TITRE

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EXEMPLE D’IMAGE

Caracterização molecular de determinantes envolvidos na tolerância à seca

Pierre Marraccini

Researcher CIRAD-UMR AGAPEmbrapa Genetic Resources and Biotechnology Brasilia – DFBrazil

E mail 1 : marraccini@cirad.frE mail 2 : pierrem@cenargen.embrapa.br

Effects of drought in coffee plants

Drought is considered to be the major environmental stress affecting coffee production (da Matta 2004)

Moderate droughtfruits malformation (↑ defects, ↓ size)↓ fruit (cup) quality

Strong droughtleaf sheddingplant death

Σ: loss of incomes for coffee producers

Coffee regions affected by drought: social, economical, environmental impacts...

How to reduce these negative effects?

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Analysis of C.canephora diversity for drought toleranceIt exists in C. canephora:

- drought-tolerant genotypes in Guinea and SG1 groups- drought-susceptible genotypes SG2 group

GuinéenC B

SG1 SG2

O

?

GuinéenC B

SG1 SG2

O

?Berthaud, 1986, Montagnon et al, 1992, Dussert et al, 1999.

Guinea

Congo

SG1: region of Kouillou > “Conilon”Region with short dry seasonRegion without dry season

Understand the genetic determinism of this tolerance to use itbreeding programs to create new cultivars and varieties

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Biochemical / physiological targets ↔ genes

drought T associated with antioxidant enzymes (against oxydative stress)superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX)

drought T: smaller stomatal conductance (gs) > rapid stomatal closureinvolment of acid abscissic (ABA) regulation pathway

drought T: maintenance of photosynthesisgenes coding chlA/B binding-protein, PSII, OEC, PSI and Rubisco

drought T: osmotic adjustment (seems to be limited in coffee)genes coding sugar and derivative metabolites

drought T: other mechanisms?other genes?

Estudos fisiológicos relacionados a tolerância a secaFábio Murilo da Matta, UFV - Brazil

Molecular determinism of droughtT in coffee?: search of candidate genes (CGs)

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EXEMPLE D’IMAGE

Molecular determinism of droughtT in coffee?: search of candidate genes (CGs)

Hypothesis: the drought T and drought S phenotypes come from differential (quantitative) expression of some important candidate genes (CGs)

What plant material ?: drought T and drought S clones of C. canephora “conilon”

What plant tissues ?: leaves (roots)

Question of research: identification of genes differentialy expressed between drought T and drought S clones of C. canephora submitted to controlled (greenhouse) water constraint (irrigated vs. non-irrigated)

What methods to isolate CGs?: transcriptomic and proteomic analyses (electronic-northern, northern-blots, qRT-PCR, macroarray screeening, 2D-gel electrophoresis...)

Compared analyses at the physiological and molecular levels

What is supposed to occur?

High expression

“A very [simplified] point of view” is looking for:genes of “tolerance”: expression drought T > drought S

genes of “sensibility”: expression drought S > drought T

Hypothesis: the drought T and drought S phenotypes come from differential (quantitative) expression of some important candidate genes (CGs)

Dogma of molecular biology: Q protein = f (Q RNA)

Low expression

•

•replication(DNA-> DNA)

se

DNA

transcription(DNA-> RNA)

A Polymerase

~RNA

translation(RNA -> Protein)

RObosome

Protein

•

RNA Protein

Clones of C. canephora “conilon” selected by Incaper- drought T clones : 14, 73 and 120- drought S clone : 22transferred at UFV and tested in greenhouse + irrigation (ΨPD = -0.2 MPa) = control- irrigation (ΨPD = -3.0 MPa) = drought-stressed

physiological analysesmolecular analyses (leaves)

What plant material?

