1. % 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 β-oxidation Lipoxygenase PDC Up-regulated expression gene LOX - Aldehydes Cte Acetaldehyde ADH ADH Non-changes in gene expression Cte Acyl-CoAs Butyl esters Hexanal Hexenal Hexanol Alcohol - Gene expression in progress AAT ? ? + Esters Concentration Concentration Concentration 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. *bdefilip@inia.cl 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. Experimental design 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). Genes analyzed: adh, lox, pdc Apricot cv. Modesto Search of ortholog sequences Maturity stages (A) (B) Full length coding sequences (RACE-PCR) Evaluation of quality attributes AF031899 ADH Pyrus communis, partial cds (1,177 bp) Search of ortholog sequences in other vegetal species related AY037946 ADH Prunus cerasus, complete cds (1,107 bp) Primers design for qPCR RNA extraction, cDNA synthesis AB218782 ADH Prunus mume, complete cds (1,119 bp) Search of conserved motifs Gene expression analyses of adh, lox, pdc Real Time PCR (qPCR) Primers design RACE-PCR Results 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. 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: adh lox pdc * Bars followed by different small letter are significantly different at p<0.05 Conclusions 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. Hexanal 2(E) Hexenal Hexyl alcohol Hexyl acetate Linalool Ethyl Octanoate * Bars followed by different small letter are significantly different at p<0.05 This work was funded by Fondecyt 1060179.