1. Caracterização molecular de determinantes
TITRE envolvidos na tolerância à seca
Pierre Marraccini
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Researcher CIRAD-UMR AGAP
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Embrapa Genetic Resources and Biotechnology
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Brasilia – DF
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Brazil
E mail 1 : marraccini@cirad.fr
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E mail 2 : pierrem@cenargen.embrapa.br
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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 death
Σ: 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 SG2
SG1: 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 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)
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What methods to isolate CGs?: transcriptomic and proteomic analyses
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(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 < 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 CO2
assimilation 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 used
neither 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 screenings
Analysis 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 electrophoresis
Analysis of proteins differentially expressed in leaves of C.canephora
“conilon” clones 14 (drought T) and 22 (drought S)
Putative protein functions
CcCA1: carbonic anhydrase

15. 3rd 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)
Putative protein functions
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
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 repair
Heat-shock proteins (CcHSP1): maintenance protein folding
Dehydrins (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 22NI
Expression increases with drought
No 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 15
Relative 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 22NI
Higher 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 components
Analysis of OEC proteins in leaves of C. canephora “conilon” clones
14 (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 22NI
Drought:
 ↓ 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 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?
 Measurements of CA activity
Adapted from Allen et al. (2011) Trends Plant Sci.

22. The ABA signaling network…
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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
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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) 32P
Examples 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 perspectives
Model plant: different clones of C. canephora in greenhouse
Analysis 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 tolerance
Necessity to integrate all the studies
 molecular analyses
 physiological analyses
 biochemical analyses
 proteomic
 metabolomic (e.g MS)
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The “Omics” cascade (Dettmers et al, 2007)

31. Genetic determinism of coffee drought tolerance
Necessity 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 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

32. Luciana P. Freire Gustavo C. Rodrigues
TITRE
Felipe Vinecky Antonio F. Guerra
Gabriel S.C. Alves Gabriel F. Bartholo
Humberto J.O. Ramos Omar C. Rocha
SoniaTexte
Elbelt Fabien de Bellis
Natalia G. Vieira Ingrid G.R. Heimbeck
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Fernanda A. Carneiro Luciano V. Paiva
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Patricia S. Sujii Carlos Bloch Jr
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Jean C. Alekcevetch Jorge A. Taquita
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Vânia A. Silva Felipe R. da Silva
Fábio M. DaMatta Pierre Marraccini
MariaTexteFerrão
A.G. Alan C. Andrade
Thierry Leroy
David Pot
Luiz G.E. Vieira EXEMPLE
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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
work
cooperation Embrapa-Cirad “Genetic determinism of drought
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tolerance in coffee” (2006-2010, 2011-2014)
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Financial supports:
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 Brazilian Coffee R&D Consortium
 FINEP
 INCT-café (CNPq/FAPEMIG)
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 Cirad
 French Ambassy in Brazil
 Fundação Araucária EXEMPLE
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34. Caracterização molecular de determinantes
TITRE envolvidos na tolerância à seca
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• TexteThanks for your attention
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Pierre Marraccini
E mail 1 : marraccini@cirad.fr
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E mail 2 : pierrem@cenargen.embrapa.br
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