Acknowledgements - GIS Biotechnologies Vertes...Guy RIBA (INRA), Alain Weil (CIRAD), Serge HAMON (IRD), André LE BIVIC (CNRS) 5. Functional Analysis. 8 Project report edition 2006

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


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


Cost: € 1,723,000

Grant: € 408,370


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.


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.


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.


11

The PHENOPSIS phenotyping platform

12


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


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


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.


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


MicroRNA Transcription and Activity: uncovering and exploiting the genes between the genes

ACRONYM: MiCROTRAC

COORdiNATOR: Olivier VOiNNET 1


Cost: € 1, 200, 000

Grant: € 420.335


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


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.


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


Involvement of non coding RNAs in the adaptation of root architecture to abiotic stress

ACRONYM: RiBOROOT

COORdiNATOR: Martin CRESPi 1


Cost: € 1,169,000

Grant: € 380,255


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


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.


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


ACRONYM: TRANSPORTOME

COORdiNATOR:

Jean-Baptiste THiBAUd1


Cost: € 1,460,124

Grant: € 590,000


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


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.


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


Cost: € 1.264.000

Grant: € 494.484


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.


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.


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


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


Cost: € 2,414,000

Grant: € 316,519


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


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.


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.


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


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


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


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.


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


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


Period: 01.01.2007 – 12.31.2009

Partners: I. PRIVAT2, P. LASHERMES3

1. CIRAD, Montpellier

2. CRD NESTLE, Tours

3. IRD, Montpellier

29


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.


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


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


Cost: € 752,000

Grant: € 382,519


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


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.


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


Cost: € 2,377,000

Grant: € 563,549


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.


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


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


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


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.


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


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


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


Cost: € 1,575,000

Grant: € 654,157


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


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.


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


Cost: € 3,548,000

Grant: € 980,755


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


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.


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


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


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


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.


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


Cost: € 1,522,000

Grant: € 684,560


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


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.


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


Cost: € 1,500,000

Grant: € 414,483


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


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.


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


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

Documents

Acknowledgements - GIS Biotechnologies Vertes...Guy RIBA (INRA), Alain Weil (CIRAD), Serge HAMON (IRD), André LE BIVIC (CNRS) 5. Functional Analysis. 8 Project report edition 2006