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TITRE
Texte• Texte
• Texte– Texte
» Texte
Texte
EXEMPLE D’IMAGE
Caracterização molecular de determinantes envolvidos na tolerância à seca
Pierre Marraccini
Researcher CIRAD-UMR AGAPEmbrapa Genetic Resources and Biotechnology Brasilia – DFBrazil
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
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)
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)
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)
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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
•
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?
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
Wi· t h - -. "pp .nedand I htJPpenlng 1...- _
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