1. Introduction
The rate of C
2
H
4
production varies with the type of plant tissue and its stage of development.
In climacteric fruits, C
2
H
4
is produced at different rates based on fruit stage of growth. Such fruit is
characterized by a low rate of C
2
H
4
production during the preclimacteric or unripe stage basal
C
2
H
4
, followed by the climacteric, a sudden in- crease in C
2
H
4
production during fruit ripening, a phenomenon referred to as autocatalytic C
2
H
4
Abeles, 1973. After the climacteric rise, C
2
H
4
production significantly declines during the post- climacteric phase Hoffman and Yang, 1980.
Nonclimacteric fruits, on the other hand, exhibit no increase in C
2
H
4
production during matura- tion and ripening Knee et al., 1977.
Autocatalytic C
2
H
4
production is a common feature of ripening in climacteric fruit, in which
increased synthesis of C
2
H
4
is triggered by exoge- nous C
2
H
4
application Burg and Burg, 1965; Abeles, 1973. Several reports however, have
demonstrated autoinhibition of C
2
H
4
production. McMurchie et al. 1972 reported that C
2
H
4
treat- ment inhibited C
2
H
4
production of banana pulp slices. Similarly, propylene treatment, which ini-
tiated ripening, suppressed C
2
H
4
production in intact green bananas. In the non-ripening stages
of sycamore fig, C
2
H
4
acts as an autoinhibitor of its own production, but this does not occur in the
ripening stages Zeroni et al., 1976. C
2
H
4
autoin- hibition was also noticed in avocado fruit Za-
uberman and Fuchs, 1973, immature tomato locule gel tissue Atta-Aly et al., 2000 and pea
segments Saltveit and Dilley, 1978.
It has been suggested that C
2
H
4
autocatalysis involves increased synthesis of ACC synthase and
the enzyme responsible for the conversion of ACC to C
2
H
4
Riov and Yang 1982; Atta-Aly et al., 2000, whereas autoinhibition involves suppres-
sion of the activity of either both enzymes Riov and Yang, 1982 or only ACC synthase Atta-Aly
et al., 2000. Ethylene, therefore, seems to play a role in regulating its own production Yang and
Hoffman, 1984. Studies involving treatment with exogenous ethylene or propylene have indicated
that fruit response to C
2
H
4
may also serve to distinguish between climacteric and nonclimac-
teric fruits McMurchie et al., 1972. The response of harvested fruit to applied C
2
H
4
depends on various factors, including tissue sensitivity and
stage of maturation, as well as whether or not the fruit is climacteric Biale and Young, 1981.
The objectives of this work, therefore, were to study C
2
H
4
feedback mechanisms autocatalysis and autoinhibition in tomato and strawberry
fruits at different developmental stages; to deter- mine the steps in C
2
H
4
biosynthesis which con- trol C
2
H
4
feedback mechanism; to examine the relation between C
2
H
4
feedback mechanism and the behaviour of both tomato and strawberry,
climacteric and nonclimacteric fruit, respectively; to determine the most suitable stage to induce
fruit ripening with exogenous C
2
H
4
application.
2. Material and methods
2
.
1
. Plant material Full size tomato Lycopersicon esculentum Mill
cv. Sunny. fruits were harvested from a commer- cial field in south Florida at immature IM,
mature-green MG and pink P stages. Blossom- end dark and light-green colours were used to
distinguish between IM and MG stages, respec- tively, since the former has no jelly-like locular
materials in any fruit locules while only one or two locules of the latter developed jelly-like mate-
rials. Strawberry Fragaria X ananassa fruits cv. Chandler, were picked from Gainesville area, FL,
at full size green G, white W and half-coloured HC stages. Tomato and strawberry fruits were
transferred to the laboratory on the same day. Fruits were washed with chlorinated water 3.4
mM NaOCl, sorted, regarding to size and devel- opmental stage, and kept at 15°C and 95 RH
overnight for treatment preparation. Experiments were repeated three times using tomato and straw-
berry fruits from the same sources.
2
.
2
. Tissue sampling Tomato outer pericarp disks were excised from
the equator of the fruit using a stainless steel cork borer at IM, MG and P stages one diskfruit.
Disks were trimmed to remove excess jelly-like locular materials which had developed only in
MG and P fruits. The presence of jelly-like mate- rials was used to distinguish between MG and IM
tomato fruit. Directly after excision, disks were placed epidermal surface down, inside glass tubes
17 ml vol.; 2 cm diam.; one disktube.
Strawberry flesh cylinders were longitudinally excised from fruit central flesh using the cork
borer after removing 0.5 cm from blossom and stem ends to obtain achene-free fleshy cylinders.
This was done to exclude the effect of auxins on C
2
H
4
biosynthesis since it is known that the ach- enes are the main source of auxins in strawberry
fruit Archbold and Dennis, 1985. Excised tissue cylinders were then placed vertically inside the
tubes, which contained 3-mm glass beads at the bottom of each tube to protect the tissue base
from anaerobic conditions. Both fruit tissues were then distributed among chemical solution treat-
ments and exposed thereafter to C
2
H
4
using a gas-flow system as described below.
2
.
3
. Chemical treatments and tissue analysis For each treatment, 100 ml of each solution was
applied to the locular surface of tomato disks or to the vascular tissue of the strawberry flesh cylin-
ders. With the exception of ACC, which was applied 3 days after continuous C
2
H
4
exposure to eliminate wound C
2
H
4
interaction, all solutions were applied immediately after excision. Chemical
solutions were applied to both tomato and straw- berry tissues as described below.
