POPSEC : molecular bases of acclimation to water deficit in poplar

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POPSEC : molecular bases of acclimation to water deficit in poplar

Marie-Béatrice Bogeat-Triboulot, David Cohen, Didier Le Thiec, Sandrine Balzergue, Marie-Laure Martin-Magniette, Jean-Pierre Renou, Philippe Label,

Marie-Claude Lesage-Descauses, Françoise Laurans, Isabelle Bourgait, et al.

To cite this version:

Marie-Béatrice Bogeat-Triboulot, David Cohen, Didier Le Thiec, Sandrine Balzergue, Marie-Laure

Martin-Magniette, et al.. POPSEC : molecular bases of acclimation to water deficit in poplar. Sémi-

naire Génoplante, Mar 2010, Mallemort-en-Provence, France. 22 p. �hal-02823287�

POPSEC : molecular bases of acclimation to water deficit in poplar

INRA Nancy - UMR EEF INRA Bordeaux - UMR Biogeco INRA Orléans - UGAPF Université d’Orléans - LBLGC URGV Evry Coord. : Marie-Béatrice Bogeat-Triboulot

Project GPLA0628G – 2006/2010

Poplars

¾ scientific interests:

ƒ perennials :

– cycles of growth, reserves, … – complementary to annual biology

ƒ woody : cambial growth

ƒ clonal propagation

ƒ genome fully sequenced

¾ ecological interests:

ƒ soil depollution

ƒ riverside biodiversity

¾ economical interests:

ƒ short rotations: “crop tree”

ƒ 1.3 Mm

wood in 2008

ƒ crates, paper pulp

ƒ bio-energy / global change

 leaf growth  gas exchange  wood growth

Â Ψ _leaf

 productivity

water deficit

Drought tolerance

several processes

several organs complexe trait drought tolerance =

Stomata

hydraulic K adjustement -> water flow -> water uptake root growth -> soil prospection stomatal closure -> water loss limitation

osmotic adjustment cell detoxication -> cell homeostasis

Root Leaves

cambial growth Wood

-> productivity

Objectives

cell homeostasis stomatal regulation

root growth cambial growth

 leaves growth  gas exchange

 wood growth

Â Ψ _leaf

 productivity

water deficit

POPSEC Consortium :

- focus on several processes involved in drought response - in several organs/tissues/cell types

- at several scales ecophysiology, transcriptomics and proteomics

¾ To better understand the molecular bases of drought tolerance in a tree

¾ Use of genetic diversity to assess

- common bases of drought response - specificity of drought tolerant cultivars

integrative

approach

Carpaccio (Monclus et al, 2006)

Soligo

1- Omics analyses in two cultivars

¾ P. deltoides x nigra : Carpaccio and Soligo

ƒ 2 commercial hybrids chosen for their

ƒ similar productivity

ƒ similar WUE

ƒ contrasted productivity maintenance under water deficit

¾ 3 water deficit treatments

2007

Genotypes drove results as much as drought

Fact. 1: 44% treatment

C.CTL

C.EAR

C.AMI C.AMO

S.CTL S.EAR

S.AMI

S.AMO

-5 -4 -3 -2 -1 0 1 2 3 4 5

-5 -4 -3 -2 -1 0 1 2 3 4 5

Fact. 2: 36 % genot ype

PCA on ecophysiological parameters

1.18 * CTL_C/S

2102 (±5) C_AMO

1864 (±3) S_AMO

0,78 * S_AMO/CTL

0 ,74 * C_AMO/CTL

2399 (±3) S_CTL

2836 (±2) C_CTL

Vessel lumen area

REW LMA

A Π

Ψ_pd growth

radial growth g_s

-1.0 -0.5 0.0 0.5 1.0

Fact. 1 : 44%

-1.0 -0.5 0.0 0.5 1.0

Fact. 2 : 36%

WUE_i

REW LMA

A Π

Ψ_pd growth

radial growth g_s

0 2 4 6 8 10 12

EAR AMI AMO

CTL CTL EAR AMI AMO

Protein content, μg. μl

Carpaccio Soligo

Transcriptomics in two cultivars

Affymetrix poplar genome array :

