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MOLECULAR AND PHYSIOLOGICAL BASES OF AROMA BIOSYNTHESIS IN
APRICOT FRUIT (Prunus armeniaca L.)
Bruno G. Defilippi1*, Mauricio González-Agüero1, Sebastián Troncoso2, Orianne Gudenschwager1,
3
1
Alejandra Moya-León , Reinaldo Campos-Vargas .
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
*[email protected]
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
Apricot cv. Modesto
Genes analyzed: aat,
adh, lox, pdc
Maturity stages
Search of ortholog
sequences
Evaluation of quality
attributes
(A)
Alcohol acyl
transferase (AAT)
Full length coding
sequences (RACE-PCR)
RNA extraction,
cDNA synthesis
Protein
Pyruvate decarboxylase
Alcohol
deshydrogenase (ADH)
(PDC)
Primers design for qPCR
Gene expression analyses of adh, lox, pdc
Lipoxygenase
(LOX)
Real Time PCR (qPCR)
Results
Amino acid
identity (%) a
Name
Size
Organism
Accession number
Pa-AAT
448
Prunus armeniaca
N.A.
----------
Pc-AAT
442
Pyrus communis
AAS48090
58
Md-AAT
459
Malus x domestica
AAS79797
58
Vv-AAT
451
Vitis vinifera
CAO66728
52
Cs-AAT
456
Citrus sinensis
ABW81204
50
Pa-ADH
267
Prunus armeniaca
EU395433
----------
Pm-ADH
267
Prunus mume
BAE48662
99
Vv-ADH
266
Vitis vinifera
CAO49038
74
Cm-ADH
266
Cucumis melo
ABC02082
71
At-ADH
266
Arabidopsis thaliana
AAM65725
57
Pa-PDC
605
Prunus armeniaca
EU395434
----------
Fa-PDC
605
Fragaria x ananassa
AAG13131
91
Lc-PDC
606
Lotus corniculatus
AAO72533
87
At-PDC
603
Arabidopsis thaliana
NP_195752
85
St-PDC
592
Solanum tuberosum
BAC23043
86
Pa-LOX
590 *
Prunus armeniaca
EU439430
----------
Ca-LOX
873
Corylus avellana
CAD10740
75
Vv-LOX
864
Vitis vinifera
CAO17594
72
St-LOX
862
Solanum tuberosum
AAB67865
71
Pd-LOX
862
Prunus dulcis
CAB94852
71
(B)
aat
adh
pdc
lox
1
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:
Firmness
TSS
TA
Ethylene
CO2
(g)
(Kgf)
(%)
(% Malic acid)
(µL C2H4 / k*h)
(mL CO2 /k*h)
31.2 c
M1
2.9 a
40.5 b
M2
1.9 b
45.1 a
M3
M4
46.2
10.1 c
14.9 b
2.0 b
a
0.4
2.2 a
1.9 a
16.9 b
c
21.3
0.0 b
a
0.8
60.2 b
0.0 b
1.5 b
70.1 a
1.4 b
c
29.5
100
58.1 b
a
55.3
b
% of Maximum
Maturity stage
Weight
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.
a
aat
100
75
75
50
50
25
b
0
pdc
100
60
Concentration (ng . Kg -1)
hexanal
a
a
a
40
600
1-hexanol
24
a
a
300
bc
bc
0
hexyl acetate
b
750
12
b
(E)-2-hexenal
a
bc
250
120
a
c
c
0
M1
M2
M3
M4
0
M1
M2
M3
M4
M1
M2
M3
M4
* Bars followed by different small letter
are significantly different at p<0,05
Maturity stages
Conclusions
linalool
a
β-oxidation
Lipids
transamination
Fatty acids
b
40
β-oxidation
M2
M3
M4
Cte
M1
M2
M3
+
Lipoxigenase
Detected volatile compound
level
b
-
M4
Up-regulated expression
gene
Acetaldehyde
-
Alcohol
Acyl-CoAs
Hexanal
Butyl esters
Hexenal
Cte
Non-changes in gene
expression
Hexanol
Maturity stages
Esters
* Different letters represent significant differences at P < 0.05 by LSD test.
LOX
Aldehydes
0
M1
Changes detected between ripening
stages
(linoleic, linolenic)
Pyruvate
c
0
0
Glycolysis
0
b
c
c
b
80
500
6
b
25
a
a
bc
8
20
a
a
16
b
0
18
50
ethyl octanoate
b
a
75
50
900
lox
100
a
75
25
b
b
b
0
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.
a
25
b
b
adh
+
This work was funded by Fondecyt 1060179.