acknowledgements - gis biotechnologies vertes...guy riba (inra), alain weil (cirad), serge hamon...
TRANSCRIPT
Acknowledgements
Among the fifty projects submitted to the ANR [French National Agency
for Research], to the call for projects launched in 2006 « Network for Plant
Genomics / GENOPLANTE 2010 », eighteen projects have been funded,
mostly focused on the development of new tools and new resources
(microarrays, new approach for transcriptomic, ..). These projects are
related to varied species: cereals, oil and protein crops, but also cotton,
flax, coffee tree, poplar.
By the way, in 2006 fourteen projects were also launched in the
framework of ERAnet Plant Genomics, in partnership with Germany and
Spain, funded by ANR for the french partners. These projects are not
presented here, because this report only deals with projects carried out
at national level.
As Chairman of the ANR Steering Committee from 2005 to 2007, I wished
to thank the ANR, and most particularly the head of the Genomics
programme, Francis Quétier, and his successor Philippe Feldmann, for
their active support of this programme, decisive for the future of our
agriculture and of the industrial sector, which should make it possible
to create the conditions for long-lasting development, thanks to new
answers in terms of environment and quality, while at the same time
preserving the food autonomy of Europe. All my thanks also go to the
members of the Evaluation Committee, chaired from 2005 to 2007 by
Georges Pelletier, and to the members of the Steering Committee, and
also to the scientific reviewers who participated in the evaluation of the
projects. I would also like to thank the teams of GIS GENOPLANTE and
of GENOPLANTE- VALOR, without whom this federative, coherent and
ambitious programme could not have been developed.
This second report, including the summaries of the results obtained,
attests to the dynamics of this programme and to the synergies between
public and private teams that must absolutely, be preserved and further
strengthened.
Michel Boucly
Chairman of the ANR Steering Committee,
Network for Plant Genomics / GENOPLANTE 2010
Table of contents
Preamble 4
Committee 5
Functional Analysis
CHAT: Cyclic nucleotide-gated cation channels involved in hypersensitive cell death
in Arabidopsis thaliana 8
DNV: Natural variation for drought tolerance:
from QTL for targeted traits to functional polymorphisms 10
ISD STARCH: Initiation, synthesis, and degradation: an integrated approach
toward the understanding of starch metabolism and formation in plants 12
MICRO TRAC: MicroRNA Transcription and Activity: uncovering and exploiting
the genes between the genes 14
RIBOROOT: Involvement of non coding RNAs in the adaptation
of root architecture to abiotic stress 16
TRANSPORTOME: The Arabidopsis plasma membrane transportome:
searching for macromolecular complexes involved in ion transport 18
New ToolsGENOLIN: Production of flax oligonucleotide microarrays 22
GLYCO-CHLOROPLAST: Identification of signals controlling the protein trafficking
between the secretory pathway and the chloroplast 24
LEGOO: A bioinformatics gateway towards integrative legume biology 26
PUCECAFE: The First 15K Coffee Microarray, a New Tool for Discovering
Candidate Genes correlated to Agronomic and Quality Traits 28
TAG: Conception and exploitation of a multi usage chip of the Arabidopsis genome 30
Oil and Protein Crops
QUALITYLEGSEED: An integrated approach using M. truncatula to identify
loci/genes controlling composition and physiological quality of legume seeds 34
SCLEROTINIA: Identification of genes involved in Sclerotinia sclerotiorum
resistance in Oilseed Rape and Sunflower 36
SNPEA: Genetic validation of functional candidate genes potentially involved
in resistance to frost and diseases in Pisum sativum 38
Cereals
PROTNBLE: Genetic analysis under N limiting conditions of the stability
of yield and grain protein content of durum and bread wheat 42
SMART: Sequencing and molecular analysis of a 12 cM locus carrying multiple
durable disease resistance genes on chromosome 3BS in hexaploid wheat 44
Other Species
COTTON-RILS: Genetic and genomic dissection of cotton fibre quality
using an interspecific RIL population 48
POPSEC: Molecular bases of acclimation and adaptation to water deficit in poplar 50
Contact list 52
Preamble
This report is the second of a collection presenting the principle results
obtained per project, for each edition of the ANR calls for plant genomics
projects.
The 2006 call for projects of the Network for Plant Genomics /
GENOPLANTE 2010 was launched on January 25, 2006, with closure on
March 15, 2006. It was rewarded with a great deal of success since fifty
projects were submitted for a total applications amount of € 24 million.
After external reviews and meetings of the Evaluation Committee and
of the Steering Committee, eighteen functional genomics projects were
supported by the ANR, for a total grants amount of € 8,82 million. An
additional project relating to the sequencing of the grapevine was also
supported.
Out of these eighteen projects, six were as a public/private partnership.
11% of the projects selected are focused on cereals, 22% concerned oil
and protein-yielding plants, 45% the model species Arabidopsis and
22% related to other species such as cotton, coffee tree, flax and poplar.
These projects were followed up by five thematic committees:
n Functional analysis
n New tools
n Oil and protein crops
n Cereals
n Other species
They were the subject of a progress report in February 2009 and of a
presentation at the Plant Genomic seminar of March 2010, with an
evaluation by the Scientific Advisory Board of GENOPLANTE, chaired by
Professor Peter Westhoff.
At the end of October 2010, the projects presented have already given
rise to 45 publications and to the filing of one patent application.
4
Committee
Evaluation Committee
Georges PELLETIER - Chairman
Hélène LUCAS, Emmanuel GUIDERDONI, Alain GHESQUIÈRE,
Dominique ROBY, Dominique JOB, Dominique de VIENNE,
Peter WESTHOFF, Pere PUIGDOMENECH, Martin KOORNNEEF,
Michel DEBRAND, Georges FREYSSINET, Xavier PINOCHET,
Michel BOULAY, Jean Jacques LEGUAY, Laurent GUERREIRO,
Patrick VINCOURT, Jean Loup RISLER
Steering Committee
Michel BOUCLY – Chairman (SOFIPROTEOL)
Daniel CHERON (LIMAGRAIN), Philippe GRACIEN (GNIS),
Georges FREYSSINET (BIOGEMMA), Jean-Jacques LEGUAY (CEA),
Guy RIBA (INRA), Alain Weil (CIRAD), Serge HAMON (IRD),
André LE BIVIC (CNRS)
5
Functional Analysis
8
Project report edition 2006 Functional Analysis
Cyclic nucleotide-gated cation channels involved in hypersensitive cell death in Arabidopsis thaliana
ACRONYM: CHAT
COORdiNATOR: Claudine BALAGUE1
ANR N°: ANR-06-GPLA-001
Cost: € 1,341,180
Grant: € 373,885
Scientists per year: 3.6
Period: 01.01.2007 – 06.30.2010
Partners: H. SENTENAC2, B. RANTY3
1. UMR 2594 CNRS/INRA Castanet Tolosan
2. UMR 5004 CNRS/INRA/ENSAM Montpellier
3. UMR 5546 CNRS/UPS Castanet Tolosan
INTRODUCTION
Pathogens cause major economic losses by reducing crop yields and
quality of plant products worldwide. One of the most efficient reactions
is the Hypersensitive Response or HR, characterized by rapid death of
plant cells facing the pathogen. Understanding the signaling networks
conditioning resistance and HR is particularly relevant for the modulation
of defense responses in crop plants. In this context, two Arabidopsis
mutants, named hlm1/dnd2/cngc4 and dnd1/cngc2, affected in the HR
and/or spontaneously developing HR-like lesions in absence of pathogen
attack, have been identified, leading to identification of two putative
signaling components, named CNGC4 and CNGC2, respectively, members
of the so-called Cyclic Nucleotide Gated Channel (CNGC) family. This family
is still poorly characterized but thought to encode cyclic nucleotide- and
calmodulin- regulated cation channels based on homology with animal
genes. The present project is aimed at deciphering the roles of CNGC2 and
CNGC4 in HR/cell death. Beyond mechanisms related to specific resistance,
this project concerns also basal resistance, since CNGCs are thought to act
downstream in the signaling pathways leading to resistance. Exploitation of
such genes, in contrast with R (for resistance) genes which can be rapidly
circumvented by microorganisms, may help to achieve durable disease
control.
RESULTS
This project is divided in three main tasks and the research program included:
(1) Placement of CNGC4 and CNGC2 in the signaling pathways leading to HR/
resistance (by crossing the mutants hlm1 and dnd1 with mutants impaired in
early resistance pathways or more downstream, salicylic acid and ethylene/
jasmonate pathways) and analyses of different elements of the defense/
resistance signaling network. (2) Functional analyses of CNGC channel
9
Project report edition 2006 Functional Analysis
activity, by patch-clamp (i) in a «green» expression system, tobacco mesophyll
protoplasts transiently transformed to express or co-express CNGC2, CNGC4 and
other members of the Arabidopsis CNGC family, and (ii) in planta, in protoplasts
prepared from wild-type, or cngc2 or cngc4 mutant plants. (3) Structure-function
analyses, with priority given to studies on the roles of the cyclic nucleotide binding
domain and the calmodulin binding domain.
In order to get an insight into the placement of CNGC2 and CNGC4 in the signalling
network leading to HR/resistance, analysis of the T3 progeny from crosses
between cngc2 and cngc4 with mutants affected in the signaling pathways leading
to resistance/defense, has been performed. The increased resistance observed
in response to pathogens in cngc2 and cngc4 was found to be ethylene (ET)-
independent, PAD4- and JAR1-dependent and to require salicylic acid (SA) production
and NDR1, a major actor of the SA signalling pathway. In addition, SA and ET but not
JA (jasmonate) pathways were required for cell death in cngc2 and cngc4 mutants.
Moreover, these two genes were differentially regulated, CNGC4 being induced
by SA but not CNGC2, whereas a slight induction of both genes was observed in
response to JA and JA/ET.
The respective role of CNGC2 and CNGC4 has been tested by complementation of
cngc2 and cngc4 mutants by the constructs pCNGC2-CNGC2 and pCNGC4-CNGC2.
Analysis of the complemented lines showed that not only the coding regions but also
the promoter regions are essential for CNGC2 and CNGC4 functions.
Functional analysis of CNGC2 and CNGC4 which present a calmodulin binding
domain in the C-terminal cytosolic region was performed by a comparative analysis
of cngc2 lines complemented with the wild type form of the channel and the mutated
channel unable to bind calmodulin. Results showed that the mutated version was
unable to complement the plant cell death phenotypes (lesion mimics) whereas the
wild type form restores the normal phenotype (plant size and absence of lesions),
providing evidence of the importance of calmodulin in the function of the channel
during programmed cell death.
Up to day, little is known on the role of the CNGCs as channels in plants. In the present
project, we encountered strong difficulties to detect channel activity by functional
expression in heterologous systems and results still needed to be confirmed. To
begin with, studies of their subcellular localization revealed for the first time that
CNGC2 and CNGC4 channels are preferentially associated with the endoplasmic
reticulum when they are (over)expressed alone, but are largely associated with
the plasma membrane in co-expression experiments. These data pave the way to
functional analyses by patch-clamp on Arabidopsis cell suspension protoplasts.
Electrophysiological recordings showed that the co-expression of CNGC2/CNGC4
in protoplasts resulted in exogenous inward currents significantly larger than the
endogenous ones.
CONCLUSION AND PERSPECTIVES
Our CHAT project allowed to decipher some of the functions of two CNGC genes in
the context of plant-pathogen interactions, and more specifically the Hypersensitive
Response, a form of programmed cell death for which we have still limited information
in terms of its regulation and execution. These studies clearly established the
placement of CNGC2 and CNGC4 in the signalling pathways leading to cell death/
resistance. They also increased our knowledge about the essential regulatory role of
CaM on channel function and brought new perspectives for the electrophysiological
analysis of the function of these two channels. Beyond these scientific advances,
the CHAT project allowed to create long-term collaborative relationships between
groups working in different and complementary areas of plant biology.
Our main objective in the near future will be the elucidation of CNGC2 and CNGC4
interactions and of their channel functions. This would help to finally shade the light
on the roles of this CNGC channel family which still represents a black box in plant
biology.
Characterization of two cation cyclic nucleotide gated
channels, CNGC2 and CNGC4 and their role in the
regulation of the Hypersensitive Response and defense
to pathogens in Arabidopsis.
Upper panel: Lesion mimic phenotype of cngc mutant
plants
Middle panel (right): increased resistance phenotype
of cngc mutants in response to Pseudomonas syringae
Midle panel( left): subcellular localization of CNGC4
in the cytoplasm
Lower panel (left and middle): localization of CNGC4
and CNGC2 in the plasma membrane after co-
expression in Arabidopsis protoplasts (left)
and in tobacco leaves (middle)
Lower panel (right): currents elicited by CNGC2
and CNGC4 expression.
Natural variation for drought tolerance: from QTL for targeted traits to functional polymorphisms
ACRONYM: dNV
COORdiNATOR: Olivier LOUdET 1
ANR N°: ANR-06-GPLA-002
Cost: € 1,723,000
Grant: € 408,370
Scientists per year: 2.7
Period: 01.01.2007 – 12.31.2009
Partners: B. MULLER 2, C. GRANIER 2
1. Institut Jean-Pierre Bourgin, UMR1318
INRA-AgroParisTech, Versailles
2. INRA, LEPSE, UMR 759 INRA-Supagro,
Montpellier
INTRODUCTION
In an agricultural context, tolerance to water deficit is the result of a trade-
off between biomass production and soil water depletion. Functions res-
ponsible for the fine tuning of this trade-off are leaf expansion, root growth,
stomatal control and flowering time, all responding to the environment as
quantitative traits. The goal of the proposed work was to analyse the genetic
architecture of some of these drought responses in Arabidopsis thaliana,
using genome-wide molecular quantitative genetics (QTL mapping and
association studies) combined with fine ecophysiological characterisation
and modelling of the plant reaction to its environment. We have proposed to
identify new loci involved in the response of natural Arabidopsis populations
as estimated by a range of quantitative traits measured in different condi-
tions and environments using high-throughput phenotyping displays. Then,
using the advantages of Arabidopsis as a model species the cloning of the
most interesting loci would be performed as a traditional positional cloning
down to the gene level and/or using candidate gene approaches. The ana-
lysis of the molecular and functional variation leading to the phenotype in
interaction with the environment provides clues as to how and where in the
pathways adaptation is shaping natural variation. Moreover, the genes and
physiological functions identified here will provide targets for crop breeding
program, as well as an integrative view of the biology of the species and its
evolution.
RESULTS
During this project, we have focused our efforts to identify loci causing na-
tural genetic variation for growth-related traits in Arabidopsis thaliana and
dissect the ecophysiological basis of adaptation to water deficit through
shoot- and root-related traits.
The RIL set derived from a cross between Cvi-0 and Col-0 has been lar-
Publications
• Tisné S., Reymond M., Vile D., Fabre J.,
Dauzat M., Koornneef M., Granier C.
(2008), Combined genetic and modeling
approaches reveal that epidermal cell area
and number in leaves are controlled by
leaf and plant developmental processes
in Arabidopsis. Plant Physiology, 148:
1117-1127.