ƒ ESTs from 13 species/genotypes :

P. tremula, P. euphratica, P. deltoides x nigra, …

ƒ gene models from P. trichocarpa

Experiment on 2 P. deltoides x nigra

mismatch of hybridization ? ¼ hybridization genomic DNA of both cultivars

61 251 probe sets

36 687 probe sets

- gDNA hybridized Soligo AND Carpaccio

- expressed in at least one condition in Soligo OR Carpaccio -346 gDNA hybridized and expressed in Soligo only

-620 gDNA hybridized and expressed in Carpaccio only -11 068 with hybridization of gDNA (C and/or S)

but never expressed -12 530 with NO hybridization of gDNA

(nor Carpaccio neither Soligo) 20%

0.6%

18%

1%

¼ Univocal comparative analysis of two genotypes

Genotypes drove results as much as drought

PCA on acid proteins

C.CTL C.EAR C.AMI

C.AMO

S.CTL S.EAR

S.AMI

S.AMO

PC2 (24 % )

PC1 (36%) genotype

treatment

Global analyses of gene expression

Normalised intensity of the 36687 probe sets

Carpaccio

EAR

AMI

AMO CTL

EAR

AMI

AMO

CTL Soligo

Number of regulated probe sets

-2000 -1500 -1000 -500 0 500 1000 1500 2000

Carpaccio Soligo

UP

DOWN

Drought-induced gene regulations

EAR AMI AMO EAR AMI AMO EAR AMI AMO

EAR AMI AMO EAR AMI AMO

(no log ratio cut-off, p < 0.05)

¾ Root apices and xylem are highly responsive

¾ Strong cultivar specificity in young xylem (wood growth maintenance?)

¾ Some strong genotype*treatment interactions - Soligo EAR - root apices

- Carpaccio EAR - stomata

Search for gene networks

distance between gene expression profiles -> combined matrix

ƒ transcriptional modules are delineated in Carpaccio

ƒ second clustering within each module in Soligo

ƒ test of co-expression relationship within each module in Soligo Differential Clustering Algorithm

Soligo

Carpaccio

ƒ Full conservation : conserved co-regulation of genes across treatments in the two cultivars

ƒ Partial conservation : some co-regulation relationship between genes are lost

¼ Identification of similarities and differences of gene regulation between cultivars

roots roots

EAR AMI AMO EAR AMI AMO

partial conservation Cluster 8 : full conservation

leaves roots leaves roots

EAR AMI AM O EAR AMI AM O EAR AMI AM O EAR AMI AM O

Carpaccio Soligo

y

a

b

Search for gene networks

In wood tissues

¾ The network contains 50 modules and 2776 probe sets covering the 12 conditions tested

ƒ A few modules propose a transcription network associated with response to stress

ƒ Ex : module 42 describes Xylem & Young Xylem in response to EAR and AMO and is enriched in genes involved in :

ƒ Hormone metabolic process

ƒ Response to stress

ƒ Transcription regulation

ƒ Perception of external stimuli

ENIGMA clustering and enrichment method

includes functional information

module 42

Transcription

regulators

Search for co-regulation of protein abundance

4 7

1540 reproducible spots

7 11

891 reproducible spots

4 7 4 7

563 reproducible spots 704 reproducible spots

47 reliably identified proteins 42 reliably identified proteins

the spots displaying a significant treatment effect or genotype x treatment interaction were identified by Nano LC-MS/MS

