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Ethylene feedback mechanisms in tomato and strawberry

fruit tissues in relation to fruit ripening and climacteric

patterns

Mordy A. Atta-Aly *, Jeffrey K. Brecht, Donald J. Huber

Horticultural Sciences Department,Uni6ersity of Florida,Gaines6ille,FL32611-0690,USA

Received 16 August 1999; accepted 18 May 2000

Abstract

Exposing pericarp tissue excised from immature tomato fruit to 4.5 mmol l−1 C₂H₄ revealed a negative C₂H₄

feedback mechanism in relation to its biosynthesis since ACC concentration and C2H4production by the tissue were

reduced. An opposite trend (positive C2H4feedback mechanism) was observed in pericarp tissue excised from fruit at

the pink stage. At the mature-green stage however, tissue showed a transition from negative to positive C2H4feedback

mechanism with the onset of tissue ripening. In strawberry tissues excised from green, white and half-coloured fruits however, C2H4application caused a short-term increase in C2H4 production followed by a sharp reduction to the

control level along with a marked reduction in ACC levels. In both tomato and strawberry fruit tissues, C2H4

application significantly induced ACC oxidase (ACO) activity at all ripening stages, as measured by in vivo ACC conversion to C2H4. This strongly suggests that ACC synthesis is the limiting step in C2H4autocatalysis and the only

limiting step in C2H4autoinhibition. In tomato pericarp tissues, C2H4 autoinhibition and autocatalysis caused by

C2H4application in immature and pink fruits, respectively, were eliminated when tissues were transferred to air and

re-occurred when tissues were returned back to C2H4. These responses did not occur in all strawberry tissues due to

the sharp reduction in C2H4production with the time course of C2H4application. Inhibiting C2H4action with STS

pretreatment inhibited both negative and positive C2H4feedback mechanisms in both tomato and strawberry tissues

indicating that C2H4feedback mechanism is one sort of C2H4action. In addition, only tomato fruit tissue showed

significant increases in CO2 production with C2H4 application. In contrast to the nonclimacteric behaviour of

strawberry fruit which exhibits only a negative C2H4 feedback mechanism, these data strongly suggest that the

transition of the C2H4feedback mechanism from negative to positive, which occurs in tomato fruit only with ripening

Keywords:Ethylene feedback mechanism; Climacteric behaviour; Tomato fruit; Strawberry fruit

www.elsevier.com/locate/postharvbio

* Corresponding author. Present and permanent address: Department of Horticulture, Faculty of Agriculture, Ain Shams University, P.O. Box 68, Hadayek Shoubra 11241, Cairo, Egypt. Tel.: +20-2-4447317; fax: +20-2-4444460.

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1. Introduction

The rate of C2H4 production varies with the

type of plant tissue and its stage of development. In climacteric fruits, C2H4is produced at different

rates based on fruit stage of growth. Such fruit is characterized by a low rate of C2H4 production

during the preclimacteric or unripe stage (basal C2H4), followed by the climacteric, a sudden

in-crease in C2H4production during fruit ripening, a

phenomenon referred to as autocatalytic C2H4

(Abeles, 1973). After the climacteric rise, C2H4

production significantly declines during the post-climacteric phase (Hoffman and Yang, 1980). Nonclimacteric fruits, on the other hand, exhibit no increase in C2H4 production during

matura-tion and ripening (Knee et al., 1977).

Autocatalytic C2H4 production is a common

feature of ripening in climacteric fruit, in which increased synthesis of C2H4is triggered by

exoge-nous C2H4 application (Burg and Burg, 1965;

Abeles, 1973). Several reports however, have demonstrated autoinhibition of C2H4 production.