Drought T Control

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8

Recursos Geneticos eBiotecn%gia

Physiological analysesComparison of clones of C. canephora “conilon” droughtTclone 14 vs. droughtS clone 22RDPWP (rate of decrease of ΨPD): 22 > 14A (net CO2 assimilation): 14D > 22Dstomatal conductance (gS): 14C < 22C

Effects of the drought on leaf pre-dawn water potential (ΨPD in MPa), rate of decrease of ΨPD (RDPWP in MPa d-1 m-2), net CO2assimilation rate (A in μmol m-2.s-1), stomatal conductance (gs in μmol m-2.s-1), internal to ambient CO2 concentration ratio (Ci/Ca),maximum photochemical efficiency of PSII (Fv/Fm), quantum yield of PSII electron transport (ФPSII), photochemical (qP) andStern–Volmer non-photochemical (qN) quenching coefficients, and the fraction of PPF absorbed in PSII antennae and used neitherin photochemistry nor dissipated thermally (PE) of clones 14 and 22 of C. canephora

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Parameters IDrought-tolerant clone (14) Drought-sensitive clone (22)

Control IDrought Control IDrought

'Ppd ~O.02±0.01 A -3.02±0.12 a ~O.03±O.OO A -3.01 ±o.11 a

RDPWP 0.67±0.04 G 1.01 ±o.04 a

A 9.40±0.34 A 2.62±0.27 a 9.35±0.17 A O.95±o.23 b

9s 60.00±5.00 B 13.00±3.50 a 105.00±9.50 A 5.00±3.00 a

C/Ca O.520±0.040 B 0.380±O.o40 b 0.670±0.040 A 0.520±o.040 a

FjFm O.840±O.o11 A 0.842±0.011 a 0.831 ±0.011 A o.800±o.011 b

Cl)PSII 0.455±0.049 AB 0.287±0.050 a,b 0.472±0.049 B o.210±0.050 b

qp 0.713±O.o51 A,B 0.495±O.o51 a,b O.697±0.051 B 0.362±0.051 b

q 0.665 ±O.056 A 0.717±O.o57 a O.642±0.056 A 0.543±0.057 a

PE 0.252±0.040 A 0.425±O.o40 a,b O.242±0.'039 A 0.508±o.039 a*

What data?Brazilian Coffee EST genome project (2002-2004)

University Campinas - SPUNICAMP - Lab. LGEhttp://bioinfo04.ibi.unicamp.br/free access

~=r'CAFES DOBRASIL

ConJordo BJoslleho de PesquilGeo Deienvolvimento do Caf"

Support

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Em~aCAFE

LinksAbout coffee

Recti/sos Gen~rJcos e Biotecno(ogie

(on50'CIO Bro5,leiro de Puquhoe De-senva'lvime.n10 do (1J1e

.­I"CAFES DO

BRASIL

TeamServices

CDFFEE GENDME PRDJECT

omaCafeAj,mnQmbl (\(

Environmmal GC'llomo

AEG

5..:..-=-J;&i'iii~~~r-_. Gene Projects

Leaf cDNA library (SH3) of drought-stressed C. canephora “conilon” (clone 14 drought T) vs.

Leaf cDNA libraries (LV) of unstressed C. arabica var. catuaí(drought S)

SH3LV

Contig 18332: no hits found!

1st method: CGs identified by “Electronic northern”

el tl-aclustecs

cada

679

"re 1:0

R 1 ~ao aopr Q

-b iorec

Mostmr BlastHits

Moslt"ar Tradul'3o nos 6 Frames

No Hits FOlUld!

Niunero de Reads: 33Lista de Read(s):

CGGACGCGTCCGCC ACGCG1CCGCCCACGCGTCCGAAATC.kGTCTTCAA':'ACA:'AAA':'!'T:'CCT1 AGCAAAAATGGGTTCCAAGACACTTCTT7TCTTTTTCAT:'TCCATGGC7GTAGTTCTAA':'GAT':'ACCTCA AGGTGGCTGC AA TCAGTTGACAAT'I'CC.ri.AGACAGT':G~

CAAATGAGGAAGG;'GAAGCCAAGTACCATGGAGGTGGCTri. GGAGGAGGCCACGGAGGA GCTACGGAGGAGGCCATGGAGGTGGCTACGGAGGAGGACATGGAGGGTACGGGGG7GGCGGCCATGGTGG7TATGG~CATGGCGGrGGCGGCCATGGTGG:TATGGACATGGTGG':l'ATG

G~CATGG'!'GGrTA': GACACGGCGGACATGGCGGT C GCCATGGTGGACATCCTGG7GAGGCTGCAGA7GCTCAGCCTCAGAACTAATC~GCCAGC':"TCT:"GC-:A'I'G'!'CATGATT.AATCATG.AATGC-:TAAA

