'Cox's Orange Pippin'

Postharvest Biology and Technology 18 (2000) 215–225 www.elsevier.com/locate/postharvbio

Control of ethylene biosynthesis and softening in ‘Cox’s Orange Pippin’ apples during low-ethylene, low-oxygen storage

J.R. Stow *, C.J. Dover, P.M. Genge

Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK

Received 18 February 1999; accepted 30 November 1999

Abstract

The response of apples (Malus domestica Borkh.) cv. Cox’s Orange Pippin (‘Cox’) to low ethylene, controlled atmosphere (CA) storage was examined in two seasons. The content of 1-aminocyclopropane-1-carboxylic acid (ACC) and the activity of ACC oxidase (measured as the capacity for formation of ethylene from exogenous ACC) in cortical tissue of apple fruits from low ethylene containers, remained constant for about the first 20 weeks of storage, before increasing. Removal of ethylene from the storage atmosphere was more effective at reducing softening in B1% B CO2 +1.25% O2 after a pretreatment with 5% CO2 +16% O2 for 15 days, than storage in 1% CO2 +0.75% O2 without a pretreatment. Low ethylene storage maintained fruit firmness by inhibiting initiation, rather than reducing the rate of softening. Fruit was no softer after more than 28 weeks of storage when ethylene removal was discontinued after 12 weeks (1989–90) or 16 weeks (1990–91) than when the removal had been continuous throughout the storage period. It was concluded that, to obtain a benefit from ethylene removal, internal ethylene concentrations must be maintained below about 4 mmol m−3 (0.1ppm). This could not be achieved with ‘Cox’ apples in 1.25% O2 by removal of ethylene without a 5% CO2 pretreatment. The onset of autocatalytic ethylene production was not delayed appreciably by the removal of ethylene and was initiated after 2–5 weeks of storage, although the production rate of ethylene increased more slowly in a low ethylene atmosphere. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Apple; Storage; Softening; Ethylene; ACC; ACC oxidase

1. Introduction pal UK cultivar, ‘Cox’s Orange Pippin’ (‘Cox’).

Reducing O2 concentrations below 2% during Flesh firmness is a major quality attribute of storage at 3.5°C has been shown to lower the rate apple and is particularly important for the princi- of loss of firmness of ‘Cox’ apples, although there was little additional benefit below 1% and ethanol * Corresponding author. Tel: +44-1732-843833; fax: +44- 1732-849067. was formed and tainting occurred at 0.5% O2 E-mail address: [email protected] (J.R. Stow) (Stow, 1989). For a number of cultivars, retention

0925-5214/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0925-5214(99)00082-4 216 J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225 of ﬁrmness in stores where concentrations of of the fruit was allocated randomly to 64 15 kg ethylene were maintained below 44 mmol m−3 samples. Sixteen storage chambers in a store at was better than in those where ethylene was al- 10°C were each loaded with four boxes of ballast lowed to accumulate (Lidster et al., 1983; Lange and either seventeen 12-fruit samples (12 cham- and Fica, 1984; Knee and Tsantili, 1988; Dover, bers, time-course experiment) or twelve 12-fruit 1989; Stow and Genge, 1990; Van Schaik, 1996). samples (four chambers, transfer experiment). Af- It was not clear whether maintaining a low ter allowing fruit to cool overnight, chambers ethylene atmosphere delayed the onset, or reduced were sealed and CO2 and N2 were admitted into the rate of softening. If the mode of action is four time-course experiment chambers and the through a delay in the onset of softening, it four transfer experiment chambers, to generate an should be possible to reduce running costs of low atmosphere of 5% CO2 +16% O2 +balance N2 ethylene storage by switching off the ethylene (the ‘5% CO2’ storage method). This atmosphere removal system after softening has been initiated. was maintained for 15 days. The chambers were

The effectiveness of ethylene removal in con- then connected to containers of dry Ca(OH)2 to trolling softening depends on the maintenance of remove CO2, and N2 was admitted to reduce the a sufﬁciently low ethylene concentration inside the O2 to 1.25%. The remaining eight chambers in the fruit. The lower the ethylene concentration re- time-course experiment were connected to quired and the higher the production rate, the Ca(OH)2 containers immediately on loading and, larger the ethylene removal system must be and after cooling overnight, were sealed and the O2 the higher the capital and running costs (Dover, concentration was reduced progressively over 9

