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a b bc bc 0 100 200 300 400 500 600 700 800 900 M1 M2 M3 M4 C oncentración (ng/K g) Concentration c a b c 0 2 4 6 8 10 12 14 16 18 M1 M2 M3 M4 C oncentración (ng/K g) Concentration c c bc a 0 100 200 300 400 500 600 700 800 M1 M2 M3 M4 C oncentración (ng/K g) Concentration b b a a 0 20 40 60 80 100 120 M1 M2 M3 M4 C oncentración (ng/K g) Concentration b b a a 0 5 10 15 20 25 30 35 40 45 50 M1 M2 M3 M4 C o n cen tració n (ng/K g) Concentration b b a a 0 5 10 15 20 25 M1 M2 M3 M4 C o n cen tració (ng/K g) Concentration MOLECULAR AND PHYSIOLOGICAL BASES OF AROMA BIOSYNTHESIS IN APRICOT FRUIT (Prunus armeniaca L.) Sebastián Troncoso 2 , Mauricio González-Agüero 1 , Orianne Gudenschwager 1 , Reinaldo Campos-Vargas 1 , Bruno G. Defilippi 1* . 1 Laboratorio de Postcosecha, Instituto de Investigaciones Agropecuarias, CRI La Platina. 2 Facultad de Química y Biología, U. de Santiago de Chile. *bdefi[email protected] Experimental design Results Conclusions A salient genetic attribute of tree fruits is the unique blend of sugar, acid, phenolic and volatile components that determine their flavor. This complex genetic trait is manifested in ripe fruit through a complex interaction of metabolic pathways and regulatory circuits that results in the unique fruit flavor composition, a key to fruit consumption. Loss of flavor, particularly the aroma attribute, is a limiting factor in apricot quality. In spite of its significance, very little is known at the molecular genetic and biochemical level of the genes and pathways that are responsible for the synthesis, accumulation and regulation of volatile compounds. In order to understand the biological basis of aroma biosynthesis, we characterized and differentiated 4 maturity stages in terms of aroma- related volatile compounds, maturity parameters and gene expression levels. We cloned and quantified by qPCR the gene(s) encoding alcohol dehydrogenase (adh), lipoxygenase (LOX) and pyruvate decarboxylase (PDC), key enzymes involved in alcohol and aldehyde synthesis. As ripening progressed, we observed an increase in ADH transcript levels simultaneously with a decrease in aldehydes (i.e. hexanal and 2(E)-hexenal). We think that further studies to be performed, within the Fondecyt project 1060179, in terms of identifying and characterizing these genes in P. armeniaca will contribute to understand overall aroma development during fruit ripening. Apricot cv. Modesto Maturity stages Evaluation of quality attributes Genes analyzed: adh, lox, pdc Search of ortholog sequences Full length coding sequences (RACE- PCR) Primers design for qPCR Gene expression analyses of adh, lox, pdc Real Time PCR (qPCR) RNA extraction, cDNA synthesis 1. Characterization of maturity stages: Maturity parameters analyzed during maturity and ripening of apricots (cv. Modesto) included: fruit firmness, total soluble solids (TSS), titratable acidity (TA), ethylene and CO 2 production rates. After evaluation we identified 4 maturity stages: Maturity stage Weight (g) Firmnes s (Kgf) TSS (%) TA (% Malic acid) Ethylene (µL C 2 H 4 / k*h) CO 2 (mL CO 2 /k*h) M1 31.2 2.9 10.1 2.2 0.0 60.2 M2 40.5 1.9 14.9 1.9 0.0 70.1 M3 45.1 2.0 16.9 1.5 1.4 58.1 M4 46.2 0.4 21.3 0.8 29.5 55.3 2. Identification and quantification of volatiles: six key aroma volatile compounds were identified by using GC-MS. Quantification was performed by GC considering internal standards for each compound. 3. Identification and cloning of adh, lox and pdc genes in P. armeniaca: For each gene analyzed we obtained the full length sequence by RACE-PCR. (A) shows the experimental procedure for cloning of the adh gene. (B) shows the aminoacidic sequence annealing for adh (267 aa) of P. armeniaca and orthologs from other species (Prunus mume, Cucumis melo, Arabidopsis thaliana). Search of ortholog sequences in other vegetal species related Primers design RACE-PCR Search of conserved motifs AF031899 ADH Pyrus communis, partial cds (1,177 bp) AY037946 ADH Prunus cerasus, complete cds (1,107 bp) AB218782 ADH Prunus mume, complete cds (1,119 bp) (A) (B) 4. Gene expression analyses for adh, lox and pdc within maturity stages: Expression patterns for the three transcripts were characterized by qPCR in 4 fruits for each maturity stage (M1 to M4). Amplification assays were performed three times. Gene expression was normalized considering an external control (Gene dap from Bacillus subtilis), and expressed as a percentage of the highest value of relative abundance. This work was funded by Fondecyt 1060179. Glycolysis β-oxidation transamination Pyruvate Aldehydes Acetaldehyde ADH PDC ADH Alcoho l AAT Esters - Cte - ? + Cte Up-regulated expression gene Non-changes in gene expression ? Gene expression in progress - + Detected volatile compound level Changes detected between maturity/ripening stages * Bars followed by different small letter are significantly different at p Hexanal 2(E) Hexenal Hexyl acetate Linalool Hexyl alcohol Ethyl Octanoate * Bars followed by different small letter are significantly different at p<0.05 b b b a 0 30 60 90 120 M1 M2 M3 M4 % d el m áxim o % of Maximum a b bc c 0 30 60 90 120 M1 M2 M3 M4 % d el m áxim o % of Maximum a a a a 0 30 60 90 120 M1 M2 M3 M4 % d el m áxim o % of Maximum adh lox pdc Lipids Fatty acids (linoleic, linolenic) β- oxidation Lipoxygenas e Acyl-CoAs Butyl esters Hexanal Hexenal Hexanol LOX
description
% of Maximum. % of Maximum. % of Maximum. Glycolysis. Changes detected between maturity/ripening stages. β -oxidation transamination. Lipids. Fatty acids (linoleic, linolenic). -. Detected volatile compound level. Concentration. Concentration. Concentration. +. Pyruvate. - PowerPoint PPT Presentation
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MOLECULAR AND PHYSIOLOGICAL BASES OF AROMA BIOSYNTHESIS IN APRICOT FRUIT (Prunus armeniaca L.)