McMurchie et al. (1972) reported that C2H4

treat-ment inhibited C2H4 production of banana pulp

slices. Similarly, propylene treatment, which ini-tiated ripening, suppressed C2H4 production in

intact green bananas. In the non-ripening stages of sycamore fig, C2H4 acts as an autoinhibitor of

its own production, but this does not occur in the ripening stages (Zeroni et al., 1976). C2H4

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 C2H4 autocatalysis

involves increased synthesis of ACC synthase and the enzyme responsible for the conversion of ACC to C2H4 (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 C2H4 may also serve to

distinguish between climacteric and nonclimac-teric fruits (McMurchie et al., 1972). The response of harvested fruit to applied C2H4 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 C2H4 feedback mechanisms (autocatalysis

and autoinhibition) in tomato and strawberry fruits at different developmental stages; to deter-mine the step(s) in C2H4 biosynthesis which

con-trol C2H4 feedback mechanism; to examine the

relation between C2H4 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 C2H4 application.

2. Material and methods

2.1. Plant material

Full size tomato (Lycopersicon esculentumMill 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

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the equator of the fruit using a stainless steel cork borer at IM, MG and P stages (one disk/fruit). 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 disk/tube).

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 C2H4 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 C2H4 using a

gas-flow system as described below. 2.3. Chemical treatments and tissue analysis

For each treatment, 100ml 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 C2H4exposure to

eliminate wound C2H4 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 C2H4

and CO2 production, immediately after excision,

with an incubation period of 30 min. This incuba-tion period was enough for measuring basal C2H4

levels and less than that required for wound C2H4

to be initiated (Atta-Aly, 1992). The second and the third groups, however, were continuously ex-posed to an air flow94.5 mmol l−1 C2H4 for 5

days, either for monitoring C2H4 and CO2

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 C2H4 which

di-minishes within 72 h of excision (Atta-Aly et al., 1987). The gas flow system removed wound C2H4

produced during the duration of the experiment and the first C2H4analysis, 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 C2H4for 3 days, when water or 100mM ACC was

added to the tissues 2 h before measuring C2H4

production as an indicator of ACO activity. 2.3.3. C2H4 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 4.5mmol l−1 C2H4 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. C2H4 and CO2

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, C2H4 and CO2 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 O2levels around the tissue. CO2

levels in the air flow were checked twice per day and its concentration was always below 0.5% throughout the experiment. The air flow94.5

mmol l−1 (100 ml l−l) C2H4 was passed through

water flasks twice before passing through tissue containers (RH 98%).

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Excised tissues were placed inside the 17-ml volume glass tubes, chemically treated and then divided into two groups for either air or C2H4

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 C2H4 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 flow94.5mmol l−1

C2H4 for 3 days, thoroughly flushed with C2H4

-free air for 60 s and exposed to the vacuum procedure described by Saltveit (1982) for measur-ing internal C2H4 concentrations. No significant

differences were found between air and C2H4

-treated tissues in internal C2H4 levels at each

developmental stage of both fruits. All tissues, therefore, were thoroughly flushed for 60 s with C2H4-free air prior to each C2H4 analysis.

In a separate experiment, tissue was exposed to 580 mmol l−1 propylene gas instead of 4.5 mmol

l−1

ethylene. C2H4 production by both tomato

and strawberry fruit tissues was similar to that obtained with C2H4 application when the tissue

was flushed with C2H4-free air for 60 s before

incubation.

2.5. C2H4, CO2 and ACC analysis

At each sampling time, the tubes were removed, thoroughly flushed with C2H4-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 C2H4 and CO2

measure-ments. A Hewlett Packard gas chromatograph Model 5080A with FID was used for C2H4

analy-sis, while a Gow Mac Model 60, with TCD (Gow Mac Instrument Co., NJ) was used for CO2

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

Immature tomato fruit tissue showed a pattern of C2H4 autoinhibition (negative C2H4 feedback

mechanism) since C2H4production by such tissue

was strongly inhibited upon exposure to exoge-nously applied C2H4 (Fig. l). An opposite trend

(C2H4 autocatalysis or positive feedback

mecha-nism) however, was observed when tomato fruit tissue at the pink stage was used (Fig. l). With the first visual sign of red colour which occurred in MG tissue 4 days after exogenous C2H4exposure,

a transition phase from C2H4 autoinhibition to

autocatalysis was detected (Fig. 1). All develop-mental stages of strawberry fruit tissues, on the other hand, showed a short-term increase in C2H4

production upon exogenous C2H4application

fol-lowed by a dramatic reduction to that of control levels after 4 days of continuous C2H4application

(Fig. 1). Since 5.8 mmol l−1 propylene has the

same impact on fruit ripening as 0.045 mmol l−1

C2H4(McMurchie et al., 1972), separate tests with

tomato and strawberry tissues were also carried out using propylene rather than C2H4. Results

showed similar levels of C2H4 production in both

treatments.

