Topo conferencia cafe conilon vitoria 2012 v1.5

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  • 1. Caracterização molecular de determinantes TITRE envolvidos na tolerância à secaPierre Marraccini Texte • TexteResearcher CIRAD-UMR AGAP • TexteEmbrapa Genetic Resources and Biotechnology – TexteBrasilia – DF » TexteBrazilE mail 1 : marraccini@cirad.fr TexteE mail 2 : pierrem@cenargen.embrapa.br EXEMPLE D’IMAGE
  • 2. Effects of drought in coffee plants Drought is considered to be the major environmental stress affecting coffee production (da Matta 2004) Moderate drought fruits malformation (↑ defects, ↓ size) ↓ fruit (cup) quality Strong drought leaf shedding plant deathΣ: loss of incomes for coffee producers Coffee regions affected by drought: social, economical, environmental impacts... How to reduce these negative effects?
  • 3. 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 Congo C B Guinéen O Guinea SG1 SG2SG1: region of Kouillou > “Conilon” ? Region with short dry season Berthaud, 1986, Montagnon et al, 1992, Dussert et al, 1999. Region without dry season Understand the genetic determinism of this tolerance to use it breeding programs to create new cultivars and varieties
  • 4. Molecular determinism of droughtT in coffee?: search of candidate genes (CGs) Estudos fisiológicos relacionados a tolerância a seca Fábio Murilo da Matta, UFV - Brazil 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 closure involment of acid abscissic (ABA) regulation pathway drought T: maintenance of photosynthesis genes 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?
  • 5. Molecular determinism of droughtT in coffee?:search of candidate genes (CGs)Hypothesis: the drought T and drought S phenotypes come fromdifferential (quantitative) expression of some important candidategenes (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 expressedbetween drought T and drought S clones of C. canephora submitted tocontrolled (greenhouse) water constraint (irrigated vs. non-irrigated) EXEMPLEWhat methods to isolate CGs?: transcriptomic and proteomic analyses D’IMAGE(electronic-northern, northern-blots, qRT-PCR, macroarray screeening,2D-gel electrophoresis...)Compared analyses at the physiological and molecular levels
  • 6. What is supposed to occur? 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) High expression Low 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
  • 7. What plant material? Clones of C. canephora “conilon” selected by Incaper - drought T clones : 14, 73 and 120 - drought S clone : 22 transferred at UFV and tested in greenhouse + irrigation (ΨPD = -0.2 MPa) = control - irrigation (ΨPD = -3.0 MPa) = drought-stressed physiological analyses molecular analyses (leaves) Drought T Control
  • 8. Physiological analyses Comparison of clones of C. canephora “conilon” droughtT clone 14 vs. droughtS clone 22 RDPWP (rate of decrease of ΨPD): 22 > 14 A (net CO2 assimilation): 14D > 22D stomatal conductance (gS): 14C < 22CEffects 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 mol m-2.s-1), stomatal conductance (gs in mol 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 usedneither in photochemistry nor dissipated thermally (PE) of clones 14 and 22 of C. canephora
  • 9. What data?Brazilian Coffee EST genome project (2002-2004) University Campinas - SP UNICAMP - Lab. LGE http://bioinfo04.ibi.unicamp.br/ free access
  • 10. 1st method: CGs identified by “Electronic northern” 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) LV SH3  Contig 18332: no hits found!
  • 11. 1st method: CGs identified by “Electronic northern” Contig 00355: galactinol synthase Ajuga reptans Contig 00367: cystein proteinase inhibitor Contig 05906: cystein proteinase inhibitor Contig 09158: Acyl-CoA-binding protein Panax ginseng Contig 12922: no hits Contig 13476: metallothionein-like protein Citrus unshiu Contig 15415: mannose 6-phosphate reductase Arabidopsis thaliana Contig 18230: chlorophyll a/b binding protein Lycopersicon esculentum Contig 18232: chlorophyll a/b binding protein Arachis hypogaea Contig 18240: no hits (CcUNK10) Contig 18244: rubisco small subunit Coffea arabica Contig 18297: catalase Gossypium hirsutum Contig 18360: no hit (EST leaves infected by Hemilia vastatrix) Contig 18378: mannose 6-phosphate reductase (NADPH-dependent) Contig 18430: no hits Contig 18470: cystein proteinase inhibitor Contig 18387: “abscisic stress ripening protein” Contig 18332: no hits
  • 12. 1st method: CGs identified by “Electronic northern” Contig 00355: galactinol synthase Ajuga reptans Contig 00367: cystein proteinase inhibitor Contig 05906: cystein proteinase inhibitor Contig 09158: Acyl-CoA-binding protein Panax ginseng Contig 12922: no hits Contig 13476: metallothionein-like protein Citrus unshiu Contig 15415: mannose 6-phosphate reductase Arabidopsis thaliana Contig 18230: chlorophyll a/b binding protein Lycopersicon esculentum Contig 18232: chlorophyll a/b binding protein Arachis hypogaea Contig 18240: no hits (CcUNK10) Contig 18244: rubisco small subunit Coffea arabica Contig 18297: catalase Gossypium hirsutum Contig 18360: no hit (EST leaves infected by Hemilia vastatrix) Contig 18378: mannose 6-phosphate reductase (NADPH-dependent) Contig 18430: no hits Contig 18470: cystein proteinase inhibitor Contig 18387: “abscisic stress ripening protein” Contig 18332: no hits
  • 13. 2nd method: CGs identified by macroarray screeningsAnalysis of genes differentially expressed in leaves of C. canephora“conilon” clones 14 (drought T) and 22 (drought S) Putative protein functions 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 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. .