• Ghandilyan A., Barboza L., Tisné S.,
Granier C., Reymond M., Koornneef M.,
Schat H., Aarts M.G.M. (2009), Genetic
analysis identifies quantitative trait loci
controlling rosette mineral concentrations
in Arabidopsis thaliana under drought.
New Phytologist, 184 :180-192.
• Tisné S., Schmalenbach I., Reymond
M., Dauzat M., Pervent M., Vile D.,
Granier C. (2010), Keep on growing under
drought: genetic and developmental bases
of the response of rosette area using a
recombinant inbred line population. Plant
Cell & Environment, Epub June 7.
Project report edition 2006 Functional Analysis
10
gely exploited in this work: we have mapped several loci
responsible for leaf area variation of 12 days-old seedlings
grown in vitro on mildly osmotic media as well as under
control conditions. One locus, SG2 (Shoot Growth – 2) was
identified on chromosome 2. This QTLs remains strongly
associated with shoot growth whatever the environmental
conditions. However, its phenotypic effect was limited and,
hence, the identification of the gene responsible for this QTL
still requires additional work. Among the loci associated with
mannitol stress response, we identified an important locus,
named EGO3 (Enhanced shoot Growth under Osmotic stress
– 3). EGO3 is a major QTL at the top of chromosome 1 reaching
a very high significance (18.5 LOD) on mannitol-containing
media, for which the Cvi allele increases shoot growth (+
20-25 %) relative to the Col allele. We were able to confirm
EGO3 in specific nearly-isogenic lines (HIF – Heterogeneous
Inbred Family) and its identification was performed essenti-
ally thanks to a map-based cloning approach. In summary,
hundreds of recombinants (from the HIF) within the initial
QTL region were studied phenotypically to reduce the extent
of the candidate region to just 10 kb. Combined with analysis
of T-DNA mutants in the three genes remaining in the interval,
this has clearly limited the candidate genes to one unknown
gene, At1g11300, predicted to be a receptor-like kinase but
not studied nor related to any phenotype yet.
A loss-of-function in At1g11300 ressembles the effect of the
Cvi allele, so that we expect that some of the three amino-acid
changes in the Cvi allele reduces the activity of the protein.
Interestingly, these polymorphisms are shared by several ac-
cessions and these accessions all segregate for a QTL very si-
milar to EGO3 when crossed to Col-0, providing independent
proof for the identity of EGO3. Even more, we found acces-
sions that only share a subset of the Cvi polymorphisms, and
these allow to shorten the list of candidate QTN (Quantitative
Trait Nucleotide = causal polymorphism). We call this original
strategy, “specific association genetics”. However it seems
that EGO3 does not underline a general response to osmotic
stress (as could be tested with different osmotic conditions),
but is linked to mannitol as a signal which could then relate to
pathogen resistance, as some fungi accumulate mannitol as
a carbon storage compound and comparing our NIL reveals
signature for biotic stress response. Indeed, the segregation
of EGO3 is accompanied by a phenotype in response to in-
festation by Botrytis cinerea, the (likely-defective) Cvi allele
being responsible for enhanced susceptibility to this fungi.
Using the PHENOPSIS phenotyping platform and the Ler x
An-1 population (chosen because An-1 is able to maintain leaf
growth under drought), main effect QTLs and QTLs in epis-
tatis were mapped controlling the number of rosette leaves,
individual leaf area, as well as epidermal cell number and
area in the leaves at two contrasted levels of soil water deficit
and two day-lengths. A major QTL and an epistatic interaction
between two QTLs affected the response of rosette area and
individual leaf area to water deficit but only with effects in
well-watered condition.
A second epistatic interaction between two QTLs control-
led the response of rosette area to drought via an increase
in the number of rosette leaves in response to drought. The
effects of the two epistatic interactions reported here were
validated by generating and phenotyping appropriate lines.
By combining quantitative genetics and a statistical model,
we have analyzed the functional relationships between these
variables.
This analysis suggests in particular that epidermal cell size
and cell number are determined to some extent by leaf num-
ber and leaf expansion. QTLs associated with this link have
been identified and confirmed by HIFs. These results suggest
a feedback effect on cell division and cell expansion of whole
plant processes thereby limiting their impact on leaf size.
Additionnaly, a quantitative genetics analysis was perfor-
med in order to explore the functional links between roots
and shoot growth. We have tested the hypothesis that root
growth QTLs could be linked to QTLs favoring large shoots,
small shoots or independent of shoot growth. Using the Bay x
Sha RILs population, we were able to validate this hypothesis
by using several statistical tools (PCA, residual) to numeri-
cally extract shoot-dependent and shoot-independent com-
ponents of root growth. These tools generated new variables
(such as PCA coordinates) that proved high heritabilities and
helped identifying shoot-growth-independent root growth
QTLs that were later validated using series of HIFs. Experi-
ments addressing the stability of these QTLs under drought
were then performed and a completely different picture emer-
ged: root growth QTLs were essentially independent of shoot
growth QTLs suggesting root growth becomes sink rather
than source limited under drought. These experiments are
now continued in a broader context using collection of acces-
sions from contrasted environments.
CONCLUSION AND PERSPECTIVES
We still have some work to do to understand the links
between shoot growth variation in the presence of mannitol
and sequence variation within the EGO3 kinase, as well as the
impact of natural variation in this new gene. Nevertheless,
this ambitious pro-
ject has led to the
identification of
some biological
and genetic mecha-
nisms underlying
response to stress,
which sheds new
light on the regu-
lation of growth
and proves how
interesting it is
to combine eco-
physiological and
quantitative gene-
tics approaches.
Project report edition 2006 Functional Analysis
11
The PHENOPSIS phenotyping platform
12
Project report edition 2006 Functional Analysis
Initiation, synthesis, and degradation: an integrated approach toward the understanding of starch metabolism and formation in plants
ACRONYM: iSd STARCH
COORdiNATOR:
Christophe d’HULST 1
ANR N°: ANR-06-GPLA-004
Cost: € 980,049
Grant: € 298, 986
Scientists per year: 3,6
Period: 01.01.2007 – 06.30.2010
Partners: V. PLANCHOT 2,
J-P. RENOU 3
1. Unité de Glycobiologie Structurale et
Fonctionnelle, UMR8576 du CNRS, UST Lille,
Villeneuve d’Ascq
2. Unité Biopolymères, Interactions,
Assemblages, Centre INRA de Nantes
3. Unité de Recherche en Génomique Végétale,
UMR INRA-CNRS, EvryINTRODUCTION
Starch, a plant polysaccharide vital for mankind but still far from being un-
der complete control
Starch is one of the major (if not the first) polymers used in the textile, food
or chemistry industries (more than 600 commercial goods are produced from
starch or its derivatives). Moreover starch is the most important source of calo-
ries for people directly or indirectly (for cattle feeding). A better understanding
of starch biogenesis and degradation in plants would lead to the production of
more elaborated polymers that better fit industrial and societal requirements.
The objectives of this project was to increase our understanding of the synthe-
sis and degradation pathways of starch but also to strengthen our knowle-
dge about the priming mechanism that leads to the synthesis of the polymers
(amylose and amylopectin) and the formation of the starch granules (these 2
processes might be or not independent). We wanted also to reveal the biolo-
gical factors that regulate this pathway and its connection with carbon meta-
bolism in the plant.
RESULTS
These objectives can be reached through the development of a genetics
approach in a model based on the analysis of mutant lines defective for the
expression of genes of the starch pathway but also on the production, by
crosses, and the analysis of combined mutant lines (double, triple or qua-
druple mutants). Therefore, we proposed to understand the contribution of
1) the SS3 and SS4 isoforms of soluble starch synthases in the process of ini-
tiation of the synthesis of the polymers (amylopectin essentially) and/or the
formation of the starch granule, 2) the plastidial amylases and glucan-phos-
13
Project report edition 2006 Functional Analysis
Publications
• Roldán I, Wattebled F, Lucas M, Delvallé
D, Planchot V, Ricardo Pérez S-J, Ball
SG, D’Hulst C, & Mérida A (2007). The
phenotype of Soluble Starch Synthase IV
defective mutants of Arabidopsis thaliana
suggests a novel function of elongation
enzymes in the control of starch granule
formation. Plant J., 49 (3): 492-504.
• Zhang X, Szydlowski N, Delvallé D,
D’Hulst C, James MG, & Myers AM
(2008). Analysis of the related roles of
starch synthase II and starch synthase
III in amylopectin biosynthesis in
Arabidopsis leaves. BMC Plant Biol., 8: 96.
• Wattebled F, Planchot V, Dong Y,
Szydlowski N, Pontoire B, Devin A, Ball S,
& D’Hulst C (2008). Further Evidence for
the Mandatory Nature of Polysaccharide
Debranching for the Aggregation of Semi-
Crystalline Starch and for Overlapping
Functions of Debranching Enzymes in
Arabidopsis Leaves. Plant Physiol., 148
(3): 1309-1323.
• Szydlowski N, Ragel P, Raynaud S,
Lucas MM, Roldán I, Montero M, Muñoz
FJ, Ovecka M, Bahaji A, Planchot V,
Pozueta-Romero P, D’Hulst C, & Mérida
A (2009). Starch granule initiation in
Arabidopsis requires the presence
of either Class IV or Class III starch
synthase. Plant Cell, 21: 2443-2457.
• D’Hulst C, & Mérida Á (2010). The
priming of storage glucan synthesis from
bacteria to plants: current knowledge
and new developments. New Phytologist,
188: 13–21
phorylases during polymers degradation. Moreover, because of a strong part
dealing with the transcriptomics analysis of mutant lines defective for one
or several genes of the starch pathway and data mining of already collected
transcriptomics studies, we wanted to evidence the input of “new” genes cur-
rently not considered to be involved in the pathway. By the way, we wanted to
discover essential genes for the regulation of the pathway and to determine
the connection of this pathway with the carbon metabolism in the plant.
Our work allowed us to confirm the essential function of one of the isoform of
soluble starch synthases, the SSIV, controlling the priming of starch synthe-
sis in the leaves of Arabidopsis thaliana. Own complementary analyses (and
recent publications) suggest that starch metabolism and the process of plas-
tid division are closely co-regulated. We believe that SSIV is directly involved
in this co-regulation since SSIV holds in its N-ter extension a domain that is
known to interact with cytoskeleton-like proteins of such as those involved in
the plastid division machinery.
CONCLUSION AND PERSPECTIVES
These results allowed us to publish 5 papers including one scientific review in
international journals. Two other manuscripts are currently under submission
or in preparation for a submission before the end of 2010. The coordinator of
this project was invited for an oral presentation at the Starch round Table in
Baltimore (Sept. 2009) and is again invited as speaker at International Sympo-
sium on “Frontiers of New Approaches for Starch Metabolism Dynamics” to be
held on November 9-11, 2010 in Akita (Japan).
αααα-1,6-(αααα-1,4 glucan)n+1 (soluble)
(αααα-1,4 glucan)n+1 (soluble)
ADP-glucose
Glucose-1-P
Insoluble/Crystalline starch
14
Project report edition 2006 Functional Analysis
MicroRNA Transcription and Activity: uncovering and exploiting the genes between the genes
ACRONYM: MiCROTRAC
COORdiNATOR: Olivier VOiNNET 1
ANR N°: ANR-06-GPLA-006
Cost: € 1, 200, 000
Grant: € 420.335
Scientists per year: 2,9
Period: 01.01.2007 – 06.30.2010
Partners: J.-P. RENOU 2,
A. BENDAHMANE 2
1. CNRS UPR 2357, Strasbourg
2. INRA URGV UMR 1165, Evry
INTRODUCTION
The intergenic or intronic DNA of many eukaryotic genomes encodes self-com-
plementary RNA molecules whose final processed forms, by the RNaseIII Dicer,
are 20-24nt-long RNAs resembling the short-interfering (si)RNA that mediate ex-
perimental RNAi. These ‘micro RNAs’ (miRNAs) are partially or fully complemen-
tary to cellular transcripts, which they regulate at the translational or stability
level. In plants and animals, miRNAs are implicated in an ever-growing number
of essential biological processes, yet our knowledge of the most basic aspects
of their biology is still in its infancy. How, when and where are miRNA transcri-
bed? What is the basis for their tissue-specific expression? How do they pair to
their target and what cellular machineries account for their repressive activities?
What is the function of the large multigene families that are often controlled by
miRNAs? What is the dynamics of the plant miRNA transcriptome and how does
it relate to the orchestration, by those molecules, of the gene regulation that
account for the execution of key physiological and developmental programs?
The proposed project was aimed at addressing those fundamental issues in the
model plant species Arabidopsis thaliana. We describe here the main outputs
of the project regarding a forward genetic screen that was designed to isolate
Arabidopsis mutants showing defects in either biogenesis or action of miRNAs.
RESULTS
1. Discovery of a widespread component of translational inhibition in the action
of plant miRNAs and siRNAs : The initial isolation of mutants (mad5 and mad6)
with defective GFP silencing, yet normal GFP mRNA suppression suggested that
translational inhibition by miR171 contributes to GFP silencing. We also descri-
bed how this conclusion had been corroborated by monitoring mRNA and pro-
tein levels of a series of endogenous miRNA targets, as well as by analysis of
mutants in genes homologous to factors implicated in miRNA guided translatio-
nal repression and decay in animals.
We have shown conclusively that the translational effect on the artificial GFP miR-
NA target really is a miRNA guided effect. This was obtained by introducing a non-
Publications
• Brodersen et al., 2008, Widespread
translational inhibition by plant miRNAs
and siRNAs. Science 320, 1185-90.
• Brodersen P. and Voinnet O. (2009).
Revisiting the principles of miRNA:target
recognition and its impact on miRNA
activities. Nature Reviews Molecular Cell
Biology. 10, 141-148.
15
Project report edition 2006 Functional Analysis
targeted GFP transgene into mad5, mad6 and vcs mutants, and
monitoring GFP protein accumulation relative to wild type. We
have also shown that RNAi in Arabidopsis also has a translational
component. Thus, a hairpin targeting a chlorophyll biosynthetic
enzyme (ChlI or “SUL”) was introduced into mutants defective in
miRNA guided translational inhibition (above), and protein and
mRNA analysis of SUL demonstrated that reduction of the mRNA
level could not account for the observed SUL silencing. Our dis-
coveries on miRNA guided translational inhibition in plants have
prompted several important questions. A top priority is now to
clarify whether plants, similar to animals, regulate highly mismat-
ched mRNAs exclusively at the level of translation. This has never
been tested, because plant miRNAs were previously believed to
act predominantly, if not exclusively via cleavage, such that only
targets perfectly complementary to miRNAs were considered
possible. We have decided to attack this problem by predicting
possible targets with central mismatches to miRNAs, expected
to prevent their cleavage. Antibodies have been raised or retrie-
ved in the literature, and a first screening of 12 putative targets
with central mismatches shows that at least 2 have substantially
higher protein accumulation in our miRNA pathway mutants. We
are continuing this work by extension of the target set, and vali-
dation of the results in mutants of specific miRNAs.
2. Identity and possible functions of the MAD genes : We des-
cribed 5 such mutants, mad1-mad6, which have all been now
cloned.