Since ACC formation and its conversion to C2H4 are the two main limiting steps in C2H4

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Fig. 1. Effect of exogenous ethylene treatment on ethylene production by tomato and strawberry fruit tissues at different developmental stages. Vertical bars superimposed on datapoints at each sampling date represent the L.S.D. values at the 5% level.

1984) and also in C2H4 feedback mechanism

(Nakatsuka et al., 1998; Atta-Aly et al., 2000), both ACC concentration and in vivo ACO activ-ity were determined in both fruit tissues at all developmental stages. While ACO activity

signifi-cantly increased in both fruit tissues at all devel-opmental stages with exogenous C2H4 treatment

(Table 1), ACC concentration showed a different pattern of response (Fig. 2). ACC increased in tomato fruit tissues as ripening initiated and

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de-Table 1

ACC oxidase activity (C2H4nmol g−1_h−1_{) in tomato pericarp and strawberry flesh tissues exposed to exogenous C2H4}_treatmenta

C2H4exposure

Treatment Fruit developmental stages

Tomato Strawberry

Immature Mature green Pink Green White Half coloured

0.04b 0.02b

H2O Air 0.12a 0.78b 0.73b 0.02b

0.06a 0.07a 0.09a 0.88a 0.92a 0.09b

C2H4

0.01a

AVG Air 0.04a 0.18a 0.31a 0.01a 0.01a

0.01a

C2H4 0.03a 0.13a 0.39a 0.01a 0.01a

0.38b 0.14b

ACC Air 2.15b 1.88b 2.07b 0.20b

0.90a 2.52a

2.80a

5.17a 0.36a 0.32a

C2H4

0.94b 0.78b

Air 0.19b

AVG+ACC 0.36b 0.10b 0.10b

0.21a 0.19a 0.53a 1.73a 1.47a 2.15a

C2H4

a_{Fruit tissues were treated with either H2O or 0.5 mM AVG immediately after excision and then exposed to either air or 4.5}

mmol

l−1_C

2H4for 3 days with H2O or 100mM ACC application 2 h before C2H4measurements. Values within each treatment for each fruit developmental stage followed by the same letter are not statistically different at the 5% level.

veloped, but decreased in immature tomato fruit tissue and in all developmental stages of straw-berry fruit (Fig. 2). Regardless of C2H4

applica-tion, ACC concentration increased the maturation of both fruit tissues (Fig. 2). A short-term increase in C2H4production occurred in all developmental

stages of strawberry fruit tissues upon C2H4

treat-ment, due mainly to induced ACO activity. This increase was diminished thereafter when ACC concentration became limiting. The reduction in ACC concentration which occurred in strawberry fruit tissues (Fig. 2) may be due not only to the high level of its consumption during the first 3 days as a result of induced ACO activity (Table 1), but also to the reduction in tissue ACC synthe-sis, since after the first 3 days, C2H4-treated tissue

contained lower ACC concentrations but pro-duced C2H4 levels at a rate equal to that of

air-treated ones (Figs. 1 and 2). These data sug-gest that both ACC and its conversion to C2H4

are limiting steps in C2H4 autocatalysis, while

only ACC is the controlling step in C2H4

auto-inhibition.

Riov and Yang (1982) suggested that autoinhi-bition of C2H4production in wounded citrus peel

tissue is attributable to the suppression of ACC formation due to the inhibition of ACC synthase formation and activity. Since the in vivo activity of the ACO enzyme relies on the available level of ACC in the tissue (Yang, 1980), it could be sug-gested that in the long term, ACC synthesis is the limiting step in C2H4 feedback mechanism.

Thus the climacteric behaviour of tomato fruit Fig. 2. Effect of exogenous ethylene treatment for 4 days on

ACC concentration in tomato and strawberry fruit tissues at different developmental stages. IM, MG and P are immature, mature-green and pink tomato while G, W and HC are green, white and half-coloured strawberry fruits, respectively. At each developmental stage, significant differences are presented at the 5% level.