  • 14. 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)Putative protein functionsCcCA1: carbonic anhydrase
  • 15. 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)Putative protein functionsCcCA1: carbonic anhydraseCcPP2C: type-2C proteinphosphataseCcPSBO: PSII O2 evolving complexCcPSBP: PSII O2 evolving complexCcPSBQ: PSII O2 evolving complexCcHSP1: heat-shock protein Σ all the methods: > 40 candidate genes (CGs) presenting differential expression profiles during drought were identified Some examples are presented…
  • 16. Effects of drought on coffee gene coding for proteins involved in the mechanisms of cell protection (1)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 10 20 20 CcGPP1 c CcHSP1 c CcDH3 b 8 b c Relative expression 15 c 15 6 10 10 4 b 5 5 2 a ab b a a 0 0 0 14I 14NI 22I 22NI 14I 14NI 22I 22NI 14I 14NI 22I 22NIExpression increases with droughtNo differences of expression profiles between the clones 14 and 22  “common responses” of these clones upon drought stress
  • 17. 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 2.0 3 2.0 20 CcCAT1 CcCAT2 CcAPX2 CcAPX1 c b c 1.5 b b 1.5 15Relative expression 2 b b b 1.0 a 1.0 10 a a 1 0.5 0.5 a 5 a a a a 0 0 0 0 14I 14NI 22I 22NI 14I 14NI 22I 22NI 14I 14NI 22I 22NI 14I 14NI 22I 22NI catalase ascorbate peroxidase Within a gene family, expression profiles differed between genes  need to analyse expression of each paralogous (allele) genes Higher expression of CcCAT2 in 14 vs. 22  slight differences of gene expression between the clones 14 and 22 regarding drought stress  Q: relation with drought T vs. drought S?
  • 18. 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? 50 15 CcAPR1 CcMPR1 b 40 Relative expression d 10 c 30 20 5 c 10 a a b a 0 0 14I 14NI 22I 22NI 14I 14NI 22I 22NIHigher expression of CcAPR1 in 14 vs. 22  Important differences of gene expression between the clones 14 and 22 regarding drought stress  Q: relation with drought T vs. drought S?  Need to performed in depth analyses of sugar metabolism (i.e. mannitol and alcohol sugars)
  • 19. Ex: effects of drought of PSII componentsAnalysis of OEC proteins in leaves of C. canephora “conilon” clones14 (drought T) and 22 (drought S) 3.0 3.0 4.0 CcPSBO CcPSBP CcPSBQ 3.0 abc Relative expression 2.0 bcd 2.0 d bc c 2.0 bc 1.0 1.0 b 1.0 a b a b a 0 0 0 14I 14NI 22I 22NI 14I 14NI 22I 22NI 14I 14NI 22I 22NIDrought: ↓ CcPSBO, CcPSBP and CcPSBQ gene expression “common responses” for these genes between drought T and drought S clones regarding to drought stress
  • 20. Effects of drought on coffee gene coding for photosynthesis components CO2 Stomatal conductance HCO3- CO2 Drought leads a reduced gene expression of rbcs1, psbO, psbP, psbQ, chlA/B and CA 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... Adapted from Allen et al. (2011) Trends Plant Sci.
  • 21. Effects of drought on coffee gene coding for photosynthesis components Abscisic acid (ABA) CO2 Stomatal conductance HCO3- CO2 carbonic anhydrase (CA)Higher levels of carbonic anhydrase (CA) in leaves of clone 14 vs. clone 22Litterature: high CA activity involved in the maintenance of photosynthesisunder droughtQ: relation between higher CA and higher A under drought in clone 14 than 22under drought?  Measurements of CA activity Adapted from Allen et al. (2011) Trends Plant Sci.
  • 22. The ABA signaling network… EXEMPLE D’IMAGE Hauser et al. (2011) Curr. Biol.
