1. 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
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
M M M M4
M M M 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
M M M M4
M M M M4
M M M M4
Maturity stages
* Different letters represent significant differences at P < 0.05 by LSD test.
This work was funded by Fondecyt