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during ripening initiation and development may be due mainly to the presence of C2H4 catalysis,

while the absence of this mechanism and the

presence of C2H4 autoinhibition are the reasons

for the nonclimacteric behaviour occurring in strawberry fruit and during the nonripening

Fig. 3. Effect of transferring tomato and strawberry fruit tissues, excised at different developmental stages from air to ethylene and back to air [air – ethylene – air; starting with 3 days in air (A) to 1 day in ethylene (B) and back to air for another day (C)] or to an opposite sequence of exposure [ethylene – air – ethylene; starting with 3 days in ethylene (D) to 1 day in air (E) and back to ethylene for another day (F)] on the levels of ethylene production at each atmosphere change. Significant differences are presented for each sampling date at the 5% level.

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stages of tomato fruit. Zeroni et al. (1976) reported that C2H4 acts as an autoinhibitor of its own

production in the immature stages of sycamore fig but not during ripening. Studies involving treat-ment of fruit with exogenous ethylene or propylene indicated that fruit response to C2H4 may also

serve to distinguish between climacteric and non-climacteric fruit (McMurchie et al., 1972). The response of harvested fruit to applied C2H4

de-pends on various features, including tissue sensitiv-ity and stage of maturation, as well as whether or not the fruit is climacteric (Biale and Young, 1981). These data suggest that exogenous C2H4

applica-tion to climacteric fruit should not be applied until fruit become mature to obtain acceptable ripening uniformity and quality, since positive C2H4

feed-back mechanism does not occur at earlier stages. To test the impact of C2H4feedback mechanism

on C2H4 production in relation to fruit

develop-mental stage and its climacteric pattern, both tomato and strawberry fruit tissues at three specific developmental stages were transferred between air and C2H4atmospheres. This was carried out in two

opposite sequences: air – C2H4– air, or C2H4– air –

C2H4, starting with 3 days in the first atmosphere

to eliminate wound C2H4 followed by one

addi-tional day for each subsequent atmosphere change. During the immature stage of tomato fruit devel-opment, C2H4 production was significantly

re-duced when tissue was exposed to C2H4 or

transferred from air to C2H4 in comparison with

that exposed to air or transferred from C2H4to air

(Fig. 3). As maturation progressed, exposure to exogenous C2H4 significantly induced C2H4

pro-duction. This induction was eliminated upon trans-ferring the tissue to air and re-occurred when tissue was returned back to C2H4 (Fig. 3). The same

pattern of response was also obtained using tomato locule gel tissue (Atta-Aly et al., 2000).

In strawberry fruit tissues however, C2H4

pro-duction strongly increased during the 3rd day of exposure to exogenous C2H4compared with those

exposed to air. After those first 3 days however, the impact on C2H4 production of transferring tissues

between air and C2H4atmospheres was diminished

(Fig. 3). Zauberman and Fuchs (1973) reported that C2H4 feedback mechanisms may last after

removing the tissue from an C2H4 atmosphere.

Table 2

C2H4and CO2production by tomato pericarp and strawberry flesh tissue excised from fruits at different developmental stages and exposed, for 3 days, to exogenous air94.5mmol l−1C2H4immediately after H2O or 0.5 mM STS applicationa

Fruit developmental stages C2H4exposure

Treatment

Tomato Strawberry

Half coloured Green White

Pink Immature Mature green

C2H4(nmol g−1_h−1₎

0.02b

0.08a 0.02b

H2O Air 0.13a 0.89b 0.77b

0.92a 0.09a 0.07a 0.06a

C2H4 0.09b 1.04a

0.14a 0.09a

STS Air 0.12a 0.84a 0.84a 0.05a

0.04a 0.11a 0.09a

0.76a

C2H4 0.14a 0.80a

CO2(mmol g −1_h−1₎

1.08a

1.96a 1.72a

H2O Air 0.68b 1.01b 1.29b

1.95a 1.19a

C2H4 0.87a 1.52a 1.55a 1.81a

1.57a 1.95a

STS Air 0.86a 0.91a 1.01a 1.91a

1.88a 1.64a

C2H4 0.80a 0.89a 1.04a 1.74a

a_{Values within each treatment for each fruit developmental stage followed by the same letter are not statistically different at the} 5% level.

