Carbon dioxide action on ethylene biosynthesis of preclimacteric and climacteric pear fruit

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1 Journal of Experimental Botany, Vol. 54, No. 387, pp. 1537±1544, June 2003 DOI: /jxb/erg159 RESEARCH PAPER Carbon dioxide action on ethylene biosynthesis of preclimacteric and climacteric pear fruit Hans P. J. de Wild 1, Els C. Otma and Herman W. Peppelenbos Agrotechnological Research Institute (ATO), PO Box 17, 6700 AA Wageningen, The Netherlands Received 21 October 2002; Accepted 26 February 2003 Abstract Ethylene production in pear fruit was studied at 2 C. Several observations showed that the inhibiting effect of CO 2 on ethylene production did not operate only via the binding site of the ethylene binding protein. Ethylene production of freshly harvested pears was stimulated by 1-methylcyclopropene (1- MCP), but unaffected or inhibited by CO 2 which points to different action sites for both molecules. In climacteric pears, where ethylene production was strongly inhibited by 1-MCP, a range of applied CO 2 partial pressures was able to inhibit ethylene production further, to an extent similar to untreated pears. In the case of pears that had been stored for a period of 25 weeks, CO 2 only had a clear effect after 1-MCP pretreatment. Respiration measurements showed that the effect of CO 2 on ethylene production did not operate via an effect on respiration. Ethylene production models based on measurements of whole pears were used to study CO 2 effects. Kinetic parameters derived from the models point to the conversion from ACC to ethylene by ACC oxidase as a possible action site for CO 2 inhibition. Key words: Enzyme kinetics, feedback regulation, 1-MCP, model, oxygen consumption, Pyrus communis L., respiration. Introduction The plant hormone ethylene regulates many physiological processes in plants. Carbon dioxide (CO 2 ) can have various effects on ethylene biosynthesis, however, the exact mode of action is unknown (Mathooko, 1996a). Key enzymes in ethylene biosynthesis are 1- aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, which catalyse the reaction from S- adenosylmethionine to ACC and from ACC to ethylene, respectively. CO 2 is an essential cofactor for ACC oxidase (Dong et al., 1992) as demonstrated both for pear fruit in vitro (Vioque and Castellano, 1994) and for pear tissue discs in vivo (Vioque and Castellano, 1998). On the other hand, CO 2 at 5 kpa and higher inhibited the autocatalytic production of ethylene in whole pear fruits (Kerbel et al., 1988; Chavez-Franco and Kader, 1993). It was suggested that inhibition by CO 2 is due to competition with ethylene at the ethylene receptor site, the ethylene binding protein (EBP) (Burg and Burg, 1967; Gorny and Kader, 1996). However, several studies point to another site of inhibition by CO 2. In preclimacteric tomato fruits, CO 2 blocked not only the expression of ethylene-dependent but also ethyleneindependent ripening-associated genes (Rothan et al., 1997). Inhibition of wound-induced ethylene by CO 2 is presumably not via the EBP (Mathooko et al., 2001). In pear fruit where the EBPs had been blocked by 1- methylcyclopropene (1-MCP), CO 2 still inhibited autocatalytic ethylene production (De Wild et al., 1999). It was concluded that the inhibiting effect of CO 2 on ethylene production could not only operate via ethylene perception. Although CO 2 is an essential cofactor for ACC oxidase, it can not be excluded that this enzyme too plays a role in the inhibition of ethylene production by CO 2. It has been shown that elevated CO 2 inhibited ethylene production without affecting ACC content in intact fruit (Li et al., 1983; Cheverry et al., 1988; Rothan and Nicolas, 1994). In addition, after the incubation of excised apple and avocado tissue discs in medium with a saturating concentration of exogenous ACC, CO 2 inhibited ethylene production (Cheverry et al., 1988). Rothan and Nicolas (1994) showed that CO 2 could either 1 To whom correspondence should be addressed. Fax: J.P.J.deWild@ato.wag-ur.nl Abbreviations: 1-MCP, 1-methylcyclopropene, EBP, ethylene binding protein. Journal of Experimental Botany, Vol. 54, No. 387, ã Society for Experimental Biology 2003; all rights reserved

