MOLECULAR AND PHYSIOLOGICAL BASES OF AROMA BIOSYNTHESIS IN APRICOT FRUIT (Prunus armeniaca L.) Bruno G. Defilippi1*, Mauricio González-Agüero1, Sebastián Troncoso2, Orianne Gudenschwager1, Alejandra Moya-León3, Reinaldo Campos-Vargas1. 1 Laboratorio de Postcosecha, Instituto de Investigaciones Agropecuarias, CRI La Platina. 2 Facultad de Química y Biología, U. de Santiago de Chile. 3 Laboratorio de Fisiología Vegetal, IBVB, Universidad de Talca, 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 four stages in terms of maturity parameters, aroma-related volatile compounds, and gene expression levels. We cloned and quantified by qPCR the genes encoding: alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), lipoxygenase (LOX) and pyruvate decarboxylase (PDC), key enzymes involved in alcohol, aldehyde and ester synthesis. As fruit ripening progressed, we observed an increase in adh and aat transcript levels simultaneously with an increase in esters (hexyl acetate) and a decrease in aldehydes (i.e. hexanal and (E)-2-hexenal) and alcohols (i.e. 1-hexanol). We think that further studies to be performed in terms of identifying and characterizing these genes in P. armeniaca will contribute to understand overall aroma development during fruit ripening. 3. Identification, cloning and characterization of aat, adh, lox and pdc genes in P. armeniaca: For each gene analyzed we obtained the full length sequence by RACE-PCR. (A) Amino acid sequence comparison between the peptides of the four aroma related genes with proteins from others species. (B) shows the schematic representation of predicted structure and the multiple alignment with closely related sequences using a Clustal software and manually alignment of selected motifs of each protein. Experimental design Genes analyzed: aat, adh, lox, pdc Apricot cv. Modesto Maturity stages Search of ortholog sequences aat Evaluation of quality attributes (A) (B) Full length coding sequences (RACE-PCR) RNA extraction, cDNA synthesis Primers design for qPCR adh Gene expression analyses of adh, lox, pdc Real Time PCR (qPCR) pdc lox Results 4. Gene expression analyses for aat, adh, lox and pdc within maturity stages: Expression patterns for the four 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: Maturity stage Weight (g) Firmness (Kgf) TSS (%) TA (% Malic acid) Ethylene (µL C2H4 / k*h) CO2 (mL CO2 /k*h) M1 31.2 c 2.9 a 10.1 c 2.2 a 0.0 b 60.2 b M2 40.5 b 1.9 b 14.9 b 1.9 a 70.1 a M3 45.1 a 2.0 b 16.9 b 1.5 b 1.4 b 58.1 b M4 46.2 a 0.4 c 21.3 a 0.8 c 29.5 a 55.3 b aat adh % of Maximum pdc lox 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. * Bars followed by different small letter are significantly different at p<0,05 M1 M2 M3 M4 M1 M2 M3 M4 Maturity stages hexanal 1-hexanol ethyl octanoate Conclusions Glycolysis β-oxidation transamination Pyruvate Aldehydes Acetaldehyde ADH PDC Alcohol AAT Esters - Cte + Lipids Fatty acids (linoleic, linolenic) β-oxidation Lipoxigenase Acyl-CoAs Butyl esters Hexanal Hexenal Hexanol LOX Cte Up-regulated expression gene Non-changes in gene expression - + Detected volatile compound level Changes detected between ripening stages Concentration (ng . Kg -1) hexyl acetate (E)-2-hexenal linalool M1 M2 M3 M4 M1 M2 M3 M4 M1 M2 M3 M4 Maturity stages * Different letters represent significant differences at P < 0.05 by LSD test. This work was funded by Fondecyt 1060179.