least 1000 independent tagging lines by the
end of 2011 and carry out gene functional studies.
References
Ayliffe MA, Pryor AJ (2011) Activation tagging and insertional mutagenesis in barley.
Methods in Molecular Biology 678: 107-128.
Wan S, Wu J, Zhang Z, et al. (2009) Activation tagging, an efficient tool for functional
analysis of the rice genome. Plant Molecular Biology 69: 69-80.
Pogorelko GV, Fursova OV, Ogarkova OA, Tarasov VA (2008) A new technique for
activation tagging in Arabidopsis. Gene 414: 67-75.
Jeong DH, An S, Park S, et al. (2006) Generation of a flanking sequence-tag database for
activation-tagging lines in japonica rice. Plant Journal 45: 123-132.
Jeong DH, An S, Kang HG, et al. (2002) T-DNA insertional mutagenesis for activation
tagging in rice. Plant Physiology 130: 1636-1644.
Keywords
Activation tagging, T-DNA tagging, Brachypodium distachyon
63

Session 3 – Tools and Bioengineering
P3.5- pBrachyTAG: A novel vector system for T-DNA tagging and trapping in
grasses
Vera Thole, Barbara Worland and Philippe Vain
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich NR4
7UH, United Kingdom.
vera.thole@bbsrc.ac.uk
Abstract
Over the past decade, Brachypodium distachyon has been established as a new
experimental and genomics model system to bridge research into temperate cereals
and to promote research in biomass crops. Recently, we have developed highly efficient
systems to produce T-DNA insertion lines (Alves et al., 2009, Vain et al., 2008) and to
retrieve flanking sequence tags (FSTs) (Thole et al., 2009) from mutant lines. Here, we
report the construction and testing of improved transformation vectors, pBrachyTAG, for
T-DNA tagging and promoter trapping in grasses. The pBrachyTAG vectors are based on
the pCLEAN dual binary vector system (Thole et al., 2007) and are designed to address
current limitations in characterising flanking plant genomic sequences and creating
functional gene fusions in trapping strategies. These new vectors contain features known
(i) to reduce vector backbone transfer, (ii) to limit the introduction of superfluous DNA
sequences, (iii) to improve transformation efficiency, and (iv) to enable more than one
FST retrieval strategy. The performance of the pBrachyTAG vectors for insertional
mutagenesis and promoter trapping will be presented, discussed and compared to
generic binary vectors such as pVec8-GFP previously used in our studies (Thole et al.,
2010).
References
Alves SC, Worland B, Thole V, et al. (2009) A protocol for Agrobacterium-mediated
transformation of Brachypodium distachyon community standard line Bd21. Nature
Protocols 4: 638-649.
Thole V, Alves SC, Worland B, et al. (2009) A protocol for efficiently retrieving and
characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA
insertional mutants. Nature Protocols 4: 650-661.
Thole V, Worland B, Snape JW, Vain P (2007) The pCLEAN dual binary vector system for
Agrobacterium-mediated plant transformation. Plant Physiology 145: 1211-1219.
Thole V, Worland B, Wright J, et al. (2010) Distribution and characterization of more than
1000 T-DNA tags in the genome of Brachypodium distachyon community standard line
Bd21. Plant Biotechnology Journal 8:734-747.
Vain P, Worland B, Thole V, et al. (2008) Agrobacterium-mediated transformation of the
temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional
mutagenesis. Plant Biotechnology Journal 6: 236-245.
Keywords
Binary vector, Agrobacterium, T-DNA tagging, T-DNA trapping, Flanking sequence tag
64

Session 4 – Biotic and Abiotic Stresses
P4.1- Genetics of Rice (Oryza sativa L.) under Normal and Water Stress
Conditions
Muhammad Ashfaq and Abdus Salam Khan
Department of Plant Breeding and Genetics, University of Agriculture Faisalabad,
Pakistan
ashfaq_qs@yahoo.com, muhammad.ashfaq@mail.mcgill.ca,
Abstract
The experiment was conducted in Department of Plant Breeding and Genetics, University
of Agriculture Faisalabad to study the genetic basis of drought tolerance in rice (Oryza
sativa). Forty diverse rice genotypes were studied under field condition for various
morphological traits in year 2008. These genotypes were evaluated for drought tolerance
on the basis of physiomorphological traits and some seed traits of rice grain. After this
from these forty, twenty genotypes were grown in polythene bags to study the root shoot
traits at seedling stage under normal and water stress condition for the selection of
diverse parents. A total 28 SSR markers were also used to see genetic diversity among the
twenty genotypes of rice. More diversity was observed between improved basmati rice
varieties and advance breeding lines. Less diversity was observed in basmati varieties in
comparison with the rice advance lines. All the 28 SSR markers showed greatest
polymorphism among the rice genotypes. But some SSR markers showed highest
polymorphism among the rice genotypes. Some highest polymorphic markers showed
more variation among the genotypes namely RM-421, RM-254, RM-235, RM-544, RM-257,
RM-224, RM-248 and RM-590. Eight parents were selected on the basis of phenotypic and
genotypic screening for the development of F1 hybrids by using diallel mating design to
see the gene action among the parents and their F1 hybrids. All the possible
combinations were made between the parents excluding reciprocals. These experiments
were conducted in the green house and various morphological traits were studied under
both normal and stress conditions. All the possible combinations were made between
the parents excluding reciprocals. These experiments were conducted in the green
house and various morphological traits were studied under both normal and stress
conditions. Stress was given at the reproductive stage. Data were analyzed by using
Hayman graphical approach and Griffing approach to study the genetic variance and
combining ability analysis among the parents and their F1 hybrids. Griffing analysis
genotypes CB-17, CB-32 and Basmati-198 were found good general combiners for
productive tillers per plant, primary branches per panicle and yield per plant, especially
under water stress condition. Maximum specific combining ability was found in Basmati-
198 × CB-17 for Productive tillers per plant, Basmati-198 × CB-42 for Primary branches per
panicle and CB-32 × CB-14 for Yield per plant.
References
Hayman, B.I. 1954. The theory and analysis of diallel crosses. Genetics 30:789-809.
Griffing, B. 1956. Concept of general and specific combining ability in relation to
diallelmating systems. Australian J. Biol. Sci. 9:463-493.
Keywords
Rice, Genetic diversity, Correlation, SSR marker, Morphological traits
65