C;;'GGC7TAC7:"TA;.,7AGTATGTACT7TGTAATGTGA7CTc;.A,ATAAG'! C::GGA':'CAG': A::CAT TAAT;"T CAC':CCATGTAC':'TGCTGGCTA7CTAGTTCAATACAAGTATA7GAGTT':GTGA7TTTGTGTC~

CAOO-XX-SH2-OO6-E 1O-EMCCOO-XX-SH3-004-B05-EMF

CCOO-XX-SH3-OO8-H05-EM.F

CCOO-XX-SH3-OO8-D02-EM.F

CCOO-XX-SHJ-Q17-B02-EMF

Contig18332

Sequencia:

CCOO-XX-SHJ-QIO-GII-EM.F

Mostrar Resultado da Montagem

CCOO-XX-SHHll O-C1O-EM.F

* - Read(s) )IIais Siguificativo(s)

.-

Total de Bibliotecas: 2

YIeulI

Bibliotecas:

E."tpressao Relatiytl emRcla~ao ao total de Readscia Biblioreca

E....press..lo Relativi\ emRcla~o aos clusters ma.isex.pressos em cadabiblioteca

Coutrole dt" BUl5

Categorias do UoigeoeEscolher Versao dosClustersExponar para 0 QBOTlmpenar do QBOTInterface \' eb do BlastLtstar BibliotecasDisponiveisLtstar Resultado doRearranjo~Oes PessoaisTeste ExaIO de Fisher

SH2 (1/4876)H3 (32/5579)

r...

Contig 00355: galactinol synthase Ajuga reptansContig 00367: cystein proteinase inhibitorContig 05906: cystein proteinase inhibitorContig 09158: Acyl-CoA-binding protein Panax ginsengContig 12922: no hitsContig 13476: metallothionein-like protein Citrus unshiuContig 15415: mannose 6-phosphate reductase Arabidopsis thalianaContig 18230: chlorophyll a/b binding protein Lycopersicon esculentumContig 18232: chlorophyll a/b binding protein Arachis hypogaeaContig 18240: no hits (CcUNK10)Contig 18244: rubisco small subunit Coffea arabicaContig 18297: catalase Gossypium hirsutumContig 18360: no hit (EST leaves infected by Hemilia vastatrix)Contig 18378: mannose 6-phosphate reductase (NADPH-dependent)Contig 18430: no hitsContig 18470: cystein proteinase inhibitorContig 18387: “abscisic stress ripening protein”Contig 18332: no hits

1st method: CGs identified by “Electronic northern”

Contig 00355: galactinol synthase Ajuga reptansContig 00367: cystein proteinase inhibitorContig 05906: cystein proteinase inhibitorContig 09158: Acyl-CoA-binding protein Panax ginsengContig 12922: no hitsContig 13476: metallothionein-like protein Citrus unshiuContig 15415: mannose 6-phosphate reductase Arabidopsis thalianaContig 18230: chlorophyll a/b binding protein Lycopersicon esculentumContig 18232: chlorophyll a/b binding protein Arachis hypogaeaContig 18240: no hits (CcUNK10)Contig 18244: rubisco small subunit Coffea arabicaContig 18297: catalase Gossypium hirsutumContig 18360: no hit (EST leaves infected by Hemilia vastatrix)Contig 18378: mannose 6-phosphate reductase (NADPH-dependent)Contig 18430: no hitsContig 18470: cystein proteinase inhibitorContig 18387: “abscisic stress ripening protein”Contig 18332: no hits

1st method: CGs identified by “Electronic northern”

2nd method: CGs identified by macroarray screenings

Membranes were hybridized with cDNA probes representing RNA extracted from leaves of C. canephora clones 22 and 14 grown with (I) or without (NI) irrigation. .