1989). However, it is not possible to determine days to 1.25% (‘1.25% O2’ storage method, four from the above reports the maximum acceptable chambers) or over 14 days to 0.75% O2 (‘0.75% level of ethylene. O2’ storage method, four chambers). These two To determine this for ‘Cox’ apples, fruits were time periods represent the time required to reduce stored in low and high ethylene conditions and O2 concentrations in good commercial stores. samples were removed at frequent intervals to Two of the chambers from each storage method measure internal ethylene concentration (IEC) and two transfer experiment chambers were con- and ﬁrmness. Earlier experiments had shown that nected to catalytic scrubbers of ethylene (Dover treatment with 5% CO2 for 15 days before storage and Stow, 1993) on loading (‘low ethylene’ treat- in 1.25% O2 was necessary for ethylene removal to ment). The atmosphere from the cabinets was reduce softening (Stow, 1988, 1990). As produc- pumped through the system for removing ethylene tion rate of ethylene was lowered by storage in at a ﬂow rate of5lh−1 per kg of fruit through-

0.75% O2 (Stow, 1989) a comparison was made of out storage, while ethylene was allowed to accu- storage in 1.25% O2 with a CO2 pretreatment and mulate in the other two chambers (‘high ethylene’ storage in 0.75% O2 with no pretreatment. treatment). Efﬁciency of conversion of ethylene always exceeded 95%. Fruit temperatures were reduced progressively to 3.5°C over 3 days from 2. Materials and methods loading in all chambers to represent cooling in good commercial stores. 2.1. Storage After the ﬁnal conditions were established, all containers were maintained at 3.5–4°C in less

‘Cox’ apples were picked on 11 September 1989 than 1% CO2 and at the O2 concentration re- (the start of the commercial harvesting period) quired (90.2%). Oxygen concentrations were from an orchard at HRI-East Malling. After maintained by a computer-based system that ad- −3 drenching in a mixture of benomyl (500g m mitted air into chambers when the O2 concentra- −3 a.i.) and CaCl2 (9000 g m , commercial flake) tion fell below that required. 232 random samples of 12 apples and 8 random The experimental procedures were repeated in samples of 80 apples were selected. The remainder the following season, with the following excep- J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225 217 tions: fruit was picked on 30 August, 1990 and 2.3. Ethylene determinations drenched in metalaxyl (100g m−3)+carbendazim −3 −3 (500g m )+20 kg m CaCl2; only the ‘5% Ethylene was measured using a gas chro- CO2’ storage method was used as this had re- matograph (Pye Unicam PU4500 with a Trivector sulted in the greatest firmness retention in the first computing integrator) fitted with a flame ioniza- year; the numbers of replicate containers were tion detector and a 300 mm long, 6 mm o.d. glass doubled to increase the precision of the experi- column filled with alumina maintained at 100°C ment. In addition, a catalytic converter with a while flushed with N2 as a carrier gas. To measure conversion rate of 1% of that fitted to the ‘low IEC, a 0.7 ml gas sample was removed from the ethylene’ chambers was placed in the return side core cavity of an apple, using a needle with the tip of the automated sampling system to reduce con- bent over to avoid blockage. The needle was tamination of ‘low ethylene’ chambers with replaced with one with a straight tip and 0.5 ml of ethylene from ‘high ethylene’ chambers. Although the sample was injected directly into the gas this additional catalytic converter slightly reduced chromatograph. the accumulation of ethylene in the ‘high ethylene’ chambers, it allowed calculation of ethylene pro- 2.4. Estimation of ethylene-forming capacity duction rate in these chambers. 2.2. Assessment of fruit A 2 mm-thick slice was cut 4 mm in from the skin of each apple of a 10-apple sample. A 20 At intervals, one 12-fruit sample was removed mm-diameter disc was cut from each slice and from each chamber of the time-course experiment placed in a 30 ml vial with 0.5 ml of 12 mM ACC, through an ‘airlock’ in the lid. Nitrogen was 600 mM glycerol in 10 mM phosphate buffer, pH admitted to the chambers while sampling was in 7.0 (Cheverry et al., 1988). Preliminary experi- progress and all chambers were returned to their ments had indicated saturation in this system at required O2 condition within4hoffruit removal. 4–8 mM ACC. After allowing 1 h for the produc- The IEC in ten of the 12 fruits from each tion of wound ethylene to cease and for ethylene sample was measured immediately on removal in the intercellular spaces to dissipate, the vials from the chamber, followed by determination of were sealed. After a further hour, the concentra- ethylene-forming capacity (1990 experiment only), tion of ethylene within the vials was determined. background colour (using a Hunter Colormeter in This estimation of ethylene-forming capacity was the L,a,b mode (1989 experiment only)) and flesh considered to be a measure of ACC oxidase firmness, using a motorised penetrometer (Top- activity. ping, 1981) fitted with an 8 mm diameter probe. Internal quality was monitored by visual in- 2.5. Ethanol and ethyl acetate determination spection of fruit cut equatorially and samples for the determination of acidity were taken by remov- Samples of chamber atmosphere were injected ing a 10 mm-thick equatorial slice and removing into a gas chromatograph (Pye Unicam PU4500) two tissue plugs from just below the peel with a 7 fitted with a flame ionization detector anda2m mm diameter cork borer. The yield from each long, 6 mm o.d. column of 5% OV3 on Gas 12-fruit sample was about 50 g of tissue, which Chrom G maintained at 50°C. The carrier gas was was frozen at −20°C until required. N2 saturated with water vapour. In the transfer experiment, samples of fruit were moved at intervals from a ‘low ethylene’ to a 2.6. Acidity determination ‘high ethylene’ chamber (the same chamber each time), to simulate switching off the ethylene Frozen tissue was allowed to thaw before ap- scrubber. No assessments were carried out on this proximately 40 g was weighed and homogenized fruit until after 29 (1989 experiment) or 33 (1990 in 50 ml of distilled water. The final volume was experiment) weeks of storage. noted and 5 ml of homogenate were diluted to 218 J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225

