Ethylene feedback mechanisms in tomato and strawberry fruit tissues in relation to fruit ripening and climacteric patterns

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1 Postharvest Biology and Technology 20 (2000) 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, Uni ersity of Florida, Gaines ille, FL , USA Received 16 August 1999; accepted 18 May 2000 Abstract Exposing pericarp tissue excised from immature tomato fruit to 4.5 mol l 1 C 2 H 4 revealed a negative C 2 H 4 feedback mechanism in relation to its biosynthesis since ACC concentration and C 2 H 4 production by the tissue were reduced. An opposite trend (positive C 2 H 4 feedback 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 C 2 H 4 feedback mechanism with the onset of tissue ripening. In strawberry tissues excised from green, white and half-coloured fruits however, C 2 H 4 application caused a short-term increase in C 2 H 4 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, C 2 H 4 application significantly induced ACC oxidase (ACO) activity at all ripening stages, as measured by in vivo ACC conversion to C 2 H 4. This strongly suggests that ACC synthesis is the limiting step in C 2 H 4 autocatalysis and the only limiting step in C 2 H 4 autoinhibition. In tomato pericarp tissues, C 2 H 4 autoinhibition and autocatalysis caused by C 2 H 4 application in immature and pink fruits, respectively, were eliminated when tissues were transferred to air and re-occurred when tissues were returned back to C 2 H 4. These responses did not occur in all strawberry tissues due to the sharp reduction in C 2 H 4 production with the time course of C 2 H 4 application. Inhibiting C 2 H 4 action with STS pretreatment inhibited both negative and positive C 2 H 4 feedback mechanisms in both tomato and strawberry tissues indicating that C 2 H 4 feedback mechanism is one sort of C 2 H 4 action. In addition, only tomato fruit tissue showed significant increases in CO 2 production with C 2 H 4 application. In contrast to the nonclimacteric behaviour of strawberry fruit which exhibits only a negative C 2 H 4 feedback mechanism, these data strongly suggest that the transition of the C 2 H 4 feedback mechanism from negative to positive, which occurs in tomato fruit only with ripening initiation and progress, may be the reason behind the climacteric behaviour of tomato fruit Elsevier Science B.V. All rights reserved. Keywords: Ethylene feedback mechanism; Climacteric behaviour; Tomato fruit; Strawberry fruit * 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.: ; fax: /00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S (00)

2 152 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) 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 increase 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 postclimacteric phase (Hoffman and Yang, 1980). Nonclimacteric fruits, on the other hand, exhibit no increase in C 2 H 4 production during maturation 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 exogenous 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 treatment inhibited C 2 H 4 production of banana pulp slices. Similarly, propylene treatment, which initiated 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 autoinhibition was also noticed in avocado fruit (Zauberman 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 suppression 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 nonclimacteric 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 determine the step(s) in C 2 H 4 biosynthesis which control 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 commercial field in south Florida at immature (IM), mature-green (MG) and pink (P) stages. Blossomend dark and light-green colours were used to distinguish between IM and MG stages, respectively, 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 materials. 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 developmental stage, and kept at 15 C and 95% RH overnight for treatment preparation. Experiments were repeated three times using tomato and strawberry fruits from the same sources Tissue sampling Tomato outer pericarp disks were excised from

3 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) 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 materials 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 C 2 H 4 biosynthesis since it is known that the achenes 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 treatments and exposed thereafter to C 2 H 4 using a gas-flow system as described below Chemical treatments and tissue analysis For each treatment, 100 l of each solution was applied to the locular surface of tomato disks or to the vascular tissue of the strawberry flesh cylinders. 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 strawberry tissues as described below 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 incubation 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 exposed to an air flow 4.5 mol l 1 C 2 H 4 for 5 days, either for monitoring C 2 H 4 and CO 2 production 3, 4 and 5 days after excision or for ACC analysis 4 days after excision. Plant tissue produces a large amount of wound C 2 H 4 which diminishes 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 In i o estimation of ACC oxidase (ACO) acti ity 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 M ACC was added to the tissues 2 h before measuring C 2 H 4 production as an indicator of ACO activity C 2 H 4 action This was achieved in two different ways as follows: 1. Tissues were treated with water and then divided into two groups. The first group was exposed to air for 3 days, then transferred to the 4.5 mol 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 atmospheres 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 Ethylene treatments Based on the highest respiratory levels of excised tomato and strawberry fruit tissues, measured 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 4.5 mol l 1 (100 l l l )C 2 H 4 was passed through water flasks twice before passing through tissue containers (RH 98%).