MAD1. As described in our previous report, the gene has been
mapped to a 40 kb region on chromosome 1, and a gene with a
missense mutation in a TIR-NBS resistance (R)-like gene in this
interval has been identified. Proof that this mutation is the cause
of the mad1 phenotype has now been obtained. We have now
conclusively demonstrated that the recessive nature of mad1 is
due to a threshold effect whereby a single copy of the mutated
R-protein is insufficient to cause interference with the miRNA
pathway. Given that no other mutations have been identified in
the mapping interval, and that mad1 mutants constitutively acti-
vate salicylic acid dependent defense signaling as measured by
constitutive PR1 and ICS1 expression, we favored the hypothesis
that the mutation in the R-gene is a gain-of-function mutation
and is the cause of the mad1 phenotype, which we have now
fully demonstrated. We also note that the clear roles of AGO1 in
innate antiviral defense, and, most likely in antibacterial defense
as well, are consistent with an intimacy between AGO1 and one
or more R proteins. As explained previously, we have taken four
approaches to clarify this and found that the MAD1 product effec-
tively appears to guard one or several key components of the
AGO1-miRNA RISC complex. Among the most recent outstanding
results is our finding that a viral suppressor of RNA silencing, P0,
and a bacterial suppressor of RNA silencing, HopT1, both acti-
vate an hypersusceptible response in Col-0 plants engineered to
express the C24 allele of MAD1, demonstrating a gene-for-gene
interaction between the R gene’s guardee and two major patho-
gen-encoded virulence factors that are both known to target
AGO1.
MAD2. We have shown that this mutant displays strongly re-
duced levels of AGO1 protein, despite wild type levels of AGO1
mRNA. Other silencing mutants defective in transposon-derived
siRNAs such as rdr2 and nrpd1a also show such AGO1 reductions.
mad2 does not belong to this class, however, because such siR-
NAs accumulate normally in mad2. mad2 may therefore be de-
fective in a factor stimulating AGO1 translation or reducing AGO1
turnover. We have generated a mapping population, and mapped
mad2 to the top of chromosome 2 using a minimal population of
32 plants. We have selected an additional 300 mutants for rough
mapping, followed by a larger population of 2400 plants for fine
mapping. This has enabled to define a 17 kb interval containing
the MAD2 locus. Sequencing of the four genes in this interval has
expedited the identification of MAD2, which encode a novel pro-
tein of unknown function, currently under study.
MAD3 and MAD4. Both genes have now been cloned and were
found to encode enzymes involved in sterol biosynthesis, at a
step that leads to the production of prenyl residues. Remarkably,
recent studies conducted in mammalian cells in our lab, and in
Drosophila in Richard Carthew’s lab, have uncovered an impor-
tant role for late endosomes and multivesicular bodies (MVB) as
sites of RISC assembly/disassembly and miRNA/siRNA loading.
Our study notably uncovered an MVB-coupled sorting mecha-
nism for the key RISC component and Ago-Hook protein, GW182,
which presumably allows the RISC to disassemble and reform on
membranes.
MAD5. MAD5 encodes the microtubule (MT)-severing enzyme
KATANIN, and similar requirements for tubulins in C. elegans
suggest that cytoskeleton dynamics is a universal though unde-
rappreciated component of miRNA action. Accordingly, we have
recently uncovered that MT depolymerization prevents MVBs
from recruiting GW182 in Human cells, leading to miRNA dysfunc-
tion. These results really illustrate how parallel work in different
model systems can illuminate the function of miRNA pathway
components
MAD6. We have just identified MAD6 to find that it encodes a
thioredoxin, indicating a key role for the redox status of the cell in
the regulation of miRNA activities. Although the exact implication
of MAD6 in the pathway is currently ill defined, we have made
the very interesting observation that the MAD6 protein interact
physically with a bacterial virulence factor, which, we have found,
suppresses RNA silencing in transgenic Arabidopsis. This result
identifies MAD6 as a novel target of a pathogen-encoded silen-
cing suppressor and further underscores the importance of the
miRNA pathway in basal defense against pathogenic bacteria.
CONCLUSION AND PERSPECTIVES
This ANR project has not only allowed the discovery that plant
and metazoan miRNA actions are in many respects much more
related than originally anticipated, but it has also allowed the
successful identification of a suite of key regulators of miRNA
action in Arabidopsis and, most likely, in metazoans as well. The
identity of these new factors both corroborates and enlarges the
spectrum of complexity found in the regulation of the miRNA pa-
thway and in its interactions with the environment. Together with
the landmark paper published in Science in 2008, the successful
identification of MAD1-6 will undoubtedly lead to at least three
additional papers in 2011, of high to very high standard.
16
Project report edition 2006 Functional Analysis
Involvement of non coding RNAs in the adaptation of root architecture to abiotic stress
ACRONYM: RiBOROOT
COORdiNATOR: Martin CRESPi 1
ANR N°: ANR-06-GPLA-011
Cost: € 1,169,000
Grant: € 380,255
Scientists per year: 3.4
Period: 01.01.2007 – 12.31.2009
Partners: L. NUSSAUME 2
1. CNRS- Institut des Sciences du Végétal
UPR 2355, Gif-sur-Yvette.
2. CEA- Laboratoire de Biologie du
Développement des Plantes, Cadarache.
INTRODUCTION
Crop yield is highly dependent on the capacity of the plant to optimise
its root system and the adaptation of root architecture to the soil
environment. The developmental plasticity of roots allows them to
modify the number and length of lateral roots in response to the abiotic
stresses encountered during root growth. However very little is known
about the genetic and molecular mechanisms involved in this quantitative
aspect of root architecture. Non-protein coding RNAs or npcRNAs are
a novel class of regulators of signal transduction pathways in diverse
developmental processes and abiotic stress responses. The goal of our
project is to determine the role of selected npcRNAs (including several
miRNAs) in the modulation of root architecture. An integrated approach
will aim to characterise transgenic plants affected in npcRNA expression
at phenotypic, molecular and physiological levels. In addition we will
examine the spatio-temporal regulation of the npcRNAs and their targets
in roots undergoing modifications in their root architecture. These results
may serve to propose novel strategies to modulate root architecture in a
controlled manner.
RESULTS
Identification of non-protein coding RNAs:
Long non-protein coding RNAs (npcRNA) represent an emerging class
of riboregulators, which act either directly in this long form or are
processed to shorter miRNA and siRNA. Plant and animals use small RNAs
(microRNAs and siRNAs) as guide for post-transcriptional and epigenetic
regulation. In plants, miRNAs and trans-acting siRNA (tasiRNA) result from
different biogenesis pathways but both interact with target transcripts to
direct their cleavage. Genome-wide bioinformatic analysis of full-length
17
Project report edition 2006 Functional Analysis
cDNA databases identified 76 Arabidopsis npcRNAs. Eleven npcRNAs
were antisense to protein-coding mRNAs, suggesting cis-regulatory roles.
Numerous 24-nt siRNA matched to five different npcRNAs, suggesting that
these npcRNAs are precursors of this type of siRNA. Abiotic stresses and
hormone treatments altered the accumulation of 22 npcRNAs, suggesting
that they are a sensitive component of the transcriptome. Overexpression
of npc536, an npcRNA antisense to a coding transcript, identified a
regulator of root growth during salt stress.
The action of miR169 and miR390 in lateral root growth and development
Modulation of miR169 (using anti-miR approaches) affected root
architecture. We prepared a series of transgenic plants showing perturbed
expression of this miRNA as well as their NFY transcription factor targets
to assay their root phenotypes. Another npcRNA expressed in root
tissues corresponded to TAS3a, a tasiRNA precursor target of miR390. We
demonstrated that miR390, TAS3-derived ta-siRNAs (trans-acting siRNAs)
and ARF4 form an auxin-responsive regulatory network controlling lateral
root growth. Spatial expression analysis using reporter gene fusions, ta-si/
miRNA sensors and mutant analysis, showed that miR390 is specifically
expressed at the sites of lateral root initiation to cleave the TAS3 precursor
and trigger the biogenesis of trans-acting siRNAs. These ta-siRNAs inhibit
ARF4 activity which represses lateral root growth. In turn, ARF4 controls
auxin-induced miR390 accumulation. This mutual cross-inhibition of
miR390 and ARF4 ensures the miR390 expression pattern and maintains
ARF4 expression in an optimal concentration range for lateral root growth.
The control of the timing of lateral root development is reminiscent of its
action during leaf development.
These results also show how small regulatory RNAs integrate with auxin
signalling to quantitatively control organ growth during development.
Finally, we showed that the npcRNA43 locus was transcribed in sense/
antisense orientation and the expression of both molecules was analysed
in several mutants affected in silencing pathways (RNA-dependent RNA
polymerases, Dicer-like genes and others). Overexpression of the NAT43
(the antisense RNA of this locus) led to a developmental phenotype and
a perturbation of the phosphate starvation responses. This may reveal
a novel antisense regulatory mechanism involved in the regulation of
phosphate starvation responses in plants. Hence, long and small npcRNAs
fine tune expression patterns of regulatory genes to modulate root
developmental plasticity.
CONCLUSION AND PERSPECTIVES
Our results suggest that the miR390/TAS3 pathway may regulate
quantitative aspects of lateral root growth and development, the
involvement of a novel dsRNA in phosphate starvation responses and a role
of miR169 through NFY transcription factors in the regulation of expression
patterns in root tissues. These results have revealed novel strategies that
plants utilize to modulate root growth and development in the soil.
Publications
• F. Merchan, A. Boualem, M. Crespi,
F. Frugier (2009), Plant polycistronic
precursors containing non-
homologous microRNAs target transcripts
encoding functionally related proteins.
Genome Biology. 10, R136.
• Ben Amor B., Wirth S., Merchan F.,
Laporte P., d’Aubenton-Carafa Y., Hirsch
J., Maizel A., Mallory A., Lucas A.,
Deragon J-M., Vaucheret H., Thermes C.
and Crespi M. (2009), Novel long
non-protein coding RNAs involved in
Arabidopsis differentiation and stress
responses. Genome Research. 19(1):57-69.
• Wirth S. and Crespi M. (2009), Point of
view: Long non-protein coding RNAs and
their impact in plant development RNA.
Biology. 6 (2): 161-164.
• Schwab* R., Maizel* A., Ruiz-Ferrer V.,
Garcia D., Bayer M., Crespi M., Voinnet O.
and Martienssen R. A. (*equally
contributing authors), (2009), Endogenous
TasiRNAs Mediate Non-cell Autonomous
Effects on Gene Regulation in Arabidopsis
thaliana. PLOS One. 4 (6): e5980.
• Lelandais-Brière C., Sorin C.,
Declerck M., Benslimane A., Crespi M.
and Hartmann C. (2010), Small RNA
diversity in plants and its impact in
development. Current Genomics. 11, 14-23.
• Marin E., Jouannet V., Herz A.,
Lokerse A.S., Weijers D., Vaucheret H.,
Nussaume L., Crespi M. and
Maizel A. (2010). miR390, Arabidopsis
TAS3 tasiRNAs, and Their AUXIN
RESPONSE FACTOR Targets Define an
Autoregulatory Network Quantitatively
Regulating Lateral Root Growth. Plant Cell
(in press).
18
Project report edition 2006 Functional Analysis
ACRONYM: TRANSPORTOME
COORdiNATOR:
Jean-Baptiste THiBAUd1
ANR N°: ANR-06-GPLA-012
Cost: € 1,460,124
Grant: € 590,000
Scientists per year: 4.6
Period: 01.01.2007 – 06.30.2010
Partners: JM. FRACHISSE2, C. LURIN3
1. CNRS UMR 5004, Montpellier
2. CNRS, UPR 2355, Gif sur Yvette
3. INRA UMR 1165, Evry
INTRODUCTION
Several hundreds of genes in Arabidopsis, encode proteins of yet
unknown functions, which appear to be associated with the plasma
membrane (membrane proteome). The goal of “TRANSPORTOME” was
to identify, among this membrane proteome, either new ion transport
systems (including transport of anions) or proteins that are partners
of known transport systems (particularly potassium channel of the
“Shaker” family) and can regulate the activity of these transport systems.
This project has been conceived while the Arabidopsis genome had
been fully sequenced (Arabidopsis Genome Initiative [2000] Analysis
of the genome sequence of the flowering plant Arabidopsis thaliana.
Nature 408:796-815) but of which the annotation remained, in 2006,
both uncertain and unachieved. To attempt, from a selected proteome,
the functional characterisation of proteins was expected to result in the
identification of new proteins involved in those transport reported at the
cell membrane but lacking molecular support. In addition this should
avoid studying pseudo-genes as addressed ORFs were deduced from
actually expressed membrane-associated polypeptides.
RESULTS
The first part of this work (work-package 1 or WP1) consisted in cloning
(within Gateway® entry vector) the ORFs encoding the selected
proteome (about 600 ORFs). Each of these ORFs was to be cloned in two
full-length forms: one without the STOP codon (“END” series) and one
with the STOP codon (“STOP” series).
The Arabidopsis plasma membrane transportome: searching for macromolecular complexes involved in ion transport
19
Project report edition 2006 Functional Analysis
The END constructs were to be used (WP2) to express fusion proteins for in
planta screening (“split-DHFR” approach) of protein-protein interactions
within this proteome or with a number of previously identified transport
proteins (namely, Shaker channels).
The “STOP” constructs were to be used (WP3) for functional expression
in appropriate contexts (Xenopus oocytes, COS or CHO cells,
protoplasts). Electrophysiological recordings (voltage-clamp or patch-
clamp) or fluorescence imaging of ion-sensitive probes were to be used
for revealing potential changes in membrane ion conductance following
expression of these ORFs.
Last, the in planta role of genes identified in this framework were to be
characterised using mutant plants, namely knock-out lines (WP4).
The ORFs targeted by this cloning project have been sorted by size in 96-
well plates. More than half of the whole set could be obtained although
the larger the ORF size the lesser was the cloning success.
The “END” series is still to exploit by “split-DHFR” due to many
unexpected problems with plant transformation. Besides, the “END”
clones have been used in systematic screening of protein-protein
interactions by the double-hybrid system in Yeast (ATOME project, in
collaboration with Marc Vidal, Boston, MA).
Screening the “STOP” series has resulted in the identification of a number
of Ca2+-dependent kinases of the CIPK or the CDPK family regulating the
activity of Shaker channels.
CONCLUSION AND PERSPECTIVES
The whole collection of ORFs cloned in the Transportome framework
has been duplicated and stored at CNRGV (Toulouse, France) where is
is available for the scientific community (information on sequences @
http://urgv.evry.inra.fr/ATOMEdb).
Publications
• Corratgé-Faillie C., Jabnoune M.,
Zimmermann S., Véry A.-A., Fizames C.,
Sentenac H. (2010), Potassium and
sodium transport in non-animal cells: the
Trk/Ktr/HKT transporter family. Cellular
and Molecular Life Sciences 67:2511-2532.
• Diatloff E., Peyronnet R., Colcombet
J., Thomine S., Barbier-Brygoo H.,
Frachisse J.-M. (2010), R type anion
channel: a multifunctional channel
seeking its molecular identity. Plant
Signaling and Behavior minireview (in
press).
• Lebaudy A., Pascaud F., Véry A.-A.,
Alcon C., Dreyer I., Thibaud J.-B.,
Lacombe B. (2010), Preferential KAT1-
KAT2 Heteromerization Determines
Inward K+ Current Properties in
Arabidopsis Guard Cells, Journal of
Biological Chemistry 285:6265–6274.
