Journal of Experimental Botany, Vol. 34, No 146, pp. 1207-1212, September 1983 Initiation of Rapid Ethylene Synthesis by Apple and Pear Fruits in Relation to Storage Temperature MICHAEL KNEE, NORMAN E. LOONEY 1, STEPHEN G. S. HATFIELI>AND STEPHEN M. SMITH East Mailing Research Station, Maidstone, Kent, ME19 6BJ, UJC. Received 15 February 1983 ABSTRACT The influence of storage temperature on the onset of rapid ethylene production was investigated for fruits of Conference pear (Pyrus communis L.) and five cultivars of apple (Malus domestica Borkh.). The time taken from harvest to rapid ethylene production was shorter and more uniform at 3 C than at 18-20 C for Conference pears and Golden Delicious apples. Increases in internal ethylene concentration, 1-amino cyclopropane-1-carboxylie acid concentration and ethylene production were simultaneous in Golden Delicious apples at 3 C. When Golden Delicious apples were held at 3 C for 48 h and then kept at 20 C the mean time of onset of ethylene production was similar to that for apples held continuously at 20 C. However, two periods of 48 h at 3 C caused earlier ethylene production. Conversely, ethylene production at 3 C was delayed by transfer to 20 C for two periods of 48 h. Cox's Orange Pippin and other apple cultivars tended to show more synchronous ethylene production at 3 C than at higher temperatures but the mean time of onset was either unaffected by temperature or slightly delayed at lower temperature. Acceleration of the onset of ethylene production by low temperature was never observed in Cox's Orange Pippin apples harvested at weekly intervals from 10 August to 17 September. Key words: Ethylene; Storage temperature; Pyrus communis; Malus domestica. INTRODUCTION Although refrigeration is the most important method of retarding post-harvest deterioration of fruits and vegetables, low temperature does not invariably suppress physiological change. Exposure to low temperature stimulates ethylene synthesis in pears, both on the tree (Wang, Mellenthin, and Hansen, 1971) and when detached (Looney, 1972; Sfakiotakis and Dilley, 1974). This accounts for the fact that many pear cultivars require a chilling treatment to induce ripening (Ulrich, 1961). Streif (1976) studied the effects of night temperature on Cox's Orange Pippin apple trees; he found that lower night temperatures resulted in earlier starch degradation and an earlier rise in ethylene production in the Fruit. This suggests that apple fruits could respond similarly to pears, but effects on the tree leading indirectly to fruit responses are also possible. 1 Permanent address: Agriculture Canada Research Station, Summerland, British Columbia, Canada, V0H 1Z0.
1208 Knee et al. Temperature and Ethylene Synthesis in Fruits We have studied the effects of temperature on ethylene production by five apple cultivars with a single pear cultivar for comparison. The rise in ethylene production is an experimentally convenient marker of the occurrence of the respiration climacteric (Reid, Rhodes, and Hulme, 1973). All measurements were made on individual fruits; composite samples were avoided because of the influence of early ripening fruits on others in the sample (Kidd and West, 1934; Knee, Smith, and Johnson, 1983). MATERIALS AND METHODS Fruit All fruit was harvested from mature trees growing at East Mailing Research Station. Conference pears (Pyrus communis L.) were grown on Quince C rootstock. The apple (Malus domestica Borkh.) rootstocks were as follows: Greensleeves on M.7, Bramley's Seedling on MM. 104, Cox's Orange Pippin on M.26, Golden Delicious and James Grieve on M.9. Treatments Immediately after harvest fruits were placed individually in 500 ml glass containers in a room at the appropriate temperature maintained within +0-5 C. Air was supplied to each container, normally at a flow rate of 0-5 1 h~', regulated by a capillary tube and barostat. Background ethylene was removed by passage through a tube containing saturated aqueous potassium permanganate on an expanded mica support Ethylene Gas samples (0-5 ml or 1-0 ml) were taken daily at the outlet of the container and injected into a gas chromatograph with a column (30 cm x 0-2 cm) of alumina (80-100 mesh) at 80 C with nitrogen carrier at 20 ml min~'. Analyses were calibrated by reference to a standard mixture and the detection limit was usually 0-005 /A I" 1. Internal ethylene concentrations were measured by insertion of a hypodermic needle to the core cavity and immediate extraction of a gas sample with a syringe. Ethylene was analysed as described above and no further measurements of ethylene production were made after this procedure. 