Caracterização molecular de determinantes envolvidos na tolerância à seca

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Caracterização molecular de determinantes

envolvidos na tolerância à seca

Pierre Marraccini

Researcher CIRAD-UMR AGAP

Embrapa Genetic Resources and Biotechnology

Brasilia – DF

Brazil

E mail 1 : marraccini@cirad.fr

Effects of drought in coffee plants

Drought is considered to be the major environmental stress

affecting coffee production (da Matta 2004)

Moderate drought

¾ fruits malformation (↑ defects, ↓ size) ¾ ↓ fruit (cup) quality

Strong drought

¾ leaf shedding

¾ plant death

Σ: loss of incomes for coffee producers

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

How to reduce these negative effects?

•

•

•

•

Analysis of C.canephora diversity for drought tolerance

It exists in C. canephora:

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

Guinéen

C

B

SG1

SG2

O

?

Guinéen

C

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 season Region without dry season

Understand the genetic determinism of this tolerance to use it

breeding programs to create new cultivars and varieties

•

Biochemical / physiological targets ↔ genes

drought T _{associated with antioxidant enzymes (against oxydative stress)}

¾ superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX)

drought T_{: smaller stomatal conductance (g}

s) > rapid stomatal closure

¾ involment of acid abscissic (ABA) regulation pathway drought T_{: maintenance of photosynthesis}

¾ genes coding chlA/B binding-protein, PSII, OEC, PSI and Rubisco

drought T_{: osmotic adjustment (seems to be limited in coffee)}

¾ genes coding sugar and derivative metabolites drought T_{: other mechanisms?}

¾ other genes?

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

Molecular determinism of drought

T

_{in coffee?:}

search of candidate genes (CGs)

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Molecular determinism of drought

T

_{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...)

What is supposed to occur?

High expression

“A very [simplified] point of view” is looking for:

¾

genes of “tolerance”: expression

drought

T

_{> drought}

S

¾

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 ~ R N A translation (RNA->Protein) RObosome Protein

•

RNA _Protein

Clones of

C. canephora “conilon”

¾

selected by

Incaper

- drought

T

_{clones : 14, 73 and 120}

- drought

S

_{clone : 22}

¾

transferred at UFV and tested in greenhouse

+ irrigation (Ψ_PD = -0.2 MPa) = control

- irrigation (Ψ_PD = -3.0 MPa) = drought-stressed

¾

physiological analyses

¾

molecular analyses (leaves)

What plant material?

Drought

T

Control

•

8

Recursos Geneticos e Biotecn%gia

Physiological analyses

Comparison of clones of

C. canephora “conilon” drought

T

clone 14 vs. drought

S

_{clone 22}

¾

RDPWP (rate of decrease of Ψ

_PD

): 22 > 14

¾

A (net CO

₂

assimilation): 14D > 22D

¾

stomatal conductance (g

_S

): 14C < 22C

Effects of the drought on leaf pre-dawn water potential (ΨPDin MPa), rate of decrease of ΨPD(RDPWP in MPa d-1m-2), net CO2

assimilation rate (A inμmol m-2_.s-1_{), stomatal conductance (g}

sinμmol 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) and

Stern–Volmer non-photochemical (qN) quenching coefficients, and the fraction of PPF absorbed in PSII antennae and used neither

in photochemistry nor dissipated thermally (PE) of clones 14 and 22 of C. canephora

•

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

Control IDrought Control IDrought

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

RDPWP 0.67±0.04G 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

What data?

Brazilian Coffee EST genome project (2002-2004)

University Campinas - SP UNICAMP - Lab. LGE

http://bioinfo04.ibi.unicamp.br/

free access

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CAFES DO BRASIL ConJordo BJoslleho de PesquilG

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CAFE Links About coffee

Recti/sosGen~rJcoseBiotecno(ogie

(on50'CIO Bro5,leiro de Puquho

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CAFES DO BRASIL Team Services

CDFFEE GENDME PRDJECT

oma

Cafe

Aj,mnQmbl (\( Environmmal GC'llomo

AEG

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₎

SH3 LV

¾ Contig 18332: no hits found!