4 154 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) 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 gasflow 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 strawberry fruit tissues, excised at each developmental stage, were exposed to the air flow 4.5 mol 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 measuring 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 mol l 1 propylene gas instead of 4.5 mol 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 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 measurements. A Hewlett Packard gas chromatograph Model 5080A with FID was used for C 2 H 4 analysis, while a Gow Mac Model 60, with TCD (Gow Mac Instrument Co., NJ) was used for CO 2 measurements. 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) Experimental design and statistical analysis Experiments were designed as factorial arrangements in completely randomized designs with five replicates each consisting of 15 samples. Experiments were repeated three times and data were subjected to combined analysis. Means were analyzed 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 C 2 H 4 autoinhibition (negative C 2 H 4 feedback mechanism) since C 2 H 4 production by such tissue was strongly inhibited upon exposure to exogenously applied C 2 H 4 (Fig. l). An opposite trend (C 2 H 4 autocatalysis or positive feedback mechanism) 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 C 2 H 4 exposure, a transition phase from C 2 H 4 autoinhibition to autocatalysis was detected (Fig. 1). All developmental stages of strawberry fruit tissues, on the other hand, showed a short-term increase in C 2 H 4 production upon exogenous C 2 H 4 application followed by a dramatic reduction to that of control levels after 4 days of continuous C 2 H 4 application (Fig. 1). Since 5.8 mol l 1 propylene has the same impact on fruit ripening as mol l 1 C 2 H 4 (McMurchie et al., 1972), separate tests with tomato and strawberry tissues were also carried out using propylene rather than C 2 H 4. Results showed similar levels of C 2 H 4 production in both treatments. Since ACC formation and its conversion to C 2 H 4 are the two main limiting steps in C 2 H 4 biosynthesis (Yang, 1980; Yang and Hoffman,

5 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) 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 C 2 H 4 feedback mechanism (Nakatsuka et al., 1998; Atta-Aly et al., 2000), both ACC concentration and in vivo ACO activity were determined in both fruit tissues at all developmental stages. While ACO activity significantly increased in both fruit tissues at all developmental stages with exogenous C 2 H 4 treatment (Table 1), ACC concentration showed a different pattern of response (Fig. 2). ACC increased in tomato fruit tissues as ripening initiated and de-

6 156 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) Table 1 ACC oxidase activity (C 2 H 4 nmol g 1 h 1 ) in tomato pericarp and strawberry flesh tissues exposed to exogenous C 2 H 4 treatment a Treatment C 2 H 4 exposure Fruit developmental stages Tomato Strawberry Immature Mature green Pink Green White Half coloured H 2 O Air 0.12a 0.78b 0.73b 0.04b 0.02b 0.02b C 2 H b 0.92a 0.88a 0.09a 0.07a 0.06a AVG Air 0.04a 0.18a 0.31a 0.01a 0.01a 0.01a C 2 H a 0.13a 0.39a 0.01a 0.01a 0.01a ACC Air 2.15b 1.88b 2.07b 0.38b 0.14b 0.20b C 2 H a 2.80a 2.52a 0.90a 0.36a 0.32a AVG+ACC Air 0.78b 0.94b 0.19b 0.36b 0.10b 0.10b C 2 H a 1.47a 1.73a 0.53a 0.19a 0.21a a Fruit tissues were treated with either H 2 O or 0.5 mm AVG immediately after excision and then exposed to either air or 4.5 mol l 1 C 2 H 4 for 3 days with H 2 Oor100 M ACC application 2 h before C 2 H 4 measurements. Values within each treatment for each fruit developmental stage followed by the same letter are not statistically different at the 5% level. 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. veloped, but decreased in immature tomato fruit tissue and in all developmental stages of strawberry fruit (Fig. 2). Regardless of C 2 H 4 application, ACC concentration increased the maturation of both fruit tissues (Fig. 2). A short-term increase in C 2 H 4 production occurred in all developmental stages of strawberry fruit tissues upon C 2 H 4 treatment, 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 synthesis, since after the first 3 days, C 2 H 4 -treated tissue contained lower ACC concentrations but produced C 2 H 4 levels at a rate equal to that of air-treated ones (Figs. 1 and 2). These data suggest that both ACC and its conversion to C 2 H 4 are limiting steps in C 2 H 4 autocatalysis, while only ACC is the controlling step in C 2 H 4 autoinhibition. Riov and Yang (1982) suggested that autoinhibition of C 2 H 4 production 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 suggested that in the long term, ACC synthesis is the limiting step in C 2 H 4 feedback mechanism. Thus the climacteric behaviour of tomato fruit

7 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) during ripening initiation and development may be due mainly to the presence of C 2 H 4 catalysis, while the absence of this mechanism and the presence of C 2 H 4 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.

