1 Laboratorio de Postcosecha, Instituto de Investigaciones Agropecuarias, CRI La Platina.
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 (*bdefi[email protected]) 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) Firmnes s (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 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 0.0 b 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 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 Alcoho l AAT Esters - Cte - + 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- oxidation Lipoxigenas e Acyl-CoAs Butyl esters Hexanal Hexenal Hexanol LOX a b b b 0 25 50 75 100 b b b a 0 25 50 75 100 a a a a 0 25 50 75 100 a b b c c 0 25 50 75 100 adh lox pdc aat % of Maximum M1 M2 M3 M4 Maturity stages M1 M2 M3 M4 a a b b 0 8 16 24 a a b b 0 40 80 120 c a b c 0 6 12 18 b b a a 0 20 40 60 c c bc a 0 250 500 750 a b bc bc 0 300 600 900 Concentration (ng Kg - 1 ) M1 M2 M3 M4 Maturity stages M1 M2 M3 M4 M1 M2 M3 M4 hexanal (E)-2-hexenal hexyl acetate linalool 1-hexanol ethyl octanoate adh lox pdc aat Protein N am e Size O rganism A ccession num ber Am ino acid identity (% ) a Pa-AAT 448 Prunus armeniaca N.A. ---------- Pc-AAT 442 Pyrus communis A A S48090 58 M d-A AT 459 Malus x domestica A A S79797 58 V v-A AT 451 Vitis vinifera CA O 66728 52 A lcoholacyl transferase (A AT) Cs-A A T 456 C itrus sinensis A BW 81204 50 Pa-A DH 267 Prunus armeniaca EU 395433 ---------- Pm -ADH 267 Prunus mume BA E48662 99 V v-ADH 266 Vitis vinifera CA O 49038 74 Cm-ADH 266 C ucumis melo A BC02082 71 A lcohol deshydrogenase (A DH) At-A DH 266 Arabidopsis thaliana A A M 65725 57 Pa-PD C 605 Prunus armeniaca EU 395434 ---------- Fa-PD C 605 F ragaria x ananassa A A G 13131 91 Lc-PD C 606 Lotus corniculatus A A O 72533 87 At-PD C 603 Arabidopsis thaliana N P_195752 85 Pyruvate decarboxylase (PD C ) St-PDC 592 Solanum tuberosum BA C23043 86 Pa-LO X 590 * Prunus armeniaca EU 439430 ---------- Ca-LO X 873 Corylus avellana CA D 10740 75 V v-LOX 864 Vitis vinifera CA O 17594 72 St-LOX 862 Solanum tuberosum A A B67865 71 L ipoxygenase (LO X ) Pd-LO X 862 Prunus dulcis CA B94852 71
description
hexanal. ethyl octanoate. 1-hexanol. Glycolysis. β -oxidation transamination. Lipids. Changes detected between ripening stages. Concentration (ng Kg -1 ). Fatty acids (linoleic, linolenic). (E)-2-hexenal. linalool. hexyl acetate. -. Change in volatile levels. β-oxidation. - PowerPoint PPT Presentation
Transcript of 1 Laboratorio de Postcosecha, Instituto de Investigaciones Agropecuarias, CRI La Platina.
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.A3. and Campos-Vargas, R.1 (*[email protected])
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
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 CO2 (respiration) production rates. After evaluation we identified 4 maturity stages:
Maturity stages Weight(g)
Firmness(Kg-f)
TSS(%)
TA(% malic acid)
Ethylene(µL C2H4 kg-1 h-1)
CO2
(mL CO2 kg-1 h-1)
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 0.0 b 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
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
AldehydesAcetaldehyde
ADH
PDC
ADHAlcohol
AATEsters
-Cte
-
+
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-oxidation Lipoxigenase
Acyl-CoAs
Butyl esters
Hexanal
Hexenal
Hexanol
LOX
a
b b b
0
25
50
75
100
b b b
a
0
25
50
75
100
aaa
a
0
25
50
75
100 a
bb c
c0
25
50
75
100
adh
loxpdc
aat
% o
f Max
imum
M1 M2 M3 M4
Maturity stages
M1 M2 M3 M4
aa
bb
0
8
16
24
a a
bb
0
40
80
120
c
a
bc
0
6
12
18
bb
aa
0
20
40
60
ccbc
a
0
250
500
750
a
b
bcbc
0
300
600
900
Con
cent
ratio
n (n
g K
g -1)
M1 M2 M3 M4
Maturity stages
M1 M2 M3 M4 M1 M2 M3 M4
hexanal
(E)-2-hexenalhexyl acetate linalool
1-hexanol ethyl octanoate
adh
lox
pdc
aat
Protein
Name Size Organism Accession number
Amino acid identity (%) a
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 Alc
ohol
acy
l tr
ansf
eras
e (A
AT
)
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
Alc
ohol
de
shyd
roge
nase
(AD
H)
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
Pyru
vate
dec
arbo
xyla
se (P
DC
)
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 Lip
oxyg
enas
e (L
OX
)
Pd-LOX 862 Prunus dulcis CAB94852 71
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