The obtained TRANSPORTOME clones (more than 500 ORFs cloned within the Gateway®
pDONR2007 entry vector) have been duplicated and stored at CNRGV (Toulouse, France) making
them accessible to the scientific community (http://cnrgv.toulouse.inra.fr/en/library/genomic_
resource/TRANSPORTOME). Cloning and sequecing data are available through the “ATOMEdb”
database (http://urgv.evry.inra.fr/ATOMEdb).
New Tools
Production of flax oligonucleotide microarrays
INTRODUCTION
Creation of flax (Linum usitatissimum) genomic tools.
Flax is an annual species cultivated for bast fibres and linseed oil. France
is the world’s leading flax producer generating 130 000 tons (57% of the
world production) of scutched flax per year. The flax fibres are traditionally
used in textiles and more recently integrated in composite materials for
the automobile and construction industries. Linseed oil is widely used as
a component of paint, varnishes, oleochemical products and polymers.
Flax seeds are also very rich in omega-3 fatty acids important in a human
health and nutrition. However, our understanding of the seed-filling and
fibre-development processes and the relationship between oil or fibre
quality and industrial processes is still limited.
The main objective is to create oligonucleotide-based arrays and genomic
tools (EST library) that will subsequently allow us to gain a better
understanding of the genetics, molecular and physiological functioning
of this plant in order to improve agriculturally and industrially-valuable
traits (productivity, oil content, fibre properties, stress tolerance).
RESULTS
The work in GENOLIN is organized in three main workpackages (WPs):
WP1: Elaboration of cDNA and EST libraries
WP2: Production of oligonucleotide-based arrays
WP3: Verification of the functionality of these microarrays and initial
expression studies.
To do that, wild type Linum usitatissimum plants (oil variety Barbara;
fibre variety Hermes) were chosen and grown under controlled conditions
in greenhouse. Roots, leaves, stem outer and inner tissues were
harvested during vegetative growth (50-60 Days After Germination) and
Project report edition 2006 New Tools
22
ACRONYM: GENOLiN
COORdiNATOR:
Brigitte THOMASSET1
ANR N°: ANR-06-GPLA-017
Cost: € 1.264.000
Grant: € 494.484
Scientists per year: 1.8
Period: 01.01.2007 – 03.31.2010
Partners: O. VAN WUYTSWINKEL2,
S. HAWKINS3, B. CHABBERT4, R.
TAVERNIER5, X. GUILLOT6, F. BERT7
1. Université de Technologie de Compiègne,
UMR-CNRS 6022, Compiègne
2. Université de Picardie Jules Verne LGFP,
EA 3900, Amiens
3. Université des Sciences et Technologie de
Lille, UMR-INRA 1281, Stress abiotiques &
différenciation des végétaux cultivés, Lille
4. INRA, UMR FARE, Reims
5. LINEA, Grandvilliers
6. Laboulet Semences, Airaines
7. Institut Technique du Lin, ITL, Ecardenville
La Campagne
at the green capsule stage (70-80 DAG), together with developing seed
(Barbara) collected 10-15, 20-30 and 40-50 Day After Flowering (DAF)
corresponding to early, maximal and late stage of storage compounds
synthesis. Nucleic acids (RNA) were extracted from these tissues and an
alternative method of high throughput pyrosequencing: 454 technology
was used to generate (EST) genetic sequence data. The production of
NimbleGen type microarrays were performed from these data.
The functionality of these tools was validated and initial expression
studies were done on different flax varieties presenting differences in oil
or in stem composition.
CONCLUSION AND PERSPECTIVES
This work has generated 1 066 481 expressed gene sequences in relation
with the developing stages of the plants. Assembling of the 454 data
has allowed to obtain 59 494 unigenes used to produce highly reliable
flax microarrays. The availability of these molecular tools will lead to
an improved understanding of flax development (seed filling and fibre
development), of the influence of culture conditions on product quality
and quantity (oil, fibre) and allow faster access to genes for breeding
(RECOLIN; PlantTeq 4; ANR-PT Flax labelled by the Pôle de compétitivité
Industrie et Agro-Ressources).
The GENOLIN was an experimental development programme allowing
the production of tools and genomic resources specific for flax that will
be used in future fundamental research and for the development of
assisted breeding.
Project report edition 2006 New Tools
23
Publications
• The unigene data produced during
GENOLIN will be submitted to the
international sequence databases and
available for the scientific community.
• A paper is submitted to BMC Journal:
Development and validation of a flax
(Linum usitatissimum L.) gene expression
oligo microarray.
Flax field trials: selection campaign
24
Project report edition 2006 New Tools
Identification of signals controlling the protein trafficking between the secretory pathway and the chloroplast
INTRODUCTION
Recently, a new route for protein trafficking between the secretory system
and the chloroplasts has been reported for two proteins. In this pathway,
proteins enter the endoplasmic reticulum where they are N-glycosylated,
they are then transported through the Golgi apparatus and finally
targeted to the chloroplast. The aim of the present project was to identify
new proteins using this new route between the secretory system and the
chloroplast and to take advantage of these new candidates to identify
the signals that control this protein trafficking. The identification of
such signals would allow pharmaceutical proteins to be expressed in
the secretory system and then to be stored in the chloroplasts, taking
advantage of (1) the post-translational modification events occurring in
the secretory pathway and (2) high capacity of protein accumulation in
higher plant chloroplasts.
RESULTS
In contrast to chloroplast-encoded proteins or to nuclear-encoded
proteins that are synthesised in the cytosol, proteins that are trafficking
between the secretory system and the chloroplasts may acquire a
glycosylation in the ER before their entry in the chloroplast. Thus, the
presence of glycans on proteins located in the chloroplast was used
as a first strategy to select candidate proteins. Various protocols were
developed to fish out glycoproteins prior to proteomic analysis. Immuno-
and affinodetection analysis of proteins from purified chloroplast
extracts using glycan-specific probes have revealed the presence of
multiple glycoproteins. However, their quantification has shown that
ACRONYM: GLYCO-CHLOROPLAST
COORdiNATOR: Patrice LEROUGE 1
ANR N°: ANR-06-GPLA-003
Cost: € 2,414,000
Grant: € 316,519
Scientists per year: 4,3
Period: 01.01.2007 – 06.30.2010
Partners: M. BARDOR1, C. RIHOUEY1,
S. LE GALL1,2, D.SALVI2, D. BERNY2,
L. MOYET2, J. JOYARD2,
N. ROLLAND2, M. FERRO3,
C. MASSELON3, S. BRUGIERE3,
M. MELLAL3, C. BRULEY3, J. GARIN3
1. EA 4358, Université de Rouen.
2. LPCV, CNRS UMR 5168 / INRA UMR 1200 /
CEA / Université de Grenoble.
3. EDyP, CEA / INSERM U 880 / Université
de Grenoble.
25
Project report edition 2006 New Tools
glycosylated proteins and as consequence proteins trafficking through
the new route between the secretory system and the chloroplasts do not
exceed 0.1 to 1% of the total chloroplast protein population. Isolation
of N-glycosylated proteins was then carried out by affinopurification on
lectins. One candidate protein was identified using this strategy.
The second strategy to select candidate proteins was based on
the generation of the most comprehensive as possible database of
chloroplast proteins and then on the search, within this database,
for candidates proteins for plastid targeting through alternative
pathways using bioinformatic tools. The AT_Chloro database (http://
prabi2.inrialpes.fr/at_chloro/; online since April 2010) for chloroplast
subcompartments (envelope, stroma, thylakoids) has been successfully
established using a new proteomic methodology based on very Accurate
Mass and chromatographic retention Time (AMT) measurements of
tryptic peptides obtained from chloroplast extracts. More than 1300
proteins have been identified with information about their sub-plastidial
localization (Ferro et al., 2010; Joyard et al., 2009, 2010). Identification of
candidate proteins within this database has been carried by searching
specific signatures using bioinformatic tools. Twenty candidate proteins,
containing putative signal peptides and glycosylation sites or lacking
classical and predictable chloroplast transit peptides, were selected.
In order to confirm their plastidial localization, the candidate proteins
(selected either by biochemical approaches or in silico), were expressed
(stable expression) in planta as GFP fusions and the localization of these
candidates was investigated by confocal microscopy. Several of these
proteins were demonstrated to be effectively targeted to the chloroplast
and some of them appear to be shared between the chloroplast and
other cell compartments.
CONCLUSION AND PERSPECTIVES
The analysis of peptide sequences of newly identified proteins will help
defining minimal sequences responsible for alternative targeting routes
to chloroplasts.
Publications
• Joyard J, Ferro M, Masselon C,
Seigneurin-Berny D, Salvi D, Garin J,
Rolland N (2009) Chloroplast proteomics
and the compartmentation of plastidial
isoprenoid biosynthetic pathways. Mol
Plant. 2: 1154-1180.
• Ferro M, Brugière S, Salvi D,
Seigneurin-Berny D, Court C, Moyet L,
Ramus C, Miras S, Mellal M, Le Gall
S, Kieffer-Jaquinod S, Bruley C, Garin
J, Joyard J, Masselon C, Rolland N
(2010) AT_CHLORO: A comprehensive
chloroplast proteome database with
subplastidial localization and curated
information on envelope proteins. Mol
Cell Proteomics 9: 1063-1084.
• Joyard J, Ferro M, Masselon C,
Seigneurin-Berny D, Salvi D, Garin
J, Rolland N (2010) Chloroplast
proteomics highlights the subcellular
compartmentation of lipid metabolism.
Prog Lipid Res. 49: 128-158.
database (Pi) :
AT-CHLORO: A comprehensive AMT
chloroplast proteome database with sub-
plastidial localization of proteins APP:
IDDN.FR.001.230022.000.D.P.2009.000.
10300. Génoplante-Valor. Authors : Ferro
M, Brugière S, Salvi D, Seigneurin-Berny
D, Court M, Moyet L, Kieffer-Jaquinod S,
Bruley C, Garin J, Joyard J, Masselon C,
Rolland N.
http://www.inra.fr/presse/at_chloro_
base_donnees_dediees_proteines_
chloroplastes
http://www2.cnrs.fr/presse/
communique/1861.htm?debut=32
http://www.ujf-grenoble.
fr/1272531773991/0/fiche___actualite/
http://www.cea.fr/le_cea/actualites/
base_de_donnee_at_chloro-34220
NEW TOOLS
- 2 -
these proteins were demonstrated to be effectively targeted to the chloroplast and some of them appearto be shared between the chloroplast and other cell compartments.
CONCLUSION AND PERSPECTIVES
The analysis of peptide sequences of newly identified proteins will help defining minimal sequencesresponsible for alternative targeting routes to chloroplasts.
Figure 1: Purification and fractionation of chloroplasts was followed by mass-spectrometry to create the AMTdatabase AT_Chloro. Stable expression of GFP-fusions in Arabidopsis allowed identifying plastid targetedproteins that lack canonical targeting peptides.
PUBLICATIONS :
Joyard J, Ferro M, Masselon C, Seigneurin-Berny D, Salvi D, Garin J, Rolland N (2009) Chloroplastproteomics and the compartmentation of plastidial isoprenoid biosynthetic pathways. Mol Plant.2: 1154-1180.
Ferro M, Brugière S, Salvi D, Seigneurin-Berny D, Court C, Moyet L, Ramus C, Miras S, Mellal M,Le Gall S, Kieffer-Jaquinod S, Bruley C, Garin J, Joyard J, Masselon C, Rolland N (2010)AT_CHLORO: A comprehensive chloroplast proteome database with subplastidial localizationand curated information on envelope proteins. Mol Cell Proteomics 9: 1063-1084.
Joyard J, Ferro M, Masselon C, Seigneurin-Berny D, Salvi D, Garin J, Rolland N (2010) Chloroplastproteomics highlights the subcellular compartmentation of lipid metabolism. Prog Lipid Res. 49:128-158.
DATABASE (PI) :
AT-CHLORO: A comprehensive AMT chloroplast proteome database with sub-plastidiallocalization of proteins APP: IDDN.FR.001.230022.000.D.P.2009.000. 10300. Génoplante-Valor. Authors : Ferro M, Brugière S, Salvi D, Seigneurin-Berny D, Court M, Moyet L, Kieffer-Jaquinod S, Bruley C, Garin J, Joyard J, Masselon C, Rolland N.
http://www.inra.fr/presse/at_chloro_base_donnees_dediees_proteines_chloroplasteshttp://www2.cnrs.fr/presse/communique/1861.htm?debut=32http://www.ujf-grenoble.fr/1272531773991/0/fiche___actualite/http://www.cea.fr/le_cea/actualites/base_de_donnee_at_chloro-34220
Figure 1: Purification and fractionation of chloroplasts was followed by mass-spec-
trometry to create the AMT database AT_Chloro. Stable expression of GFP-fusions
in Arabidopsis allowed identifying plastid targeted proteins that lack canonical
targeting peptides.
26
Project report edition 2005 New Tools
A bioinformatics gateway towards integrative legume biology
INTRODUCTION
The integration of numerous and heterogeneous data (from genomics to
post-genomics) produced by the legume community represents a challenge
that needs to be overcome to fully exploit the considerable investment and
progress made by many laboratories worldwide. Our goal is to provide and
to ensure the interoperability of bioinformatics resources that are required
to integrate and interpret such data.
RESULTS
LEGOO is a web portal which is based on the protocol BioMoby both
to permit the interoperability between the different resources and to
make available data and software within the bioinformatics community.
The portal provides a unified entry point to numerous bioinformatics
resources including a knowledge base that represents and structures
published results. It provides a comparative genome browser to
facilitate knowledge transfer between models and crops, which can be
achieved by integrating genetic maps and genomic sequences as well
as overlaying expression data. The portal also provides several genomic
tools to exploit and combine the results of various technologies that
have been developed in recent years. Finally, several workflows are
provided allowing data and software to be combined to address frequent
requests, such as promoter sequences analysis for sets of co-regulated
genes.
LEGOO is publicly available and is accessible by the link http://www.
legoo.org. The portal illustrates the power of developing service oriented
architecture (SOA) in bioinformatics. As it relies on BioMoby web services
to integrate dedicated bioinformatics resources, it both, takes advantage
of the power of specific tools for in depth analysis and provides the
ACRONYM: LEGOO
COORdiNATOR: Jérôme GOUZY1
ANR N°: ANR- 06-GPLA-005
Cost: € 679,000
Grant: € 214,475
Scientists per year: 2.3
Period: 01.01.2007 – 12.31.2009
Partners: T. FARAUT2, K. GALLARDO3
1. Laboratoire des Interactions Plantes Micro-
organismes (LIPM), UMR441/2954 INRA/CNRS,
Castanet Tolosan
2. Laboratoire Génétique Cellulaire UMR444,
INRA/ENVT, Castanet Tolosan
3. Unité de Recherche en Génétique et
Ecophysiologie des Légumineuses à Graines,
INRA, Dijon
27
Project report edition 2005 New Tools
users with a unified gateway between the resources allowing a global
discovery strategy which is the aim of integrative biology approaches.
