Transcript Harvest 2 4 7 1 - Platina

INHIBITION OF ETHYLENE IN APRICOT FRUIT: ACTION VERSUS
BIOSYNTHESIS
1,2
M .,
González-Agüero,
and Defilippi, B.G.1,2*.
Rubio,
3
P. ,
Pizarro,
3
M. ,
1
O .Campos-Vargas,
Gudenschwager,
1,2
R.
1
Instituto de Investigaciones Agropecuarias, INIA-La Platina. Casilla 439-3, Santiago
2 The Plant Cell Biotechnology Millennium Nucleus (PCB-MN)
3 Facultad de Ciencias Agronómicas, Universidad de Chile
Apricot (Prunus armeniaca L.) fruit is highly susceptible to flesh softening and loss of flavor, particularly during postharvest storage. Most of these changes are under
ethylene regulation. During the last two seasons we have studied the effect of 1-methylcyclopropene and aminoethoxyvinylglycine (an ethylene action and an
ethylene synthesis inhibitor, respectively) on quality attributes of Patterson apricot cultivar. Both ethylene inhibitors have been effective in reducing ethylene
production, fruit softening and color development. On the other hand, soluble solids concentration and titratable acidity have shown an ethylene-independent pattern.
In order to understand the effect of both inhibitors in the ethylene biosynthetic pathway, we identified, cloned and characterized the expression pattern of the key
genes involved in ethylene synthesis and perception (ACS, ACO, ETR and ERS). The expression profile was characterized by qPCR as ripening progressed at 20
°C after harvest. The significance of the changes measured during apricot ripening is discussed.
Experimental design
Apricot cv. Patterson
Genes analyzed: acs,
aco, etr and ers
Treatments *
20 °C
Season 1
Season 2
Evaluation of
quality attributes
- Firmness
- Ethylene
A
Search of ortholog
sequences
Evaluation of
quality attributes
Full length coding
sequences (RACE-PCR)
RNA extraction,
cDNA synthesis
Primers design for
qPCR
B
Methionine
AVG
ACC synthase 1,2 y 3 (ACS)
Search of
conserved
motifs
ACC
ACC oxidase (ACO)
Gene expression analyses of acs, aco, etr and ers
Primers
design
RACE-PCR
1-MCP
Real Time PCR (qPCR)
X
Hydrophobic
domain
Cys-4
Receptors (ETR, ERS)
* Treatments performed in season 1:
* Treatments performed in season 2:
T2
1000 nL L-1 1-MCP (SmartFresh™)
C
T3
10000 nL L-1 1-MCP (SmartFresh™)
1-MCP 10000 nL L-1 1-MCP (SmartFresh™)
T4
100 mg L-1 AVG (Retain® 15% p/p)
AVG
T5
1000 mg L-1 AVG (Retain® 15% p/p)
ACS2
ACS1
acs1
a
120
a
a
a
kg-f
% of Maximum
c
a ab ab b b
bc c
30
30
5
a a a
COSECHA
Harvest
30
30++ 1
momento de evaluación
a b a
b
DIA 4
2
a
ab
a
ab a
b
b
c bc
b
b
b
c
b
4
2020++ 4
30
30
30 ++ 11
30
TESTIGO
AVG
a
1 MCP
b
DIA
4 4
angle
Hue
Hue value
bc
d
b
cd
a a ab
a
b
CONSUMO
71
a
80
a a
a
a a
c
abc
bc
ab a
a a a
10000 nL/L 1-MCP
70
100 mg/L AVG
1000 mg/L AVG
65
60
20
20
2020+ +44
3030
momento de evaluación
Evaluation time (days)
a
30++11
30
DIA
22
20
b
a
a
a
a
80
2
a
b
a
b
a
20
0
DIA 4
4
Momento de evaluación
71
CONSUMO
Evaluation time (days)
b
CONSUMO
71
a
a
80
TESTIGO
60
AVG
40
1 MCP
20
T
a
a
a
a a
a
0
COSECHA
Harvest
DIA 2
2
DIA 4
4
Momento de evaluación
COSECHA
Harvest
DIA
2 2
DIA
4 4
CONSUMO
71
Momento de evaluación
Evaluation time (days)
1
a
100
60
AVG
a
4
Momento de evaluación
120
a
TESTIGO
a
ers
ERS
a
100
DIA 4
DIA 2
COSECHA
Harvest
a
120
ab
A
0
CONSUMO
71
DIA
44
T
ab
a
etrETR
a
2
40
Momento de evaluación
1 MCP
40
DIA 2
a
AVG
1 MCP
b b
b
CONSUMO
71
Evaluation time (days)
LLEGADA
Harvest
DIA 2
2
DIA 4
4
71
CONSUMO
Momento de evaluación
Evaluation time (days)
* Different letters represent significant differences at P < 0.05 by LSD test.
