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. 3.

symposium franco-suédois UPRA, Nov 2009, Champenoux, France. 26 p. �hal-02817980�

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. : MB Bogeat-Triboulot

Project GPLA0628G – 2006/2010

Objectives

ª Integrative study of the response of poplar to water deficit

ƒ combining several approaches : ecophysiology, transcriptomics and proteomics

ƒ studying different tissues in order to focus on different processes – mature leaves -> CO

assimilation, cell metabolism – guard cells -> stomatal conductance regulation – wood and growing xylem -> cambial growth

– root apices -> primary growth

ª Identify genes, gene networks and transcription factors involved in drought

tolerance

UPRA meeting - Nancy, November 2009 - 3

Poplar genomic toolkit

Diversity in drought tolerance

between clones Genomic plateforms

Expertise in transcriptomic Expertise in proteomic

Expertise in ecophysiology

2 poplar clones: 1 tolerant and 1 sensitive to drought

growing root apex mature root differentiating xylem leaf stomata Transcriptome

Proteome

Transcriptome Proteome

Transcriptome Proteome

Transcriptome

Identification of genes involved in drought tolerance

Fonctional validation Transformation?

Selection of some candidate genes by:

- Nature of gene

- Regulation in several organs

qPCR on other clones of different drought tolerance

Leaf T and P

guard cells Transcriptome Wood formation

Stem mechanics & hydraulics

Root growth

Gas exchange, WUE

Genetics wood

Controlled water deficit and fine

ecophysiology characterisation

Carpaccio

(Monclus et al, 2006)

Soligo

Experimental design

ª 2 genotypes P. deltoides x nigra : Carpaccio and Soligo chosen for

ƒ similar productivity

ƒ similar WUE

ƒ contrasted productivity maintenance under water deficit

ª Design

ƒ 1 batch for repeated ecophysiological measurements

ƒ 1 batch for molecular analyses

– 2 biological replicates (pools of 3 plants) per modality

ª 3 water deficit treatments

ƒ Control of soil volumetric water content

ª Characterisation of water status, growth, gas exchange, wood anatomy, δ13C (WUE) ª Tissues harvesting for transcriptome and

proteome analyses

ƒ mature leaves

ƒ wood and growing xylem

ƒ root apices

UPRA meeting - Nancy, November 2009 - 5

Carpaccio

Date

21/05 28/05 04/06 11/06

Growth (cm day-1)

0 1 2 3 4 5 6 7

CTL EAR AMI Sev

Soligo

Date

21/05 28/05 04/06 11/06 0 1 2 3 4 5 6 7

Kinetics

Soligo

23/05 27/05 31/05 04/06 08/06 12/06 16/06 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Carpaccio 22/05 26/05 30/05 03/06 07/06 11/06 15/06

Stomatal conductance, mmol.m

.s

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

CTL EAR AMI AMO 10

10,5 11 11,5

12 12,5

13 13,5

14 14,5

15

22/5 23/5 24/5 25/5 26/5 27/5 28/5 29/5 30/5 31/5 1/6 2/6 3/6 4/6 5/6 6/6 7/6 8/6 9/6 10/6 11/6 12/6 13/6 14/6 15/6 16/6

Date (2007)

Stem diameter(mm)

Water Deficit (12 d) Rewatering (6 d)

Ecophysiological response (D day)

CTL EAR AMI AMO Clone trait interac.

Carpaccio -0.13 -0.11 -0.11 -0.13

Soligo -0.23 -0.26 -0.29 -0.28

Carpaccio 92 93 93 93

Soligo 91 91 91 92

Carpaccio 627 618 648 667

Soligo 616 624 676 716

Carpaccio 4.6 4.8 3.4 3.1

Soligo 4.5 4.7 3.4 3.5

Carpaccio 0.168 0.121 0.182 0.146

Soligo 0.198 0.146 0.182 0.131

Carpaccio 53.1 54.6 57.9 55

Soligo 54.8 57.9 65.6 63.3

Carpaccio 12.5 10.9 12.3 9.4

Soligo 19.0 17.4 18.0 12.6

Carpaccio 0.68 0.37 0.32 0.15

Soligo 0.85 0.38 0.47 0.23

Carpaccio 20.7 30.0 43.8 66.7

Soligo 23.2 56.0 41.0 57.0

0.05 <0.001

0.29 <0.001 0.012 1.0 <0.001 0.92

0.002 0.49

<0.001

0.53 <0.001 0.24

0.7 Primary growth, cm

day-1

Radial growth, mm day-1

0.19

0.11 <0.001 0.27

0.013 0.047 0.59

Wi (A/g), %°

pd, MPa

Leaf RWC, %

FT, mosmol/kg

LMA, g m-2

A, µmol m-2 s-1

gs, mol m-2 s-1

<0.001 0.59

Absolute values p value

<0.001 0.53 0.22

UPRA meeting - Nancy, November 2009 - 7

Ecophysiological response

ª moderate water deficit => small but significant physiological responses

ƒ primary and secondary growth, gs, CO

assimilation rate, δ

C

ƒ significant effect of EAR on gs

ƒ differences between AMI and AMO (radial growth, A, gs, WUE, Π

)