With the LEGOO portal we provide the legume community with a
coherent set of resources which allow to (i) integrate high-throughput
sequence data produced by successive generations of “omics” tools (ii)
extract knowledge from these data and associated literature, organize
and represent it to help deciphering molecular mechanisms acting at
cell, tissue or organism level (iii) transfer knowledge between model and
crop species.
CONCLUSION AND PERSPECTIVES
In the next few years, the use of next-generation DNA sequencing
techniques will generate huge amounts of sequence data, including
whole genome from multiple lines or accessions of the same species,
in order to tap natural diversity for traits of interest through strategies
such as association genetics. In addition sequences will be generated
from legume crop species that have so far been poorly studied (orphan
legume crops) enabling the development of translational genomics and
genomics-assisted breeding in these legumes. The comparative genomics
tools of the LEGOO platform will be developed to take advantage of
these new resources. New user interfaces and additional web-services
will be developed to retrieve, search and assemble sequence data
(with or without reference sequence) including short reads generated
by next generation sequencers. In parallel, we will keep feeding the
knowledgebase in the short term.
In a middle term, our hope is that more and more biologists will feed
the database with their own publications and that our role will move to
that of moderators and curators. In the future, we will develop the portal
to integrate data and knowledge related to regulation by small RNAs.
Several RNASeq projects are in progress in the legume community;
new key molecules involved in the regulations will be identified and the
portal will be developed in order to define and represent comprehensive
regulatory networks whatever the nature of the actors involved. Building
such networks is a prerequisite for the ultimate goal of most of the
bioinformatics providers that is the in silico modelling of living cells and
organisms.
Publications
• Courcelle E, Beausse Y, Letort S,
Stahl O, Fremez R, Ngom-Bru C,
Gouzy J, Faraut T: Narcisse: a mirror
view of conserved syntenies. Nucleic
Acids Res 2008, 36:D485-490.
• Neron B, Menager H, Maufrais C,
Joly N, Maupetit J, Letort S, Carrere S,
Tuffery P, Letondal C: Mobyle: a new
full web bioinformatics framework.
Bioinformatics 2009, 25:3005-3011.
28
Project report edition 2006 New Tools
The ‘PUCE CAFE’ Project: the First 15K Coffee Microarray, a New Tool for Discovering Candidate Genes correlated to Agronomic and Quality Traits
INTRODUCTION
Developing a DNA CHIP
Coffee is a product of mass consumption, with world consumption of more
than 2.2 billion cups per day. Coffee is largely produced by small farmers and
provides a livelihood for over 125 million people in more than 50 countries.
The development of the most advanced biotechnological tools to help
improve the species is justified by its economic and social importance. For
biologists and agronomists, it is a question of adapting a species of African
origin so that the crop can be grown in tropical regions throughout the world.
In the context of global warming, the crop has to meet the huge challenges
of increasing disease proliferation and the product needs to adapt to more
and more different uses. In order to gear quality to the needs of the industry,
it is important to understand all the stages of fruit formation that determine
its biochemical composition. The project consisted in developing a DNA
chip, which notably means that it is possible to conduct large-scale studies
of gene expression during fruit development in addition to other types of
research. The Coffee Microarray Project is based on scientific collaboration
between NESTLE and CIRAD/IRD granted by ANR (National Research
Agency) via GENOPLANTE. PUCECAFE project has two main objectives 1)
Create the first Coffee 16 K oligo microarray and 2) validate and use this new
tool to analyze gene expression patterns during coffee grain maturation in
Coffea arabica (Arabica) and Coffea canephora (robusta).
RESULTS
To validate the spotted 70-mer oligo-gene microarray ‘PuceCafé’ we
compared three different coffee tissues (flowers, beans and leaves).
ACRONYM: PUCE CAFE
COORdiNATOR: Benoît BERTRANd1
ANR N°: ANR- 06-GPLA-014
Cost: € 1,009,000
Grant: € 495,172
Scientists per year: 2.7
Period: 01.01.2007 – 12.31.2009
Partners: I. PRIVAT2, P. LASHERMES3
1. CIRAD, Montpellier
2. CRD NESTLE, Tours
3. IRD, Montpellier
29
Project report edition 2006 New Tools
Through statistical and biological approaches we estimated the
repeatability of the signals. We verified also that the regulation of sugars,
lipids and proteins metabolism transcriptome were in accordance with
the published results available.
Then, three experiments were performed and completely analyzed.
In the first experiment, the polyploid Coffea Arabica that derived from
two diploid species c. canephora and c. eugenioides was compared
in two growth temperature conditions with the two parental species
through a transcriptomic approach using the PuceCafé. We showed
that the divergence of patterns expression between natural but recent
allopolyploid species in comparison with its two relatives is environment-
specific. These results reinforce the hypothesis of a better functional
plasticity of the allopolyploids in comparison to the diploids species
related and consequently the evolutionary advantage of this genome
architecture. We also demonstrated that the PuceCafé can be used for
other coffee species than c. canephora.
In the second experiment, we decipher the transcriptional networks
associated with seed maturation, achievement of germination capacity
and partial acquisition of desiccation tolerance in the coffee seeds,
since by contrast with orthodox seeds, intermediate seeds as coffee
seeds withstand only partial drying and their conservation remains
problematic. The coffee seeds are also characterized by the absence of
an abrupt water loss during the maturation phase of their development.
Data provided by the PuceCafé in combination with desiccation tolerance
and hormone measurements throughout seed development, provides a
new global picture of the intermediate seed physiology.
In the third experiment, Comparison with two c. canephora genotypes
was performed to analyze transcriptome evolution during bean
maturation. We identified genes with unknown function differentially
expressed during bean development. This set of genes could be
correlated with unsuspected metabolic pathways or regulating genes
correlated to coffee cup quality. Confrontation between agronomic and
physiological data in front of transcriptomic data will allow us to define
adapted breeding criteria to be used in future quantitative genetic
studies, or by breeders in breeding programmes. The work also includes
new knowledge acquisition on Arabidopsis nitrogen nutrition during
post flowering period taking into account the fact that most of the results
are coming from early development stages of the plant.
CONCLUSION AND PERSPECTIVES
Three other experiments namely, i) Comparison of C. arabica and C
canephora transcriptomes for a few stages of fruit development and ii)
& iii) comparison of the leaf and roots transcriptome of four C. canephora
ecotypes under abiotic stress conditions – ii) high temperatures or iii)
drought stress- , are under biological interpretation. The data obtained
along the fruit development and ripening phase will undoubtedly allow
a better understanding of several metabolisms in the fruit and allow the
identification of new candidate genes for compounds of interest. For
other part, the abiotic stress experiments will give precious information
on specific defense mechanisms /pathways used by coffee plant for
stress tolerance.
Publications
• Joët T., Laffargue A., Salmona J.,
Doulbeau S., Descroix F., Bertrand B.,
de Kochko A. and Dussert S., (2009),
Metabolic pathways in tropical
dicotyledonous albuminous seeds: Coffea
arabica as a case study. New Phytologist,
182:1, 146-162.
• Bardil A., Combes M.C., Lashermes P.
and Bertrand B. (submitted), Massive
nonadditive gene expression in the
natural allopolyploid Coffea arabica is
affected by growth temperature. BMC
Biology.
• Privat I., Bardil A., Severac D.,
Dantec C., Fuentes I., Mueller L. ,
Joet T., Pot D., Foucrier S., Dussert
S., Leroy T., Journot L., de Kochko A.,
Combes M.C., Lashermes P. (submitted),
The ‘PUCE CAFE’ Project: the First
15K Coffee Microarray, a New Tool for
Discovering Candidate Genes correlated
to Agronomic and Quality Traits. BMC
Genomics.
The First 15K Coffee Microarray, a New Tool for
Discovering Candidate Genes correlated to Agronomic
and Quality Traits.
Comparisons of RNA collected from two species,
three tissues and different stages of coffee bean
development.
30
Project report edition 2006 New Tools
Conception and exploitation of a multi usage chip of the Arabidopsis genome
INTRODUCTION
During the last decades molecular tools have been used to study genes
on a one by one approach. It was recently observed that gene position in
the genome and the presence of transposons in their vinicity has profound
influence on gene expression. Furthermore the study of the so called
“epigenome” requires pan genomic tools to describe the different processes
by which the expression of a gene is modulated. Two types of tools can be
used: the deep sequencing approach and the genome scale tiling arrays.
We have chosen the tiling arrays due to their versatility and low cost of
exploitation thus facilitating comparative studies.
RESULTS
The goal of the project was to design and to validate a genome scale
tiling array covering the Arabidopsis genome with 2X720 000 probes
of size 50-70 nt (One probe for each 200nt window). This array was set
up in collaboration with NimbleGen company. Data are embedded in
FLAGdb++exploited by utilization of an information system developed
at INRA URGV and derived from Flagdb. This tool is coupled with a new
and efficient module of statistical treatment of the data (MultiChIPmix).
Arrays can be used by simple labeling or by dye swap for comparative
studies. Applications have been made for different uses including
DNAmethylation, ChIP/, CGH, detection of non coding transcripts, as
illustrated above. These tools provide data which are exploitable in
combination with FLAGdb and CAT db, for instance to explore intergenic
regions and define new gene models.
This whole-genome tiling microarray platform is now routinely used for
transcriptome analysis (including the detection of sense and antisense
transcripts), comparative genomic hybridization (for the detection of
ACRONYM: TAG
COORdiNATOR: Michel CABOCHE 1
ANR N°: ANR-06-GPLA-010
Cost: € 752,000
Grant: € 382,519
Scientists per year: 3,1
Period: 01.01.2007 – 06.30.2010
Partners: S.AUBOURG1,
J.-P. RENOU1, V.COLOT2,
M.-L. MARTIN-MAGNIETTE3
1. INRA, UMR 1165 URGV, Evry
2. ENS Ulm, UMR CNRS 8197-INSERM U1024
Paris
3. Agro Paris Tech, UMR518 MIA Paris
31
Project report edition 2006 New Tools
Copy Number Variants or CNVs) and epigenomic mapping of chromatin
modifications in wild type and mutant backgrounds. For example,
epigenomic profiling of the chromatin marks H3K4me3 and H3K27me3
indicated that they represent antagonist marks, which occur at any one
time over approximately 20% and 50% of Arabidopsis genes, respectively.
Furthermore, analysis of the distribution of these marks in a mutant
affected in Polycomb Repressive Complex 2 (PRC2) activity revealed an
almost complete abolition of the H3K27me3 mark over genes, with no
alteration of the H3K4me3 deposition pattern. In another application,
numerous CNVs, corresponding mainly to transposable elements were
identified by CGH between the Col-0, C24 and Cvi accessions.
CONCLUSION AND PERSPECTIVES
The whole-genome tiling microarray platform validated under the TAG
project is now being used in two collaborative projects financed by ANR
(ANR Génomique des plantes 2008 REGENEOME, Coord. J.-D. Faure;
and ANR Blanc 2008 DDB1, Coord. C. Bowler), as well as in several other
projects.
Publications
• Martin-Magniette ML, Mary-Huard
T, Bérard C, Robin S. (2008) ChIPmix:
mixture model of regressions for two-
color ChIP-chip analysis. Bioinformatics.
2008 Aug 15;24(16):i181-6.
• Roudier F, Teixeira FK, Colot V (2009).
Chromatin indexing in Arabidopsis: an
epigenomic tale of tails and more. Trends
in genetics : TIG, 25:511-7.
Raw and analyzed data generated using TAG chips are imported, visualized and
explored through a specific FLAGdb++ module based on a client Apache Derby
database.
Oil and Protein Crops
An integrated approach using M. truncatula to identify loci/genes controlling composition and physiological quality of legume seeds
ACRONYM: QUALiTYLEGSEEd
COORdiNATOR: Karine GALLARdO1
ANR N°: ANR-06-GPLA-008
Cost: € 2,377,000
Grant: € 563,549
Scientists per year: 9.3
Period: 01.01.2007 – 04.30.2010
Partners: J. BUITINK2,
A. BENDAHMANE3, J.-M. PROSPERI4,
J. GOUZY5, T. HUGUET6
1. UMR LEG 102 INRA/ENESAD, INRA Dijon
2. UMR PMS 1191 Univ. Angers/Agrocampus
Ouest/INRA, Angers
3. URGV 1165, INRA Evry
4. INRA UMR DGPC, Mauguio
5. UMR 441/2954 INRA/CNRS, Castanet
Tolosan
6. BAP Pôle INP ENSAT, Castanet TolosanINTRODUCTION
The project took place in the context of a socio-economic need to
improve seed quality and competitiveness of European grain legumes. It
was aimed at studying possible associations between seed composition
and physiological seed traits (longevity, germination and seedling
performance under stress), and identifying loci/genes controlling these
characters. This project was based on the use of the legume model
species Medicago truncatula, for which extensive genomic sequences
are available, along with post-genomics resources describing the
transcriptome of seeds at different stages of development. The work was
organised in three parts: (1) QTL mapping for physiological seed traits,
seed size and composition; (2) in silico mapping of gene expression
profiles to allow the selection of candidate genes colocalizing with these
QTL; and (3) functional study of positional candidate genes through
reverse genetics and association studies in M. truncatula and P. sativum.
One fascinating aspect of this project has been to allow a comparison of
physiological traits of the seed with its protein composition.
RESULTS
Combining genetics and post-genomics studies to identify relevant
candidate genes related to seed quality in legumes.
A QTL search for seed longevity, germination vigor, seedling performance
(on water, under water stress and on nitrate), and seed weight,
was performed in M. truncatula using the recombinant inbred line
population LR4 (and LR1 for some traits such as seed sugar content and
germination). In parallel, the innovative PQL (protein quantity loci) and
34
Project report edition 2006 Oil and Protein Crops
NIRS (near-Infrared spectroscopy) approaches were employed to identify
chromosomal regions controlling seed protein content and composition.
The LEGOO software developed in the ANR project GPLA06026G was
used to localize on the physical map of M. truncatula an exhaustive set
of expressional candidate genes arising from previous transcriptomic
surveys on seed development and germination. Because the LR4 map
is composed of genetic markers anchored to the physical map, it offers
the opportunity to explore the genes in QTL/PQL regions. Several
hundred seed-expressed genes were localized in silico on this map and
the positions of a number of them were confirmed by genetic mapping.
Candidate genes overlapping with QTL/PQL for seed traits were selected
for functional studies using association genetics and induced mutants in
both legume species M. truncatula and pea.
Certain PQL for seed proteins and QTL for sugar content overlapped
perfectly with QTLs for physiological seed traits, thus highlighting some
proteins and sugars as possible indicators of longevity or germination
performance.
12 000 genes were anchored to the genetic map, of which 5 400 were
preferentially expressed in seeds. By exploring the genes behind those
QTLs, regulatory networks for the accumulation of proteins possibly
related to longevity or germination were proposed, and the link between
a number of these genes and seed quality has been validated by mean
of mutants.