100 ml with distilled water before titration to pH 3. Results 8.1 with 0.1 M NaOH (Smith, 1985). 3.1. Concentrations of ethylene in storage chambers

2.7. Soluble pectin and ACC analysis In 1989–90, for the first 8 weeks of storage in chambers without converters of ethylene, the con- Soluble pectin and 1-aminocyclopropane-1-car- centrations of ethylene increased at similar rates. boxylic acid (ACC) were extracted as described by Thereafter concentrations in ‘0.75% O2’ remained m −3 Bartley et al., (1982) except that the extraction constant at about 6600 mol m . In chambers of medium was 0.1 M HCl. Soluble pectin was pre- the other two storage methods (‘5% CO2’ and cipitated from 80% acetone and estimated as an- ‘1.25% O2’), accumulation continued, reaching a m −3 hydro-galacturonic acid using 3-phenyl-phenol maximum of 26 000 mol m after 24 weeks, (Blumenkrantz and Asboe-Hansen, 1973). ACC and then declined over the next 8 weeks to 17 600 m −3 was estimated directly from the extract by the mol m . In 1990–91, in ‘high ethylene’ cham- m −3 method of Lizada and Yang (1979). bers, concentrations rose to about 40 mol m within 7 days of loading, increasing to about 2200 mmol m−3 at 18 weeks and 2600–3100 mmol m−3 after 32 weeks. The slower rise and generally 2.8. Rates of respiration and production of lower concentration of ethylene in 1990–91 was a ethylene consequence of the removal of ethylene by the automatic sampling system (see Section 2.1). At the end of the time-course experiment, ap- In both years, the concentrations were lower in proximately 1.5 kg of ballast apples from each ‘low ethylene’ chambers than in ‘high ethylene’ storage chamber were placed in a sealed jar at chambers, reaching a maximum of less than 35 3 −1 mmol m− in ‘low ethylene’ chambers by the end 3.5°C flushed with 5 l h of 1.25% O2 or 0.75% of the storage period (Fig. 1A). In both years, the O2, as appropriate, and the content of ethylene concentration of ethylene started to increase after and CO2 of the effluent was monitored.