  • 23. Regulation of ABA network… Three-components system of regulation:  ABA receptors (PIR/PYL/RCAR)  PP2Cs (protein phosphatase s2C) = negative regulators  SnRK2s (SNF1-related protein kinases ) = positive regulators = no stress = stress P P The genes involved Expression of in the reduction of genes involved to drought effects ARE reduce drought NOT expressed effects adapted from Cutler et al. (2010) Ann Rev Plant Biol
  • 24. Regulation of ABA network… Three-components system of regulation:  ABA receptors (PIR/PYL/RCAR)  PP2Cs (protein phosphatase s2C) = negative regulators  SnRK2s (SNF1-related protein kinases ) = positive regulators = no stress = stress P P The genes involved Expression of in the reduction of genes involved to drought effects ARE reduce drought NOT expressed effects adapted from Cutler et al. (2010) Ann Rev Plant Biol
  • 25. What about PP2C in our coffee model?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 3.0 CcPP2C d - decreased under drought Relative expression 2.0 c  “less ABA inhibitor” : easier to activate 1.0 b the ABA pathway under drought a in the clone 14 vs. clone 22 0 14I 14NI 22I 22NI  Q: The differences observed between the clones 14 and 22 for the CcPP2C protein contents could explain the phenotypical differences regarding to drought stress?
  • 26. Transduction pathway of drought (abiotic stress) signal EXEMPLE D’IMAGE Is the transduction pathway of drought signal altered in drought T vs. drought S clones of C. canephora “conilon” ? Shinozaki et Yamaguchi-Shinozaki 2007 J. Exp. Bot. 58: 221-227
  • 27. Expression profiles of CcDREB2 gene 12 CcDREB2 CcDREB2 expression clone 14 > clone 22 9 d expression of CcDREB2 gene very low and poorly 6 induced by drought in the drought S vs. drought T ± clones 3 abc a bc 0 14I 14NI 22I 22NI 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?
  • 28. Other (“no-hit”) genes are also very interesting to study…Genes coding putative protein with “unknown (UNK)” function Hybridation 08 02 07 Sonde # 18240 (10) 32PExamples of CcUNK1 and CcUNK10 Gel Northern 27-1  highly induced by drought 14CcUNK1022 73 120 I 14NI 22I 22NI 14I NI I NI I NI I N  expression clone 22 > clone 14 CcUNK1 200 50 d d 40 Relative expression 150 30 c 100 c 20 50 b 10 b a a 0 0 14I 14NI 22I 22NI 14I 14NI 22I 22NI In “a very [simplified] point of view”:  genes CcUNK1 and CcUNK10 = gene of “sensibility” to drought?  molecular marker of drought S ?
  • 29. On going work ….and perspectivesModel plant: different clones of C. canephora in greenhouseAnalysis of C. canephora and C. arabica in the field Leaf transcriptomic Meristem transcriptomic 454 sequencing Embrapa Cerrados experimental fields Planaltina -DF Roots transcriptomic 454 sequencing e.g: roots 14 (I / NI) and 22 (I / NI) Coffee WGS Sequencing DNAg 14 and 22
  • 30. Genetic determinism of coffee drought toleranceNecessity to integrate all the studies  molecular analyses  physiological analyses  biochemical analyses  proteomic  metabolomic (e.g MS) EXEMPLE D’IMAGE The “Omics” cascade (Dettmers et al, 2007)
  • 31. Genetic determinism of coffee drought toleranceNecessity to integrate all the studies  molecular analyses  physiological analyses  biochemical analyses  proteomic  metabolomic (e.g MS)Necessity to analyse the coffee genetic diversity  Coffee populations/genotypes in the field  Genetic analysesNecessity to analyse the genomes (WGS)  Identification of new molecular markers (e.g. SNP)Better understanding of the genetic determinism of drought toleranceHelp (accelerate) the creation of new varieties/cultivars
  • 32. Luciana P. Freire Gustavo C. Rodrigues TITREFelipe Vinecky Antonio F. GuerraGabriel S.C. Alves Gabriel F. BartholoHumberto J.O. Ramos Omar C. RochaSoniaTexte Elbelt Fabien de BellisNatalia G. Vieira Ingrid G.R. Heimbeck • TexteFernanda A. Carneiro Luciano V. Paiva • TextePatricia S. Sujii Carlos Bloch Jr – TexteJean C. Alekcevetch Jorge A. Taquita » TexteVânia A. Silva Felipe R. da SilvaFábio M. DaMatta Pierre MarracciniMariaTexteFerrão A.G. Alan C. AndradeThierry LeroyDavid PotLuiz G.E. Vieira EXEMPLE D’IMAGE
  • 33. Marraccini et al., 2011 BMC Plant Biol. TITRE Marraccini et al., 2012 J. Exp. Bot.This Textewas carried out under the project of scientific workcooperation Embrapa-Cirad “Genetic determinism of drought • Textetolerance in coffee” (2006-2010, 2011-2014) • Texte – TexteFinancial supports: » Texte Brazilian Coffee R&D Consortium FINEP INCT-café (CNPq/FAPEMIG) Texte Cirad French Ambassy in Brazil Fundação Araucária EXEMPLE D’IMAGE
  • 34. Caracterização molecular de determinantes TITRE envolvidos na tolerância à seca Texte • Texte • TexteThanks for your attention – Texte » TextePierre MarracciniE mail 1 : marraccini@cirad.fr TexteE mail 2 : pierrem@cenargen.embrapa.br EXEMPLE D’IMAGE