8 158 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) stages of tomato fruit. Zeroni et al. (1976) reported that C 2 H 4 acts as an autoinhibitor of its own production in the immature stages of sycamore fig but not during ripening. Studies involving treatment of fruit with exogenous ethylene or propylene indicated that fruit response to C 2 H 4 may also serve to distinguish between climacteric and nonclimacteric fruit (McMurchie et al., 1972). The response of harvested fruit to applied C 2 H 4 depends on various features, including tissue sensitivity and stage of maturation, as well as whether or not the fruit is climacteric (Biale and Young, 1981). These data suggest that exogenous C 2 H 4 application to climacteric fruit should not be applied until fruit become mature to obtain acceptable ripening uniformity and quality, since positive C 2 H 4 feedback mechanism does not occur at earlier stages. To test the impact of C 2 H 4 feedback mechanism on C 2 H 4 production in relation to fruit developmental stage and its climacteric pattern, both tomato and strawberry fruit tissues at three specific developmental stages were transferred between air and C 2 H 4 atmospheres. This was carried out in two opposite sequences: air C 2 H 4 air, or C 2 H 4 air C 2 H 4, starting with 3 days in the first atmosphere to eliminate wound C 2 H 4 followed by one additional day for each subsequent atmosphere change. During the immature stage of tomato fruit development, C 2 H 4 production was significantly reduced when tissue was exposed to C 2 H 4 or transferred from air to C 2 H 4 in comparison with that exposed to air or transferred from C 2 H 4 to air (Fig. 3). As maturation progressed, exposure to exogenous C 2 H 4 significantly induced C 2 H 4 production. This induction was eliminated upon transferring the tissue to air and re-occurred when tissue was returned back to C 2 H 4 (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 2 H 4 production strongly increased during the 3rd day of exposure to exogenous C 2 H 4 compared with those exposed to air. After those first 3 days however, the impact on C 2 H 4 production of transferring tissues between air and C 2 H 4 atmospheres was diminished (Fig. 3). Zauberman and Fuchs (1973) reported that C 2 H 4 feedback mechanisms may last after removing the tissue from an C 2 H 4 atmosphere. Table 2 C 2 H 4 and CO 2 production by tomato pericarp and strawberry flesh tissue excised from fruits at different developmental stages and exposed, for 3 days, to exogenous air 4.5 mol l 1 C 2 H 4 immediately after H 2 O or 0.5 mm STS application a Treatment C 2 H 4 exposure Fruit developmental stages Tomato Strawberry Immature Mature green Pink Green White Half coloured C 2 H 4 (nmol g 1 h 1 ) H 2 O Air 0.13a 0.89b 0.77b 0.08a 0.02b 0.02b C 2 H b 1.04a 0.92a 0.09a 0.07a 0.06a STS Air 0.12a 0.84a 0.84a 0.14a 0.09a 0.05a C 2 H a 0.80a 0.76a 0.11a 0.09a 0.04a CO 2 ( mol g 1 h 1 ) H 2 O Air 0.68b 1.01b 1.29b 1.96a 1.08a 1.72a C 2 H a 1.52a 1.55a 1.95a 1.19a 1.81a STS Air 0.86a 0.91a 1.01a 1.91a 1.57a 1.95a C 2 H a 0.89a 1.04a 1.88a 1.64a 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.

9 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) To test that a C 2 H 4 feedback mechanism is dependent on C 2 H 4 sensitivity, STS was used to inhibit C 2 H 4 action before exposing both fruit tissues either to air or C 2 H 4 atmosphere. Data presented in Table 2 show C 2 H 4 autoinhibition in immature tomato tissue but C 2 H 4 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 CO 2 production 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.