Analysis of genes differentially expressed in leaves of C. canephora“conilon” clones 14 (drought T) and 22 (drought S)

tumour necrosis factor receptor (TNFR)-associated factor

prephenate dehydrogenase

no hits

dehydrin

enhanced disease resistance

heat shock protein

mannose 6-phosphate reductase

ubiquitine: constitutive expression

Putative protein functions 22NI22I14NI

~

:J

Gene 14I

CcTRAFl

CcPDHl

CcUNKB

CcDH3

CcEDRl

CcHSPl

CcMPRl

CcUBQ10

3rd method: CGs identified by 2D-gel electrophoresisAnalysis of proteins differentially expressed in leaves of C.canephora“conilon” clones 14 (drought T) and 22 (drought S)

CcCA1: carbonic anhydrasePutative protein functions

CcCA1

0.6D ,41

14NI0.5 D 221

0 > 22NI'#-- 0.4eog~"U

0.3c::~

.0~

.~ 0.2!!:e

0 0 a..0.11

0'Cc-CA1

3rd method: CGs identified by 2D-gel electrophoresisAnalysis of proteins differentially expressed in leaves of C.canephora“conilon” clones 14 (drought T) and 22 (drought S)

CcCA1: carbonic anhydraseCcPP2C: type-2C protein phosphataseCcPSBO: PSII O2 evolving complexCcPSBP: PSII O2 evolving complexCcPSBQ: PSII O2 evolving complexCcHSP1: heat-shock protein

Putative protein functions

Σ all the methods: > 40 candidate genes (CGs) presenting differentialexpression profiles during drought were identified

Some examples are presented…

A Cc:CA1

0.6D 141

14NI0.5 D 221

® 0 > 22NI'::;!!e....0.4..g

c.s"0 0.3c;:).0-('IS

.~ 0.2~e

0 0 a...0.1

0'CcCA11

Effects of drought on coffee gene coding for proteins involved in the mechanisms of cell protection (1)

Relative expression

14I 14NI0

5

10

15

20

22I 22NI

CcHSP1

ab

c

b

c

14I 14NI0

5

10

15

20

22I 22NI

CcDH3

a

b

a

b

14I 14NI 22I 22NI0

4

6

8

2

10

a

c

b

cCcGPP1

Glycin-rich proteins (CcGRP1): cell wall, reinforcement, and repairHeat-shock proteins (CcHSP1): maintenance protein foldingDehydrins (CcDH3): preventing the denaturation of macromolecules

proteins preventing cellular damages

Expression increases with droughtNo differences of expression profiles between the clones 14 and 22

“common responses” of these clones upon drought stress

Effects of drought on coffee gene coding for proteins involved in the mechanisms of cell protection (2)

Catalase (CcCAT1, CcCAT2)Ascorbate peroxidase (CcAPX1, CcAPX2)

proteins reducing oxidative burst caused by drought

Within a gene family, expression profiles differed between genesneed to analyse expression of each paralogous (allele) genes

Higher expression of CcCAT2 in 14 vs. 22slight differences of gene expression between the clones 14 and 22 regardingdrought stressQ: relation with drought T vs. drought S?

Relativeexpression

0.5

1.0

1.5

2.0

14I 14NI 22I 22NI0

CcCAT1

b

a

b

a

14I 14NI0

0.5

1.0

1.5

2.0

22NI

CcAPX2

22I

b

a

c

a

14I 14NI 22I 22NI0

CcCAT23

1

2

a

c

a

b

0

5

10

15

20CcAPX1

14I 14NI 22I 22NI

a

b

a

b

catalase ascorbate peroxidase

• I

1----- j-----

I I

Effects of drought on coffee gene coding for proteins involved in the mechanisms of cell protection (3)

Mannose-6 P reductase (CcMPR1)Aldose-phosphate reductase (CcAPR1)

synthesis of sugars (and derivatives) = osmoprotectors?

Relative expression

0

10

20

40

50CcAPR1

14I 14NI 22I 22NI

30

b

d

a

c

14I 14NI0

5

10

22I 22NI

a

b

a

c

15CcMPR1

Higher expression of CcAPR1 in 14 vs. 22Important differences of gene expression between the clones 14 and 22regarding drought stressQ: relation with drought T vs. drought S?Need to performed in depth analyses of sugar metabolism (i.e. mannitoland alcohol sugars)

,-;:.- --,- -,------,

Ex: effects of drought of PSII componentsAnalysis of OEC proteins in leaves of C. canephora “conilon” clones14 (drought T) and 22 (drought S)

Relativeexpression

14I 14NI0

1.0

2.0

3.0

22I 22NI

CcPSBO

bc

a

bcd

b

14I 14NI0

1.0

2.0

3.0

22I 22NI

CcPSBP

c

a

d

b

14I 14NI0

1.0

2.0

3.0

4.0

22NI

CcPSBQ

22I

bc

a

abc

b

Drought:

↓ CcPSBO, CcPSBP and CcPSBQ gene expression

“common responses” for these genes between drought T anddrought S clones regarding to drought stress

Effects of drought on coffee gene coding for photosynthesis components

Adapted from Allen et al. (2011) Trends Plant Sci.