Session 4 – Biotic and Abiotic Stresses
P4.2- Metabolic reprogramming in pre-symptomatic phases of Magnaporthe
grisea infection of susceptible and resistant Brachypodium distachyon plants
Hassan Zubair, Manfred Beckmann, Kathleen Tailliart and John Draper
Institute of Biological, Environmental and Rural Sciences, Aberystwyth University,
Ceredigion, UK
hhz@aber.ac.uk
Abstract
Metabolite fingerprinting using flow infusion electrospray MS in combination with
machine learning data mining provided a primary discovery tool to investigate the reprogramming
of plant’s early defence metabolism in pre-symptomatic leaf tissues during
Magnaporthe grisea (M. grisea) infection of Brachypodium distachyon (B. distachyon).
Two B. distachyon ecotypes, one susceptible (ABR1) and other resistant (ABR5) to blast
infection were used to elucidate pathogen-induced changes. Explanatory m/z signals
were annotated using ultra-high accurate mass analysis by Fourier-transform ion
cyclotron resonance (FT-ICR) MS and the accurate mass database MZedDB searched at
< 1ppm mass accuracy.
Susceptible interactions of M. grisea with ABR1 plants showed an early metabolic shift
during penetration of the fungal pathogen into epidermal cells 24-28 h after inoculation.
A disruption of the TCA cycle and energy metabolism, as well as amino acid biosynthesis
was evident during this phase. Effects on trisaccharide and flavonoid synthesis were also
evident. However, most of these changes were not observed in the infection of resistant
ABR5 plants.
A metabolic response to M. grisea infection in ABR5 started < 8 h after inoculation. By 24
h (pre-penetration stage) infected ABR5 plants had already accumulated specific
antifungal compounds (e.g. sphingofungin E) at significantly high levels. A range of other
compounds accumulated in ABR5 plants by 24 h included phospholipids such as
PC(18:2), PC(18:3) and 2-16:1-lysoPG, along with pantothenate and melibionate.
These metabolome alterations in pathogen-challenged leaf tissues (both ABR1 and ABR5
plants) prior to penetration of first epidermal cell, and during penetration itself were
striking and suggests that M. grisea is able to manipulate host’s defence metabolism in
susceptible plants in a very early phase of the infection process. Host enzyme-activities,
potentially modulated by the invading pathogen, are currently being explored.
Keywords
Metabolic re-programming, Magnaporthe grisea, Brachypodium distachyon, presymptomatic
infection, susceptible and resistance responses
66

Session 4 – Biotic and Abiotic Stresses
P4.3- Brachypodium distachyon diversity and drought response: establishing
a reference experiment for automated phenotyping
John Draper, Alan Gay, Hassan Zubair, Gordon Allison, Manfred Beckmann, Kathleen
Tailiart and John Doonan
Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,
Ceredigion, UK
hhz@aber.ac.uk
Abstract
Automated, comprehensive phenotyping of plant genotype collections is expected to
offer new insight into genetic diversity and functional genomics. The UK National Plant
Phenomics Centre based at IBERS will be working with partners internationally to refine
protocols, calibrate, and where possible standardise cross-platform phenotyping
approaches suitable for future data integration. The phenotyping approach in
Aberystwyth is based on a LemnaTec conveyor system that offers opportunity to
measure non-invasively dynamic aspects of growth architecture and development (both
aerial and underground) as well as monitor aspects of water physiology, temperature
control and stress responses. Specific sampling stations allow for destructive sampling of
plant tissue for further analysis , such as transcriptomics and metabolomics.
We describe the functional aspects of the National Plant Phenomics Centre and outline
plans for characterising the growth, development and responses to drought stress in a
small collection of diverse B. distachyon ecotypes. Specifically we intend to image a
selection of highly diverse B. distachyon genotypes collected from a range of habitats
differing in altitude, average temperature and rainfall.
One aspect of EU FP7 plans to use automated phenotyping in systems level biology is to
integrate data with that derived from comprehensive ‘chemical phenotyping’. With this
in mind metabolite fingerprinting using flow infusion electrospray MS in combination with
machine learning data mining will provide a primary discovery tool to investigate the reprogramming
of plant metabolism in response to drought.
Keywords
Automated phenotyping, Imaging, Metabolic re-programming, Brachypodium
distachyon, drought stress
67

Session 5 – Plant Development, Metabolism and Physiology
P5.1- Describing and modelling root and shoot growth and development in
Brachypodium distachyon (L.) Beauv.
Delory Benjamin*, Delaplace Pierre, Gfeller Aurélie, Fauconnier Marie-Laure, du Jardin
Patrick
University of Liège, Gembloux Agro-Bio Tech, Plant Biology Unit, Gembloux, Belgium
* Benjamin.Delory@student.ulg.ac.be
Abstract
Due to its small size, its short developmental cycle and its close phylogenetic relationship
with the Triticeae tribe, Brachypodium distachyon (L.) Beauv. has been proposed as a
model species for temperate cereals [1-3]. In this context, this work aims to describe and
model root and shoot growth and development of B. distachyon (Bd21-1) grown under
controlled environmental conditions [22°C, 65% RH, 20h light, 95 µmol.m -2.s -1 (PAR, LED
lighting)]. For this purpose, vernalized caryopses were sown in a substrate consisting of
vermiculite and compost (80/20, v/v). Growth and development of the above and
belowground parts were monitored for 70 days. Dry and fresh masses of plant organs
were measured every seven days from sowing. Biomasses of adventitious and seminal
roots were measured separately. The number of spikelets on the main stem and on tillers
was also counted on plants aged of 70 days.
The modelling of root and shoot growth was achieved by calibrating sigmoidal growth
models to the mean biomass values measured at each day of analysis. For each plant
organ, the growth model selected was the one with the lowest residual variance. Finally,
developmental stages identified for B. distachyon were compared with those defined for
cereal crops by Zadoks et al. (1974) [4].
Maximum rates of fresh and dry shoot biomass production were 29,5 and 14,1 mg.day -1
respectively. Based on modelling, these values seem to be reached 49 and 72 days after
sowing. Results also show that the fresh mass of adventitious roots at day 42 is significantly
higher than that of seminal roots. Maximum rates of fresh and dry root biomass
production were 6,9 and 0,8 mg.day -1 respectively, and were reached after 37 and 43
days.
References
1. Draper J, Mur LAJ, Jenkins G, et al. (2001) Brachypodium distachyon. A new model
system for functional genomics in grasses. Plant Physiology 127: 1539-1555.
2. Garvin DF (2007) Brachypodium: a new monocot model plant system emerges.
Journal of the Science of Food and Agriculture 87: 1177-1179.
3. Watt M, Schneebeli K, Dong P, Wilson IW (2009) The shoot and root growth of
Brachypodium and its potential as a model for wheat and other cereal crops. Functional
Plant Biology 36: 960-969.
4. Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the growth stages of
cereals. Weed Research 14: 415-421.
Keywords
root growth, shoot growth, Brachypodium distachyon, growth modelling
68