10 160 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) 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 CO 2 production at each atmosphere change. Significant differences are presented for each sampling date at the 5% level. 3 days of continuous C 2 H 4 exposure. Both C 2 H 4 autoinhibition and autocatalysis were diminished when STS was used. Riov and Yang (1982) reported that silver ion blocked the autocatalytic effect of C 2 H 4. Inhibiting C 2 H 4 action in the climacteric tomato fruit with diazocyclopentadiene (DACP) application at different ripening stages depressed C 2 H 4 production (Sisler and

11 M.A. Atta-Aly et al. / Posthar est Biology and Technology 20 (2000) 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 2 H 4 autoinhibition is shifted to C 2 H 4 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 2 H 4 action using silver ion blocks both C 2 H 4 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 2 production therefore, was determined in both fruit tissues at three developmental stages during continuous exposure to either air or C 2 H 4 atmosphere. Exogenous C 2 H 4 application induced CO 2 production by tomato fruit tissue while it had no effect on strawberry as ripening progressed (Fig. 4). Inhibiting C 2 H 4 action with DACP application reduced tomato fruit respiration (Sisler and Lallu, 1994; Tian et al., 1997a), while no effect was found in strawberry (Tian et al., 1997b). When both fruit tissues were transferred between air and C 2 H 4 atmospheres, CO 2 production by tomato significantly increased in the exogenous C 2 H 4 atmosphere. This increase diminished upon transfer to air and re-occurred when tissues were returned to the C 2 H 4 atmosphere. In strawberry however, none of these differences occurred (Fig. 5). The stimulation of tomato fruit respiration caused by exogenous C 2 H 4 application did not occur when C 2 H 4 action 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 2 H 4 feedback mechanism and fruit climacteric respiration. This correlation was absent in the nonclimacteric strawberry fruit. In conclusion, it is suggested that a negative C 2 H 4 feedback mechanism may be the reason for the nonclimacteric behaviour of strawberry fruit and immature tomato fruit, while a positive C 2 H 4 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 2 H 4 feedback mechanisms, since exogenous C 2 H 4 treatment induced ACO activity regardless of fruit species and physiological age. References Abeles, F.B., Ethylene in Plant Biology. Academic Press, New York, p Archbold, D.D., Dennis, F.G., Strawberry receptacle growth and endogenous IAA content as affected by growth regulator application and achene removal. J. Am. Soc. Hort. Sci. 110, Atta-Aly, M.A., Ethylene production by different tomato fruit tissues at different ripening stages. Egypt. J. Hort. 19, Atta-Aly, M.A., Saltveit, M.E., Hobson, G.E., Effect of silver ions on ethylene biosynthesis by tomato fruit tissue. Plant Physiol. 83, Atta-Aly, M.A., Brecht, J.K., Huber, D.J., Ripening of tomato locule gel tissue in response to ethylene, Postharvest Biol. Technol. 19 (3), Biale, J.B., Young, R.E., Respiration and ripening in fruits retrospect and prospect. In: Friend, J., Rhodes, M.J. (Eds.), Recent Advances in the Biochemistry of Fruits and Vegetables. Academic Press, New York, pp Burg, S.P., Burg, E.A., Ethylene action and the ripening of fruit. Science 148, Hoffman, N.E., Yang, S.F., Changes in L-aminocyclopropane-L-carboxylic acid content in ripening fruits in relation to their ethylene production rates. J. Am. Soc. Hort. Sci. 105, Knee, M., Sargent, J.A., Osborne, D.J., Cell wall metabolism in developing strawberry fruit. J. Exp. Bot. 28, Little, T.M., Hills, F.J., Agricultural Experimentation. Wiley, New York, pp Lizada, C., Yang, S.F., A simple and sensitive assay for L-amino cyclopropane-l-carboxylic acid. Anal. Biochem. 100, McMurchie, E.J., McGlasson, W.B., Eaks, I.L., Treatment of fruit with propylene gives information about the biogenesis of ethylene. Nature (Lond.) 235, 237. Nakatsuka, A., Murachi, S., Okunishi, H., Shiomi, S., Nakano, R., Kubo, Y., Inaba, A., Differential expression and internal feedback regulation of 1-aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1- carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening. Plant Physiol. 118, Riov, J., Yang, S.F., Autoinhibition of ethylene production in citrus peel disks: suppression of L-aminocyclopropane-L-carboxylic acid synthesis. Plant Physiol. 69,

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