2 1538 de Wild et al. stimulate or inhibit ethylene synthesis of kiwifruit tissue, depending on the internal ACC level. They concluded from Lineweaver±Burk plots that when the ACC concentration was high, CO 2 stimulated ethylene synthesis, but when ACC concentration was low (at a level present in fruits), CO 2 would inhibit ethylene synthesis. Therefore, it is likely that the inhibition of ethylene production by CO 2 is operating (at least partially) at the level of ACC oxidase. To study ACC oxidase as a possible site of CO 2 inhibition, whole fruit responses to CO 2 were measured in the present research. There are several advantages of using whole fruit. First, the operation of the enzyme ACC oxidase in vivo is possibly not re ected by the operation in vitro (John, 1997). The plasma membrane may interact with the activity of ACC oxidase (Ramassamy et al., 1998). Second, the use of the whole product allows CO 2 effects to be studied over an extensive period. Third, it excludes any interference of wound ethylene caused by cutting, as in excised tissue. On the other hand, using whole fruit excludes the possibility for direct enzyme studies. However, by using an ethylene production model that is based on enzyme kinetics, ACC oxidase activity can be studied indirectly. As ACC oxidase catalyses the nal step in ethylene biosynthesis, its activity is directly related to ethylene production rate. The model describes the relationship between ethylene production and O 2. The in uence of CO 2 on this relationship was studied in the present experiment at both preclimacteric and climacteric pears. A possible indirect effect on ACC oxidase is through the effect of CO 2 on respiration (De Wild et al., 1999). Both ethylene production and respiration can be affected by CO 2 and for both processes the effect of CO 2 depends on CO 2 level, duration of CO 2 exposure and temperature (Mathooko, 1996a, b). Data on simultaneously measured responses of respiration and ethylene production to various CO 2 partial pressures can improve the understanding of a possible relationship between these two processes. To study this relationship, in the present experiment, pears were exposed to a range of CO 2 partial pressures. In a second experiment, ethylene production models for whole fruit were used to study CO 2 effects. Pears were pretreated with 1-MCP to exclude the possibility of a CO 2 effect on ethylene binding. An extra advantage of using 1-MCP is that the physiological phase of the pears can be indicated. The negative feedback loop in ethylene biosynthesis, which is present before the induction of autocatalytic ethylene production, can be uncoupled by 1-MCP (Anderson et al., 1997). In that case, 1-MCP treatment results in increased ethylene synthesis as observed in grapefruit (Mullins et al., 2000). During autocatalytic ethylene production, 1-MCP treatment results in decreased ethylene production (De Wild et al., 1999). Materials and methods Effect of CO 2 on ethylene production and respiration (experiment 1) Plant material: Pears (Pyrus communis L. cv. Conference) were harvested in September 1999 on the date that was optimal for commercial long-term storage (based on rmness, starch and soluble solids content of the fruits) and stored for 4 weeks in a cold room (± 0.5 C) (normal procedure, in practice, to reduce development of storage disorders) and subsequently under controlled gas conditions (±0.5 C, 2.0 kpa O 2 and <0.7 kpa CO 2 ) until the start of the experiment. This experiment started in February 2000 after 20 weeks of storage. Pears were placed in a temperature controlled room at 2 C. The fresh weight of the pears was measured. After warming up for 11 h, the pretreatment with 1-MCP started. Pretreatment with 1-MCP: Pears were treated with a saturating concentration of 50 ppm 1-MCP for 24 h at 2 C in 20 l desiccators. Control pears were left in normal air. To prevent the accumulation of CO 2, KOH pellets were placed inside the desiccators. The active 1- MCP gas for each desiccator was produced in 100 ml asks at 20 C by adding 60 ml of water to 1.5 g of Ethylbloc (Biotechnologies for Horticulture, Inc., Walterboro, South Carolina). The headspace of the asks was injected into the desiccators through a rubber septum. After 12 h, the desiccators were opened brie y to refresh the air and, on closing again, fresh 1-MCP was applied. Experiments were performed at 2 C to ensure minimal physiological change of the pears during the experimental period. 