1

st

_{method: CGs identified by}

_{“Electronic northern”}

el tl-a clustecs cada 6 79 "re 1:0 R 1 ~ao ao pr Q -b iorec Mostmr BlastHits

Moslt"ar Tradul'3o nos 6 Frames No HitsFOlUld! Niunero de Reads: 33 Lista de Read(s): CGGACGCGTCCGCC ACGCG1CCGCCCACGCGTCCGAAATC.kGTC TTCAA':'ACA:'AAA':'!'T:'CCT1 AGCAAAAATGGGTTCCAAGACAC TTCTT7TCTTTTTCAT:'TCCATGGC7GTAGTTCTAA':'GAT':'ACCT CA AGGTGGCTGC AA TCAGTTGACAAT'I'CC.ri.AGACAGT':G~ CAAATGAGGAAGG;'GAAGCCAAGTACCATGGAGGTGGCTri. GGAG GAGGCCACGGAGGA GCTACGGAGGAGGCCATGGAGGTGGCTACG GAGGAGGACATGGAGGGTACGGGGG7GGCGGCCATGGTGG7TATG G~CATGGCGGrGGCGGCCATGGTGG:TATGGACATGGTGG':l'ATG G~CATGG'!'GGrTA':GACACGGCGGACATGGCGGT C GCCATG GTGGACATCCTGG7GAGGCTGCAGA7GCTCAGCCTCAGAACTAAT C~GCCAGC':"TCT:"GC-:A'I'G'!'CATGATT.AATCATG.AATGC-:TAAA C;;'GGC7TAC7:"TA;.,7AGTATGTACT7TGTAATGTGA7CTc;.A,ATA AG'! C::GGA':'CAG': A::CAT TAAT;"T CAC':CCATGTAC':'TGCT GGCTA7CTAGTTCAATACAAGTATA7GAGTT':GTGA7TTTGTGTC ~ CAOO-XX-SH2-OO6-E 1O-EM CCOO-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 ResultadodaMontagem

CCOO-XX-SHHllO-C1O-EM.F

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

.-Total de Bibliotecas: 2

YIeulI

Bibliotecas:

E."tpressaoRelatiytlem

Rcla~aoao total de Reads ciaBiblioreca

E....press..lo Relativi\ em Rcla~oaos clusters ma.is ex.pressos em cada biblioteca Coutrole dt" BUl5 Categorias do Uoigeoe Escolher Versao dos Clusters

Exponar para0QBOT

lmpenar doQBOT Interface \' eb do Blast Ltstar Bibliotecas Disponiveis Ltstar Resultado do Rearranjo ~OesPessoais TesteExaIOde Fisher

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

¾ Contig 00355: galactinol synthase Ajuga reptans ¾ Contig 00367: cystein proteinase inhibitor

¾ Contig 05906: cystein proteinase inhibitor

¾ Contig 09158: Acyl-CoA-binding protein Panax ginseng ¾ Contig 12922: no hits

¾ Contig 13476: metallothionein-like protein Citrus unshiu

¾ Contig 15415: mannose 6-phosphate reductase Arabidopsis thaliana ¾ Contig 18230: chlorophyll a/b binding protein Lycopersicon esculentum ¾ Contig 18232: chlorophyll a/b binding protein Arachis hypogaea

¾ Contig 18240: no hits (CcUNK10)

¾ Contig 18244: rubisco small subunit Coffea arabica ¾ Contig 18297: catalase Gossypium hirsutum

¾ Contig 18360: no hit (EST leaves infected by Hemilia vastatrix)

¾ Contig 18378: mannose 6-phosphate reductase (NADPH-dependent) ¾ Contig 18430: no hits

¾ Contig 18470: cystein proteinase inhibitor

¾ Contig 18387: “abscisic stress ripening protein” ¾ Contig 18332: no hits

¾ Contig 00355: galactinol synthase Ajuga reptans ¾ Contig 00367: cystein proteinase inhibitor

¾ Contig 05906: cystein proteinase inhibitor

¾ Contig 09158: Acyl-CoA-binding protein Panax ginseng ¾ Contig 12922: no hits

¾ Contig 13476: metallothionein-like protein Citrus unshiu

¾ Contig 15415: mannose 6-phosphate reductase Arabidopsis thaliana

¾ Contig 18230: chlorophyll a/b binding protein Lycopersicon esculentum

¾ Contig 18232: chlorophyll a/b binding protein Arachis hypogaea

¾ Contig 18240: no hits (CcUNK10)

¾ Contig 18244: rubisco small subunit Coffea arabica

¾ Contig 18297: catalase Gossypium hirsutum

¾ Contig 18360: no hit (EST leaves infected by Hemilia vastatrix)

¾ Contig 18378: mannose 6-phosphate reductase (NADPH-dependent) ¾ Contig 18430: no hits