CONCLUSION AND PERSPECTIVES
Among the outputs of the project are: (1) the construction of a consensus
genetic-expression map for the selection of seed-specific candidate
genes colocalized with QTL for seed traits; (2) the identification on
this map of the bZIP transcription factor ABI5 as a central regulator
of stress proteins and longevity, arising from the comparison of QTL
for seed longevity with PQL for seed proteins; (3) the role of raffinose
family oligosaccharides in seed and seedling vigor highlighted by QTL
mapping for physiological seed quality and seed sugar content; and
(4) the characterization of a subtilisine-like serine protease influencing
seed weight in legumes. The perspectives include the fine mapping of
QTL conserved between M. truncatula and pea for seed weight and/or
protein composition in the frame of the ongoing ANR-Génoplante project
“GENOPEA” (coord. J. Burstin).
35
Project report edition 2006 Oil and Protein Crops
Pods and seeds of the parental lines of the LR4 population used for QTL and PQL
mapping (left), and reference map of the seed proteome of LR4 showing variations
in spot intensity or position between two contrasted lines (right).
Identification of genes involved in Sclerotinia sclerotiorum resistance in Oilseed Rape and Sunflower
ACRONYM: SCLEROTiNiA
COORdiNATOR:
Bruno GREZES-BESSET1
ANR N°: ANR-06-GPLA-015
Cost: € 1,984,320
Grant: € 688,198
Scientists per year: 5
Period: 01.01.2007 – 04.30.2010
Partners: D. ROBY2, D. BRUNEL3,
F. VEAR4
1. BIOGEMMA – Mondonville
2. LIPM, UMR INRA/CNRS – Auzeville
3. INRA - (EPGV) – Evry
4. UMR ASP-INRA - Clermont Ferrand
INTRODUCTION
Oilseed rape and sunflower are the main oilseed crops in Europe. These
crops are susceptible to a major pathogen: Sclerotinia sclerotiorum. This
fungus can attack many plant species and induce economic losses on
many crops legumes and field crops. The fungus can survive in soil for a
long period as much as 10 years, and become a limiting growing factor
for susceptible crops.
In oilseed rape chemical protection was systematically used and presence
of resistant isolates raised a major problem. In sunflower fungicides are
inefficient and only genetic resistance was used to limit disease impact.
Genetic resistance to this pathogen exists in oilseed rape and sunflower.
However this kind of resistance, polygenic and partial, and the
strong climate incidence on disease development make difficult the
development of breeding programs using classical breeding methods.
The main target of this project is the identification of major genes
involved in Sclerotinia resistance to optimize the exploitation of natural
variability by identification of favorable allelic sequences inside the
genetic resources of oilseed rape and sunflower.
RESULTS
The project is divided in three main tasks.
The first one aims at the identification of signaling pathways and
candidate genes supporting the disease resistance process. For this
purpose, different approaches were developed in the cultivated crops
(sunflower – oilseed rape) and in the model plant Arabidopsis thaliana.
Concerning the cultivated crops, the process consists mainly in the use
36
Project report edition 2006 Oil and Protein Crops
of gene expression technology (transcriptomic analysis) coupled with
bioanalysis work exploiting genetic results ( QTL for résistance) and
the oilseed rape Arabidopsis syntheny. The work done on Arabidopsis
consists mainly in the exploitation of large mutant collections to define
precisely the signaling pathways mobilized during the infection process.
This first task allowed the identification of a gene list then analyzed in
detail during the second project step.
In this second work package, two types of work were developed in
parallel. First a fine gene expression analysis using qRT-PCR technology
was done. In a second work, selected genes In a second work, selected
genes were mapped to study their co localization with major QTL for
resistance.
Finally in a third work, functional validation tools were developed : (i)
in oilseed rape transgenic plants expressing RNAi constructs were
obtained, (ii) a diversity panel was developed in sunflower for future
genomic association studies, (iii) finally, a functional validation tool
using Arabidopsis insertion mutant was developed.
As results, the Sclerotinia project allowed:
n Identification of signaling pathways and major genes network
involved in Sclerotinia resistance
n Definition of a candidate gene list showing differential expression
during infection in susceptible and resistant sunflower and oilseed
rape lines.
n Identification of 2 candidate genes co-localized with major QTLs for
resistance to Sclerotinia in oilseed rape
n Development of tools for candidate genes functional validation: (i)
oilseed rape RNAi technology, (ii) Sunflower: genetic diversity panel
for genetic association study, (iii) Arabidopsis: a mutant phenotyping
tool for Sclerotinia resistance evaluation.
CONCLUSION AND PERSPECTIVES
Sclerotinia project produced major results and tools in the resistance
gene identification process to the Sclerotinia disease. At the end of
the project, candidate genes are available as well as efficient tools to
validate their function. Thanks to scientific publication and congress
communication, this project helped the partners to create links with
other labs working on this Sclerotinia disease. A new project following
the Sclerotinia project was proposed in response to the 2010 plant KBBE
call, this project involved German, Spanish and French partners. Main
future work will consist in functional gene resistance validation and
genetic resources screening to improve resistance to this major pathogen
ion oilseed rape and sunflower.
37
Project report edition 2005 Oil and Protein Crops
Publication
• Perchepied L., Balagué C., Riou C.,
Claudel-Renard C., Rivière N.,
Grèzes-Besset B. and Roby D. (2010),
NO participates to the complex interplay
of defense-related signalling pathways
controlling disease resistance to Sclerotinia
sclerotiorum in Arabidopsis thaliana.
Mol. Plant Microbe Interact. Vol 29, No 7,
pp 846-860.
Oilseed rape with Sclerotinia.
38
Project report edition 2006 Oil and Protein Crops
Genetic validation of functional candidate genes potentially involved in tolerance to frost and resistance to diseases in Pisum sativum
INTRODUCTION
Tolerances to frost and resistance to diseases (namely Mycosphaerella
pinodes and Aphanomyces euteiches) are required for the development
of the winter pea crop in northern Europe.The challenge of this project is
to identify associated molecular markers to assist breeding. QTL mapping
has formerly provided an overview of the pea genomic regions controlling
the studied traits. The partners have taken benefit from the numerous
functional candidate ESTs produced in the frame of Genoplante phase I
and II projects to undertake a candidate genes approach. In this context,
the aim of the SNPEA project is to identify and validate expressional «winter
pea candidate genes»: (i) through transcript profiling using quantitative RT-
PCR (ii) through genetic mapping, looking at colocalisations with previously
identified QTLs (iii) through association genetics, looking at correlations
between allelic frequencies of candidate genes and quantitative variation
for traits of interest and (iv) through the characterization of induced allelic
variation, evaluating the phenotype of induced mutants targeted to genes
of interest.
RESULTS
Our main results are :
Development of an EST-based functional mapping resource
Quantitative RT-PCR experiments have been performed (Biogemma) in
order to help choosing the best candidates among ESTs that were formerly
identified (using SSH libraries, macroarray hybridization and preliminary
qRT-PCR in the GOP-PeaG project) for their differential expression during
ACRONYM: SNPEA
COORdiNATOR:
isabelle LEJEUNE-HENAUT1
ANR N°: ANR-06-GPLA-019
Cost: € 1,575,000
Grant: € 654,157
Scientists per year: 3,45
Period: 01.01.2007 – 12.31.2010
Partners: A. BARANGER2,
A. BENDAHMANE3, C. BLASSIAU4,
A. BLUTEAU1, G. BOUTET2, J.
BURSTIN6, A.-S. CANOY5, M.
DALMAIS3, G. DENIOT2,
R. DEVAUX1, G. DUC6, V. FONTAINE1,
O. JAMINON1, C. LE SIGNOR6,
J.-B. MAGNIEN-ROBERT6,
G. MARQUE1, H. MITEUL2, J. MORIN2,
F. MUEL7, M.-L. PILET-NAYEL2,
N. RIVIERE5, J. WILMER5
1. INRA, UMR 1281 Stress Abiotiques
et Différentiation des Végétaux cultivés,
Estrées-Mons
2. INRA, UMR 0118 Amélioration des Plantes
et Biotechnologies Végétales, Le Rheu
3. INRA, UMR 1165 Génomique Végétale, Evry
4. USTL, UMR 1281 Stress Abiotiques et
Différentiation des Végétaux cultivés,
Villeneuve d’Ascq
5. Biogemma, Mondonville
6. INRA, UMR 0102 Génétique et Ecophysiologie
des Légumineuses, Dijon
7. UNIP, Union Nationale Interprofessionnelle
des Plantes riches en Protéines, Paris
Publications
• Dumont et al., 2009, Theor. Appl. Genet.
118:1561-1116
• Lejeune-Hénaut et al., 2008, Theor. Appl.
Genet. 116:1105–1116
39
Project report edition 2006 Oil and Protein Crops
the establishment of tolerance to frost, or resistance to
Mycosphaerella pinodes (Mp) and Aphanomyces euteiches
(Ae). The SNPEA qRT-PCR experiments have been performed
within 3 biological repetitions for 188, 92 and 92 ESTs related
to the tolerance to frost, Mp and Ae respectively.
The selected ESTs have been integrated in a mapping strategy
relying on three successive complementary techniques to
reveal polymorphism within intronic regions : (i) optimized
SSCP (Single Strand Conformation Polymorphism), using
forward and reverse strand fluorescence labelling, test of
3 migration temperatures (i.e. 18, 25 and 30°C), and non
denaturing (CAP) or denaturing (POP) polymer (ii) High
Resolution Melting (HRM) which has proved to detect
polymorphism even when SSCP has failed (iii) direct
sequencing and development of CAPS markers. The first
results of a comparison of the 3 approaches on a set of 31
ESTs has shown that HRM and SSCP allow together to reveal
polymorphism for 56% of the ESTs. By the end of 2010, the
sequencing results will allow to estimate the proportion of
undetected polymorphism.
Polymorphism was searched among 12 pea parental
accessions for 7 RIL mapping populations segregating for
the tolerance or resistance traits studied, and mapping was
carried out using a unique population.
When possible, ESTs were mapped preferentially on the
Champagne x Terese derived reference map (Loridon et al,
2005), which carries QTLs for tolerance to frost (Lejeune-
Hénaut et al., 2008 ; Dumont et al., 2009). Otherwise, they
were mapped on one of the 6 other maps, which carry QTLs
for resistance to Ae and Mp (Pilet-Nayel et al, 2005 ; Prioul et
al, 2004 ; Hamon, 2010). Up to now, 76, 49 and 51 ESTs have
been mapped on one of the 7 populations for frost tolerance,
Ae and Mp resistances respectively.
SNPlex production for valuable ESTs
Fourty-eight ESTs selected for their position under a QTL
of interest and/or their differential expression are being
sequenced for a set of 40 accessions including pea parental
accessions for RIL mapping populations. Identified SNPs will
allow to constitute a stress-dedicated SNPlex.
Identification of mutant variation by TILLING
As a proof of concept, 4 genes, chosen in the literature for
their potential role in disease resistance (DCL2, DCL3 and
MPK4) or frost tolerance (FVE), have entered the tilling
process.
Double mutants have been produced by crossing a DCL2 stop
mutant with different DCL3 mutants inducing an amino acid
change. This strategy is based on the observation that only
the double mutant has a visible phenotype in Arabidopsis.
Twelve double-mutant DCL2-DCL3 lines are available, among
which 10 lines having enough seeds to allow phenotyping for
resistance to Mp and Ae in 2010. 2 more lines have still to be
multiplied.
Screening of the pea TILLING population on the PsMPK4 gene
has revealed a stop mutant. Unfortunately, this homozygous
stop mutant did not have the expected phenotype in
comparison to the Arabidopsis mpk4 mutant. The pea mutant
did not show any activation of the basal defense system as
expected. We suspect a functional redundancy of this gene
on the pea genome.
The DCL2, DCL3 and MPK4 genes will be mapped in 2010 using
sequencing of the 12 parental accessions and development of
CAPS markers.
The FVE mutation affects both the switch from the vegetative
to the reproductive stage and the tolerance to frost in
Arabidopsis. In pea, FVE is localized in the confidence interval
of a QTL implied in tolerance to frost and to Mp. From the 8
mutant families initially identified, only 6 were predicted to
have a modified protein function, and only 5 were also fertile.
Finally, 1 stop mutant and 3 non redundant missense mutants
have been produced : from the initial M3 or M4 seeds, 2
crosses have been performed with the reference genotype
Cameor in order to go back to this genetic background.
Four FVE lines, including the stop mutant, are now fixed at
the homozygous state (mutant and control) and have been
multiplied in 2010 to allow phenotyping for flowering and
frost tolerance.
Preparation of the tools for ECOTILLING
Seed increase and DNA extractions of the French reference
collection of biological resources (371 lines) have allowed :
(i) phenotyping for tolerance to frost across 4 environments
in the field and 1 environment under controlled conditions,
(ii) phenotyping for resistance to Mp (1 isolate) and Ae
(two isolates) – these phenotyping actions constituting a
comprehensive data set for further association studies , (iii)
the identification of Endo1 mismatches with Cameor for a part
of the FVE sequence.
CONCLUSION AND PERSPECTIVES
The project will be finished at the end of 2010. This fourth
year will be dedicated to i) scientific valorization (SSCP/HRM
comparison; construction of the EST mapping resource), ii)
phenotyping of the produced tilling mutants, iii) ecotilling
analyses and iv) SNPlex production for valuable ESTs.
A pea Tilling mutant for the FVE gene.
© I
NR
A
Cereals
Project report edition 2006 Cereals
42
Genetic analysis under N limiting conditions of the stability of yield and grain protein content of durum and bread wheat
INTRODUCTION
The main objective of the project is to identify the genetic and ecophysiological
determinants of (1) the deviation from the negative genetic relationship between
Grain Yield (GY) and Grain Protein Concentration (GPC) and of (2) N remobilization
and absorption during the reproductive phase.
RESULTS
Main results are presented by workpackage:
– WP1 : Quantitative genetics studies
Genetic maps have been built and QTL detections performed for 2 bread wheat
and 1 durum wheat populations. All these populations are composed of connected
populations issued from diallel with 5 (MetaPop), 3 (GPD) and 6 (durum wheat)
parents respectively. The parents of the GPD (Grain Protein Deviation) population
departe from (+ or -) of the negative GY/GPC relation (GPD+ or GPD-). QTL detection
was carried out with phenotypic data produced in ProtNblé and in previous
research programs. QTL of interest were identified and Near isogenic lines are
under construction. An eQTL detection has also been done using transcriptomic
data produced on selected lines of the GPD population (cf WP4). A meta-analysis
is on going to valorize all these data. On durum wheat, the genetic map will be
obtained soon and QTLs detection will follow.
Fine mapping was also performed using material created in previous GNP programs.
it allow us to obtain one of the major result of the project: the identification of a
candidate gene for a QTL on chromosome 3B. These results have been submitted
for publication to PNAS and also for a patent. Another QTL on chr. 5B has also been
validated in the field on BC1F6 material.
– WP2: New tools to predict N traits
A detailed ecophysiological study of N dynamics within the plant during post-
anthesis has been carried in the field for two parents of the GPD populations.
