Genetic and molecular determinism
of diterpenes metabolism in Coffea spp
Phenotypic diversity
➜ Diterpenes contents were analyzed in
green beans of:
• 3 C. canephora, and 3 C. arabusta
genotypes. For each genotype, 3 clonal replicates were analyzed separately.
• C. congensis, C. sessiliflora, C. liberica var
liberica and C. liberica var dewevrei. For each
species, 3 plants were analyzed separately.
➜ Diterpenes quantification was carried out
according to the method of Dias et al. (2007).
➜ Significant intraspecific (within C. canephora
and C. arabusta) and interspecific variability were observed (Fig. 1).
➜ According to these results analysis of
genotypes representative of the diversity of
C. canephora and C. arabica is on-going.
Genes involved in the cafestol/kahweol
biosynthesis
➜ Information generated by the
Genoma Café project
(http://www.lge.ibi.unicamp.br/cafe/) allowed the full length
sequencing of the genes encoding CPS, KO and KS (Fig. 3).
➜ For CPS and KS, only one isoform
was isolated whereas two isoforms
(KO_C1 and KO_C2) presenting 86% identity at the protein level were
identified for KO.
Expression patterns of CPS, KO
and KS during fruit development
Expression of the genes were analyzed in perisperm (green bars) and endosperm (brown bars) along fruit development in one plant of C. arabica cv Iapar59 by
qPCR (Fig. 4). Among the 5 housekeeping genes tested (Actin2, GAPDH1,
Cyclohylin1, ADP1, Cathepsin1), Actin2 and Cathepsin1 were selected for the normalization of the data according to the results obtained using GeNorm
(Vandesompele et al., 2002). Qbase (http://medgen.ugent.be/qbase) was used to analyze expression evolution:
➜ CPS is highly differentially regulated between tissues and presents an
overexpression in the first stage of perisperm development.
➜ KS expression was stable between tissues and between stages.
➜ The two KO isoforms exhibit slightly different patterns of expression.
Conclusion
References
Inter and intraspecific variability were detected for diterpenes contents. The patterns of accumulation during fruit development were defined suggesting transfers between the perisperm and the endosperm. Finally, CPS seems to be one of the
key enzymes controlling diterpenes accumulation.
Evolution of diterpenes concentrations
during fruit development
➜ Fruits of Coffea arabica cv Iapar59 (3 plants) and Coffea canephora cv RL R2
4-I-6 (3 plants) were harvested every 4 weeks between flowering and complete maturation and immediately stored in liquid nitrogen.
➜ Perisperm, and endosperm of each stage/plant were dissected and analyzed
separately for kahweol and cafestol contents using the method of Dias et al. (2007) (Fig. 2).
➜ Kahweol concentration increases in the perisperm of C. arabica between
60 and 100 DAF. Following this peak, a continuous decrease simultaneous to an increase in endosperm was observed.
➜ Almost the same patterns were observed for cafestol in C. arabica and
C. canephora: rapid increase between 60 and 120 DAF (150 for C. canephora)
followed by a gradual decrease in perisperm accompanied by an accumulation in the endosperm.
CIRAD
French Agricultural Research Centre for InternationalDevelopment CIRAD - August 2008 0.00 2.00 4.00 6.00 8.00 C. ca n_12 6 C. ca n_11 9 C.ca n_19 7 C. a rabus ta_133 8 C. a rabu sta_133 4 C. a rabus ta_130 7 C.co ngen sis C.ses siliflo ra C. lib erica liber ica C.lib erica dewe vrei g/ kg o f d ry w ei gh t Cafestol Kahweol 16 OMC 0 10 20 Pe ri _1 1 Pe ri _1 2 Pe ri _1 Pe ri _2 Pe ri _3 Endo_1 2 Endo _1 Endo _2 Endo _3 Endo_ 4 A Endo_4 B Endo _5 0 10 20 Pe ri _1 1 Pe ri _1 2 Pe ri _1 Pe ri _2 Pe ri _3 Endo_1 2 Endo_ 1 Endo_ 2 Endo_ 3 Endo_4 A Endo_4 B Endo_ 5 0 10 20 30 40 50 60 Pe ri _1 1 Pe ri _1 2 Pe ri _1 Pe ri _2 Pe ri _3 En do _1 2 En do_ 1 En do_ 2 En do_ 3 En do _4 A En do _4 B En do_ 5
CPS
KO_C1
KO_C2
0 10 20 Pe ri _1 1 Pe ri _1 2 Pe ri _1 Pe ri _2 Pe ri _3 En do _1 2 End o_ 1 End o_ 2 End o_ 3 En do _4 A En do _4 B End o_ 5KS
Figure 1. Diterpenes concentrations in a few genetic units of the Coffea genus.
Figure 3. Biosynthesis pathway of cafestol and kahweol.
Perisper
m
Endosper
m
Kahweol (mg/100 g of fresh tissue) Cafestol (mg/100 g of fresh tissue)
150 250 350 450 550 650 40 70 100 130 160 190 220 150 250 350 450 550 650 40 70 100 130 160 190 220 50 100 150 200 250 50 100 150 200 250 300 350
Figure 2. Evolution of kahweol and cafestol concentrations during fruit development (Days After Flowering (DAF) on the X axis) in endosperm and perisperm of C. arabica (green lines) and C. canephora (brown lines).
Figure 4. Evolution of gene expression during fruit development.
Peri = Perisperm, Endo = Endosperm; the numbers correspond to the month of collection.
CPS : Copapyl diPhosphate Synthase KS : ent-Kaurene Synthase
KO : ent-Kaurene Oxydase
Dias R.C.E., Campanha F.G., Pot D., Ferreira L.P., Benassi M.T., 2007. Validação de metodologia cromatográfica para determinação de caveol e cafestol em café torrado
e tecidos de Coffea. Simpósio Latino Americano de Ciência de Alimentos, Novembro 4-7 2007, Campinas, SP, Brasil.
Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric
averaging of multiple internal control genes. Genome Biology [On line], 3 (7),<URL: http://genomebiology.com/2002/3/7/research/0034.1>.
Cafestol
and
kahweol
are two coffee-specific diterpenes.
Although various effects of these compounds have been shown or
hypothesized on human health, little information is currently
available on the genetic and molecular bases of their metabolism.
This work presents data on diterpenes accumulation during fruit
development and analysis of gene expression for three key
enzymes of the diterpenes biosynthesis pathway.
Financial support: Consórcio Brasileiro de Pesquisa e Desenvolvimento do Café and French Ministry of Foreign Affairs.
Pot David1,2, Ferreira Lucia P.2, Rafael Dias C.E.3,
Durand Noël4, Guyot Bernard4, Ramos Juliana2, Perthuis Bernard1,
Sandrin Paula2, Benassi Marta T.3, Marraccini Pierre1,
Perreira Luiz F.P.5, Leroy Thierry1, Vieira Luiz G.E.2
1. CIRAD, UMR DAP, Montpellier, F-34398 France 2. IAPAR, LBI-AMG, Londrina (PR) Brazil
3. UEL, CTA, Londrina, (PR) Brazil
4. CIRAD, UMR Qualisud, Montpellier, F-34398 France 5. Embrapa Café, Londrina (PR) Brazil