Drought leads a reduced gene expression of rbcs1, psbO, psbP, psbQ,and CAchlA/B

CO2

HCO3- CO2

Stomatal conductance

This is in accordance with the decrease of A (net CO2 assimilation)observed with drought for the clones 14 and 22 de C. canephorano major differences between clones except...

Il:mbryophyte leukaryote) Arabidopsis thaliana

ElElElEl

(

BtCP47)

PS 11 fPSb}IDlmer)

•

•

•

PS 11 (p.sa)IMono r)

Effects of drought on coffee gene coding for photosynthesis components

Adapted from Allen et al. (2011) Trends Plant Sci.

Higher levels of carbonic anhydrase (CA) in leaves of clone 14 vs. clone 22

Litterature: high CA activity involved in the maintenance of photosynthesisunder drought

Q: relation between higher CA and higher A under drought in clone 14 than 22under drought?

Measurements of CA activity

CO2

HCO3- CO2

Stomatal conductance

carbonic anhydrase(CA)

Abscisic acid (ABA)

I.. LS

(s

NIADIFl'H

~~\

LS

~..,.......-

Ru'sceII

0.6D 141

14N0.5 D 221

> 22N~ r=r=r=r=J:5!......0.4(iD

g

~L

('U it:-0c: 0.3

~~ H~..0m

lre::: 0.2'Q)e0..

0.1

0'CcCA1

EXEMPLE D’IMAGE

The ABA signaling network…

Hauser et al. (2011) Curr. Biol.

TranscriptIon factors llnd protoJn mOdifIcatIon

AtSIN3

CAMTA

AiBCG25,40

ABAE

ABA met.abollsm and transport

AOa.iIt'" ..

AtBG1

\ABA

ABA3 __ .:1 AA03

,,' ""''''"..'.-- ~....

...........-

ABA1 ------t> NeED -----io-ABA2f ~

. .

MYBIJ

...

:1::1-<~-:.--"-'

-------- -...... ' ...---:.,. ..----' ABI5

SIZ ------ A .. ~

/ ..// \..I : '.,- : .,

AFP1 .,".... ABI3 __~_

IIW5 .'ATHB5 WRKY2 T

A1P2

MV84

ATM:YB2.

ATHB6

ATMYC2

Regulation of ABA network…

Three-components system of regulation:ABA receptors (PIR/PYL/RCAR)PP2Cs (protein phosphatase s2C) = negative regulatorsSnRK2s (SNF1-related protein kinases ) = positive regulators

= no stress = stress

P

P

The genes involved in the reduction of

drought effects ARE NOT expressed

Expression of genes involved to reduce drought

effects

adapted from Cutler et al. (2010) Ann Rev Plant Biol

ABANo.ABAa

•

Regulation of ABA network…

Three-components system of regulation:ABA receptors (PIR/PYL/RCAR)PP2Cs (protein phosphatase s2C) = negative regulatorsSnRK2s (SNF1-related protein kinases ) = positive regulators

= no stress = stress

P

P

The genes involved in the reduction of

drought effects ARE NOT expressed

Expression of genes involved to reduce drought

effects

adapted from Cutler et al. (2010) Ann Rev Plant Biol

ABANo.ABAa

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What about PP2C in our coffee model?

Relativeexpression

Q: The differences observed between the clones 14 and 22 for theCcPP2C protein contents could explain the phenotypical differencesregarding to drought stress?

14I 14NI0

1.0

2.0

3.0

22I 22NI

CcPP2C

c

a

d

b

CcPP2C: type-2C protein phosphatase

CcPP2C protein level:- clone 14 < clone 22- “less ABA inhibitor” in 14 vs. 22

Gene expression:- expression CcPP2C: 14 < 22- decreased under drought

“less ABA inhibitor” : easier to activatethe ABA pathway under droughtin the clone 14 vs. clone 22

CcPP2C0.6 • •D 141

• 14NI 0 00.5 D 221;;:- 1- "I • 22NI~.!1,...0.4~

g«lu

0.3c~

.0«l

0c 0.2 0'q;ea.