ª intrinsic differences between clones

ª almost no significant interaction genotype x treatment :

ƒ the difference in WD tolerance not due to large contrast in one or several process(es)

ƒ tolerance = result of the time integration of weak differences

Leaf transcriptome

Affymetrix Poplar GeneChip microarray

partial annotation -> completion of probe set annotation

36,687 out of the 61,000 probe sets of the array were validated in our comparative set-up (DNA hybridization, ESTs)

R egulated probe sets

-300 -200 -100 0 100 200

300 ^{U P}D O W N

ª short term response : mainly up-regulations

ª long term response : mainly down-regulations

ª genotype specificity : sensitive genotype Soligo showed

– contrasted response between the two levels of long term WD – more numerous gene regulations in comparison with Carpaccio

EAR AMI AMO EAR AMI AMO

Carpaccio Soligo

2,195 DW responsive probe sets

(no Log 2 ratio cut-off, p<0.05)

normalised intensity of the 1334 probe sets with complete expression

pattern

EAR

AMI

AMO CTL

EAR

AMI

AMO CTL

Carpaccio

Soligo

Leaf transcriptome

ª Expression strongly contrasted between genotypes

ª Rank conservation among treatments within genotypes

WD markers and candidates for WD tolerance in leaves

common to EAR, AMI, AMO

specific of the long term response (AMI et AMO) specific of the short

term response (EAR)

Homeobox-leucine zipper protein*

Galactinol synthase 1 RCI 2A - like

Protein phosphatase 2C (2)*

Xyloglucan endotransglycosylase*

Stress markers

S C

(84 + 36) AREB

ABA-induced-like protein SNARE-interacting protein

Aquaporin TIP / PIP NADPH oxidase Galactinol synthase 2

LEA (4)

ERD protein-related Rapid alkalinization factor putative serine protease (2)

(13 + 43) Polyphenol oxidase Superoxide dimutase Alcohol dehydrogenase

WAK-Like (6) Sucrose synthase (3) NBS-LRR family protein (6) Unknown protein EAR AMI AMO

ABA 8'-hydroxylase (2) ABC transporter family protein

Receptor-like protein kinase LRR receptor-like protein kinase

NBS-LRR type

Candidates for water deficit tolerance

concordant with litterature

(Bray, 2004; Bogeat-Triboulot, 2007)

UPRA meeting - Nancy, November 2009 - 11

Leaf and root transcriptome meta-analysis

ª Root apex transcriptome more responsive than the mature leaves one

ƒ number of regulations

ƒ higher fold change

Data set: 6725 probe sets regulated at least once among the 12 combinations

Leaf root

Log2 ratio

-800 -600 -400 -200 0 200 400 600 800 1000 1200

Up-regulated Down-regulated

EAR AMI AMO

Carpaccio Soligo Carpaccio Soligo

EAR AMI AMO EAR AMI AMO EAR AMI AMO

Leaf Root Apex

Number

Leaf and root transcriptome meta-analysis

ª Leaf and root common regulated genes : common stress marker

ƒ Homeobox-leucine zipper protein (atHB12-like)

ƒ Protein phosphatase 2C

ƒ NCED, caroten dioxygenase activity

ƒ HSP, HSP-binding

ƒ Bet-v1 allergen (Pyr-like protein)

ƒ Etc ….

ª Leaf specific regulated genes

ƒ Galactinol synthase-llike

ƒ B-glucosidase (other photosynthesis-associated gene)

ƒ Etc ….

ª Root specific regulated genes

ƒ PIP1-4

ƒ Beta-caroten hydrolase

ƒ ABC transporter

ƒ Asparagine synthetase

ƒ Early responsive to dehydration (AtERD15-like)

ƒ ACC oxidases

ƒ Transferases

ƒ

UPRA meeting - Nancy, November 2009 - 13

Transcriptional regulation of the aquaporins family

ª More numerous and stronger regulation in root apices as

compared to leaves (similar to the whole genome response)

ª Co-regulations detection

ª Induction detection

ƒ TIP1.4, PIP2.5 in leaves

ª Identification of AQPs potentially involved in drought tolerance

expression pattern in

control

AMO

AMI

EAR EAR AMO AMO

2008 AMI 2008 AMI AMI AMO EAR EAR

TIP SIP XIP

PIP NIP

root apex mature leaf

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ª Expression in controls

ƒ 10 genes not expressed

ƒ 12 genes root-specific

ƒ 2 genes leaf-specific

Guard cell transcriptomics

Laser microdissection (1500 guard cell complexes)

