Transcript Regulation of flavor and texture in apple fruit genetically

Regulation of flavor and texture in apple fruit genetically
modified for ethylene biosynthesis
Bruno G. Defilippi, Gianni Teo, Sandra L. Uratsu, Adel A. Kader and
Abhaya M. Dandekar
Department of Pomology, University of California, Davis, CA 95616
INTRODUCTION
A salient genetic attribute of tree fruits is the unique blend of sugar, acid and volatile components that determine their flavor, a hallmark of the quality of each kind of
fruit (e.g., apple, peach, orange). 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 (FFC) a key to fruit consumption. In spite of its significance, very little is known at the molecular genetic level of the genes
and pathways that are responsible for the synthesis, accumulation and regulation of FFC. FFC is key to marketing fresh fruits and their products as it greatly affects
consumer preferences, which in turn, impact on the livelihood of fruit growers, and indirectly on the nutritional quality of consumer diets. Fruit that are silenced for
ethylene biosynthesis described here show a unique phenotype that can be used to investigate FFC as it affects the volatile flavor component of FFC without having
an effect on the sugar-acid component. These fruit show a distinct phenotype in their texture and shelf-life. In the future ethylene suppressed apple fruit such as
reported here can be used to study the channeling and regulation of metabolic pathways that lead to the manifestation of a complex trait like fruit quality. This new
knowledge could lead to the development of more precise diagnostics for quality control leading to a more consistent and high quality fruit for the consumer.
OBJECTIVES
The goal this year was to evaluate the role of ethylene in regulating fruit quality (the complex relationship
between sugar, acid, texture and volatile components) using transgenic apple fruit modified in their capacity to
synthesize endogenous ethylene.
RESULTS
Table 1. Ethylene biosynthesis of transgenic apples as a percentage of that
observed for the untransformed lines*
Line
ACS
activity
ACC
concentration
ACO
activity
EXPERIMENTAL STRATEGY
Figure
1.
The
Agrobacterium
binary
vectors
pDU93.0114/pDU93.0128 for sense/antisense expression of apple
ACC synthase (ACS) and pDU93.0412/pDU93.0402 for
sense/antisense expression of apple ACC oxidase (ACO)
respectively in apple.
Methionine
Ethylene
production rate
SAM
ACS
GS
100
100
100
100
ACC
(control)
190 B
35
60
120
40
55 G
68
nd
1.7
8
80 G
72
1,200
0.8
6
68 G
62
3,200
0.4
4
130 Y
10
12
158
Silencing (sense,
antisense)
ACO
Ethylene
6
2Values
of control: ACS activity = 2.5 nmolmg-1(protein) h-1, ACC concentration = 1.6
nmoles ACC g-1 (tissue), ACO activity= 205nLC2H4 mg-1(protein)h-1, ethylene
production=73 uL C2H4 kg-1h-1
nd – Not determined
Table 2. Content of major volatile compounds in Greensleeves fruits derived
from different lines. Fruits were evaluated after 12 days at 20°C
Compound (nL L-1)
GS
68G
130Y
Aldehydes
273 ± 47
398 ± 23
320 ± 35
Hexanal
420 ± 30
290 ± 40
300 ± 30
(E) 2-Hexenal
Alcohols
12 ± 2
3± 1
8± 1
Butanol
6± 2
Methyl 2-butanol
NP
NP
74 ± 7
54 ± 7
73 ± 6
Hexanol
Esters
55 ± 5
45 ± 4
36 ± 5
Butyl butanoate
40 ± 7
8±2
7±2
Butyl 2-methylbutanoate
12 ± 2
Hexyl acetate
NP
NP
41 ± 6
20 ± 4
Hexyl propanoate
NP
340 ± 20
100 ± 25
98 ± 9
Hexyl butanoate
85 ± 17
34 ± 9
40 ± 13
Alfa-farnesene
Figure 2. Northern analysis of transgenic lines
suppressed for ethylene biosynthesis.
Table 3. Quality attributes at harvest and after storage for 12 days at 20°C.
Line
Firmness
(Newton)
GS
68 G
130 Y
78  3.91
93  3.2
90  8.2
At Harvest
Soluble solids Hue angle
(°Brix)
15.7  0.9
13.7  0.7
13.9  0.6
104  0.8
108  2.0
107  0.5
are means  SE of 3 replicates of 5 fruits each.
2No differences in titratable acidity were detected.
-Very low ethylene producing transgenic apple lines
have been identified that are suppressed for either
ACS (ACC Synthase) or ACO (ACC Oxidase)
expression.
16.8  0.1
16.1  1.6
17.2  0.4
97  0.8
103  1.0
105  0.7
-To determine the role of ethylene in texture
development
and
accumulation
of
carbohydrates and acids in transgenic apple
fruit silenced for ethylene biosynthesis.
-ACS/ACO transgenic fruits that make very low
ethylene are significantly suppressed in their
capacity to make volatile esters.
-To understand the role of ethylene in overall
flavor in apples, including sugars, organic
acids and phenolic compounds.
-Color changes in transgenic lines is delayed
relative to control fruit.
a
FUTURE WORK
-To determine the role of ethylene in the
pattern
of
volatile
components
and
biosynthesis in transgenic apple fruit silenced
for ethylene biosynthesis.
-ACS/ACO suppressed fruits are firmer than the
controls but show no significant differences In
soluble solid content or acid accumulation.
show
50  2.6
82  6.5
78  5.5
1Values
SUMMARY
-Ethylene suppressed lines
reduction in alfa-farnesene.
After 12 days at 20°C
Firmness Soluble solids
Hue
(°Brix)
(Newton)
angle
marked
Figure 4. a) three year old transgenic apple tree of the line 80G. b) ACO silenced apple
1 month after storage at room temperature. c) wild type GS apple 1 month after
storage. d) ACO silenced apple 3 months after storage at room temperature and e) wild
type 3 months after storage at room temperature.