Session 5 – Plant Development, Metabolism and Physiology
P5.2- Endosperm development in Brachypodium
Philip Hands 1, Magdalena Opanowicz 3, Donna Betts 1, Mary L. Parker 2, Geraldine A.
Toole 2, E. N. Claire Mills 2, John H. Doonan 3, Sinéad Drea 1
1 Department of Biology, University of Leicester, University Rd, Leicester LE1 7RH, UK.
2 Institute of Food Research, Norwich, NR4 7UA, UK
3 The Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth
University, Penglais, Aberystwyth, Ceredigion, SY23 3DA
psh14@le.ac.uk
Abstract
Brachypodium distachyon has become an important model system for the temperate
grasses. It is the first member of the economically important Pooideae group and the first
wild grass species to be sequenced. This comparative analysis of grain structure and
development in B. distachyon to the cultivated cereal grains provides insight into the
nature of temperate cereal grain development and to the effects of the domestication
process. Analyses reveal differences in endosperm tissue organisation, cell structure and
grain storage reserves between Brachypodium and wheat. Molecular marking of
domains within the developing Brachypodium grain using mRNA in situ hybridisation
reveals distinct differences in endosperm differentiation. Most notably, the aleurone
layer is not as distinct or regionally differentiated as in wheat and a modified aleurone
region is absent. Cell walls in the central endosperm and nucellar epidermis are
unusually prominent and the large nucellar epidermis layer is persistent throughout grain
development, suggesting a role in grain filling mechanisms. These significant grain
structural differences may reflect Brachypodium’s phylogenetic position and its position
intermediate between the triticeae and rice.
References
Opanowicz M, Hands P, Betts D, Parker ML, Toole GA, Mills ENC, Doonan JH, Drea S (2011)
Endosperm development in Brachypodium distachyon. J Exp Bot 62: 735–748
Keywords
Grain development, endosperm, aleurone
69

Session 5 – Plant Development, Metabolism and Physiology
P5.3- Unraveling the functional diversity of the CYP73 family in Brachypodium:
is there more than 4-hydroxylation of cinnamic acid?
Hugues Renault 1, Jean-Etienne Bassard 1, Richard Sibout 2, Martine Schmitt 3, François
André 4, Danièle Werck-Reichhart 1
1Institut de Biologie Moléculaire des Plantes – CNRS UPR2357, Université de Strasbourg, 28
rue Goethe, 67083 Strasbourg, France
2Institut Jean-Pierre Bourgin – UMR1318 INRA-AgroParisTech, Route de Saint-Cyr (RD10),
78026 Versailles, France
3Laboratoire d’Innovation Thérapeutique (LIT) – CNRS, Université de Strasbourg, Faculté
de Pharmacie, 74 route du rhin, 67400 Illkirch, France
4Service de Bioénergétique, Biologie Structurale et Mécanismes (SB 2SM) – CEA, CNRS
URA2096, Gif-sur-Yvette, France
hugues.renault@ibmp-cnrs.unistra.fr
Abstract
Plant phenolic compounds contribute to the agronomic, industrial and nutritional
performances of agricultural and forest resources (Vogt, 2010). Genes in the phenolic
metabolism are usually present in single or low copy number. Duplications are conserved
across taxa, which is indicative of gene specialization and branching in the pathway.
Cytochromes P450 of the CYP73 family catalyze the 4-hydroxylation of cinnamic acid
(C4H activity), an early and obligatory step in the biosynthesis of most phenolic
compounds such as lignins monomers, flavonoids, coumarins, stilbenes, lignans and
tannins. Growing evidence, based on protein alignment and phylogenetic analysis,
suggest that two classes of CYP73 occur in angiosperms (Ehlting et al., 2006). Class I
CYP73s are mainly expressed in lignified tissues and are meant to play function in the
general phenylpropanoid pathway. Class II CYP73s are atypical in that they have a
longer N-terminal membrane anchor and preferential expression in flower and roots. In
Brachypodium genome, we identified three CYP73 genes. Two paralogs encode class I
C4Hs while the third one encodes a class II protein. Orthologs of the three genes are
found in all sequenced monocot genomes. The PHENOWALL ANR project is aimed at
clarifying the specific functions of the three C4Hs. Each protein will be expressed in yeast
for testing catalytic properties with a large set of natural and synthetic substrates.
Resulting data will help elaborating substrate pharmacophores to be docked in 3D
model of each protein. Enzyme functions in planta will be addressed by investigating
phenotype of TILLING mutants and silenced/overexpressor lines. Likewise, promoter-GUS
lines are being produced for fine description of the expression pattern of each CYP73.
References
Ehlting J, Hamberger B, Million-Rousseau R, Werck-Reichhart D (2006) Cytochromes P450
in phenolic metabolism. Phytochemistry 5: 239-270
Vogt T (2010) Phenylpropanoid biosynthesis. Molecular Plant 3: 2-20
Keywords
Phenolic metabolism, lignin, cell wall, cytochrome P450, gene duplication
70