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To test that a C2H4 feedback mechanism is

dependent on C2H4 sensitivity, STS was used to

inhibit C2H4 action before exposing both fruit

tissues either to air or C2H4 atmosphere. Data

presented in Table 2 show C2H4autoinhibition in

immature tomato tissue but C2H4autocatalysis in

mature-green and pink tomato as well as in white and half-coloured strawberry fruit tissues after the

Fig. 4. Effect of exogenous ethylene treatment on CO2production by tomato and strawberry fruit tissues at different developmental stages. Vertical bars superimposed on data points at each sampling date represent the L.S.D. values at the 5% level.

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Fig. 5. Effect of transferring tomato and strawberry fruit tissues, excised at different developmental stages from air to ethylene and back to air [air – ethylene – air; starting with 3 days in air (A) to 1 day in ethylene (B) and back to air for another day (C)] or to an opposite sequence of exposure [ethylene – air – ethylene; starting with 3 days in ethylene (D) to 1 day in air (E) and back to ethylene for another day (F)] on the levels of CO2 production at each atmosphere change. Significant differences are presented for each sampling date at the 5% level.

3 days of continuous C2H4 exposure. Both C2H4

autoinhibition and autocatalysis were diminished when STS was used. Riov and Yang (1982) re-ported that silver ion blocked the autocatalytic

effect of C2H4. Inhibiting C2H4 action in the

climacteric tomato fruit with diazocyclopentadi-ene (DACP) application at different ripening stages depressed C2H4 production (Sisler and

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Lallu, 1994; Tian et al., 1997a), while opposite results were obtained in the nonclimacteric strawberry fruit (Tian et al., 1997b). It was also evident that C2H4 autoinhibition is shifted to

C2H4 autocatalysis in tomato fruit as ripening is

initiated and progresses by stimulating both ACC synthase and ACO (Nakatsuka et al., 1998; Atta-Aly et al., 2000). Data presented in this work, therefore, indicate that inhibiting C2H4 action using silver ion blocks both C2H4

positive and negative feedback mechanisms. Since the pattern of fruit respiration during ripening initiation and development is another strong feature to distinguish between climacteric and nonclimacteric fruit behaviour, CO2

produc-tion therefore, was determined in both fruit tis-sues at three developmental stages during continuous exposure to either air or C2H4

atmo-sphere. Exogenous C2H4 application induced

CO2 production by tomato fruit tissue while it

had no effect on strawberry as ripening pro-gressed (Fig. 4). Inhibiting C2H4 action with

DACP application reduced tomato fruit respira-tion (Sisler and Lallu, 1994; Tian et al., 1997a), while no effect was found in strawberry (Tian et al., 1997b). When both fruit tissues were trans-ferred between air and C2H4 atmospheres, CO2

production by tomato significantly increased in the exogenous C2H4 atmosphere. This increase

diminished upon transfer to air and re-occurred when tissues were returned to the C2H4

atmo-sphere. In strawberry however, none of these differences occurred (Fig. 5). The stimulation of tomato fruit respiration caused by exogenous C2H4 application did not occur when C2H4

ac-tion and subsequently its feedback mechanism was blocked by STS application (Table 2). This means that during tomato fruit ripening there was a positive correlation between a positive C2H4 feedback mechanism and fruit climacteric

respiration. This correlation was absent in the nonclimacteric strawberry fruit.

In conclusion, it is suggested that a negative C2H4 feedback mechanism may be the reason

for the nonclimacteric behaviour of strawberry fruit and immature tomato fruit, while a posi-tive C2H4 feedback mechanism is the reason for

tomato fruit climacteric behaviour during

ripen-ing initiation and development. ACC formation is possibly the limiting step for either positive or negative C2H4 feedback mechanisms, since

ex-ogenous C2H4 treatment induced ACO activity

regardless of fruit species and physiological age.