ACRONYM: PROTNBLé
COORdiNATOR: Stéphane LAFARGE1
ANR N°: ANR-06-GPLA-016
Cost: € 3,548,000
Grant: € 980,755
Scientists per year: 9,7
Period: 01.01.2007 – 30.06.2010
Partners: M. THROUDE1, S. PRAUD1,
J.-P. PICHON1, N. RIVIERE1,
F. TORNEY1, J. LE GOUIS2,
J. SALSE2, P. MARTRE2, V. ALLARD2,
E. HEUMEZ3, L. GUERREIRO4,
K. BEAUCHENE4, P. ROUMET5,
J.B. BEAUFUME6, C. DUQUE6
1. Biogemma, Clermont-Ferrand
2. INRA-UBP, UMR1095 Genetics, Diversity
& Ecophysiology of Cereals, Clermont-Ferrand,
3. INRA/USTL, UMR 1091 Abiotic Stress
and Vegetal Development, Mons-Péronne
4. ARVALIS-Institut du végétal, Paris
5. INRA UMR DIA-PC, Mauguio
6. Limagrain Europe, Verneuil l’Etang
Project report edition 2006 Cereals
43
The results of this experiment showed that the distribution of N in the plant and the
translocation of vegetative N to grains is coordinated at the plant level and depends
on (i) the local light environment; (2) the N status of the different organs at anthesis.
These results allowed us t o develop a wheat ecophysiological simulation model
(SiriusQuality2) where the growth and N concentration of plant organs are emergent
properties of the model dynamics. A global sensitivity analysis of the model has been
carried out. This analysis allowed us to identify 24 key parameters explaining observed
Genotype x Environment x Management interactions for grain yield and protein
concentration.
New phenotyping tools have been tested: hyperspectral imaging system, near infrared
spectroscopy to determine correlation between imaging analysis and field data. A first
QTL detection has been performed on the GPD population using such data.
– WP3: Bioinformatic, Bioanalysis and data mining
Meta-analysis of transcriptomic data has been performed using data produced in the
previous GénoPlante B4 program. We have generated a list of 157 candidate genes. Part
of them were validated by qPCR.
– WP4: Expression studies & mapping
One hundred and eighty genes have been analyzed by qPCR on a collection of 260 plant
samples with different N supplies. After statistical analysis, we identified 62 genes with
an expression level correlated to the nitrogen status. Annotations of these genes (N and
C metabolism, transcription factor…) were promising. So, to valorize this discovery, they
were integrated in the SNP discovery/mapping pipeline. In parallel, a transcriptome
analysis (affymetrix microarrays) was performed on 20 GPD+/- lines. It generated a
list of 517 candidate genes differentially expressed. These data were used for eQTL
detection. Among the 517 genes, 168 are linked to an eQTL and 12 of them co-localized
with a GPD QTL.
All in all, more than 75 candidate genes have been analyzed for SNPs discovery and
mapping. We have identified 33 polymorphic markers / 124 SNPs and mapped 48
genes.
– WP5: Functional validation
Phenotyping of Glutamine Synthetase (GS) and Nitrate transporters (NRT2) transgenic
plants is underway. Creation of new transgenic plants was initiated for two new
candidates.
An association panel has been created by gathering 220 French elite lines registered
in the last 10 years. Field trials were performed in 2008 and 2009 in two locations
and different N supplies. Phenotypic data concerning GY, GPC, GPD, N absorbed and
remobilized will be used for association studies with SNP from our main candidate
genes. Some association between markers and traits has been found in both years.
These results have to be verified.
CONCLUSION AND PERSPECTIVES
From this large collection of data, we would like to prioritize chromosomic areas and
candidate genes by connecting results coming from different sources, species or
techniques. For example:
n Selection of QTL regions coming from different populations and concerning different
traits (MetaPop, GPD, eQTLs…), and search for correspondence to association study
results in order to decrease the confidence interval and identify potential candidate
genes using genome sequences available within sequencing programs (3B-Seq, 4A,
Triticeae Genome,…).
n Use of candidates genes from transcriptome data analysis to discover SNP and
perform an association scan to determine their role and possibly deliver useful
markers for breeding.
n Construction of new genetic materials (back-crosses and transgenic plants) for
further molecular characterization and phenotyping.
All these data will be finally combined to extend our knowledge of NUE.
Publications
• Bertheloot J, Martre P, Andrieu B
(2008) Dynamics of light and nitrogen
distribution during grain filling within
wheat canopy. Plant Physiology
148:1707-1720.
• Bertheloot J, Andrieu B, Fournier C,
Martre P (2008) A process-based model
to simulate nitrogen distribution in wheat
(Triticum aestivum) during grain-filling.
Functional Plant Biology 35: 781-796.
• Ferrise R, Stratonovitch P, Triossi A,
Bindi M Martre P (2010) Sowing date and
nitrogen fertilisation effects on dry matter
and nitrogen dynamics for durum wheat:
an experimental and simulation study.
Field Crops Research 117: 245-257.
• Lacaze X, David J, Roumet P (2010)
Heritable basis of phenotypic plasticity:
a hidden genetic variation in durum
wheat. Theoretical and Applied Genetics
(submitted).
• Lacaze X, Roumet P Stage specific
climatic characterization for studying QTL
by Environment Interactions. Heredity
(submitted).
• Massod Quraishi M, Abrouk M, Murat
F, Pont C, Bolot S, Confolent C, Touret
L, Charmet G, Murigneux A, Guerreiro
L, Lafarge S, Le Gouis J, Feuillet C,
Salse J (2010) Cross-genome map based
cloning of a nitrogen use efficiency
ortho-metaQTL on wheat chromosome
3B unravels new evidence for concerted
cereal genome evolution. The Plant
Journal (accepted).
Patent:
• (02/2010) Improvement of the grain
filling of wheat through the modulation
of NADH-Glutamate Synthase activity.
44
Project report edition 2006 Cereals
Sequencing and molecular analysis of a 12 cM locus carrying multiple durable disease resistance genes on chromosome 3BS in hexaploid wheat
INTRODUCTION
Significant advances in gene discovery and in the management and exploitation
of genetic resources are needed to improve wheat breeding. Despite significant
advances in wheat genomics in the past, gene isolation remains time consuming
and labor intensive and very little is known about the structure and function of the
wheat genome. The SMART project, which has been performed in collaboration
with 6 laboratories involved in map-based disease resistance gene cloning, aimed
at the establishment of the Minimal Tiling Path, the large scale sequencing and the
analysis of a region of about 20Mb carrying a number of fungal disease resistance
genes on the short arm of chromosome 3B of hexaploid wheat cv. Chinese Spring.
It was based on the exploitation of the physical map of this chromosome that was
under construction in the PI’s laboratory at the beginning of the project and is the
foundation of a larger project that aims at using chromosome 3B as a model to
study the structure, function and evolution of the hexaploid wheat genome. After
the establishment of the MTP in the target region, a number of contigs were selected
for sequencing within the SMART project and by international collaborators. The
objectives were to generate data that should enable the identification of all genes
in the sequenced regions and lead to the development of perfect markers for
marker-assisted selection (MAS) of the resistance genes located in this region. In
addition, Megabase-level sequencing was expected to provide original and new
data on the wheat genome structure, function and evolution at a scale that has
never been studied in wheat so far.
RESULTS
Establishment of a physical contig spanning the 12 cM region
Physical mapping of the R locus, which is located between the SSR markers
gwm389 and gwm493 at the distal end of the short arm of chromosome 3B, has
ACRONYM: SMART
COORdiNATOR: C. FEUiLLET1
ANR N°: ANR-06-GPLA-009
Cost: € 956,000
Grant: € 460,000
Scientists per year: 3.5 (in France)
Period: 01.01.2007 – 12.31.2009
Partners: F. CHOULET1, B. GILL2,
B. KELLER3, E. LAGUDAH4,
R. APPELS5, S. GOODWIN6,
F. DEDRYVER7
1. INRA GDEC UMR 1095, Clermont-Ferrand
2. Kansas State University, USA
3. University of Zurich, Switzerland
4. CSIRO, Australia
5. Murdoch University, Australia
6. USDA-ARS, Purdue University, USA
7. INRA UMR INRA-Agrocampus APBV, France
Publications
• Choulet F., Wicker T., Rustenholz
C., Paux E., Salse J., Leroy P., Schlub
S., Le Paslier M.-C., Magdelenat G.,
Gonthier C., Couloux A., Budak H.,
Breen J., Pumphrey M., Liu S., Kong X.,
Jia J., Gut M., Brunel D.,
Anderson J.-A., Gill B.-S., Appels R.,
Keller B., and Feuillet C. (2010), Megabase
level sequencing reveals contrasted
organization and evolution patterns of
the wheat gene and transposable element
spaces. Plant Cell 22, 1686-1701.
• Breen J.M., Wicker T., Kong X.,
Zhang J., Ma W., Paux E., Feuillet C.,
Appels R., Belgard M. (2010), A highly
conserved gene island of three genes
on chromosome 3B of hexaploid wheat:
diverse gene function and genomic
structure maintained in a tightly linked
block BMC Plant Biology 2010, 10:98
• Hao C.Y., Perretant M.R., Choulet F.,
Wang L.F., Paux E., Sourdille P.,
Zhang X.Y., Feuillet C., and Balfourier F.
(2010), Genetic diversity and linkage
disequilibrium studies on a 3.1-Mb
45
Project report edition 2006 Cereals
been completed based on the anchoring of BAC contigs from the 3B physical map.
In total, 15 BAC contigs accounting for 18 Mb of sequence were identified in this
interval. During the anchoring, 98 molecular markers have been developed and
22 of them have been genetically mapped onto a single Chinese Spring x Renan
F2 population of 381 individuals enabling to order the 15 contigs along the region.
A Minimal Tiling Path (MTP) has been established for all 15 contigs and the four
largest contigs were selected for sequencing by the different partners according to
their interest in map-based cloning and evolutionary studies projects.
Sequencing the Minimal Tilling Path on 3B and development of markers for R
genes map based cloning
In the framework of SMART, INRA GDEC focused on sequencing and analyzing the
largest contig (3.1 Mb) that carries markers for the fusarium head blight disease
resistance QTL Fhb1 and also encompasses the region orthologous of the leaf rust
(Puccinia hordei) Rph7 resistance locus identified previously in barley. The 26 BAC
clones comprising the MTP were fully sequenced using the Sanger technology at
8X coverage.
In order to get a final single scaffold sequence from the 237 unordered sequence
contigs initially assembled, INRA GDEC developed a bioinformatics toolbox for 1)
comparing multiple sequence contigs between neighbour BACs (CtgAssembler,
CtgAligner, MergeCtg), 2) assembling neighbour BAC sequences into a single super-
contig (BacAssembler), and 3) extracting sub-sequences ready to be annotated
(BacFromScaff). A single super-contig of 3.110 Mb has been finally assembled.
Annotation of the sequences, identification of genes and markers
Annotation revealed the presence of 41 protein encoding genes and 11 pseudogenes representing 2.3% of the 3.1 Mb contig sequence.
In addition, 526 transposable elements were identified, representing 63.2% of the region. Genes were mainly clustered into small islands
containing 3 to 4 genes separated by block of repetitive elements. Functional annotation indicated the presence of a number of gene family
members such as protein kinases and glycosyltranferases that were likely amplified by tandem duplications. A single resistance gene
analog (NBS-LRR type) has been identified which, however, is not included in the genetic interval spanning the Fhb1 QTL. Comparative
genomics revealed that only 18 genes are syntenic with rice and Brachypodium chromosomes 1 and 2, respectively. The remaining 34
genes and pseudogenes share homology with rice and Brachypodium genes that are located at non-syntenic positions. This revealed that
this region of the wheat genome has recently been subject to numerous rearrangements through interchromosomal gene duplication/
translocation (Choulet et al, 2010). Several programs dedicated to automated marker design were also developed during the project:
IsbpFinder for ISBP marker development and SsrDesign for microsatellites (Paux et al, 2010). In total, 131 ISBP, 140 SSR, 18 STS and 8 SNP
markers were developed for the 3.1 Mb sequence. Annotation, comparison and marker data have been integrated into the URGI GnpIS
information system. These data were used as a pilot project for testing and improving the interoperability of the GnpIS system that should
allow easily navigating between the different databases. The whole annotation and the marker sequences are now accessible through the
Wheat3B Gbrowse interface (http://urgi.versailles.inra.fr/cgi-bin/gbrowse/wheat3B_RPH7_pub/). In addition, three additional contigs,
including a contig of 800 kb spanning the Sr2 disease R locus, were sequenced and analyzed by R. Appels group (Partner 5, Breen et al,
2010) and a diagnostic marker was developed for MAS of Sr2 (Mc Neil et al, 2008).
Establishment of homoeologous physical contigs in target regions on chromosomes 3A and 3D
A screening of the 3AS specific BAC library was performed using 41 probes designed from the genes identified on the 3.1 Mb sequenced
region of chromosome 3B. Five BAC contigs were identified and 28 MTP BAC clones were sequenced and analyzed by B. Gill’s group
(Partner 2). First comparative analyses revealed that most of the genes are highly conserved between 3B and 3A. Further sequencing is
in progress to produce a high quality super-contig sequence. The same probes were used to screen a BAC library from Aegilops tauschii
(diploid wheat ancestral donor of the D genome) and 5 BAC contigs spanning the homoeologous region have been reconstructed by the
group of J. Dolezel (Institute of Experimental Botany, Czech Republic). MTP BAC clones were sequenced in collaboration between the group
of J. Dolezel and our group (INRA Clermont-Ferrand). Comparative and evolutionary analyses are currently in progress.
CONCLUSION AND PERSPECTIVES
The present study gave better insights into the wheat genome structure, composition and evolution at a scale never reached so far with the
analysis of the first Mb-sized sequence produced in wheat. The SMART project also allowed identifying genes potentially involved in fungal
disease resistance and developing molecular markers for several of the target loci. In addition, with the development of bioinformatics
tools for assembling, annotating and analyzing wheat genomic sequences, for comparative genomics studies and for automatic marker
development, the SMART project contributed to lay the foundation of a larger sequencing effort comprising 10 additional Mb sized contigs
distributed along chromosome 3B (Choulet et al, 2010) as well as the complete sequencing of the wheat 3B chromosome (1 Gb) which
began in 2010 in the framework of the flagship ANR project 3BSEQ.
genomic region of chromosome 3B in
European and Asian bread wheat (Triticum
aestivum L.) populations. Theor Appl Genet,
DOI: 10.1007/s00122-010-1382-x.
• Paux E., Faure S., Choulet F., Roger D.,
Gauthier V., Martinant J.-P., Sourdille P.,
Balfourier F., Le Paslier M.-C., Chauveau A.,
Cakir M., Gandon B., and Feuillet C. (2010),
Insertion site-based polymorphism markers
open new perspectives for genome saturation
and marker-assisted selection in wheat.
Plant Biotechnol J 8, 196-210.
• McNeil M.D., Kota R., Paux E., Dunn D.,
McLean R., Feuillet C., Li D., Kong
X., Lagudah E., Zhang J.C., Jia J.Z.,
Spielmeyer W., Bellgard M. and Appels R.
(2008), BAC-derived markers for assaying
the stem rust resistance gene, Sr2, in wheat
breeding programs. Mol. Breeding, 22:15-24.
• Saintenac C., Faure S., Choulet F., Ravel C.,
Remay A., Paux E., Feuillet C., Sourdille P.
Variation in crossover rates correlates with
coding sequences in bread wheat (Triticum
aestivum L.). Submitted to Genetics.