Session 5 – Plant Development, Metabolism and Physiology
P5.4- Starch bioengineering in Brachypodium distachyon
Vanja Tanackovic 1, Jan T. Svensson 1, Mikkel A. Glaring 2, Massimiliano Carciofi 3, Susanne
Langgård Jensen 1, Andreas Blennow 1
1 VKR Research Centre Pro-Active Plants, Department of Plant Biology and
Biotechnology, Faculty of Life Sciences, University of Copenhagen, DK-1871,
Frederiksberg C, Denmark
2 Department of Agriculture and Ecology, Faculty of Life Sciences, University of
Copenhagen, DK-1871, Frederiksberg C, Denmark
3 Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus
University, DK-4200 Slagelse, Denmark
vtana@life.ku.dk
Abstract
Brachypodium distachyon was recently introduced as a model plant for temperate
cereals (Opanowicz et al., 2008; IBI 2010). In order to explore pre-domesticated and
novel features of cereal starch metabolism we aim to establish Brachypodium as a
model. Bioinformatics analysis identified starch biosynthesis genes including seven soluble
starch synthases (SS), two granule bound starch syntheses (GBSS), four starch branching
enzymes (SBE), two glucan- and one phosphoglucan- water dikinases (GWD, PWD).
Transit peptides and putative carbohydrate-binding modules (CBMs) of the families
CBM20, CBM45, CBM48 and CBM53 were identified. The gene setup for starch
biosynthesis is very similar to barley and a phylogenetic analysis based on the SS genes
provided evidence for a close relation to barley and wheat.
To investigate the starch structural features, grain starch from two lines, Bd21 and Bd21-3
were characterized. Microscopic, chemical and structural data including amylopectin
chain length distribution, phosphate content, amylose content of the starch granules
provided evidence for a close structural relationship to temperate cereals even though
kernel starch content and starch granule size were considerably lower and -glucan
content was much higher than that for barley (Hordeum vulgare). Small-angle and wideangle
X-ray scattering (SAXS and WAXS) show low crystallinity of Brachypodium starch
granules as compared to barley. These data were confirmed by differential scanning
calorimetry (DSC) data.
Further steps include Agrobacterium-mediated and biolistic transformations and mutant
analyses of starch biosynthesis genes of interest to provide evidence for their specific
action in this grass as compared to domesticated cereals.
Our data show that Brachypodium distachyon can provide a valuable and efficient
model for starch bioengineering in temperate cereals.
References
Opanowicz, M, Vain, P, Draper, J, Parker D and Doonan, JH (2008) Brachypodium
distachyon: making hay with a wild grass, Trends Plant Sci. 13, 172-177.
The International Brachypodium Initiative (2010) Genome sequencing and analysis of the
model grass Brachypodium distachyon, Nature 463, 763–768.
Keywords
Brachypodium distachyon, model plant, grain starch, starch biosynthesis genes, starch
bioengineering
71

AUTHORS INDEX
72

S: Speakers’ abstracts P: Posters’ abstracts
ADAM G. S4.4 GAO C. -
ANTELME S. S2.2, S3.6, GARVIN D.F. S4.6, S4.8
S4.7, P2.4, GAYMARD F. -
P3.3 GIRALDO P. P1.2
ASHFAQ M. P4.1 GONNEAU M. P2.6
BAKAN B. - GONZALEZ DE LA CALLE V. H. S5.2
BARATINY D. - GONZALEZ GARCIA M. de la C. -
BARRIERE Y. P2.1, P2.8 GOUJON T. -
BELCRAM H. - GUBAEVA E. -
BENAVENTE E. S4.1, P1.2 HANDS P. S5.4, P5.2
BERGER A. - HARDTKE C. S1.6
BERQUIN P. - HARRINGTON M. P2.2
BERTOLINI E. S4.2 HAZEN S. S2.5, P2.5
BHALLA P. - HIMURO Y. -
BILU A. - HOFTE H. S2.7, S3.4,
BLONDET E. - S3.6, P2.2,
BLUEMKE A. S4.9 P2.6
BOSCH M. P2.9, P3.1, HO-YUE-KUANG M.S. -
P3.2 IDZIAK D. S3.2
BOUCHABKE-COUSSA O. S2.7, P3.3 JAMET E. S2.6
BOUCHEZ D. - JØRGENSEN B. -
BOUVIER D'YVOIRE M. S2.2, S3.6, JOUANIN L. S2.2, S3.6,
P3.3 P2.4
BOUYER D. - JUNG J.-H. P3.4
BREIMAN A. S4.1, P1.1 JURANIEC M. -
CATALAN P. S1.2 KRAPP A. -
CAZALIS R. - KUMLEHN J. S3.7
CHATEIGNER-BOUTIN A.-L. S5.1 LAPIERRE C. S2.2, S3.6,
CHUPEAU M.-C. - P2.2, P2.4,
CHUPEAU Y. S2.7 P2.6
CLAUSEN S.S. - LARRE C. S5.1
COURTIAL A. P2.1 LE BRIS P. -
DALMAIS M. S3.6 LECOMTE P. -
DARRACQ O. S2.2, S3.6, LEGAY S. P2.4
P2.4, P2.6 LEPINIEC L. -
DAVID L. - LEVI BAR SHALOM A. -
DELAPLACE P. S5.3, P5.1 LOUDET O. -
DELL'ACQUA M. - MACADRE C. S4.3
DELORY B. P5.1 MAILLET F. -
DINH THI V. H. - MARCEL T.C. S4.7, S4.8
DOOHAN F. S4.5 MARION-POLL A. -
DOONAN J. S5.4, P4.3, MARRIOTT P. S2.8
P5.2 MARTIN M. -
DOUCHE T. S2.6 MASCLAUX-DAUBRESSE C. -
DUBREUCQ B. S5.1 MAZEL J. -
DUFRESNE M. S4.3, S4.7 MAZZAMURRO V. S4.8
DURAND-TARDIF M. - MECHIN V. P2.8
EL GUEDDARI N.E. - MICA E. S4.2
ENARD C. - MOCHIDA K. S3.3
FERRARIO-MERY S. - MORIN H. -
GANDOUR M. P1.3 MOSCOU M. -
73