Fig. 1. Ethylene concentration in the storage atmosphere (A) and internal ethylene concentration (B), of ‘Cox’ apples stored in ‘low B ethylene’ chambers using different methods. 1989–90: ‘1.25% O2’ method: 1% CO2 +1.25% O2 throughout ( ); ‘5% CO2’ B B method: 5% CO2 +16% O2 for 15 days followed by 1% CO2 +1.25% O2 ( ); ‘0.75% O2’ method: 1% CO2 +0.75% O2 B throughout ( ). 1990–91: ‘5% CO2’ method: 5% CO2 +16% O2 for 15 days followed by 1% CO2 +1.25% O2 ( ). Upper vertical bar: SED ((a) 81 df, (b) 48 df), 1989–90. Lower vertical bar: SED ((a) 78 df, (b) 66 df), 1990–91. J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225 219

Fig. 2. Distribution of internal ethylene in fruits stored in ‘low ethylene’ chambers. Fruit was stored in 5% CO2 +16% O2 for 15 B days followed by 1% CO2 +1.25% O2. 1989–90 ( ); 1990–91( ). Dotted lines: left-hand axis. Solid lines: right-hand axis.

about 5 weeks where the 5% CO2 treatment had in 1990–91, this rise was delayed for about 14 been applied and after about 2–3 weeks where it weeks (Fig. 1B). By the end of storage, the aver- had not. In 1989–90 the rate of accumulation was age IEC was 260–1100 mmol m−3. There was also similar in ‘1.25% O2’ and ‘0.75% O2’ and slowest a seasonal variation in the change in the distribu- in ‘5% CO2’, for about the ﬁrst 10 weeks of tion of internal ethylene. In 1989–90 95% of fruits storage, after which the concentration in ‘0.75% stored using the ‘5%’ ‘low ethylene’ method had m −3 O2’ chambers increased very little and at the end an IEC of less than 4 mol m after 5 weeks. of storage was less than half the concentration in After a further 13 weeks all fruits had an IEC in m −3 the ‘5% CO2’ chambers. excess of 22 mol m (Fig. 2). Although, in 1990–91, fruit IEC remained predominantly below 4 mmol m−3 for longer, this transition from 3.2. Internal ethylene low IEC to high IEC took only 6 weeks.

In ‘high ethylene’ chambers in 1989–90 and 1990–91, the IEC of fruit increased from week 1 3.3. Production of ethylene with a pattern similar to that of the accumulation in the chamber atmosphere. The IEC was slightly Ethylene production in ‘low ethylene’ ‘5% CO2’ higher than concentration in the chamber atmo- fruit increased from 0.4 to 40 pmol kg−1 s−1 over sphere in both years (data not shown). the storage period in 1989—90 and from 0.3 to In 1989–90 the IEC of apples from ‘low 19.5 pmol kg−1 s−1 in 1990–91 (Fig. 3). In ethylene’ chambers increased after 3–5 weeks but, 1990–91 ethylene production in ‘high ethylene’ 220 J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225

‘5% CO2’ samples increased from 0.85 to 61 pmol 3.4. Ethylene–forming capacity and ACC content kg−1 s−1. Although in the ‘high ethylene’ ‘5%

CO2’ and ‘low ethylene’ ‘5% CO2’ samples a The capacity for forming ethylene from exoge- maximum rate was observed after about 25 weeks nous ACC (a measure of ACC oxidase activity) in of storage, the rate was lower in ‘low ethylene’ ‘low ethylene’ ‘5% CO2’ apples remained constant fruit. for the ﬁrst 22 weeks of storage, then increased

Fig. 3. Ethylene production rate of ‘Cox’ apples stored using different methods. 1989–90: ‘low ethylene’ only, ‘1.25% O2’ method: B B 1% CO2 +1.25% O2 throughout ( ); ‘5% CO2’ method: 5% CO2 +16% O2 for 15 days followed by 1% CO2 +1.25% O2 ( ); B ‘0.75% O2’ method: 1% CO2 +0.75% O2 throughout ( ). Vertical bar: SED (48 df). 1990–91: ‘high ethylene’ ‘5% CO2’ method

( ); ‘low ethylene’, ‘5% CO2’ method ( ). Upper vertical bar: SED (66 df), ‘high ethylene’. Lower vertical bar: SED (66 df), ‘low ethylene’.

Fig. 4. Ethylene-forming capacity (A) and the content of ACC in fruit ﬂesh (B) of ‘Cox’ apples during storage in high and low B ethylene. Fruit was held in 5% CO2 +16% O2 for 15 days followed by 1% CO2 +1.25% O2. 1990–91: ‘high ethylene’ ( ); ‘low ethylene’ ( ). Vertical bar is SED ((A) 51 df, (B) 48 df). J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225 221

ally higher than in ‘low ethylene’ fruit. In ‘high

ethylene’ ‘5% CO2’ chambers, ACC content of fruit had increased above harvest levels after about 8 weeks of storage and reached a maximum after about 18 weeks (Fig. 4B). In ‘low ethylene’ fruit, concentrations remained at initial levels until 20 weeks had elapsed, then rose sud- denly to values similar to those of ‘high ethylene’ samples.