Other Species
48
Project report edition 2006 Other Species
Genetic and genomic dissection of cotton fibre quality using an interspecific RIL population
INTRODUCTION
Cotton (Gossypium sp.) is the most important source of natural textile fibre and
a commodity of key economic importance in both developed and developing
countries worldwide. The ever-increasing possibilities of plant biotechnology
(genetic engineering, genomics and transcriptomics) and molecular breeding
techniques increasingly offer practical solutions to both field production
constraints and to the improvement of fibre quality.
The major objective of this project is the genetic and genomic dissection of
important cotton fibre quality characteristics. A combination of “classical” QTL
mapping and of gene expression QTL mapping (eQTL) will permit us to identify key
candidate genes. The project is centred on the characterization of a population of
interspecific G. hirsutum x G. barbadense recombinant inbred lines (RILs), created
by CIRAD, that will be concurrently studied by each of the 3 partners of the project,
CIRAD, Bayer CropScience and CSIRO. The major workpackages relate to the
genotyping of the RIL population, the phenotyping of fibre characteristics and the
gene expression studies for large numbers of the RILs.
RESULTS
n Genotyping (Publication 1)
We report the development of a new interspecific cotton recombinant inbred
line (RIL) population of 140 lines deriving from an interspecific cross between
Gossypium hirsutum (Gh) and G. barbadense (Gb). Two marker systems,
microsatellites and AFLPs, were used. An important feature of the RIL population
was its marked segregation distortion with a genome-wide bias to Gh alleles
(parental genome ratio is 71/29). The RIL map displays an excellent colinearity with
the formerly published BC1 map, although it is severely contracted in terms of map
size. Existence of 255 loci in common (between 6 and 14 per chromosome) allowed
the integration of the 2 data sets and a consensus BC1-RIL map based upon 215
individuals (75 BC1 + 140 RIL) was built. It consisted of 1745 loci, spanned 3637
ACRONYM: COTTON-RiLS
COORdiNATOR: Jean-Marc LACAPE1
ANR N°: ANR-06-GPLA-018
Cost: € 1,522,000
Grant: € 684,560
Scientists per year: 2.3
Period: 01.01.2007 – 12.31.2010
Partners: J. JACOBS2, D. LLEWELLYN3
1. UMR-DAP, CIRAD, Montpellier
2. Bayer SAS, Gand
3. CSIRO, Canberra
Publications
• Lacape J.-M., J. Jacobs, T. Arioli,
R. Derijcker, Forestier-Chiron, J. Jean,
D. Llewellyn, E. Thomas and C. Viot.
2009, A new interspecific, Gossypium
hirsutum _ G. barbadense, RIL population:
towards a unified consensus linkage
map of tetraploid cotton. Theoretical and
Applied Genetics, 119 (2): 281-292.
• Lacape J. –M. D. Llewellyn, J. Jacobs,
Y. Al-Ghazi, S. Liu, T. Arioli, O. Palai, S.
Georges, M. Giband, H. de Asunção, C.
Viot, J. Jean, M. Claverie, G. Gawryziak
and M. Vialle. 2010, Meta-analysis of
cotton fibre quality quantitative trait loci,
QTL, across diverse environments in
an inter-specific Gossypium hirsutum x
G. barbadense recombinant inbred line
population. BMC Plant Biology, 132
49
Project report edition 2006 Other Species
cM, intermediate between the sizes of the 2 component maps, and
constituted a solid framework to cross align cotton maps using
common markers.
n Phenotyping (Publication 2)
We evaluated the RIL population for fibre characteristics in 11
independent experiments under field and glasshouse conditions.
Sites were located on 4 continents and 5 countries and over
multiple years at some locations. The population displayed a large
variability for all major fibre traits. QTL analyses were performed on
a per-site basis by composite interval mapping. One hundred and
sixty seven fibre QTLs exceeding permutation based thresholds
were discovered. Coincidence in QTL localization across data sets
was assessed using 651 LOD peaks exceeding LOD2 as a relaxed
threshold for the different fibre trait categories including strength,
elongation, length, length uniformity, fineness/maturity, and
colour. A meta-analysis across all data sets (more than a thousand
LOD peak positions or QTLs) was conducted with MetaQTL
software to integrate data (LOD>2) from the RIL and from 3
backcross populations (from the same parents) and to compare
them with the literature. Although the global level of congruence
across experiments and populations was generally moderate,
the QTL clustering was possible for 30 trait x chromosome
combinations (5 traits in 19 different chromosomes) where an
effective co-localization of unidirectional (sign of additivity) LOD
peaks from at least 5 different data sets was observed. Among the
26 clusters (or meta-clusters), most consistent were identified in
the case of fibre colour on c6, c8 and c25, fineness on c15, and
fibre length on c3. Meta-analysis provided a reliable means of
integrating phenotypic and genetic mapping data across multiple
populations and environments for complex fibre traits. The
consistent chromosomal regions contributing to fibre quality traits
constitute good candidates for the further dissection of the genetic
and genomic factors underlying important fibre characteristics.
n Gene expression and eQTL mapping
Quantitative cDNA-AFLP (64 BstYI/MseI primer pairs used) was
used at CIRAD to study the accumulation kinetics of 3164 transcript-
derived fragments (TDFs) in the 2 parents of the population
throughout fibre development (12 time-points between 6 and
28 days post anthesis, dpa). STEM software was used for serial
analysis to define significant co-regulated expression patterns.
Lists of differentially regulated TDFs between the 2 parents in the
different typical expression patterns will be established (analysis
underway).
Regarding expression studies for eQTL mapping we undertook
broad transcript profilings of fibre cDNAs of 88 to 100 RILs that
were either hybridized to cDNA microarrays (at CSIRO from both
field-grown and glasshouse grown
plants) or analyzed by cDNA-AFLP (at CIRAD from glasshouse-
grown plants). Transcriptome analysis concentrated on fibres at
10-11 dpa in both platforms but also included a second stage, 22
dpa, in the cDNA-AFLP experiments.
After cDNA-AFLP profiling, we used QuantarPro (Keygene)
programme to quantify from scanned radiograms the segregation
of 3263 and 1204 TDFs (63 BstYI/MseI primer pairs used),
respectively, for the 2 developmental time-points. Normalized
intensity ratios were analyzed for QTL analyses by composite
interval mapping with QTL Cartographer. Two third of all TDFs
appear to be controlled by between 1 and 6 eQTLs (LOD>3.5
as a threshold). 3665 and 1375 eQTLs in total were mapped on
the RIL map from the 10 and 22 dpa experiments respectively.
Distribution of eQTLs among and within chromosomes was not
uniform. Chromosome 21 had a higher density as compared to
other chromosomes in both data sets. Distribution of eQTLs
along chromosomes was assessed along bins of 2 cM, indicating
that 132 bins (or 13% of the 1020 bins) hosted significantly high
number of eQTLs (total 1835 or 36% of the 5040 eQTLs). Within
chromosomes, hotspots of presumably trans-acting eQTLs (up to
73 LOD peaks, and as high as 140 overlapping eQTL confidence
intervals, in a single bin of 2 cM) controlling multiple genes.
Most populated hotspots were identified on c12, c15 and c21 for
10 dpa and c19 and c24 for 22 dpa samples. The localization of
eQTL hotspots was compared to the localization of phenotypic
QTLs (phQTL) for fibre characteristics of the RILs (article in
preparation). This research is being reported in an article
submitted in July 2010.
A total of 231 slides of the 24K microarray used at CSIRO were
hybridized to 11 dpa old fibre cDNAs corresponding to 102 different
RILs grown in France or in Australia as well as the parental controls.
As a preliminary result (analyses underway) from QTL analyses
with Rqtl, a lower number of eQTLs were mapped as compared
to cDNA-AFLP: 3018 transcripts mapped 3455 eQTLs. Hotpots
of eQTLs were also detected with the microarray platform (on
chromosomes c5 and c12 accounting for 40% of all the eQTLs)
but they did not necessarily correspond to the ones detected with
the cDNA-AFLP platform (c12 in common but hotspot localization
differed). In addition a large correlation matrix has been generated
from the expression of individual transcripts on the arrays and
each of the fibre quality data sets for the RILs, and is being mined
for genes with significant correlations to specific fibre quality
traits. This will provide an alternative way of identifying candidate
genes underlying those fibre traits.
Finally, both AFLP-based and hybridization-based profiling of the
RILs is being tentatively validated by quantitative population-
wide RT-PCR analysis at CIRAD and CSIRO for a limited number
of cloned AFLP bands or genes present on the microarray (around
25 in total). Initial results indicate that eQTLs are only partially
confirmed by qPCR.
CONCLUSION AND PERSPECTIVES
After 3 years, the Cotton-RIL project has demonstrated the
innovative nature of the approach and has shown novelty in
several of its results. The “genetical genomics” approach only
documented in a limited number of plant model systems has
been successfully applied for the first time in cotton. A general
preliminary conclusion to be drawn from the very high number of
phQTLs and eQTLs is the inherent complex genetic architecture of
cotton fibre quality as a trait of interest.
Further dissection of the genes underlying some of the most
important regions of ph- and eQTLs will be facilitated by the on-
going pyro-sequencing (1 run of 454) of 4 cDNA libraries (2 parents
x 2 development time points). An important amount of fibre gene
expression data has been generated by the project that remains
a useful resource for further data mining and exploitation (2
additional publications in preparation).
50
INTRODUCTION
In temperate zones, the global change will include longer and more severe
drought episodes. As long-living organisms, trees will be faced with
these droughts without the possibility to adapt through the generational
selection. Increasing our knowledge of the molecular mechanisms involved
in drought acclimation in woody perennials is a key for a sustainable forest
management and the adjustment of plantation planning.
Drought tolerance requires the regulation of many processes occurring in
different organs. This complex trait has been addressed by an integrative
approach seeking 5 key-tissues of the tree: the growing root apex, the
young and the mature xylem, the mature leaf and the stomata. In order to
discriminate genes presenting a potential adaptative character from genes
responding passively to the constraint, we compared the responses of two
genotypes contrasting for their drought tolerance, at different stages and
intensities of water deficit. The objective was to identify genes involved
in drought acclimation in poplar, the model tree species for molecular
biology.
RESULTS
The ecophysiological side allowed applying controlled and reproducible
water deficits and characterizing the tree responses, which were
consistent with the level and the duration of the applied water regimes.
The transcriptional regulation playing a major role in the control of
numerous biological processes, we searched for drought-responsive
genes (Affymetrix technology). The analyses showed that the molecular
responses were largely tissue – as well as genotype-dependent. Wood
tissues and growing root apices showed more numerous gene regulations
and more drought-responsive proteins than the leaf tissue. In Carpaccio –
ACRONYM: POPSEC
COORdiNATOR: Marie-Béatrice
BOGEAT-TRiBOULOT 1
ANR N°: ANR-06-GPLA-007
Cost: € 1,500,000
Grant: € 414,483
Scientists per year: 3.8
Period: 01.01.2007 – 06.30.2010
Partners: C. Plomion2, J.-C. Leple3,
F. Brignolas4, J.-P. Renou5
1. UMR1137 Ecologie et Ecophysiologie
Forestières, INRA - Nancy Université, IFR 110
EFABA, Champenoux
2. UMR1202 Biodiversité, Gènes et
Ecosystèmes, INRA Université Bordeaux I,
Cestas
3. UR588, Amélioration, Génétique et
Physiologie Forestières, INRA Orléans, Olivet
4. Laboratoire de Biologie des Ligneux et des
Grandes Cultures, Université d’Orléans, USC
INRA 230, Orléans
5. UMR1165, Unité de Recherche en Génomique
Végétale, INRA, Evry
Project report edition 2006 Other Species
Molecular bases of acclimation and adaptation to water deficit in poplar
51
the most tolerant genotype –, the transcriptional remodeling implied the
activation of functions specifically associated with water stress while in
Soligo the stress responses were more stress-generic. Among drought-
responsive proteins (identified by two-dimensional electrophoresis), 15%
were implied in stress response. Others were mostly tissue-specific: energy
metabolism and photosynthesis in leaves, phenylpropanoide biosynthesis
and protein metabolism in wood and hormone biosynthesis in root apices.
Overall we identified stress markers (orthologs of RCI2A, galactinol
synthase, stachyose synthase, Athb12, Pyl 4, PP2C, …) and genes of interest
for drought acclimation (dead-box RNA helicase, polyphenoloxidase, SOD,
NBS-LRR proteins, glutathione S-transferase, …).
In order to test the robustness of some candidates, this screening approach
was completed by an analysis of their expression by RT-qPCR in a wider
range of genotypes submitted to drought.
CONCLUSION AND PERSPECTIVES
The multi-dimension and multi-disciplinary approach provided an
integrative view of the drought responses. They were strongly dependent
of the genotype, showing that the genetic variability must be taken into
account in the future studies. The interest of considering growing tissues,
which showed a strong molecular responsiveness, and of focusing on
cell types was highlighted. Results suggest that a better tolerance could
be linked to the ability to respond rapidly and specifically to water deficit
instead of developing a slow and stress-generic response, and, to better
cope with the drought-induced energy deficit. In the future, we seek to
estimate the genetic variability of the molecular plasticity in response to
water deficit. We will focus on the control of growth, which is crucial for
productivity maintenance under drought. The knowledge issued from this
project will benefit to other forest tree species of important economical
interest. Results will allow the development of tools to select species or
genotypes better adapted to unfavorable water availability.
Plantlets of Populus deltoides x nigra (3 month-old) grown in controlled
conditions
Project report edition 2006 Other Species
GENOPLANTE
28, rue du Docteur Finlay
75015 Paris - France
Contact: Dominique Laborde / [email protected]
Design and production: La maison d’été
Photos: © INRA / Julien Abbaretz (p. 10, 12, 14, 16, 22, 24, 30, 34, 38, 42, 44, 50)
© INRA / André-Paul Jacques (p. 8, 18, 26, 28, 36, 48)
Printed by Imprimerie Peau (Imprim’vert), december 2010,
with inks from plant sources and paper from sustainably managed forests
Contact list
52
COORDINATOR EMAIL PROJECT PAGE
n BALAGUE Claudine [email protected] CHAT 8
n BERTRAND Benoît [email protected] PUCECAFE 28
n BOGEAT-TRIBOULOT Marie-Béatrice [email protected] POPSEC 50
n CABOCHE Michel [email protected] TAG 30
n CRESPI Martin [email protected] RIBOROOT 16
n D’HULST Christophe [email protected] ISD-STARCH 12
n FEUILLET Catherine [email protected] SMART 44
n GALLARDO Karine [email protected] QUALITYLEGSEED 34
n GOUZY Jérôme [email protected] LEGOO 26
n GREZES-BESSET Bruno [email protected] SCLEROTINIA 36
n LACAPE Jean-Marc [email protected] COTTON-RILS 48
n LAFARGE Stéphane [email protected] PROTNBLE 42
n LEJEUNE Isabelle [email protected] SNPEA 38
n LEROUGE Patrice [email protected] GLYCO-CHLOROPLAST 24
n LOUDET Olivier [email protected] DNV 10
n THIBAUD Jean-Baptiste [email protected] TRANSPORTOME 18
n THOMASSET Brigitte [email protected] GENOLIN 22
n VOINNET Olivier [email protected] MICROTRAC 14