1. DIFFERENTIAL EXPRESSION LEVELS OF AROMA BIOSYNTHETIC GENES DURING RIPENING OF APRICOT (Prunus armeniaca L.) Defilippi, B.G. 1,*, González-Agüero, M. 1, Troncoso, S. 2, Gudenschwager, O. 1, Valdés, H. 1, Moya-León, M.A 3. and Campos-Vargas, R. 1 (*bdefilip@inia.cl) 1 Laboratorio de Postcosecha, Instituto de Investigaciones Agropecuarias, CRI La Platina. 2 Facultad de Química y Biología, U. de Santiago de Chile. 3 Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Chile Experimental design Results Conclusions One of the most important limiting factors in apricot quality is the loss of flavor after harvest, especially during long term storage. Flavor in 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. Despite the importance of aroma in fruit quality, limited information is available 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 a decrease in aldehydes (i.e. hexanal and (E)-2-hexenal) and alcohols (i.e. 1-hexanol), and an increase in esters. Further studies are being performed in terms of characterizing gene expression levels under different environmental conditions during storage. These studies will contribute to understand overall aroma development during apricot ripening. Apricot cv. Modesto 4 maturity stages Evaluation of quality attributes Analyzed genes: aat, adh, lox, pdc Search of ortholog sequences Full length coding sequences (RACE-PCR) Primers design for qPCR Gene expression analyses of adh, lox, pdc and aat Real Time PCR (qPCR) RNA extraction, cDNA synthesis 1. Characterization of maturity stages: Parameters analyzed during maturity and ripening of apricots (cv. Modesto) included: fruit firmness, total soluble solids (TSS), titratable acidity (TA), ethylene and CO 2 (respiration) production rates. After evaluation we identified 4 maturity stages: Maturity stages Weight (g) Firmness (Kg-f) TSS (%) TA (% malic acid) Ethylene (µL C 2 H 4 kg -1 h -1 ) CO 2 (mL CO 2 kg -1 h -1 ) M1 31.2 c2.9 a10.1 c2.2 a0.0 b60.2 b M2 40.5 b1.9 b14.9 b1.9 a0.0 b70.1 a M3 45.1 a2.0 b16.9 b1.5 b1.4 b58.1 b M4 46.2 a0.4 c21.3 a0.8 c29.5 a55.3 b 2. Identification and quantification of volatiles: six key aroma volatile compounds were identified by using GC-MS. Quantification was performed by GC considering standards for each compound. 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. (A)(B) 4. Gene expression analyses for aat, adh, lox and pdc within maturity stages: Expression patterns for the four transcripts were characterized by qPCR in fruit from 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 Alcohol AAT Esters - Cte - + Up-regulated expression gene Non-changes in gene expression - + Change in volatile levels Changes detected between ripening stages * Bars followed by different small letters are significantly different at p<0,05 * Different letters represent significant differences at P < 0.05 by LSD test. Lipids Fatty acids (linoleic, linolenic) β-oxidationLipoxigenase Acyl-CoAs Butyl esters Hexanal Hexenal Hexanol LOX adh loxpdc aat % of Maximum M1 M2 M3 M4 Maturity stages M1 M2 M3 M4 Concentration (ng Kg -1 ) M1 M2 M3 M4 Maturity stages M1 M2 M3 M4 hexanal (E)-2-hexenal hexyl acetate linalool 1-hexanol ethyl octanoate adh lox pdc aat