3.5. Firmness

3.5.1. Time–course experiment In 1989–90 there was little effect of the storage method on softening of ‘high ethylene’ samples and for clarity, only average firmness values Fig. 5. Firmness of ‘Cox’ apples, 1989–90, during storage for the three storage treatments are presented in using different methods. ‘High ethylene’: ( ) (average of the 3 Fig. 5. In samples from all chambers, there was B methods). ‘Low ethylene’: ‘1.25% O2’ method: 1% CO2 + an initial decline in firmness over the first 4 1.25% O2 throughout ( ); ‘5% CO2’ method: 5% CO2 +16% B weeks of storage. In ‘high ethylene’ chambers O2 for 15 days followed by 1% CO2 +1.25% O2 ( ); B this continued for about another 16 weeks, ‘0.75% O2’ method: 1% CO2 +0.75% O2 throughout ( ). Vertical bar is SED (96 df). when softening ceased. For the ‘low ethylene’

‘5% CO2’ fruit, ﬁrmness remained constant for 16 weeks following the initial decline, then de- creased until the experiment ended at 32 weeks.

The ‘1.25% O2’ and ‘0.75% O2’ ‘low ethylene’ fruit were intermediate in softening between the

‘high ethylene’ and ‘low ethylene’ ‘5% CO2’ samples. In 1990–91 a similar result was obtained (Fig. 6), with an initial decline in ﬁrmness in all samples, followed by a further decrease in ‘high ethylene’ samples and a cessation of softening in ‘low ethylene’ samples for a further 18 weeks before softening restarted.

3.5.2. Transfer experiment In the 1989–90 experiment, fruit held in ‘low ethylene’ storage for less than 12 weeks before transfer to ‘high ethylene’ storage were softer at Fig. 6. Firmness of ‘Cox’ apples, 1990–91, during storage in the end of storage (32 weeks) than those held in high and low ethylene. Fruit was stored in 5% CO2 +16% O2 B ‘low ethylene’ storage for 12 weeks or longer for 15 days followed by 1% CO2 +1.25% O2. ‘High ethylene’ ( ); ‘low ethylene’ ( ). Vertical bar is SED (64 df). before transfer (Fig. 7). A similar result was obtained in 1990–91, when no further beneﬁt of 7–fold by week 32 (Fig. 4A). Ethylene–forming continued ‘low ethylene’ storage was obtained capacity also increased in ‘high ethylene’ ‘5% after fruits had been held in a low ethylene at-

CO2’ apples after about 27 weeks, and was usu- mosphere for more than 16 weeks. 222 J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225

3.6. Soluble pectin

In ‘high ethylene’ chambers in 1990–91, soluble pectins of fruit increased above harvest levels after about 4 weeks from harvest and continued to do so for the next 12 weeks (Fig. 8). Soluble pectin in ‘low ethylene’ samples did not start to increase until after 22 weeks of storage. Where the firmness was less than 40 N, the concentration of soluble pectin was correlated negatively with firmness, with correlation coefficients (18 df) of − 0.63 for ‘low ethylene’ samples and −0.87 for ‘high ethylene’ samples. However, when treatments were combined, there was no overall correlation between soluble pectin and firmness.

3.7. Acidity Fig. 7. Firmness of ‘Cox’ apples at the end of storage, 29 (1989–90) or 32 (1990–91) weeks, following transfer from ‘low ethylene’ to ‘high ethylene’ at different times during storage. Fruit titratable acidity in 1989–90 was 10 g −1 Fruit was stored in 5% CO2 +16% O2 for 15 days followed by malic acid equivalent kg fresh weight at har- B 1% CO2 +1.25% O2. 1989–90 ( ); 1990–91 ( ). Upper vest; it declined linearly to 6 g after 32 weeks vertical bar is SED (27 df), 1990–91. Lower vertical bar is storage. Storage method or ethylene removal did SED (6 df), 1989–90. not affect acid loss (data not presented).