0.1

o l...- ....CcPP2C

EXEMPLE D’IMAGE

Shinozaki et Yamaguchi-Shinozaki 2007 J. Exp. Bot. 58: 221-227

Transduction pathway of drought (abiotic stress) signal

Is the transduction pathway of drought signal altered in drought T

vs. drought S clones of C. canephora “conilon” ?

'Ge' e Produ.cts InvoJved· I Stress Response and Tolelra ce

DREB11JCBF(AP2!ERF)

DREB2(AP2IERF)

\

1,ER01

NACAREB/ABF HD-ZI

(Ib P)

,ABRE(ACGTGGC),

RD29S~ FlDZOA

D ou ht, HOg sal"nity Colld

~ ~Signal Perception

/ AaA independent pathwa¥S

L J !

!NAC

(RD'26)

~

MYB2, MYC2(MY!, YC)

!MYBRS, YCR

(YAACR" CA I NTG),

RD22 Gly

B'o ic slressand wound1ng

genef nctio'

Cis~ctil 9e emenls

geneexpession

'transcriptionfactos

Si:gnaltransductio

•

Expression profiles of CcDREB2 gene

CcDREB2 expression clone 14 > clone 22expression of CcDREB2 gene very low and poorlyinduced by drought in the drought S vs. drought T

clones

14I 14NI0

3

6

9

12

22I 22NI

CcDREB2

abc

d

a bc

±

Sequencing of DREB2 promoter regions from the clones 14 and 22

great sequence differences observed in the DREB2 promoter regions ofclones 14 and 22Q: sequences differences related with the variation of gene expressionobserved for the DREB2 gene between clone 14 and 22?

•

•

•

3S0370160350140no320

- -GCAACC'GCTGGl'AAAAAGCCATAAGAATCA'ITAGCJlGTJlGTJlCTATAAAGAGAACAACTTGC'ITCTG- - --T-GCAACC'GCTGGl'll.AA.AASCCATAAGAATCATIAGTCGTA"GTACTATAAAGAGAACAACT"I'GCTTCTG- - - --

CC'GCTGGl'AAAAAGCCATAAGAATCA'ITAGCAGTAGTJlCTATAAAGAGAACAACTTGC'ITCTG- - - - T-GCAACC'GCTGGl'MAAAGCCATAATAATCATl'AGTAGTAGTACTATAAAGAGAACAACTTGCTTc.-rG- - - - T

- -GCAACC'GCTGGl'AAAAlWCCATAAGAATCATTAGYAGTAGTACTATAAAGAGAACAACTTGCTTc.-TG- - - - T

no

- -GCAACC'GCTGGl'AAAAAGCCATAAGAATCA'ITAGTAGTAGTJlCTATAAAGAGAACAACTTGC'ITCTG- - --T- -GCAACC'GCTGGl'.AAAAAECCATAAGAATCATI'AGCAGTA:GTAcrATAAAGAGAACAACTTGCTTCTG- - --T

GCAACC'GCTGGl'.AAAA.AGCCl\TAAGA.ATCATI'.AGT~TlL:GTACTATAAAGAGAACAACTTGCTTC'TG

CC'GATGGT.AAA.AA9CCATAAGA.ATCATl'AGTAGTAGTA.cTATAAAGAGAACAACT'I'GCTTCTG- TT- - - - - - - - - - CAACC'GCTGGl'.AAA.AASCCATAAGA.ATCAITAGTAGTGGTACTATAAAGAGAACAACTTGG'M'CTG-TI'TT

- - -GCAACC'GCTGGl'AAGAAGCCATAAGAATCA'ITAGTAGTAGTJlCTATAAAGAGAACAACTTGC'ITCTG- - - - T- - - - - - CAACC'GCTGGl'MAAAGCCATAAGAATCATl'AGTAGTGGTACTATAAAGAGAACAACTTGGTTC'TGTI'TTT