Sebastián Troncoso2, Mauricio González-Agüero1, Orianne Gudenschwager1, Reinaldo Campos-Vargas1, Bruno G. Defilippi1*.
1Laboratorio de Postcosecha, Instituto de Investigaciones Agropecuarias, CRI La Platina.2Facultad de Química y Biología, U. de Santiago de Chile. *[email protected]
Experimental design
Results
Conclusions
A salient genetic attribute of tree fruits is the unique blend of sugar, acid, phenolic and volatile components that determine their flavor. This complex genetic trait is manifested in ripe fruit through a complex interaction of metabolic pathways and regulatory circuits that results in the unique fruit flavor composition, a key to fruit consumption. Loss of flavor, particularly the aroma attribute, is a limiting factor in apricot quality. In spite of its significance, very little is known at the molecular genetic and biochemical level of the genes and pathways that are responsible for the synthesis, accumulation and regulation of volatile compounds. In order to understand the biological basis of aroma biosynthesis, we characterized and differentiated 4 maturity stages in terms of aroma-related volatile compounds, maturity parameters and gene expression levels. We cloned and quantified by qPCR the gene(s) encoding alcohol dehydrogenase (adh), lipoxygenase (LOX) and pyruvate decarboxylase (PDC), key enzymes involved in alcohol and aldehyde synthesis. As ripening progressed, we observed an increase in ADH transcript levels simultaneously with a decrease in aldehydes (i.e. hexanal and 2(E)-hexenal). We think that further studies to be performed, within the Fondecyt project 1060179, in terms of identifying and characterizing these genes in P. armeniaca will contribute to understand overall aroma development during fruit ripening.
Apricot cv. Modesto
Maturity stages
Evaluation of quality attributes
Genes analyzed: adh, lox, pdc
Search of ortholog sequences
Full length coding sequences (RACE-PCR)
Primers design for qPCR
Gene expression analyses of adh, lox, pdc
Real Time PCR (qPCR)
RNA extraction, cDNA synthesis
1. Characterization of maturity stages: Maturity parameters analyzed during maturity and ripening of apricots (cv. Modesto) included: fruit firmness, total soluble solids (TSS), titratable acidity (TA), ethylene and CO2 production rates. After evaluation we identified 4 maturity stages:
Maturity stageWeight
(g)
Firmness(Kgf)
TSS(%)
TA(% Malic acid)
Ethylene(µL C2H4 / k*h)
CO2
(mL CO2 /k*h)
M1 31.2 2.9 10.1 2.2 0.0 60.2
M2 40.5 1.9 14.9 1.9 0.0 70.1
M3 45.1 2.0 16.9 1.5 1.4 58.1
M4 46.2 0.4 21.3 0.8 29.5 55.3
2. Identification and quantification of volatiles: six key aroma volatile compounds were identified by using GC-MS. Quantification was performed by GC considering internal standards for each compound.
3. Identification and cloning of adh, lox and pdc genes in P. armeniaca: For each gene analyzed we obtained the full length sequence by RACE-PCR. (A) shows the experimental procedure for cloning of the adh gene. (B) shows the aminoacidic sequence annealing for adh (267 aa) of P. armeniaca and orthologs from other species (Prunus mume, Cucumis melo, Arabidopsis thaliana).
Search of ortholog sequences in other
vegetal species related
Primers design
RACE-PCR
Search of conserved
motifs
AF031899 ADH Pyrus communis, partial cds (1,177 bp)
AY037946 ADH Prunus cerasus, complete cds (1,107 bp)
AB218782 ADH Prunus mume, complete cds (1,119 bp)
(A) (B)
4. Gene expression analyses for adh, lox and pdc within maturity stages: Expression patterns for the three transcripts were characterized by qPCR in 4 fruits for each maturity stage (M1 to M4). Amplification assays were performed three times. Gene expression was normalized considering an external control (Gene dap from Bacillus subtilis), and expressed as a percentage of the highest value of relative abundance.
This work was funded by Fondecyt 1060179.
Glycolysis β-oxidation
transamination
Pyruvate
AldehydesAcetaldehyde
ADH
PDC
ADHAlcohol
AATEsters
-Cte
-
? +
Cte
Up-regulated expression gene
Non-changes in gene expression
?Gene expression in progress
-
+
Detected volatile compound level
Changes detected between maturity/ripening stages
* Bars followed by different small letter are significantly different at p<0.05
Hexanal 2(E) Hexenal
Hexyl acetate Linalool
Hexyl alcohol
Ethyl Octanoate
* Bars followed by different small letter are significantly different at p<0.05
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