3.8. Ethanol and ethyl acetate

After 10 weeks of storage, the concentrations of ethanol and ethyl acetate in the atmosphere of

‘0.75% O2’ ‘high ethylene’ chambers was equivalent to 400 and 8 mg kg−1 fresh weight in the fruit ﬂesh, respectively. The concentrations of these compounds declined to 150 and 0.5 mg kg−1 fresh weight equivalent after 32 weeks. Nei- ther compound was detected in ‘low ethylene’

chambers or chambers maintained at 1.25% O2. However, the catalytic converters of ethylene oxi- dised ethanol and ethyl acetate in the vapour phase. 3.9. Respiration acti6ity

There was no effect of storage method or ethylene removal during storage on rate of pro-

duction of CO2 after storage for 32 weeks (Table Fig. 8. Soluble pectin in the ﬂesh of ‘Cox’ apples stored in 1). Overall, ‘low ethylene’ samples had a lower 1990–91 in high and low ethylene. Fruit was stored in 5% B B production rate of ethylene (P 0.05) and the CO2 +16% O2 for 15 days followed by 1% CO2 +1.25% lowest production rate was in samples from O2. ‘High ethylene’ ( ); ‘low ethylene’ ( ). Vertical bar is SED (46 df). ‘0.75% O2’. J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225 223

Table 1

Rates of production of CO2 and ethylene at 3.5°C by ‘Cox’s Orange Pippin’ apples after storage at 3.5°C for 32 weeks, 1989–90

−1 −1 −1 −1 Storage method Ethylene removal CO2 (nmol kg s ) Ethylene (pmol kg s )

‘1.25% O2’ Yes 14.6 45.1 No 14.6 58.6

‘5% CO2’ Yes 12.2 39.0 No 15.9 46.4

‘0.75% O2’ Yes 14.6 11.0 No13.4 19.5 SED (18 df) 0.98 6.69

3.10. Disorders and ground colour when the transfer was carried out later than 12 weeks (1989–90) or 16 weeks (1990–91), when Disorders were not found in 1989–90 but core concentration of ethylene in the ‘low ethylene’ flush, brownheart and flesh breakdown were ob- chambers was about 2 mmol m−3. It was con- served in 1990–91 after storage for 32 weeks. In cluded that switching off the converter after such ‘low ethylene’ samples, 20% of apples had devel- a concentration was reached was a means of oped core flush and 5% were affected by flesh reducing running costs while still retaining the breakdown. In ‘high ethylene’ samples, 5% had benefits of ‘low ethylene’ storage. brownheart and 10% flesh breakdown. These dis- The time course study of softening indicates orders were all in the ‘slight’ category and were that the effect of ethylene removal was predomi- not commercially significant. There were no ef- nantly to delay the onset of softening, rather than fects of storage method or ethylene removal on to reduce the rate of softening. The longer delay ground colour in 1989–90. Measurements were in the onset of softening in 1990–91 than in not made in 1990–91. 1989–90 is consistent with the more effective control of ethylene production by fruit stored in an atmosphere low in ethylene in 1990–91. The ear- 4. Discussion lier start but longer duration of the transition in distribution of IEC in 1989–90, suggests that the It had been considered that transferring fruit less effective control was a result of a greater from a ‘low ethylene’ to a ‘high ethylene’ chamber initial range in the rate of ethylene production for was equivalent to switching off the system for individual fruit rather than an overall increase in removing ethylene. It was calculated from mea- responsiveness to ethylene. Factors responsible sured production rates of ethylene by apples in for the onset of softening are uncertain. It is

‘low ethylene’ ‘5% CO2’ chambers that, if the possible that softening was initiated when the IEC converter of ethylene was switched off, concentra- reached a particular value. The linearity of re- tion of ethylene would have reached 44 mmol m−3 sponse to time in an atmosphere low in ethylene within three days early in storage and within one (transfer experiment) and the apparent abrupt day at the time of later fruit transfer. This rate of cessation of further benefit suggest that softening increase suggests that fruit would respond simi- is initiated when a threshold is reached and that larly to the effects of transfer and to the switching low ethylene slows, but does not inhibit, this off of the ethylene converter. process. From the transfer experiment, the time in Transferring fruit from a ‘low ethylene’ cham- ‘low ethylene’ storage after which no further ber to a ‘high ethylene’ chamber did not affect benefit of ethylene removal was obtained was firmness of fruit at the end of the storage period 8–12 weeks in 1989–90 and 14–16 weeks in 224 J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225

1990–91. At this time, the average IEC in ‘5% ethylene in ‘Cox’ apples can be reduced by in-