MOUILLE G. S2.7, S2.2, VALDIVIA E. S5.6
S2.4 VARIN S. S5.3
MRAVEC J. S2.7 VEDELE F. -
MULLINS E. S4.5 VERBRUGGEN N. -
MUR L.A.J. S1.2, S4.1, VERNHETTES S. P2.6
P2.9
MUROZUKA E. S4.5 VOGEL J.P. S2.4, S3.5,
MUTWIL M. S3.4 P1.4
MUYLLE H. P2.3, P2.7 VOIGT C. S4.9, S2.1
NEJI M. P1.3 VOOREND W. P2.7
NG C. S5.5 VOZABOVA T. -
NICHOLSON P. S1.3 WANG Y. -
O'DRISCOLL A. S4.5 WARD E. -
ORIA N. S3.6, P2.7 WIESENBERGER G. -
PACHECO VILLALOBOS D. S1.6 WORLAND B. P3.5
PANNETIER C. - WULFF B. -
PARK C.-M. P3.4 ZHANG Y. P2.8
PASQUET J.-C. S4.3 ZUBAIR H. P4.2, P4.3
PECCHIONI N. S4.8
PELLETIER G. -
PERALDI A. S1.3
PERSSON S. S3.4
POIRE R. S3.5
PUENTE P. -
RASMUSSEN S.K. -
RENAULT H. P5.3
REYMOND M. P2.1
RIZZOLATTI C. -
SALSE J. S1.1
SANGUINET OSMONT K. S2.5, P2.5
SAUNIER DE CAZENAVE M. S5.3
SAVAS G. -
SCAGNELLI A. -
SCHNITTGER A. -
SCHULMAN A. -
SEO P.J. P3.4
SIBOUT R. S2.2, S2.7,
S3.4, S3.6,
S4.7, P2.2,
P2.4, P2.6,
P2.7, P3.3,
P5.3
SOULHAT C. P3.3
SRIVASTAVA V. S1.4
SUER S. S1.5
TANACKOVIC V. P5.4
THEVENIN J. -
THOLE V. S3.1, S5.4,
P3.5
TIMPANO H. P2.6
TORRES-ACOSTA J.A. S4.4
TUNA M. P1.4
VAIN P. S3.1, S5.4,
P3.5
74

LIST OF PARTICIPANTS
75

ADAM Gerhard
Univ. of Natural Res. & Life Sci. (BOKU),
Konrad Lorenz Str. 24,
3430 Tulln , Austria
gerhard.adam@boku.ac.at
ANTELME Sébastien
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr
78000 Versailles, France
sebastien.antelme@versailles.inra.fr
ASHFAQ Muhammad
Department of Plant Breeding and
Genetics, University of Agriculture,
Room No 6-D Z.A, Hashmi Hall,
38000 Faisalabad, Pakistan
muhammad.ashfaq@mail.mcgill.ca
BAKAN Bénédicte
INRA BIA, Rue de la Géraudière, BP71627,
44316 Nantes, France
bakan@nantes.inra.fr
BARATINY Davy
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
davy.baratiny@versailles.inra.fr
BARRIERE Yves
INRA Lusignan, RD 150,
86600 Lusignan, France
yves.barriere@lusignan.inra.fr
BELCRAM Harry
INRA URGV - Génomique Végétale,
2 rue Gaston Crémieux,
91000 Evry, France
belcram@evry.inra.fr
BENAVENTE Elena
Universidad Politecnica de Madrid, Dpto.
Biotecnoloía, ETSIA
28040 Madrid, Spain
e.benavente@upm.es
BERGER Adeline, INRA IJPB Institut Jean-
Pierre Bourgin - umr 1318, Rd 10 - Route de
Saint-Cyr,
78000 Versailles, France
adeline.berger@versailles.inra.fr
BERQUIN Patrick
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr
78000, Versailles, France
patrick.berquin@versailles.inra.fr
BERTOLINI Edoardo
Scuola Superiore Sant'Anna of Pisa, c/o
ENEA RC Casaccia V. Angiullarese 301,
123 Rome, Italy
e.bertolini@sssup.it
BHALLA Prem
The University of Melbourne, Grattan
Street,
VIC 3010 Parkville, Australia
premlb@unimelb.edu.au
BILU Alon
Evogene Ltd., Gad Fainstiain 13,
Rehovot, Israel
alonb@evogene.com
BLONDET Eddy
INRA URGV - Génomique Végétale, 2 rue
Gaston Crémieux,
91000 Evry, France
blondet@evry.inra.fr
BLUEMKE Antje
University of Hamburg, Biocenter Klein
Flottbek, Ohnhorststr. 18,
22609 Hamburg, Germany
antje.bluemke@botanik.uni-hamburg.de
BOSCH Maurice
Institute of Biological, Environmental &
Rural Sciences (IBERS), Aberystwyth
University, Gogerddan, Aberystwyth,
SY23 3EB, Aberystwyth, United Kingdom
bosch.maurice@gmail.com
76

BOUCHABKE-COUSSA Oumaya
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Oumaya.Bouchabke-Coussa@versailles.inra.fr
BOUCHEZ David
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
david.bouchez@versailles.inra.fr
BOUVIER D'YVOIRE Madeleine
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
madeleine.bouvier-dyvoire@versailles.inra.fr
BOUYER Daniel
CNRS - IBMP Institut de Biologie Moléculaire
des Plantes, 12, Rue General Zimmer,
67084 Strasbourg, France
daniel.bouyer@ibmp-cnrs.unistra.fr
BREIMAN Adina
Tel Aviv University, Dept of Plant Sciences,
69978 Tel Aviv, Israel
adina@post.tau.ac.il
CATALAN Pilar
High Polytechnic School of Huesca,
University of Zaragoza, Ctra. Cuarte km 1,
22071 Huesca, Spain
pilar.catalan09@gmail.com
CAZALIS Roland
URBV, University of Namur,
61 rue de Bruxelles,
5000 Namur, Belgium
roland.cazalis@fundp.ac.be
CHATEIGNER-BOUTIN Anne-Laure
INRA BIA, Rue de la Géraudière, BP71627,
44316 Nantes, France
anne-laure.chateigner-boutin@nantes.inra.fr
CHUPEAU Marie-Christine
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
marie-christine.chupeau@versailles.inra.fr
CHUPEAU Yves
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
yves.chupeau@versailles.inra.fr
CLAUSEN Signe Sandbech
Risø National Laboratory for Sustainable
Energy, BIO-309, P.O. Box 49,
Frederiksborgvej 399,
4000 Roskilde, Denmark
sicl@risoe.dtu.dk
COURTIAL Audrey
UPS LRSV UMR5546 UPS/CNRS,
Pôle de Biotechnologies Végétales 24,
Chemin de Borde Rouge, BP42617,
31326 Castanet-Tolosan, France
courtial@lrsv.ups-tlse.fr
DALMAIS Marion
INRA URGV - Génomique Végétale,
2 rue Gaston Cremieux,
91000 Evry, France
m.dalmais@evry.inra.fr
DARRACQ Olivier
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
o_darracq@hotmail.com
DAVID Laure
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Laure.David@versailles.inra.fr
DELAPLACE Pierre
University of Liège,
Gembloux Agro-Bio Tech,
2, Passage des Deportes,
5030 Gembloux, Belgium
pierre.delaplace@ulg.ac.be
77

