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DIFFERENTIAL EXPRESSION LEVELS OF AROMA BIOSYNTHETIC
GENES DURING RIPENING OF APRICOT (Prunus armeniaca L.)
1,*
B.G. ,
1
M. ,
2
S. ,
Defilippi,
González-Agüero,
Troncoso,
Gudenschwager,
Moya-León, M.A3. and Campos-Vargas, R.1 (*[email protected])
1
O. ,
Valdés,
1
H. ,
1Laboratorio
de Postcosecha, Instituto de Investigaciones Agropecuarias, CRI La Platina.
2Facultad de Química y Biología, U. de Santiago de Chile.
3Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Chile
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.
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
Analyzed genes: aat,
adh, lox, pdc
4 maturity stages
Search of ortholog
sequences
Evaluation of
quality attributes
(A)
Alcohol acyl
transferase (AAT)
Full length coding
sequences (RACE-PCR)
Pyruvate decarboxylase
(PDC)
Alcohol
deshydrogenase (ADH)
Primers design for
qPCR
RNA extraction,
cDNA synthesis
Protein
Gene expression analyses of adh, lox, pdc and aat
Lipoxygenase
(LOX)
Real Time PCR (qPCR)
Results
(B)
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
aat
adh
pdc
lox
1
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.
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 CO2
(respiration) production rates. After evaluation we identified 4 maturity
stages:
Firmness
TSS
TA
Ethylene
CO2
(g)
(Kg-f)
(%)
(% malic acid)
(µL C2H4 kg-1 h-1)
(mL CO2 kg-1 h-1)
M1
31.2 c
M2
40.5 b
M3
45.1 a
M4
46.2 a
2.9 a
10.1 c
1.9 b
2.2 a
14.9 b
2.0 b
1.9 a
16.9 b
0.4 c
0.0 b
21.3 a
70.1 a
1.4 b
0.8 c
aat
58.1 b
29.5 a
75
50
50
25
b
b
25
b
0
0
100
100
pdc
a
75
hexanal
Concentration (ng Kg -1)
a
1-hexanol
a
40
a
bc
bc
75
18
750
hexyl acetate
12
b
b
(E)-2-hexenal
M1
M2
M3
M4
0
Glycolysis
120
transamination
linalool
a
bc
c
c
M4
(linoleic, linolenic)
-
b
β-oxidation
40
M2
M3
M4
Cte
Acetaldehyde
-
M1
M2
M3
Lipoxigenase
+
Change in volatile
levels
b
0
M1
Changes detected between
ripening stages
Fatty acids
-
LOX
Aldehydes
Acyl-CoAs
M3
M4
* Bars followed by different
small letters are significantly
different at p<0,05
β-oxidation
c
M2
M1
Lipids
Pyruvate
0
M1
M3
c
b
b
0
M2
25
0
80
250
c
0
b
Conclusions
a
500
6
a
bc
8
a
a
a
a
16
20
0
25
50
a
b
0
lox
ethyl octanoate
b
300
a
Maturity stages
24
a
600
b
b
b
55.3 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.
60
a
adh
75
50
900
100
60.2 b
0.0 b
1.5 b
a
100
% of Maximum
Maturity stages
Weight
M4
Maturity
stages
* Different letters represent significant differences at P < 0.05 by LSD test.
Alcohol
Butyl esters
Esters
+
Up-regulated
expression gene
Hexanal
Hexenal
Cte
Non-changes in
gene expression
Hexanol
This work was funded by Fondecyt 1060179