UPRA meeting - Nancy, November 2009 - 15

RNA extraction Amplification

Hybridization on microarrays

Guard cell transcriptomics

CEAR vs CCTL

CAMI vs CCTL

CAMO vs CCTL

SEAR vs SCTL

SAMI vs SCTL

SAMO vs SCTL

ª Rapid and strong induction response in the tolerant genotype

ª Strong regulation levels

1,196 DW responsive probe sets (no Log 2 ratio cut-off, p<0.05)

Deeper analysis currently running

Wood histology

stress onset

end of stress

STRESS 13 days

REIIRIGATION 6 days

Stem diameter(mm)

Water Deficit (12 d) Rewatering (6 d)

CTL and AMO wood characteristics

Fiber (mean area) Vessel (mean area) F (% CSA) V (%CSA)

C_CTL 67,55 (4,47) 2836,68 (2,47) 17,51 (4,79) 19,92 (1,08)

C_AMO 52,67 (5,06) 2102,12 (5,01) 16,07 (4,16) 20,12 (1,8)

S_CTL 76,01 (5,25) 2399,34 (3,41) 23,05 (4,91) 16,71 (2,36)

S_AMO 49,41 (3,24) 1864,07 (2,79) 15,80 (2,69) 16,40 (8,78)

Ratio (P value)

C_AMO versus C_CTL 0,78 (0,019)* 0 ,74 (0,0008)* 0,91 (0,356) 1,01 (0,738) S_AMO versus S_CTL 0,65 (0,0004)* 0,78 (0,0012)* 0,68 (0,0006)* 0,98 (0,884)

ª Differences in vessel mean CSA and in vessel total CSA fraction between clones

ª Fiber and vessel mean CSA reduced under WD

ª Vessel total CSA fraction insensitive to WD

ª Conservation of genotype differences under WD

UPRA meeting - Nancy, November 2009 - 17

Gene networks in wood

ª 484 genes classified in 15 modules

ª 7 modules covering 51 % of gene regulation

ª Main functional modules :

ƒ GH17, GH3, GT2, pectine esterase laccase …. Cell wall construction

ƒ Snare-like, IMP, Aquaporins, ABC transporter : Cell membrane

associated proteins

TF (out of module)

presence of TF biding

sites

presence in other modules

Interaction relations

Ontology

Functional

classification

Enigma (Maere et al. 2008)

Poplar genomic toolkit

Diversity in drought tolerance

between clones Genomic plateforms

Expertise in transcriptomic Expertise in proteomic

Expertise in ecophysiology

2 poplar clones: 1 tolerant and 1 sensitive to drought

growing root apex mature root differentiating xylem leaf stomata Transcriptome

Proteome

Transcriptome Proteome

Transcriptome Proteome

Transcriptome

Identification of genes involved in drought tolerance

Fonctional validation Transformation?

Selection of some candidate genes by:

- Nature of gene

- Regulation in several organs

qPCR on other clones of different drought tolerance

Leaf and guard cells T Leaf Proteome Wood formation

Stem mechanics & hydraulics

Root growth

Gas exchange, WUE

Genetics

wood

UPRA meeting - Nancy, November 2009 - 19

Carpaccio Soligo

0 2 4 6 8 10 12

EAR AMI AMO

CTL CTL EAR AMI AMO

μg. μl-1

G***, T

, GxT

Leaf proteome

ª higher protein content in Soligo

ª higher spots number in Soligo (1200) than in Carpaccio (600) Æ comparison of 563 reproducible spots

pH4 pH7 pH4 pH7

10kDa 100kDa

Soligo Carpaccio

300 µg of proteins

ANOVA FDR

p-value ≤ 0.05 q-value ≤ 0.05

G 400 400

T 40 0

G x T 43 0

ANOVA FDR

p-value ≤ 0.05 q-value ≤ 0.05

G 361 361

T 59 0

G x T 55 0

ANOVA FDR

p-value ≤ 0.05 q-value ≤ 0.05

T 53 1

ANOVA FDR

p-value ≤ 0.05 q-value ≤ 0.05

T 27 0

ª strong genotype effect

ª after FDR, no significantly WD-regulated protein was discovered

Root apex proteome

ª 3*40 root apices, i.e. about 500 mg fresh weight

ª Required protocol optimisation of several steps :

ƒ extraction

ƒ electrophoresis conditions : small pH range (4-7 & 7-11)