Reduced abundance under prolonged drought

Mature leaves Wood Root apices

Primary Metabolism Biosynthesis of

plant hormones Glycolysis/

Gluconeogenesis Metabolism of amino acids

Protein metabolism

Other Glutathione Metabolism Glycolysis/

Gluconeogenesis Metabolism of amino acids Protein metabolism

TCA and pentose phosphate pathway

Biosynthesis of phenylpropanoids

Metabolism of nitrogen

Other Mature leaves

Root apices Wood

Functional protein groups involved in drought response

Glutathione Metabolism

Glycolysis/

Gluconeogenesis Metabolism of

amino acids Protein metobolism

TCA and pentose phosphate pathway

Metabolism of nitrogen

Photosynthesis

Other

Main functional groups of genes involved in drought response

revealed by iterative group analysis

Carpaccio Soligo

E A R A M I A M O E A R

ABA signalling (NCED, PP2C, ABI5-binding like, PYR like)

Water channel

Signal transduction (less focused on ABA) Transcription and metabolic processes Photosynthesis

A M I

Dead-box helicase like

A M O

ABA signalling

ABA response (XERICO- like, PP2C, RCI 2A)

E A R

Water channel ( ↑ ) Detoxication ABA metabolism

Water channel ( ↓ )

Flavonoid biosynthesis (↓) Transcription

A M

I Cell wall thickening Response to gibberellin Cell wall (↓)

A M Redox homeostasis

Cell wall (↓)

Abiotic stress/drought ABA signalling

Abiotic

stress/drought ABA signalling Transcription Stomata

Detoxication (SOD, Glutaredoxin,

metallothionein, germin) Cell rescue

(RCI2A-like)

Detoxication

(galactinol synthase) Transporter

Secondary metabolism Mature

leaves

Abiotic stress/drought :

ABA biosynthesis and signalling (NCED, PP2C, ABA-responsive TF, PYR) Cell rescue / redox homeostasis (DnaJ, HSP,

Glutathion-S-transferase, metallothionein)

Hypoxia (ADH, Pyruvate decarboxylase, LOB41-like protein) Growth :

Expansion (aquaporins, pectine esterase)

Meristem activity (chromsome organisation, DNA replication, constituent of ribosome) apices Root

Number of transcription

factors

Carpaccio Soligo

EA R

Response to stress (biotic, abiotic, oxydative) Jasmonic acid metabolic process

response to water deprivation (NCED, ABA-responsive TF, Nitrate transporter)

Response to cold

AM I

Auxin transport,

Protein amino acid phosphorylation,

cell wall, regulation of cell size, cellulose biosynthetic process

photosynthesis, light reaction

Whole xylem

AM O

flavonoid metabolic process response to water deprivation apoplastic response

Cell wall and plasma membrane compartments, microtubule polymerization,

response to stress (oxidative, salt, cold)

EA R

Response to stress (biotic, abiotic, oxydative) jasmonic acid metabolic process,

trehalose biosynthetic process, transcription factor activity

Plasma membrane compartment,

response to hormone stimulus (ABA, auxin), trehalose-phosphatase activity,

transcription factor activity

AM I

sesquiterpenoid biosynthetic process, ATP synthesis coupled proton transport, cell wall pectin metabolic process

Chloroplast et apoplastic compartments Response to stress (chemical stimulus) Response to water

Hormone stimulus (gibberellin 20-oxidase activity)

Young Xylem

AM O

Plasma membrane and apoplast compartments, Auxin biosynthesis,

response to cadmium ion,

regulation of ethylene biosynthetic process

Plasmamembrane and apoplast

Response to water deprivation, osmosensor activity cellular carbohydrate biosynthetic process

proteolysis, transporter

Main functional groups of genes involved in drought response

revealed by topology based method (Topology-Elim, Ontologizer)

2- Drought response in 8 cultivars

Omics ^Æ Transcriptome remodelling analyses and selection of genes of interest Diversity ^Æ Test the validity of these genes

Control Water-deficit Rei-irrigated

B

B C B

C C D

D D S

S

S

F F

I

K

-3 -2 -1 0 1 2 3

Fact. 2 : 19.7%

-5 -4 -3 -2 -1 0 1 2 3 4 5

A I K A

PCA on ecophysiological traits

¾ 2008 : independent experiment on 8 cultivars Prolonged water deficit (10 days at 20% REW)