302S0270260

•

C!one14 SlS2 R D06 021.abl(6S>5SB) ~ TrCGTAATCAATTA cc--- -----CLONll22 Si R F07 OlO.abl (10,540) ••••+-............... .. CCACC-----

clone22 SlS2 !? FOJ 02J.ab1(102,716) -+ =TJlATCAATTA C-----clone22 SlS2 R COJ 030 .ab1 (59,617) -+ =TJlATCAATTA CCAAClone22 SlS2 R COB 030_abl(60,,487) ...-.. TTCGTAATCAATTA C---------Clone22 SlS2 R POB 027 .abl (60,,69:0) ~ TTCGTAATCAATTA CC-AA

=TAATCAATTA CC---------

CLONllH Si R D06 C09.abl (59,492) ••••+-............... .. CCACC--------­

CLONIlH Si R H06 !?OJ.abl (2,,503) ••••+-............... .. CCACC---------

CloneH SlS2 !? C06 022.abl(100,n6) -+ =TJlATCAATTA.~Eiffi~~jCC---------Clone14 SlS2 po H06 017.abl(lOl"SJO) ~ 'I'TCGTAATCAATTA CC---------Clone14 SlS2 R A06 024 .abl (122)~a9) ~ Tl'CGTAATCAATTA CC- - -- - ----

•

Other (“no-hit”) genes are also very interesting to study…

Relative expression

14I 14NI0

50

100

150

200

22I 22NI

ab

c

d

14I 14NI 22I 22NI

CcUNK10

14NI 22I 22NI

b

c

d

14I0

20

30

40

10

50

a

CcUNK1

Genes coding putative protein with “unknown (UNK)” function

Examples of CcUNK1 and CcUNK10highly induced by droughtexpression clone 22 > clone 14

In “a very [simplified] point of view”:genes CcUNK1 and CcUNK10 = gene of “sensibility” to drought?molecular marker of drought S ?

•

Leaf transcriptomic

Meristem transcriptomic

Roots transcriptomic

454 sequencing

454 sequencing

e.g: roots 14 (I / NI) and 22 (I / NI)

Coffee WGS Sequencing DNAg 14 and 22

On going work ….and perspectivesModel plant: different clones of C. canephora in greenhouseAnalysis of C. canephora and C. arabica in the field

Embrapa Cerrados experimental fieldsPlanaltina -DF

~ ~ ..

". ............ K

EXEMPLE D’IMAGE

Genetic determinism of coffee drought tolerance

Necessity to integrate all the studiesmolecular analysesphysiological analysesbiochemical analyses

proteomicmetabolomic (e.g MS)

The “Omics” cascade (Dettmers et al, 2007)

e "0 le' C. s'ca e,I

,ha can happen

What' Jr ar' tobe happening

Wha m kesithappen

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Genetic determinism of coffee drought tolerance

Necessity to integrate all the studiesmolecular analysesphysiological analysesbiochemical analyses

proteomicmetabolomic (e.g MS)

Necessity to analyse the coffee genetic diversityCoffee populations/genotypes in the fieldGenetic analyses

Necessity to analyse the genomes (WGS)Identification of new molecular markers (e.g. SNP)

Better understanding of the genetic determinism of drought tolerance

Help (accelerate) the creation of new varieties/cultivars

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Luciana P. FreireFelipe VineckyGabriel S.C. AlvesHumberto J.O. RamosSonia ElbeltNatalia G. VieiraFernanda A. CarneiroPatricia S. SujiiJean C. AlekcevetchVânia A. SilvaFábio M. DaMattaMaria A.G. FerrãoThierry LeroyDavid PotLuiz G.E. Vieira

Gustavo C. RodriguesAntonio F. Guerra Gabriel F. BartholoOmar C. RochaFabien de BellisIngrid G.R. HeimbeckLuciano V. PaivaCarlos Bloch JrJorge A. TaquitaFelipe R. da SilvaPierre MarracciniAlan C. Andrade

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This work was carried out under the project of scientific cooperation Embrapa-Cirad “Genetic determinism of drought tolerance in coffee” (2006-2010, 2011-2014)

Financial supports:Brazilian Coffee R&D ConsortiumFINEPINCT-café (CNPq/FAPEMIG)CiradFrench Ambassy in BrazilFundação Araucária

Marraccini et al., 2011 BMC Plant Biol.Marraccini et al., 2012 J. Exp. Bot.

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Caracterização molecular de determinantes envolvidos na tolerância à seca

Pierre MarracciniE mail 1 : marraccini@cirad.frE mail 2 : pierrem@cenargen.embrapa.br

Thanks for your attention