1-MCP concentration was saturating as a lower concentration (25 ppm) and a shorter exposure time (16 h) had equal effects on ethylene production (data not shown). Application of a range of CO 2 partial pressures: Untreated pears and pears that had been treated with 50 ppm 1-MCP for 24 h were used. Application of a range of CO 2 partial pressures (0, 1, 2.5, 5, 10, and 20 kpa, all in combination with 20 kpa O 2 )at2 C started 24 h after the end of the 1-MCP treatment. Pears were placed in 1.5±1.8 l cuvettes (two fruits per cuvette) which were connected to a owthrough system in which pure N 2,O 2 and CO 2 were mixed at a total ow rate of 250 ml min ±1 using mass ow controllers (Brooks 5850 TR series). Two replicates were used per treatment. The gas was directed through a water ask prior to entry into each cuvette, resulting in a relative humidity close to saturation (>97%). Ethylene production rate and O 2 consumption rate were measured at days 1 (20 h after connection to the ow-through system), 2, 5, and 8. Measurement of ethylene production and O 2 consumption: The ethylene production rate of pears was measured by closing the cuvettes temporarily. Samples were taken using syringes and subsequently analysed by gas chromatography (Packard model 437A equipped with a packed alumina column and a FID detector, Varian-Chrompack, Bergen op Zoom, The Netherlands). The rst sampling of ethylene was quickly followed by measurements of O 2, N 2 and CO 2. These measurements were done as described by De Wild and Peppelenbos (2001) with the following exceptions: the time period between the rst and second measurements was 5.5 h. The total pressure in each cuvette was measured between the rst sampling of ethylene and the rst sampling of headspace O 2,CO 2 and N 2. This pressure reading was corrected for the pressure drop caused by the latter sampling. This corrected pressure was used to convert the measured initial and nal gas mole fractions (%, ppb) to partial pressures. The pressure change due to gas exchange was assumed negligible as the absolute change of gas partial pressures was small (maximum change of 0.4 kpa O 2 ) and the respiratory quotient was close to 1.

3 As a measure of respiration, the O 2 consumption was taken (De Wild and Peppelenbos, 2001). The free volume of the cuvettes was calculated by subtracting the estimated pear volume (fresh weight divided by density) from, respectively, desiccator and cuvette volume. Density was calculated by measuring the weight and volume of pears that were not included in the experiment (Baumann and Henze, 1983). Check for internal damage: Long-term exposure to high CO 2 can lead to internal damage of pears (Veltman et al., 1999). To exclude any interference by damaged tissue to the production of ethylene, pears were checked for damage at the end of the experiments. Therefore, pears were cut into pieces and visually checked for necrotic tissue. Statistical analysis: The effect of CO 2 on O 2 consumption rate was analysed by analysis of variance (ANOVA). When signi cant differences between CO 2 treatments were found, comparisons between pairs of data were made using the least signi cant differences between means (LSD). The signi cance level used was 95%. (ANOVA was not performed for ethylene production rates as residuals were not normally distributed). Effect of 1-MCP and CO 2 during preclimacteric and climacteric stage (experiment 2) Plant material: Pears (Pyrus communis L. cv. Conference) were picked on ve different days in August and September 2000 at intervals of one week. The third harvest took place on the date that was advised for commercial long-term storage (based on rmness, starch and soluble solids content of the fruits). After each harvest, pears were stored at 2 C for measurements. Pears from the third harvest were also placed at ±0.5 C for 4 weeks under ambient air and subsequently under controlled gas conditions (±1 C, 2.0 kpa O 2 and <0.7 kpa CO 2 ) until measurements in December 2000 (13 weeks storage) or March 2001 (25 weeks storage). In total, seven identical experiments were performed. Experiments were performed shortly after harvest on pears from all ve harvest days and after 13 and 25 weeks of storage on pears from the third harvest. Pretreatment with 1-MCP: Some pears were treated with 1-MCP, other pears were left untreated. In total, 64 pears were divided into four desiccators for treatment with 1-MCP. Treatment with 1-MCP at 2 C started 1 d after harvest or 1 d after the end of storage. Treatment was done in 20 l desiccators with 50 ppm 1-MCP for 16 h. Application of 1-MCP was carried out as described for experiment 1. The 1-MCP treatments were stopped by opening the desiccators (referred to as day 0). To prevent the accumulation of CO 2, KOH pellets were placed inside the desiccators. The estimated decrease in O 2 partial pressure did not exceed 0.5 kpa. This estimation was based on O 2 uptake measurements of a number of pears (performed as described in De Wild and Peppelenbos, 2001). Such a decline is considered physiologically not important at the initial high O 2 partial pressure. CO 2 effect and determination of kinetic parameters: To study kinetic parameters, pears were