DELL'ACQUA Matteo
Scuola Superiore Sant'Anna of Pisa,
Piazza martiri della Libertà, 33 ,
56127 Pisa, Italy
m.dellacqua@sssup.it
DELORY Benjamin
University of Liège, Gembloux Agro-Bio
Tech, 2, Passage des Deportes,
5030 Gembloux, Belgium
Benjamin.Delory@student.ulg.ac.be
DINH THI Vinh Ha
INRA URGV - Génomique Végétale,
2 rue gaston crémieux,
91057 Evry, France
dinhthi@evry.inra.fr
DOOHAN Fiona
University College Dublin,
Belfield, Dublin 4,
Dublin, Ireland
fiona.doohan@ucd.ie
DOONAN John
National Plant Phenomics Centre,
Gogerddan campus, Aberystwyth
University,
SY23 3EB Aberystwyth, United Kingdom
john.doonan@aber.ac.uk
DOUCHE Thibaut
UPS LRSV UMR5546 UPS/CNRS, Pôle de
Biotechnologies Végétales,
24, chemin de Borde Rouge, BP42617,
31326 Castanet-Tolosan, France
douche@lrsv.ups-tlse.fr
DUBREUCQ Bertrand
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
bertrand.dubreucq@versailles.inra.fr
DUFRESNE Marie
Institut de Biologie des Plantes,
rue Noetzlin,
91405 Orsay cedex, France
marie.dufresne@u-psud.fr
DURAND-TARDIF Mylène
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Mylene.Durand-Tardif@versailles.inra.fr
EL GUEDDARI Nour Eddine
IBBP, Hindenburgplatz 55,
48143 Münster, Germany
guedari@uni-muenster.de
ENARD Corine
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Corine.Enard@versailles.inra.fr
FERRARIO-MERY Sylvie
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Sylvie.Ferrario@versailles.inra.fr
GANDOUR Mhemmed
Center of Biotechnology of Borj-Cédria,
BP 901 hammam lif,
2050 Hammam-lif, Tunisia
gandourmed@yahoo.fr
GAO Caixia
Institute of Genetics and Developmental
Biology, Da Tun Lu Rd,
Chaoyang District,
100101 Beijing, P.R.China
cxgao@genetics.ac.cn
GARVIN David F.
USDA-ARS Plant Science Research Unit,
St. Paul, MN 55108, Unit. States of America
David.Garvin@ars.usda.gov
GAYMARD Frédéric
INRA Dept BV, Bat 7, 2 Place Viala,
34060 Montpellier, France
bv@supagro.inra.fr
78

GIRALDO Patricia
E.T.S.I. Agronomos. Universidad Politecnica
de Madrid,
Av. Complutense s/n. ,
28040 Madrid, Spain
patricia.giraldo@upm.es
GONNEAU Martine
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Martine.Gonneau@versailles.inra.fr
GONZALEZ DE LA CALLE Virginia
Centro de Biotecnología y Genómica de
Plantas, ETSI Agrónomos,
Universidad Politécnica de Madrid,
Campus de Montegancedo,
28223 Pozuelo de Alarcón, Spain
virginia.gonzalez@upm.es
GONZALEZ GARCIA María de la Cruz
Instituto de Bioquímica Vegetal y
Fotosíntesis, Avda. Americo Vespucio 49,
41092 Seville, Spain
maricruz@ibvf.csic.es
GOUJON Thomas
INRA Dept BV, 46 Allée d'Italie,
69364 Lyon cedex 07, France
thomas.goujon@ens-lyon.fr
GUBAEVA Ekaterina
Westfalische Wilhelms University,
Hindenburgplatz 55,
48143 Muenster, Germany
i_guba01@uni-muenster.de
HANDS Philip
University of Leicester, University Rd,
LE1 7RH, Leicester, United Kingdom
psh14@le.ac.uk
HARDTKE Christian
Dept. Plant Mol. Biol., Biophore Bldg.,
DBMV,
CH-1015 Lausanne, Switzerland
christian.hardtke@unil.ch
HARRINGTON Michael
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
mjhphd@gmail.com
HAZEN Samuel
University of Massachusetts, 221 Morrill
Science Center,
1003 Amherst, United States of America
hazen@bio.umass.edu
HIMURO Yasuyo
RIKEN Tsukuba Institute,
3-1-1 Koyadai,
Tsukuba-shi, Ibaraki,
305-0074 Tsukuba, Japan
yasuyo.himuro@riken.jp
HOFTE Herman
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
herman.hofte@versailles.inra.fr
HO-YUE-KUANG Marie Séverine
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
severine.hyk@gmail.com
IDZIAK Dominika
University of Silesia,
ul. Bankowa 12,
Katowice, Poland
didziak@us.edu.pl
JAMET Elisabeth
UPS LRSV UMR5546 UPS/CNRS, Pôle de
Biotechnologies Végétales
24, chemin de Borde Rouge, BP42617,
31326 Castanet-Tolosan, France
jamet@lrsv.ups-tlse.fr
JØRGENSEN Bodil
Department of Agriculture and Ecology,
Thorvaldsensvej 40,
1871 Frederiksberg, Denmark
boj@life.ku.dk
79