¾ Contig 18470: cystein proteinase inhibitor

¾ Contig 18387: “abscisic stress ripening protein” ¾ Contig 18332: no hits

2

nd

_{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 14NI 22I 22NI

~

:J

Gene 14I CcTRAFl CcPDHl CcUNKB CcDH3 CcEDRl CcHSPl CcMPRl CcUBQ10

3

rd

_{method: CGs identified by 2D-gel electrophoresis}

Analysis of proteins differentially expressed in leaves of C.canephora

“conilon” clones 14 (drought

T

_{) and 22 (drought}

S

₎

CcCA1: carbonic anhydrase Putative protein functions

CcCA1 0.6 D ,41 14NI 0.5 _{D 221}

0

>_'#- 22NI

-

_eo 0.4 g ~ "U 0.3 c: :~ . 0 ~ .~ _0.2 !!: e

0

0

a.. 0.11 0 'Cc-CA1

3

rd

_{method: CGs identified by 2D-gel electrophoresis}

Analysis of proteins differentially expressed in leaves of C.canephora

“conilon” clones 14 (drought

T

_{) and 22 (drought}

S

₎

CcCA1: carbonic anhydrase CcPP2C: type-2C protein phosphatase

CcPSBO: PSII O₂ evolving complex CcPSBP: PSII O₂ evolving complex CcPSBQ: PSII O₂ evolving complex CcHSP1: heat-shock protein

Putative protein functions

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

Some examples are presented…

A Cc:CA1 0.6 D 141 14NI 0.5 _{D 221}

®

0

>'::;!! 22NI e.... 0.4

..

g c.s " 0 0.3 c ;:) .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 14NI 0 5 10 15 20 22I 22NI CcHSP1 ab c b c 14I 14NI 0 5 10 15 20 22I 22NI CcDH3 a b a b

14I 14NI 22I 22NI

0 4 6 8 2 10 a c b c CcGPP1

Glycin-rich proteins (CcGRP1): cell wall, reinforcement, and repair

Heat-shock proteins (CcHSP1): maintenance protein folding

Dehydrins (CcDH3): preventing the denaturation of macromolecules

¾ proteins preventing cellular damages

Expression increases with drought

No differences of expression profiles between the clones 14 and 22

Effects of drought on coffee gene coding for proteins

involved in the mechanisms of cell protection (2)

Catalase (CcCAT1, CcCAT2)

Ascorbate peroxidase (CcAPX1, CcAPX2)

¾ proteins reducing oxidative burst caused by drought

Within a gene family, expression profiles differed between genes ¾ need to analyse expression of each paralogous (allele) genes Higher expression of CcCAT2 in 14 vs. 22

¾ slight differences of gene expression between the clones 14 and 22 regarding drought stress

¾ Q: relation with drought T _{vs. drought} S_?

Relative expression 0.5 1.0 1.5 2.0

14I 14NI 22I 22NI

0 CcCAT1 b a b a 14I 14NI 0 0.5 1.0 1.5 2.0 22NI CcAPX2 22I b a c a

14I 14NI 22I 22NI

0 CcCAT2 3 1 2 a c a b 0 5 10 15 20 CcAPX1

14I 14NI 22I 22NI

a b

a b

catalase ascorbate peroxidase

• I

1---

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)

¾ synthesis of sugars (and derivatives) = osmoprotectors?

Relative  expression 0 10 20 40 50 CcAPR1

14I 14NI 22I 22NI

30 b d a c 14I 14NI 0 5 10 22I 22NI a b a c 15 CcMPR1

Higher expression of CcAPR1 in 14 vs. 22

¾ Important differences of gene expression between the clones 14 and 22

regarding drought stress

¾ Q: relation with drought T _{vs. drought} S_?

¾ Need to performed in depth analyses of sugar metabolism (i.e. mannitol

and alcohol sugars)

Ex: effects of drought of PSII components

Analysis of OEC proteins in leaves of C. canephora “conilon” clones

14 (drought

T

_{) and 22 (drought}

S

₎

Relative expression 14I 14NI 0 1.0 2.0 3.0 22I 22NI CcPSBO bc a bcd b 14I 14NI 0 1.0 2.0 3.0 22I 22NI CcPSBP c a d b 14I 14NI 0 1.0 2.0 3.0 4.0 22NI CcPSBQ 22I bc a abc b

Drought:

¾

↓ CcPSBO, CcPSBP and CcPSBQ gene expression

¾

“common responses” for these genes between drought

T

_and

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 CA chlA/B CO₂ HCO₃- _CO 2 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. canephora no major differences between clones except...

Il:mbryophyte leukaryote)Arabidopsis thaliana

ElElElEl ( B tCP47) PS11 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 photosynthesis under drought

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

under drought? ¾ Measurements of CA activity CO₂ HCO₃- _CO 2 Stomatal conductance carbonic anhydrase (CA)

Abscisic acid (ABA)

I.. L S s _NIADIFl'H

(

~~\

L S ~..,...-Ru'sce I I 0.6 D 141 14N 0.5 _{D 221} > _22N ~ r=r=r=r=J :5!... 0.4 (iD g

~

L ('U it: - 0 c: 0.3

~

~ _H~ ..0 m

l

re::: 0.2 'Q)

e

0.. 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-ABA2

f ~

.