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Sisler, E.C., Lallu, N., 1994. Effect of diazocyclopentadiene (DACP) on tomato fruits harvested at different ripening stages. Postharvest Biol. Technol. 4, 245 – 254.

Tian, M.S., Bowen, J.H., Bauchot, A.D., Gong, Y.P., Lallu, N., 1997a. Recovery of ethylene biosynthesis in diazocyclopen-tadiene (DACP)-treated tomato fruit. Plant Growth Regul. 22, 73 – 78.

Tian, M.S., Gong, Y.P., Bauchot, A.D., 1997b. Ethylene

biosynthesis and respiration in strawberry fruit treated with diazocyclopentadiene and IAA. Plant Growth Regul. 23, 195 – 200.

Yang, S.F., 1980. Regulation of ethylene biosynthesis. HortScience 15, 238 – 243.

Yang, S.F., Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155 – 189.

Zauberman, G., Fuchs, Y., 1973. Ripening processes in avocado stored in ethylene atmosphere in cold storage. J. Am. Soc. Hort. Sci. 98, 477 – 480.

Zeroni, M., Galil, I., Ben-Yehoshua, S., 1976. Autoinhibition of ethylene formation in nonripening stages of the fruit of sycamore fig (Ficus sycomorusL.). Plant Physiol. 57, 647 – 650.

(1)

during ripening initiation and development may

be due mainly to the presence of C

H

catalysis,

while the absence of this mechanism and the

presence of C

H

autoinhibition are the reasons

for the nonclimacteric behaviour occurring in

strawberry fruit and during the nonripening

(2)

stages of tomato fruit. Zeroni et al. (1976) reported

that C

H

acts as an autoinhibitor of its own

production in the immature stages of sycamore fig

but not during ripening. Studies involving

treat-ment of fruit with exogenous ethylene or propylene

indicated that fruit response to C

H

may also

serve to distinguish between climacteric and

non-climacteric fruit (McMurchie et al., 1972). The

response of harvested fruit to applied C

H

de-pends on various features, including tissue

sensitiv-ity and stage of maturation, as well as whether or

not the fruit is climacteric (Biale and Young, 1981).

These data suggest that exogenous C

H

applica-tion to climacteric fruit should not be applied until

fruit become mature to obtain acceptable ripening

uniformity and quality, since positive C

H

feed-back mechanism does not occur at earlier stages.

To test the impact of C

H

feedback mechanism

on C

H

production in relation to fruit

develop-mental stage and its climacteric pattern, both

tomato and strawberry fruit tissues at three specific

developmental stages were transferred between air

and C

H

atmospheres. This was carried out in two

opposite sequences: air – C

H

– air, or C

H

– air –

C

H

, starting with 3 days in the first atmosphere

to eliminate wound C

H

followed by one

addi-tional day for each subsequent atmosphere change.

During the immature stage of tomato fruit

devel-opment, C

H

production was significantly

re-duced when tissue was exposed to C

H

or

transferred from air to C

H

in comparison with

that exposed to air or transferred from C

H

to air

(Fig. 3). As maturation progressed, exposure to

exogenous C

H

significantly induced C

H

pro-duction. This induction was eliminated upon

trans-ferring the tissue to air and re-occurred when tissue

was returned back to C

H

(Fig. 3). The same

pattern of response was also obtained using

tomato locule gel tissue (Atta-Aly et al., 2000).

In strawberry fruit tissues however, C

H

pro-duction strongly increased during the 3rd day of

exposure to exogenous C

H

compared with those

exposed to air. After those first 3 days however, the

impact on C

H

production of transferring tissues

between air and C

H

atmospheres was diminished

(Fig. 3). Zauberman and Fuchs (1973) reported

that C

H

feedback mechanisms may last after

removing the tissue from an C

H

atmosphere.