JOUANIN Lise
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
lise.jouanin@versailles.inra.fr
JUNG Jae-Hoon
Séoul University,
Gwanak _ 599 Gwanak-ro, Gwanak-gu,
151-742 Seoul, Korea
hanbari22@korea.com
JURANIEC Michal
Laboratoire de Physiologie et de
Génétique Moléculaire des Plantes,
Université Libre de Bruxelles,
Campus Plaine- CP242,
1050 Brussels, Belgium
juraniec@univ.rzeszow.pl
KRAPP Anne
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
anne.krapp@versailles.inra.fr
KUMLEHN Jochen
IPK Gatersleben,
Corrensstrasse 3,
6466 Gatersleben, Germany
kumlehn@ipk-gatersleben.de
LAPIERRE Catherine
AgroParisTech, UMR1318,
Route de St Cyr,
78026 Versailles, France
catherine.lapierre@versailles.inra.fr
LARRE Colette
INRA BIA, Rue de la Géraudière,
BP71627,
44316 Nantes, France
larre@nantes.inra.fr
LE BRIS Philippe
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France, philippe.l
bris@versailles.inra.fr
LECOMTE Philippe
INRA - UMR GDEC,
234, avenue du Brézet,
63100 Clermont-Ferrand, France
philippe.lecomte@clermont.inra.fr
LEGAY Sylvain
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
sylvain.legay@versailles.inra.fr
LEPINIEC Loïc
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
loic.lepiniec@versailles.inra.fr
LEVI BAR SHALOM Avishag
Evogene Ltd.,
13 Gad Feinstein street,
76121 Rehovot, Israel
avishaglb@evogene.com
LOUDET Olivier
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Olivier.Loudet@versailles.inra.fr
MACADRE Catherine
Institut de Biologie des Plantes,
rue Noetzlin,
91405 Orsay cedex, France
catherine.macadre@u-psud.fr
MAILLET Fabienne
INRA LIPM,
Chemin de Borde Rouge, BP 52627,
31326 Castanet-Tolosan, France
maillet@toulouse.inra.fr
MARCEL Thierry
INRA BIOGER, UR1290 BIOGER-CPP,
Avenue Lucien Brétignières, BP01,
78850 Thiverval-Grignon, France
Thierry.Marcel@versailles.inra.fr
80

MARION-POLL Annie
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
annie.marion-poll@versailles.inra.fr
MARRIOTT Poppy
Univeristy of York, CNAP, Department of
Biology, University of York,
YO10 5DD York, United Kingdom
pm518@york.ac.uk
MARTIN Marjolaine
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
marjolaine.martin@hotmail.fr
MASCLAUX-DAUBRESSE Céline
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
masclaux@versailles.inra.fr
MAZEL Julien
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
julien.mazel@versailles.inra.fr
MAZZAMURRO Valentina
Dipartimento di Scienze Agrarie e degli
Alimenti, Università di Modena e Reggio
Emilia, Via Amendola, 2 - Padiglione Besta,
42122 Reggio Emilia, Italy
valentina.mazzamurro@unimore.it
MECHIN Valérie
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
vmechin@versailles.inra.fr
MICA Erica
Scuola Superiore Sant'Anna of Pisa, Piazza
martiri della Libertà, 33 ,
56127 Pisa, Italy
erica.mica@sssup.it
MOCHIDA Keiichi,
RIKEN Biomass Engineering Program, 1-7-
22, Suehiro-cho, Tsurumi-ku,
230-0045 Yokohama, Japan
mochida@psc.riken.jp
MORIN Halima
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
halima.morin@versailles.inra.fr
MOSCOU Matthew
The Sainsbury Laboratory,
Norwich Research Park,
NR4 7UH Norwich, United Kingdom
matthew.moscou@tsl.ac.uk
MOUILLE Grégory
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
gregory.mouille@versailles.inra.fr
MRAVEC Jozef
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
jmravec@versailles.inra.fr
MULLINS Ewen
Teagasc, Dept. Crop Science,
Oak Park,
Carlow, Ireland
ewen.mullins@teagasc.ie
MUR Luis A. J.
Aberystwyth University,
"IBERS" - Institute of Biological,
Environmental and Rural Sciences.
Edward Llwyd Building,
Aberystwyth SY23 3DA, Wales, United
Kingdom
lum@aber.ac.uk
MUROZUKA Emiko
Faculty of Life Sciences, University of
Copenhagen,
Thorvaldsensvej 40, opg. 8, 3rd floor,
DK-1871 Copenhagen, Denmark
emikomu@life.ku.dk
81

MUTWIL Marek
Max-Planck-Institute for Molecular Plant
Physiology, Am Mühlenberg 1,
14476 Potsdam, Germany
mutwil@mpimp-golm.mpg.de
MUYLLE Hilde
ILVO, Caritasstraat 21
9090 Melle, Belgium
Hilde.Muylle@ilvo.vlaanderen.be
NEJI Mohamed
Center of Biotechnology of Borj-Cédria,
Borj cedria Hammam Lif,
2050 Tunis, Tunisia
mnmedneji@gmail.com
NG Carl
University College Dublin, School of Biology
and Environmental Science, Dublin 4,
Dublin, Ireland
carl.ng@ucd.ie
NICHOLSON Paul
John Innes Centre, Norwich Research Park,
NR4 7UH Norwich, United Kingdom
paul.nicholson@bbsrc.ac.uk
O'DRISCOLL Aoife
Teagasc, Dept. Crop Science,
Oak Park,
Carlow, Ireland
aoife.odriscoll@teagasc.ie
ORIA Nicolas
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
nicolas.oria@versailles.inra.fr
PACHECO VILLALOBOS David
Department of Plant Molecular Biology,
University of Lausanne, Biophore Building,
CH-1015 Lausanne, Switzerland
David.PachecoVillalobos@unil.ch
PANNETIER Catherine
INRA-CIRAD, IJPB UMR1318, Bât 2,
Route de St-Cyr (RD10),
78026 Versailles, France
catherine.pannetier@versailles.inra.fr
PARK Chung-Mo
Séoul University, Gwanak _ 599 Gwanak-ro,
Gwanak-gu,
151-742 Seoul, Korea
cmpark@snu.ac.kr
PASQUET Jean-Claude
IBP, rue Noetzlin,
91405 Orsay cedex, France
jean-claude.pasquet@u-psud.fr
PECCHIONI Nicola
Dipartimento di Scienze Agrarie e degli
Alimenti, Università di Modena e Reggio
Emilia, Via Amendola, 2 - Padiglione Besta
42122 Reggio Emilia, Italy
nicola.pecchioni@unimore.it
PELLETIER Georges
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
georges.pelletier@versailles.inra.fr
PERALDI Antoine
John Innes Centre, Norwich Research Park,
NR4 7UH Norwich, United Kingdom
antoine.peraldi@bbsrc.ac.uk
PERSSON Staffan
Max-Planck-Institute for Molecular Plant
Physiology, Am Mühlenberg 1,
14476 Potsdam, Germany
persson@mpimp-golm.mpg.de
POIRE Richard
CSIRO Plant Industry Black Mountain
Laboratories, Clunies Ross Street,
2601 Canberra, Australia
richard.poire@csiro.au
PUENTE Pilar
BASF SE, Speyerer St 2,
67117 Limburgerhof, Germany
pilar.puente@basf.com
RASMUSSEN Søren K.
University of Copenhagen,
Thorvaldsensvej 40,
1871 Frederiksberg, Denmark
skr@life.ku.dk
82