subjected to various gas conditions shortly after the end of the 1-MCP treatment. Therefore, pears were placed in 1.8 l cuvettes (two fruits per cuvette). Cuvettes were connected to a ow-through system in which pure N 2,O 2 and CO 2 were mixed at a total ow rate of 250 ml min ±1 using mass ow controllers. A range of O 2 partial pressures was applied (0.5, 1, 2.5, 6, and 20 kpa) in combination with 0 and 5 kpa CO 2. Two replicates were used per treatment. The gas was directed through a water ask prior to entry into each cuvette, resulting in a relative humidity close to saturation (97±99%). Ethylene production rate was measured on days 3 and 4. Ethylene production values of these days were averaged for each cuvette as there were no signi cant differences between the two days. Ethylene measurements: The ethylene production rate of pears in the cuvettes was measured as described for experiment 1. Two samples were taken over a time span of 4±5 h. The volume of each pear was measured separately. The free volume of the cuvettes was calculated by subtracting the pear volume from the cuvette volume. Ethylene production models: Ethylene production in response to O 2 was described by an ethylene production model. The ethylene production model, as described by De Wild et al/em>. (1999), was extended with a term `h'. This improves the description of the ethylene production response of whole products (MG Sanders and HPJ De Wild, unpublished data). The use of this term was based on enzyme kinetics (Copeland, 1996). V C2H4 ˆ Vm C2H4 p e O h 2 K C2H4 h p e O h 2 where V C2H4 is the ethylene production rate (pmol kg ±1 s ±1 ), V C2H4 m is the maximum ethylene production rate (pmol kg ±1 s ±1 ), p e O 2 is the external oxygen partial pressure (kpa), h is the Hill coef cient, and K C2H4 is the oxygen partial pressure for half maximum ethylene production (kpa). When optimization of the three parameters could not be performed satisfactorily, then the model was used with h=1. The ethylene production at 0 kpa O 2 was not measured, but was assumed to be 0 as has been found in previous experiments (De Wild et al/em>., 1999). Statistical analysis: The measured data were compared with the model using the facilities for non-linear regression in the statistical package Genstat (release 4.2). Results CO 2 action on ethylene biosynthesis in pear 1539 Effect of CO 2 on ethylene production and respiration (experiment 1) Pears stored for 20 weeks did not develop visual damage during the experiments so interference from damaged tissue with ethylene production is not to be expected. Compared with untreated pears, pretreatment with 1- MCP reduced ethylene production (Fig. 1A, B, respectively). Although 1-MCP had reduced ethylene production, very similar effects of CO 2 were found. In both cases, a 1 d exposure of pears to 1±20 kpa CO 2 resulted in an ethylene production that was higher than at 0 kpa CO 2. Later, in the case of 2.5±20 kpa CO 2, there was a reduction of ethylene production (days 5 and 8). The strongest decline occurred at the highest CO 2 partial pressures. The effect of CO 2 on O 2 consumption rate depended on 1-MCP pretreatment, while the period of CO 2 exposure did not have a statistically signi cant effect (Fig. 2). Application of CO 2 inhibited O 2 consumption rate of the pears without 1-MCP pretreatment, but no statistical signi cant differences were observed between the various

4 1540 de Wild et al. Fig. 1. Ethylene production rate (pmol kg ±1 s ±1 ) at 2 C of pears without 1-MCP pretreatment (A) and with 1-MCP pretreatment (B) during 8 d exposure to various CO 2 partial pressures (experiment 1). CO 2 partial pressures (Fig. 2A; O 2 consumption data are means over days). For 1-MCP-treated pears none of the applied CO 2 partial pressures signi cantly inhibited O 2 consumption rate compared to 0 kpa CO 2 (Fig. 2B). Effect of 1-MCP and CO 2 during preclimacteric and climacteric stage (experiment 2) The ethylene production rate of pears (harvest date 6 September) at 20 kpa O 2 had greatly increased from 0.23 pmol kg ±1 s ±1 shortly after harvest to more than 30 pmol kg ±1 s ±1 after 13 and 25 weeks of storage. The large differences in ethylene production rate between control pears directly after harvest and after storage were con rmed by the differences in V m values as calculated by the model (Tables 1, 2). For the relationship between ethylene production and O 2 partial pressures of pears after harvest, equation 1 with h=1 was used (Table 1). After 13 and 25 weeks of storage, h was estimated (Table 2; Fig. 3). Treatment with 1-MCP stimulated ethylene production of freshly harvested pears at 20 