Carpaccio Soligo I214 Dorskamp Koster Flévo Beaupré 717-1B4 (A)

P. deltoides x nigra P. trichocarpa P. tremula x alba

Consistency of drought regulation in mature leaves

Number of probe sets

-500 0

500 ^Carpaccio

Soligo

2008 2007

AMI AMO

2007 : 2120 drought- responsive

probe sets 323 1797

415 2008 :

drought- 783 responsive probe sets

28

191 104

(Carpaccio, 219) (Soligo, 132)

¾ Strong environmental effect

¾ 276 genes robustly responsive to prolonged drought

¾ Sense of regulation and cultivar specificity conserved

Transcriptome of mature leaves of Carpaccio & Soligo

Selected gene expression in 8 cultivars

2008

0,00 0,20 0,40 0,60 0,80 1,00 1,20

A S B D I K C F

TUB8

0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 1,80 2,00

A S B D I K C F

LRR3

0,00 0,50 1,00 1,50 2,00 2,50

A S B D I K C F

XYL

¾ Reference genes

¾ Expression measured by RT-qPCR in Young Xylem

¾ Sense of regulation conserved between 2007 and 2008 (for Carpaccio and Soligo)

¾ Homogeneousness or diversity of expression depending on tested genes

UBI PP2A

Technical issues

¾ Hybridization of genomic DNA when using a multispecies chip

¾ _Protocols to get access to the proteome of root apices (small amount of material)

to the transcriptome of stomata (lyophilisation, laser microdissection)

¾ Interconnected transcriptome databases dedicated to poplar (ForOrthology, ForExpress)

¾ Implementation of proteome database (ProticDB)

¾ Improvement of statisitical procedures for Affymetrix analyses (normalisation)

¾ New insights in gene and protein functions in poplar

Emerging perspectives

¾ Continue with integrative approaches & improve quantitative phenotyping of the response

ƒ Multiscale approach gave an integrative view of processes involved in drought tolerance – Difference of responsiveness of organs/tissues

– Tissue dependency/independency of enriched functional groups Æ Meta-analysis of the 5 “organs/tissues/cell types”

¾ Better take diversity into account

ƒ Genotypes drove results as much as drought (physiological traits, histology, transcripts and proteins)

ƒ High throughput RNA sequencing should become an important tool

¾ Continue the search for the molecular bases of drought tolerance

ƒ Focus the questions on highlighted cultivar-contrasted processes :

– rapid and water deficit-specific vs delayed and generic stress response – responsiveness of cambial tissue

ƒ Track the recovery phase in addition to drought acclimation

Ä Stepping stone for studies of other woody / perennial species

¾ INRA NANCY (UMR EEF)

ƒ Irène HUMMEL

ƒ David COHEN

ƒ Didier LE THIEC

ƒ Nathalie NINGRE

ƒ Erwin DREYER

¾ INRA ORLEANS (UAGPF)

ƒ Jean-Charles LEPLE

ƒ Philippe LABEL

ƒ Marie-Claude LESAGE-DESCAUSES

ƒ Françoise LAURANS

ƒ Isabelle BOURGAIT

ƒ Nelly DESPLAT

ƒ Annabelle DEJARDIN

ƒ Gilles PILATE

¾ ORLEANS UNIVERSITY (LBLGC)

ƒ Ludovic BONHOMME

ƒ Franck BRIGNOLAS

ƒ Domenico MORABITO

¾ INRA BORDEAUX (UMR Biogeco)

ƒ Delphine VINCENT

ƒ Christophe PLOMION

¾ URGV Evry

ƒ Sandrine BALZERGUE

ƒ Marie-Laure MARTIN-MAGNIETTE

ƒ Jean-Pierre RENOU

¾ INRA Nancy (UMR IAM)

ƒ Emilie TISSERANT

¾ Ecogenomic plateform of INRA Nancy

Thank you for your attention