Table 2

C2H4and CO2production by tomato pericarp and strawberry flesh tissue excised from fruits at different developmental stages and

exposed, for 3 days, to exogenous air94.5mmol l−1C2H4immediately after H2O or 0.5 mM STS applicationa

Fruit developmental stages C2H4exposure

Treatment

Tomato Strawberry

Half coloured Green White

Pink Immature Mature green

C2H4(nmol g−1h−1)

0.02b

0.08a 0.02b

H2O Air 0.13a 0.89b 0.77b

0.92a 0.09a 0.07a 0.06a

C2H4 0.09b 1.04a

0.14a 0.09a

STS Air 0.12a 0.84a 0.84a 0.05a

0.04a 0.11a 0.09a

0.76a

C2H4 0.14a 0.80a

CO2(mmol g −1_h−1₎

1.08a

1.96a 1.72a

H2O Air 0.68b 1.01b 1.29b

1.95a 1.19a

C2H4 0.87a 1.52a 1.55a 1.81a

1.57a 1.95a

STS Air 0.86a 0.91a 1.01a 1.91a

1.88a 1.64a

C2H4 0.80a 0.89a 1.04a 1.74a

a_{Values within each treatment for each fruit developmental stage followed by the same letter are not statistically different at the}

(3)

To test that a C

H

feedback mechanism is

dependent on C

H

sensitivity, STS was used to

inhibit C

H

action before exposing both fruit

tissues either to air or C

H

atmosphere. Data

presented in Table 2 show C

H

autoinhibition in

immature tomato tissue but C

H

autocatalysis in

mature-green and pink tomato as well as in white

and half-coloured strawberry fruit tissues after the

Fig. 4. Effect of exogenous ethylene treatment on CO2production by tomato and strawberry fruit tissues at different developmental

(4)

sampling date at the 5% level.

3 days of continuous C

H

exposure. Both C

H

autoinhibition and autocatalysis were diminished

when STS was used. Riov and Yang (1982)

re-ported that silver ion blocked the autocatalytic

effect of C

H

. Inhibiting C

H

action in the

climacteric tomato fruit with

diazocyclopentadi-ene (DACP) application at different ripening

(5)

Lallu, 1994; Tian et al., 1997a), while opposite

results

were

obtained

in

the

nonclimacteric

strawberry fruit (Tian et al., 1997b). It was also

evident that C

H

autoinhibition is shifted to

C

H

autocatalysis in tomato fruit as ripening is

initiated and progresses by stimulating both

ACC synthase and ACO (Nakatsuka et al.,

1998; Atta-Aly et al., 2000). Data presented in

this work, therefore, indicate that inhibiting

C

H

action using silver ion blocks both C

H

positive and negative feedback mechanisms.

Since the pattern of fruit respiration during

ripening initiation and development is another

strong feature to distinguish between climacteric

and nonclimacteric fruit behaviour, CO

produc-tion therefore, was determined in both fruit

tis-sues

at

three

developmental

stages

during

continuous exposure to either air or C

H

atmo-sphere. Exogenous C

H

application induced

CO

production by tomato fruit tissue while it

had no effect on strawberry as ripening

pro-gressed (Fig. 4). Inhibiting C

H

action with

DACP application reduced tomato fruit

respira-tion (Sisler and Lallu, 1994; Tian et al., 1997a),

while no effect was found in strawberry (Tian et

al., 1997b). When both fruit tissues were

trans-ferred between air and C

H

atmospheres, CO

production by tomato significantly increased in

the exogenous C

H

atmosphere. This increase

diminished upon transfer to air and re-occurred

when tissues were returned to the C

H

atmo-sphere. In strawberry however, none of these

differences occurred (Fig. 5). The stimulation of

tomato fruit respiration caused by exogenous

C

H

application did not occur when C

H

ac-tion and subsequently its feedback mechanism

was blocked by STS application (Table 2). This

means that during tomato fruit ripening there

was a positive correlation between a positive

C

H

feedback mechanism and fruit climacteric

respiration. This correlation was absent in the

nonclimacteric strawberry fruit.

In conclusion, it is suggested that a negative

C

H

feedback mechanism may be the reason

for the nonclimacteric behaviour of strawberry

fruit and immature tomato fruit, while a

posi-tive C

H

feedback mechanism is the reason for

tomato fruit climacteric behaviour during

ripen-ing initiation and development. ACC formation

is possibly the limiting step for either positive or

negative C

H

feedback mechanisms, since

ex-ogenous C

H

treatment induced ACO activity

regardless of fruit species and physiological age.

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