3 11

1500 detected spots

4 7

2500 spots

7 11

1000 spots

TCA/acetone

4 7 4 7

pI PM

Phenol/ammonium acetate

ª Acid and basic proteins

ª Statistical analysis

ƒ PCA

ƒ Hierarchical clustering

Acid proteins Basic proteins

UPRA meeting - Nancy, November 2009 - 21

Acid proteins

1540 reproducible spots

C.CTL CEAR C.AMI

C.AMO

S.CTL S.EAR

S.AMI

S.AMO

PC2 (23.6%)

PC1 (36.4%) génotype

traitement

0 10 20 30 40

1 2 3 4 5 6 7 8 PC

R2

60%

Root apex proteome

ª 1st source of variation of protein quantities: genotype

ª 2nd source of variation of protein quantities: treatment

x 87 x

6

x 3377 x 3383 3379x x36 x 1513 2999 2998x x x2402

x x

2393 x 2395 2394 x

56 37

476

x

x

x72

x x x 175 31 3373 x

x x

58

115 62

x3441

x 430

x x x x 3515 3519

3518 3512 x x

x x

370 376

2562 3864

x 3545 x 3658

x x x x 64

2662

2650 2652 3079x x 3254 x

x x 3256

34293426 3913 3837x x x 3909

3457x x3471 x3477 x

3027 x2962

x 3492 x x 3790 3788

x x x274

191 205 x234 360

x 466

x

Heat Shock Proteins (HSP70, HSP82) 14-3-3 protein

2 APX, PRX2B, GST etc…

ª Excision of selected proteins

ƒ 61 acid proteins

ƒ 33 basic proteins

ª LC-MS/MS identification

Poplar genomic toolkit

Diversity in drought tolerance

between clones Genomic plateforms

Expertise in transcriptomic Expertise in proteomic

Expertise in ecophysiology

2 poplar clones: 1 tolerant and 1 sensitive to drought

growing root apex mature root differentiating xylem leaf stomata Transcriptome Transcriptome

Proteome

Transcriptome Proteome

Transcriptome Proteome

Identification of genes involved in drought tolerance

Fonctional validation Transformation?

Selection of some candidate genes by:

- Nature of gene

- Regulation in several organs

qPCR on other clones of different drought tolerance

Leaf T and P

guard cells transcriptome Wood formation

Stem mechanics & hydraulics

Root growth

Gas exchange, WUE

Genetics

cambium

UPRA meeting - Nancy, November 2009 - 23

Expression in other clones

ª Independent experiment on 8 genotypes:

ƒ Carpaccio, Soligo

ƒ 4 others P. deltoides x nigra :

I214, Dorskamp, Koster, Flévo

ƒ P. trichocarpa Beaupré

ƒ P. tremula x alba 717-1B4

ª long-term water deficit (10 days at 20% REW)

ª RT-qPCR analyses

ƒ housekeepers genes

ƒ currently running

UBI

PP2A

2008

Conclusions from this integrative study

ª Validated water deficit “markers”

ƒ previously identified in other species or poplar genotypes

ª Identified candidates genes for drought tolerance in poplar

ª Focused on cell types and specialised tissues :

ƒ stomata vs whole leaf

ƒ growing xylem vs whole wood

ª Between genotype differences higher than drought response

ƒ for physiological traits, histology, transcripts and proteins

ƒ also pointed out by Wilkins et al (2009, mature leaves) – this point has to be addressed by diversity analyses

ª Root apices more “responsive” than mature leaves

ƒ seen at the scale of the whole genome as well as at the scale of one multigene family

ƒ seen at the transcript level as well as at the protein level – growing vs mature tissue ?

– root : closer to the constraint ?

– organ specificity ?

UPRA meeting - Nancy, November 2009 - 25

Collective work :

ª INRA NANCY (UMR EEF)

ƒ Irène Hummel

ƒ David Cohen

ƒ Rémy Merret

ƒ Didier Le Thiec

ƒ Nathalie Ningre

ƒ Erwin Dreyer

ª INRA ORLEANS (UAGPF)

ƒ Jean-Charles Leplé

ƒ Philippe Label

ƒ Gilles Pilate

ƒ Annabelle Déjardin

ƒ Isabelle Bourgait

ª ORLEANS UNIVERSITY (LBLGC)

ƒ Franck Brignolas

ƒ Domenico Morabito

ƒ Ludovic Bonhomme

ª INRA BORDEAUX (UMR Biogeco)

ƒ Delphine Vincent

ƒ Christophe Plomion

ª URGV Evry

ƒ Sandrine Balzergue

ƒ Marie-Laure Martin-Magniette

ƒ Jean-Pierre Renou

ª INRA Nancy (UMR IAM)

ƒ Emilie Tisserant

ª Ecogenomic plateform of INRA Nancy

Thank you Irène and David for your

support on my way to genomics…

Thank you for your attention