.

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:

¾ ABA receptors (PIR/PYL/RCAR)

¾ PP2Cs (protein phosphatase s2C) = negative regulators

¾ SnRK2s (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

ABA No.ABA

a

Regulation of ABA network…

Three-components system of regulation:

¾ ABA receptors (PIR/PYL/RCAR)

¾ PP2Cs (protein phosphatase s2C) = negative regulators

¾ SnRK2s (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

ABA No.ABA

a

What about PP2C in our coffee model?

Relative

expression

¾ Q: The differences observed between the clones 14 and 22 for the

CcPP2C protein contents could explain the phenotypical differences regarding to drought stress?

14I 14NI 0 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

¾ “less ABA inhibitor” : easier to activate the ABA pathway under drought

in the clone 14 vs. clone 22

CcPP2C 0.6

•

•

D 141 • 14NI

0

0

0.5 _{D 221} ;;:- 1- _{"I • 22NI} ~ .!1,... 0.4 ~ g «l u 0.3 c ~ .0 «l

0

c _0.2

0

'q; e a. 0.1 ol...- .... CcPP2C

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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· IStress Response and Tolelra ce

DREB11JCBF (AP2!ERF) DREB2 (AP2IERF)

\

1

,ER01 NAC AREB/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" CAI NTG), RD22 Gly B'o ic slress and wound1 ng gene f nctio' Cis~ctil 9 e emenls gene expession 'transcription factos Si:gnal transductio

•

Expression profiles of CcDREB2 gene

CcDREB2 expression clone 14 > clone 22

expression of CcDREB2 gene very low and poorly induced by drought in the drought S _{vs. drought} T

clones 14I 14NI 0 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 of clones 14 and 22

Q: sequences differences related with the variation of gene expression observed for the DREB2 gene between clone 14 and 22?

•

•

•

3S0 370 160 350 140 no 320 - -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 30 2S0 270 260

•

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 CCAA

Clone22 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 14NI 0 50 100 150 200 22I 22NI a b c d

14I 14NI 22I 22NI

CcUNK10

14NI 22I 22NI

b c d 14I 0 20 30 40 10 50 a CcUNK1

Genes coding putative protein with “unknown (UNK)” function Examples of CcUNK1 and CcUNK10

¾ highly induced by drought

¾ expression clone 22 > clone 14

In “a very [simplified] point of view”:

¾ genes CcUNK1 and CcUNK10 = gene of “sensibility” to drought? ¾ 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 perspectives

Model plant: different clones of C. canephora in greenhouse

Analysis of C. canephora and C. arabica in the field

Embrapa Cerrados experimental fields Planaltina -DF

~ ~ ..

EXEMPLE

D’IMAGE

Genetic determinism of coffee drought tolerance

Necessity to integrate all the studies

¾ molecular analyses ¾ physiological analyses ¾ biochemical analyses

¾ proteomic

¾ metabolomic (e.g MS)

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

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

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Genetic determinism of coffee drought tolerance

Necessity to integrate all the studies

¾ molecular analyses ¾ physiological analyses ¾ biochemical analyses

¾ proteomic

¾ metabolomic (e.g MS)

Necessity to analyse the coffee genetic diversity

¾ Coffee populations/genotypes in the field ¾ Genetic analyses

Necessity to analyse the genomes (WGS)

¾ 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. Freire

Felipe Vinecky

Gabriel S.C. Alves

Humberto J.O. Ramos

Sonia Elbelt

Natalia G. Vieira

Fernanda A. Carneiro

Patricia S. Sujii

Jean C. Alekcevetch

Vânia A. Silva

Fábio M. DaMatta

Maria A.G. Ferrão

Thierry Leroy

David Pot

Luiz G.E. Vieira

Gustavo C. Rodrigues

Antonio F. Guerra

Gabriel F. Bartholo

Omar C. Rocha

Fabien de Bellis

Ingrid G.R. Heimbeck

Luciano V. Paiva

Carlos Bloch Jr

Jorge A. Taquita

Felipe R. da Silva

Pierre Marraccini

Alan C. Andrade

ad

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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:

¾

Brazilian Coffee R&D Consortium

¾

FINEP

¾

INCT-café (CNPq/FAPEMIG)

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Cirad

¾

French Ambassy in Brazil

¾

Fundação 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 Marraccini

E mail 1 : marraccini@cirad.fr

E mail 2 : pierrem@cenargen.embrapa.br