kpa O 2 while it strongly inhibited ethylene production of pears after 13 weeks of storage. Inhibition was less strong after 25 weeks. This was again re ected in calculated V C 2H 4 m values (Tables 1, 2). Some remarkable effects of CO 2 on kinetic parameters as calculated by the model were found. Regarding Fig. 2. O 2 consumption rate (nmol kg ±1 s ±1 )at2 C of pears without 1-MCP pretreatment (A) and with 1-MCP pretreatment (B) at various CO 2 partial pressures (experiment 1). Data are means of several days. Means with different letters are signi cantly different. measurements directly after harvest, CO 2 did not affect V C 2H 4 m values of pears from harvest dates 23 August and 30 August, but inhibited V C 2H 4 m values of pears from later harvest dates (Table 1). However, after 1-MCP treatment, CO 2 also inhibited V C 2H 4 m values on the rst two harvest dates. Also, a strikingly different effect of CO 2 was found between the two storage periods. Although the V C 2H 4 m values of untreated fruits were similar between these periods, additional CO 2 strongly inhibited V C 2H 4 m values after 13 weeks storage, but had no effect after 25 weeks storage. Remarkably, after 1-MCP treatment, CO 2 had an inhibiting effect in both cases. The estimated K C 2H 4 values had, in general, high standard errors (Tables 1, 2). In pears without 1-MCP pretreatment, CO 2 only had a clear effect on K C 2H 4 values at harvest day 6 September. After 1-MCP pretreatment, CO 2 clearly affected K C 2H 4 values of pears from the rst three harvest days. Discussion The observed stimulation of ethylene production by 1- MCP shortly after harvest re ects the phase of autoinhibition of ethylene production by ethylene (preclimacteric stage), while the observed inhibition by 1-MCP after prolonged storage showed that pears were in the phase with autocatalytic ethylene production (climacteric stage). This change to the climacteric stage was also indicated by the increase in absolute level of ethylene production. The

5 CO 2 action on ethylene biosynthesis in pear 1541 Table 1. Results of the regression analysis for ethylene production of pears directly after harvest (experiment 2) r 2 =percentage variance accounted for, V C2H4 m the maximum ethylene production rate (pmol kg ±1 s ±1 ) and K C2H4 = Michaelis±Menten constant for ethylene production (kpa), Est=estimated values, SE=standard error. Treatment r 2 V C2H4 m K C2H4 Est SE Est SE Harvest 23 August Control CO MCP MCP+CO Harvest 30 August Control CO MCP MCP+CO Harvest 6 September Control CO MCP MCP+CO Harvest 13 September Control CO MCP MCP+CO Harvest 20 September Control CO MCP MCP+CO Table 2. Results of the regression analysis for ethylene production of pears (harvest date 6 September) after 13 and 25 weeks of storage (experiment 2) r 2 = percentage variance accounted for, V C2H4 m = the maximum ethylene production rate (pmol kg ±1 s ±1 ), K C2H4 is the oxygen partial pressure for half maximum ethylene production (kpa), h is the Hill coef cient, Est= stimated values, SE=standard error. r 2 V C2H4 m K C2H4 Est SE Est SE Est SE 13 weeks storage Control CO MCP a 1-MCP+CO weeks storage Control CO MCP MCP+CO h a Model used with h=1. basal low rate of ethylene production in the rst stage is often referred to as system 1 ethylene, while system 2 is the high rate of ethylene production, during ripening (McMurchie et al., 1972). In system 1, negative feedback regulated genes are involved leading to auto-inhibitory ethylene production while in system 2 positive feedback regulated genes are involved leading to auto-stimulatory ethylene production (Nakatsuka et al., 1998; Barry et al., 2000). The rise in ethylene production of untreated pears between the ve subsequent harvest dates indicated the start of system 2 (Table 1). The inhibitory effect of 1-MCP on autocatalytic ethylene production was less strong after 25 weeks compared tot 13 weeks storage (experiment 2). This may

6 1542 de Wild et al. Fig. 3. Ethylene production rate (pmol kg ±1 s ±1 )at2 C of pears after 13 weeks of storage (A) and after 25 weeks of storage (B) (experiment 2). In both cases pears are without and with 1-MCP pretreatment and exposed to several O 2 partial pressures in combination with 0 kpa CO 2 and 5 kpa CO 2. The symbols represent the mean values. The curves are tted using the parameters of the ethylene production model. be due to changes in ACC synthase and/or ACC oxidase gene expression. Both ACC synthase and ACC oxidase are encoded by multigene families and ACC synthase and ACC oxidase gene family members are differentially expressed in different stages of development (Fluhr and Mattoo, 1996; Nakatsuka et al., 1998; Barry et al., 2000). 