CO2’ ‘low ethylene’ apples were 4.4–13.2 and creasing the rate of removal of ethylene from 2.6–8.8 mmol m−3 respectively, for 1989–90 and storage containers, but this was only found to 1990–91 and the distribution of IEC was chang- reduce softening where ethylene production was ing from predominantly low (B4.4 mmol m−3)to reduced (Dover and Stow 1993). Any further im- predominantly high (\22 mmol m−3). Knee et al. provements must await the development of meth- (1983) found a range of IEC of 0.4–4.4 mmol ods for further reducing the rate of production of m−3 in apples on the tree just prior to a sustained ethylene. rise in the IEC. Thus it is possible that for ‘Cox’ Concentrations of soluble pectin in apple fruit apples in 1.25% O2, biochemical events that lead increase during softening (Knee and Bartley, to softening are initiated when IEC reaches about 1981) and it has been suggested that this is a 4.4 mmol m−3 and the first decline in the pen- result of the degradation of cell wall pectin (Bart- etrometer firmness value occurs about 8 weeks ley, 1978). Softening in this study did not corre- later. late well with soluble pectin even though the onset The effectiveness of maintaining firmness of of softening, at 22 weeks, in ‘low ethylene’ fruit ‘Cox’ apples in a CA store by removing ethylene coincided with an increase in soluble pectin. For thus seems to depend on the successful mainte- the first 5–6 weeks of storage in the 1990–91 nance of an IEC below about 4.4 mmol m−3.A experiment, fruit from the ‘high ethylene’ and positive feedback system is thought to function in ‘low ethylene’ containers softened, though little apples and other climacteric fruits, whereby change in soluble pectin was evident. Further, ethylene concentration in the fruit affects ACC when softening started in fruit from ‘low ethylene’ synthase (Bufler, 1984) and oxidase (Bufler, 1986) containers, the absolute concentration of soluble activity. This corresponds to the system 2 de- pectin was higher than for ‘high ethylene’ fruit at scribed by McMurchie et al. (1972). It has also a similar firmness. Knee and Sharples (1981) simi- been suggested that there is a two-phase receptor larly found a poor correlation between softening system in apples (Knee 1985), an hypothesis sup- and soluble pectin accumulation in ‘Cox’, and ported by studies on Arabidopsis mutants (Chang, Knee (1989) reported softening of Bramley apples 1996). Maintaining a low ethylene CA environ- without an increase in soluble pectin. ment should prevent the internal ethylene rising The lack of effect of storage in low ethylene on sufficiently to trigger autocatalytic production. the ground colour of apples has been reported This has been achieved with ‘Gloster 69’ apples previously (Knee, 1986; Stow, 1990; Stow and (Knee and Tsantili, 1988). However, it appears Genge, 1990) and Streif and Bangerth (1976) con- that in ‘Cox’, the positive feedback system is cluded that breakdown of chlorophyll in tomato operational shortly after harvest as, even in ‘low fruit is affected more by O2 than ethylene. ethylene’ samples, ethylene production increased From the data presented here it can be con- from 5 weeks after the start of storage, although cluded that removal of ethylene from the storage ACC did not increase until 15 weeks and atmosphere of ‘Cox’ apples delays the onset of ethylene-forming capacity did not increase until softening for about 20 weeks. This delay was only after 22 weeks. In 1990–91, storage in an atmo- achieved when the ethylene production rate was sphere low in ethylene reduced the gain of the reduced by treatment with 5% CO2 for 15 days at positive feedback system (slope of log ethylene the beginning of storage. Further, to obtain a production rate) until approximately 15 weeks, firmness benefit from storage in a low ethylene when the gain increased to a level similar to that atmosphere, in was necessary to maintain an IEC exhibited by ‘high ethylene’ fruit early in storage. of less than 4 mmol m−3, which corresponded to It was expected that the ‘low ethylene’ treatment an ethylene concentration in the storage atmo- should subsequently appear to have no further sphere of less than 2 mmol m−3. It is possible that effect on softening (transfer experiment, 1990– the failure of some other varieties to respond to 91). The rate of the accumulation of internal low-ethylene storage resulted from a lack of a J.R. Stow et al. / Posthar6est Biology and Technology 18 (2000) 215–225 225 similar reduction in ethylene production rate and NATO ASI Series, vol. H35, Springer-Verlag, Berlin, a consequent loss of control of IEC. Heidelberg, pp. 145–154. Knee, M., Bartley, I.M., 1981. 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