b
75nL/L 1-MCP
1000
a
COSECHA
Harvest
a
COSECHA
c
Testigo
ab b
71
0
b
a a a
a
60TESTIGO
Conclusions
Momento de evaluación
85
a
20
40
ab
DIA
2 2
a
a
Harvest
b
1000
nL/L 1-MCP
3,00
10000 nL/L 1-MCP
2,00
100 mg/L AVG
1,00
1000 mg/L AVG
0,00
COSECHA
Harvest
momento de evaluación
b b
71
140MCP
a a
60
a
a
Testigo
4,00
a
Momento de evaluación
80
CONSUMO
4
b
aco
ACO
20
5,00
b
84
82
80
78
76
DIA 2
6,00
ab b
b
Momento de evaluación
a
4
100
0
2020++44
2
120
b
80
60
AVG
CONSUMO
DIA 4
DIA 2
COSECHA
Harvest
1 MCP
10
100
0
AVG
1000 mg/L AVG
b b
a
a
a
0
%
(µL C2H4 kg-1 h-1)
(µL C2H4 / k*h)
a b b b b
C
Hue angle
20
Kg
-f
Kg f
uL C2H4/ kg*hr
(µL C2H4 / k*h)
kg - f
a
c
20
a
%
60
a
120
80
TESTIGO
TESTIGO
1000015
nL/L 1-MCP
100 mg/L AVG
20
20
Hue angle
25
1000 nL/L 1-MCP
bc
20
20
74
72
70
68
66
64
30
Testigo
45
30
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
a
a
100
40
35
acs3
120
%
Season 2
ACS3
acs2
80
%
Season 1
75
60
COOH
3. Gene expression analyses performed in season 2 for acs, aco, etr and
ers. Expression patterns for the 6 transcripts were characterized by qPCR in 4
fruits for each treatment. 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.
%
1000 mg L-1 AVG (Retain® 15% p/p)
100
ab
N G1 F G2
Control
1. Evaluation of quality attributes from two seasons trial. Maturity
parameters analyzed included: ethylene production rate (A), fruit
firmness (B) and color (C). For treatments information see above.
105
90
H
Hys-353
Results
a
GAF
Cys-6
Control
120
HK
domain
NH2
T1
B
AF316534 ERS Prunus persica,
complete cds (1,935 bp)
Ethylene (C2H4)
- Etc.
15
0
EF585295 ERS1 Prunus salicina,
complete mRNA (2,470 bp)
SAM
X
- Color
A
AJ890091 ERS1 Prunus domestica,
complete cds (1,929 bp)
Search of ortholog
sequences in other
vegetal species
related
%
0°C
0°C + 20°C
2. Schematic representation of AVG and 1-MCP effect on ethylene
biosynthetic pathway (A). For each gene analyzed we obtained the full length
sequence by RACE-PCR. (B) shows the experimental procedure for cloning of
the etr gene (example) and possible structure of Pa-etr from P. armeniaca.
- Patterson apricots applied with AVG and 1-MCP displayed a lower ethylene
production rate than the non-applied fruit.
- Despite that both inhibitors have a different mode of action, we did not
TESTIGO
AVG observe differences at transcripcional level in the genes studied.
1 MCP
- Only acs2 would be regulated by ethylene, reducing its expression
significantly with 1-MCP and AVG applications. For 1-MCP, the low level of
expression of acs2 could be due to the importance of ethylene action in the
autocatalytic production system of climacteric fruits.
* Different letters represent significant differences at P < 0.05 by LSD test.
Funded by Fondecyt 1060179 and PBCT PSD03
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