1-MCP is known to affect the expression of the members of the gene families for ACC synthase and ACC oxidase differently (Nakatsuka et al., 1998). Nakatsuka et al. (1998) also demonstrated the existence of certain genes that were expressed in tomato fruit throughout development and ripening, irrespective of treatment with 1-MCP or propylene (a substitute for ethylene), thus irrespective of the mode of feedback regulation. The presence of such genes can explain why ethylene production of stored pears in the present research could not be totally reduced by 1- MCP. The smaller effect of 1-MCP on ethylene production after 25 weeks storage compared to 13 weeks storage indicated a gene expression that was less sensitive to 1- MCP. There are various clues that the effect of CO 2 did not operate via the EBP binding site. In experiment 1, pears were used at which the maximum reduction of ethylene production by 1-MCP had been reached. Elevated CO 2 was able to inhibit ethylene production of the 1-MCP pretreated pears to an extent very similar to untreated pears. It can be concluded that CO 2 is still able to inhibit ethylene production when 1-MCP had already reached its maximal effect. The fact that CO 2 is still able to inhibit ethylene production of 1-MCP treated pears con rmed observations by De Wild et al. (1999). As 1-MCP blocks the EBP binding sites (Sisler and Serek, 1997) it is unlikely that CO 2 was able to inhibit ethylene production through an effect on ethylene perception. Another indication that the effect of CO 2 was not via the EBP binding site was observed in experiment 2. As in experiment 1, CO 2 still had a large effect after 1-MCP treatment of stored pears (13 and 25 weeks storage). Additionally, in the case of 25 weeks storage, CO 2 only had an effect after 1-MCP treatment. Another indication is given by the fact that ethylene production directly after harvest was stimulated by 1-MCP, but unaffected or inhibited by CO 2 which points to different action sites for both molecules. This is consistent with experiments by AB Bleecker (personal communication), who found that there was no effect of CO 2 on 14 C 2 H 4 binding on the ETR1 receptor expressed in yeast. Also experiments with tulip bulbs (De Wild et al., 2002) showed that competition between ethylene and CO 2 at the receptor binding-site was unlikely. Effects of CO 2 on various intermediates and enzymes, and on gene expression involved in ethylene biosynthesis have been found (Mathooko, 1996a; Rothan et al., 1997). However, it must be taken into account that an initial effect by CO 2 on one certain point in the ethylene biosynthetic route can result in effects elsewhere through feedback regulation by ethylene. Thus, the primary action site of CO 2 remained unclear. By using 1-MCP in the current experiments, this feedback is prevented. As ATP is involved in the conversion of ACC to ethylene (Apelbaum et al., 1981; Gorny and Kader, 1996), the CO 2 effect on ethylene production may operate via the inhibition of respiration. The inverse relationship has also been suggested. Kubo et al. (1990) stated that respiratory responses to high CO 2 might be mediated by the effect of CO 2 on ethylene biosynthesis and/or action. In the present research, the inhibition of ethylene production by CO 2 was not always accompanied by a reduction of O 2 consumption (respiration). Differences in ethylene production in response to various CO 2 partial pressures were not re ected in differences in respiration. It can be concluded

7 that there was no clear relationship between the inhibition of ethylene production and the inhibition of respiration. The effect of CO 2 on ethylene production depended on the applied CO 2 partial pressure and on the duration of exposure. Elevated CO 2 initially stimulated ethylene production (experiment 1, day 1), which may re ect a fast direct effect of CO 2 on ACC oxidase. Later (days 5 and 8) CO 2 inhibited ethylene production with the exception of 1 kpa CO 2. This points to a relatively slow indirect effect of CO 2 regarding inhibiting properties, for instance, an effect on gene regulation. It is unlikely that this relatively slow effect of CO 2 could have been due to delayed diffusion of CO 2 into the cell sap considering that the effect of CO 2 on O 2 consumption was already in steadystate at day 1. An initial stimulation of ethylene production followed by inhibition by CO 2 has been shown before for pear (De Wild et al., 1999). Also stimulation by 1 kpa CO 2, but inhibition by higher CO 2 partial pressures con rmed earlier observations on pear (Chavez-Franco