2
.
3
.
1
. Control treatments These tissues were divided into three groups.
The first group was used to measure initial C
2
H
4
and CO
2
production, immediately after excision, with an incubation period of 30 min. This incuba-
tion period was enough for measuring basal C
2
H
4
levels and less than that required for wound C
2
H
4
to be initiated Atta-Aly, 1992. The second and the third groups, however, were continuously ex-
posed to an air flow 9 4.5 mmol l
− 1
C
2
H
4
for 5 days, either for monitoring C
2
H
4
and CO
2
pro- duction 3, 4 and 5 days after excision or for ACC
analysis 4 days after excision. Plant tissue pro- duces a large amount of wound C
2
H
4
which di- minishes within 72 h of excision Atta-Aly et al.,
1987. The gas flow system removed wound C
2
H
4
produced during the duration of the experiment and the first C
2
H
4
analysis, therefore, was carried out after 3 days of excision.
2
.
3
.
2
. In 6i6o estimation of ACC oxidase ACO
acti6ity Tissues were treated with water or 0.5 mM
AVG directly after excision and then exposed to C
2
H
4
for 3 days, when water or 100 mM ACC was added to the tissues 2 h before measuring C
2
H
4
production as an indicator of ACO activity.
2
.
3
.
3
. C
2
H
4
action This was achieved in two different ways as
follows: 1. Tissues were treated with water and then di-
vided into two groups. The first group was exposed to air for 3 days, then transferred to
the 4.5 mmol l
− 1
C
2
H
4
atmosphere for 1 day, then returned to air for another day, while the
second group was exposed to the above atmo- spheres in the opposite order. C
2
H
4
and CO
2
production were analyzed at the time of each atmosphere transfer.
2. STS silver thiosulfate; 0.5 mM was applied to the tissues while water was the control
treatment. After 3 days of gas treatments, C
2
H
4
and CO
2
produced by the tissues were analyzed.
2
.
4
. Ethylene treatments Based on the highest respiratory levels of ex-
cised tomato and strawberry fruit tissues, mea- sured 1 day ahead, an air flow system was
calculated and adjusted to a rate of 3.5 l h
− 1
for supplying normal O
2
levels around the tissue. CO
2
levels in the air flow were checked twice per day and its concentration was always below 0.5
throughout the experiment. The air flow 9 4.5 m
mol l
− 1
100 ml l
− l
C
2
H
4
was passed through water flasks twice before passing through tissue
containers RH 98.
Excised tissues were placed inside the 17-ml volume glass tubes, chemically treated and then
divided into two groups for either air or C
2
H
4
treatment. Each group was placed inside 10-l gas- flow containers. The containers were kept at 20°C
and 95 RH throughout the experiment. Time between tissue excision and gas exposure for each
treatment was less than 30 min.
Since applied C
2
H
4
may emanate during tissue incubation and interfere with the measurement of
endogenous levels, 100 g of tomato and straw- berry fruit tissues, excised at each developmental
stage, were exposed to the air flow 9 4.5 mmol l
− 1
C
2
H
4
for 3 days, thoroughly flushed with C
2
H
4
- free air for 60 s and exposed to the vacuum
procedure described by Saltveit 1982 for measur- ing internal C
2
H
4
concentrations. No significant differences were found between air and C
2
H
4
- treated tissues in internal C
2
H
4
levels at each developmental stage of both fruits. All tissues,
therefore, were thoroughly flushed for 60 s with C
2
H
4
-free air prior to each C
2
H
4
analysis. In a separate experiment, tissue was exposed to
580 mmol l
− 1
propylene gas instead of 4.5 mmol l
− 1
ethylene. C
2
H
4
production by both tomato and strawberry fruit tissues was similar to that
obtained with C
2
H
4
application when the tissue was flushed with C
2
H
4
-free air for 60 s before incubation.
2
.
5
. C
2
H
4
, CO
2
and ACC analysis At each sampling time, the tubes were removed,
thoroughly flushed with C
2
H
4
-free air for 60 s, and then sealed with rubber stoppers. After 30
min of incubation at 20°C, 1-ml gas samples were withdrawn and used for C
2
H
4
and CO
2
measure- ments. A Hewlett Packard gas chromatograph
Model 5080A with FID was used for C
2
H
4
analy- sis, while a Gow Mac Model 60, with TCD Gow
Mac Instrument Co., NJ was used for CO
2
mea- surements. After withdrawing the gas samples the
tubes holding tissues were unsealed and returned to the gas flow containers.
For ACC analysis, fruit tissues were removed after 4 days of continuous exposure, frozen in
liquid nitrogen and kept at − 20°C. Two grams of the frozen tissues were homogenized in 10 ml
0.2 mM trichloroacetic acid TCA Atta-Aly et al., 1987. The mixture was centrifuged at 1000 ×
g for 10 min and the supernatant decanted. Aliquots were assayed for ACC with a modified
version of the procedure used by Lizada and Yang 1979.
2
.
6
. Experimental design and statistical analysis Experiments were designed as factorial arrange-
ments in completely randomized designs with five replicates each consisting of 15 samples. Experi-
ments were repeated three times and data were subjected to combined analysis. Means were ana-
lyzed for statistically significant differences using the LSD test at the 5 level Little and Hills,
1978.
3. Results and discussion
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