RENAULT Hugues
CNRS - IBMP Institut de Biologie Moléculaire
des Plantes, 28 rue Goethe,
67083 Strasbourg, France
hugues.renault@ibmp-cnrs.unistra.fr
REYMOND Matthieu
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
mreymond@versailles.inra.fr
RIZZOLATTI Carine
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Carine.Rizzolatti@versailles.inra.fr
SALSE Jérôme
Laboratory ‘Plant Paleogenomics for Traits
Improvement’, UMR INRA-UBP 1095,
Domaine de Crouelle,
234 Avenue du Brézet,
63100 Clermont-Ferrand, France
jsalse@clermont.inra.fr
SANGUINET OSMONT Karen
UMass-Amhert, 611 N. Pleasant St.,
1002 Amherst, United States of America
ksosmont@bio.umass.edu
SAUNIER DE CAZENAVE Magdalena
University of Liège, Gembloux Agro-Bio
Tech, 2 Passage des Deportes,
5030 Gembloux, Belgium
m.saunierdecazenave@ulg.ac.be
SAVAS Gulsemin
Namik Kemal University - Faculty of
Agriculture - Department of Field Crops,
De irmenaltı Campus,
59030 Tekirdag, Turkey
SCAGNELLI Aurélie
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
aurelie.scagnelli@versailles.inra.fr
SCHNITTGER Arp
CNRS - IBMP Institut de Biologie Moléculaire
des Plantes, 28 rue Goethe
67083 Strasbourg, France
Arp.Schnittger@ibmp-cnrs.unistra.fr
SCHULMAN Alan
MTT & University of Helsinki, PO Box 65,
FIN-00014 Helsinki, Finland
alan.schulman@helsinki.fi
SEO Pil Joon
Séoul University, Gwanak _ 599 Gwanak-ro,
Gwanak-gu,
151-742, Seoul, Korea
dualnt83@snu.ac.kr
SIBOUT Richard
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Richard.Sibout@versailles.inra.fr
SOULHAT Camille
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Camille.Soulhat@versailles.inra.fr
SRIVASTAVA Vibha
University of Arkansas,
115 Plant Science bldg,
72704 Fayetteville, Unit. States of America
vibhas@uark.edu
SUER Stefanie
Gregor Mendel Institute of Molecular Plant
Biology, Dr. Bohr-Gasse 3,
1030 Vienna, Austria
stefanie.suer@gmi.oeaw.ac.at
TANACKOVIC Vanja
Faculty of Life Sciences, Department of
Plant Biology and Biotechnology,
Thorvaldsensvej 40, opg. 10, 1,
1871 Copenhagen, Denmark
vtana@life.ku.dk
83

THEVENIN Johanne
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Johanne.Thevenin@versailles.inra.fr
THOLE Vera
John Innes Centre, Norwich Research Park,
NR4 7UH Norwich, United Kingdom
vera.thole@bbsrc.ac.uk
TIMPANO Hélène
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
helene.timpano@versailles.inra.fr
TORRES-ACOSTA Juan Antonio
Universität für Bodenkultur Department für
Angewandte Genetik und Zellbiologie,
Konrad Lorenzstrasse 24,
3430 Tulln , Austria
juan-antonio.torres-acosta@boku.ac.at
TUNA Metin
Namik Kemal University - Faculty of
Agriculture - Department of Field Crops,
De irmenaltı Campus,
59030 Tekirdag, Turkey
metintuna66@yahoo.com
VAIN Philippe
Department of Crop Genetics, John Innes
Centre, Norwich Research Park,
Norwich NR4 7UH, United Kingdom
philippe.vain@jic.ac.uk
VALDIVIA Elene
Univ Santiago de Compostela ,
Departamento de Fisiología Vegetal ,
15782 Santiago de Compostela , Spain
elenevaldivia@gmail.com
VARIN Sébastien
University of Liège, Gembloux Agro-Bio
Tech, 2 Passage des Deportes,
5030 Gembloux , Belgium
seb.varin@orange.fr
VEDELE Françoise
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
francoise.vedele@versailles.inra.fr
VERBRUGGEN Nathalie
Laboratoire de Physiologie et de
Génétique Moléculaire des Plantes,
Université Libre de Bruxelles, Campus
Plaine- CP242,
1050 Brussels, Belgium
nverbru@ulb.ac.be
VERNHETTES Samantha
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
samantha.vernhettes@versailles.inra.fr
VOGEL John P.
USDA-ARS Western Regional Research
Center,
Albany, CA, 94710, Unit. States of America
john.vogel@ars.usda.gov
VOIGT Christian
Biocenter Klein Flottbek,
Ohnhorststr. 18,
22609 Hamburg, Germany
voigt@botanik.uni-hamburg.de
VOOREND Wannes,
ILVO,
Caritasstraat 21,
9090 Melle, Belgium
wannes.voorend@ilvo.vlaanderen.be
VOZABOVA Tereza
Laboratory of Plant Breeding, PO box 386,
Droevendaalsesteeg,
6708PB Wageningen, The Netherlands
T.Vozabova@seznam.cz
WANG Yin
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
yin.wang@versailles.inra.fr
84

WARD Eric
Two Blades Foundation, P.O. Box 51291,
27717 Durham, United States of America
erw@2blades.org
WIESENBERGER Gerlinde
Univ. of Natural Res. & Life Sci. (BOKU),
Konrad Lorenz Str. 24,
3430 Tulln, Austria
gerlinde.wiesenberger@boku.ac.at
WORLAND Barbara
John Innes Centre,
Norwich Research Park,
NR4 7UH Norwich, United Kingdom
barbara.worland@bbsrc.ac.uk
WULFF Brande
The Sainsbury Laboratory,
Norwich Research Park,
NR4 7UH Norwich, United Kingdom
brande.wulff@sainsbury-laboratory.ac.uk
ZHANG Yu
INRA IJPB Institut Jean-Pierre Bourgin - umr
1318, Rd 10 - Route de Saint-Cyr,
78000 Versailles, France
Yu.Zhang@versaillesinra.fr
ZUBAIR Hassan
IBERS, Aberystwyth University, Edward
Llwyd Building,
SY23 3DA Aberystwyth, United Kingdom
hhz@aber.ac.uk
85