and Kader, 1993). The effect of CO 2 also depended on ripening stage and 1-MCP pretreatment. There was no inhibition in the earlier stages ( rst two harvests). During the autocatalytic stage, CO 2 clearly inhibited after 13 weeks, but had only a small effect after 25 weeks of storage. After 25 weeks of storage CO 2 clearly inhibited ethylene production after 1-MCP pretreatment. A possible explanation for these observations is that CO 2 may affect expression of members of the ACC synthase and/or ACC oxidase gene family differently. The expression of the different members depends on ripening stage and on 1-MCP pretreatment (Fluhr and Mattoo, 1996; Nakatsuka et al., 1998; Barry et al., 2000). An indication that CO 2 can affect gene expression, but not via affecting ethylene binding, was shown in preclimacteric tomato fruits, where CO 2 blocked the expression of both ethylene-dependent and also of ethylene-independent ripening-associated genes (Rothan et al., 1997). In peach, the abundance of ACC synthase member PP-ACS1 after wounding was blocked by CO 2, but stimulated by 1-MCP (Mathooko et al., 2001). The ethylene production rate in relation to external O 2 partial pressures describes the real (not the maximum) activity of ACC oxidase in response to O 2 as substrate. Thus, the parameter values of the ethylene production model give information about ACC oxidase. A change in the parameter values (e.g. by CO 2 ) may, besides a direct effect on the ACC oxidase enzyme, also re ect a change in ACC oxidase content or in substrates other than O 2.A higher K mo2 value means a lower af nity of ACC oxidase for O 2. This af nity can be in uenced by other substrates. Yip et al. (1988) showed in in vivo experiments that the K mo2 value increased as the ACC concentration decreased. If the af nity of ACC oxidase for O 2 depends only on ACC concentration, the model parameters of the current experiments give the following indications about the inhibition site of CO 2. (1) In several cases CO 2 inhibited ethylene production. With some exceptions directly after harvest (6 September without 1-MCP pretreatment and 23 August, 30 August and 6 September with 1-MCP pretreatment) treatment with CO 2 never clearly affected K mo2 CO 2 action on ethylene biosynthesis in pear 1543 or h values. This would mean that CO 2 did not affect the level of ACC or other substrates. This con rms earlier observations that elevated CO 2 inhibited ethylene production without affecting ACC content in intact fruit (Li et al., 1983; Cheverry et al., 1988; Rothan and Nicolas, 1994). Observations point to the formation of ACC and to ACC oxidase as the inhibition site for CO 2. (2) In some cases CO 2 did have a clear effect on K mo2 values. This was the case both for untreated and for 1-MCP treated fruits. However, the fact that the K mo2 value decreased due to CO 2 suggests an increase in ACC. This points to ACC oxidase as the inhibition site for CO 2. ACC oxidase as the inhibition site for CO 2 could be explained by a diminishing effect of CO 2 on ACC oxidase content. A direct inhibiting effect of CO 2 on ACC oxidase is unlikely as CO 2 is a cofactor for ACC oxidase (Dong et al., 1992). Also the response of ethylene production to CO 2 was relatively slow compared to respiration (experiment 1). However, it can not be excluded that under certain circumstances CO 2 can directly inhibit the enzyme activity, depending on substrate levels. Rothan and Nicolas (1994) found that CO 2 could inhibit ethylene production only at low ACC concentration. In experiment 2 it is likely that the ACC content increased during storage under low O 2 as has been shown for apples (Li et al., 1983), resulting in a smaller effect of CO 2. Also 1-MCP is likely to change substrate levels, which may subsequently cause an altered effect of CO 2. Conclusions The effect of CO 2 on ethylene production of pear depended on exposure time, CO 2 level, ripening stage, and 1-MCP pretreatment. The inhibiting effect of CO 2 on ethylene production did not operate via the EBP binding site. Respiration was not involved in the inhibitory effect of CO 2. Kinetic parameters derived from ethylene production models based on measurements in vivo point to the conversion from ACC to ethylene by ACC oxidase as a possible site for CO 2 inhibition. Acknowledgements The authors would like to thank CLJ Roelofsen and MG Staal for technical assistance, LACJ Voesenek for critically reading the manuscript, and MG Sanders for the stimulating discussions. The research presented has been funded in part by The Ministry of LNV, The Netherlands.

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