1-Amino cyclopropane-1-carboxylic acid (ACC) Concentrations of ACC in whole individual apples were estimated by the technique of Lizada and Yang (1979). Analysis of data The onset of ethylene producdon was defined as the day on which it first exceeded a particular rate of production, usually 0-1 fi\ kg" 1 h~', and was followed by a sustained rise. The number of days from the beginning of the experiment to the onset was recorded for each of the fruits (usually 8) in a treatment. The mean time of onset was calculated. Variation in this time has been expressed as a 95% quartile; because it is derived from the variance of all data it is a more reliable estimate of the spread of times of onset than a record of first and last apples. It represents the time interval on either side of the mean in which 95% of the fruits would have been expected to commence rapid ethylene production. RESULTS Progress of ethylene production An example of the primary data from which later results are derived is shown in Fig. 1. At the beginning of all experiments ethylene production by fruits was at or below the limit of detection. After a variable time interval it began to increase by three or four orders of magnitude. The rate of increase and the final rate were directly related to temperature. The time of onset was defined so as to be early on the rise but clearly above the detection limit Effect of temperature Rapid ethylene production began earlier at 3 C than at 18 20 C in Conference pears and Golden Delicious apples (Table 1). The timing of ethylene production by Golden Delicious at 12 C relative to other temperatures was variable (Table 1) as was the delay in
Knee et al. Temperature and Ethylene Synthesis in Fruits 1209 100- B r 10- o o.i- 0.01-100- 10-1.0-0.1-0.01-0 10 20 0 10 Time (d) 20 30 FIG. 1. Time course of ethylene production for single Golden Delicious apples harvested 29/9/80 and stored at (A) 3 C or (B) 20 C. The horizontal bars show the 95% quartile on either side of the mean time when apples first showed a rate of production higher than 0-1 /il kg" 1 h~'. production at 18-20 C in different years (Tables 1 and 2). In four other apple cultivars the mean time of onset of ethylene production was relatively unresponsive to temperature but the timing for individual fruits was usually less variable at 3 C than at higher temperatures (Table 1). In most experiments constant temperature regimes were maintained but in one experiment the effect of changing temperatures was investigated. Golden Delicious apples were subjected to the following treatments immediately after harvest: 48 h at 3 C, then held at 20 C (initial 48 h); 48 h at 20 C, 48 h at 3 C then held at 20 C (48 h break); 48 h at 20 C, 48 h at 3 C, 48 h at 20 C, 48 h at 3 C then held at 20 C (2 x 48 h break); apples held mainly at 3 C were given similar timetables of exposure to 20 C. Only the 2 x 48 h break at 3 C caused earlier and more synchronous ethylene production relative to apples held continuously at 20 C (Table 2). Any period at 20 C seemed to retard subsequent ethylene production at 3 C although synchrony was not affected. A similar experiment with Cox apples showed no effects of this kind. TABLE 1. Mean time and variability of onset of ethylene production in apples and pears at different storage temperatures Figures in parenthesis show the variability in terms of the 95% quartile; *20 C, b 4 C, C 22 C. Fruit variety Conference pear Bramley apple Greensleeves apple James Grieve apple Golden Delicious apple Cox's Orange Pippin apple Harvest A a*a Gate 14/9/79 4/10/79 29/9/80 21/8/80 4/10/78 2/10/79 9/10/80 13/9/79 8/9/80 Time of onset after harvest (d) 4-9 (1-7) 10-8 (3-4) 7-3 (2-0) 8-9 (l-6) b 13-4 (2-6) 6-9 (11) 6-9 (2-2) 7-6 (1-4) 10-1 (2-4) 12 C 10-9(7-4) 9-6(4-6) 5-7(3-6) 19-4(11-7) 8-3 (4-9) 6-1(4.1) 8-9(9-8) 18 C 24-6(36-5)" 11 Cl 1-5)- 7-5(3-5) 7-3 (4-7) c 51-7(75-2) 23-7(15-7) 26-5 (23) 7-8(7-4) 7-5 (3-6)
1210 Knee et al. Temperature and Ethylene Synthesis in Fruits TABLE 2. Mean time after harvest (d) to onset of ethylene production by single Golden Delicious apples harvested 13/10/81 and exposed to alternating temperatures Figures in parenthesis show variability in terms of the 95% quartile. Normal temperature Exposure to alternate temperature (3 C or 20 ' C) None Initial 48 h 48 h Break 2x48h Break 20 C 12-9(10-1) 7-4(2-3) 11-3(12-4) 8(0) 13-6(9-4) 9(1-1) 7-3(3-1) 10(3-0) Internal ethylene andacc levels The rise in ethylene production by Golden Delicious apples at 3 C was matched by increases in ethylene concentration in the internal gas space of the fruit and by increases in the ethylene precursor, ACC; within the limits of accuracy and frequency of sampling all three changes occurred simultaneously (Fig. 2). Ethylene production, internal ethylene concentration and ACC levels remained low in apples at 18 C. Effect of temperature in relation to fruit development In most experiments the fruit was harvested at the normal date for storage. In one experiment Cox apples were harvested at weekly intervals from 50 d before the normal date. Fruits were held in an oxygen atmosphere as well as in air because of reports that this would enhance ethylene production (Sfakiotakis and Dilley, 1973). In this experiment ethylene production was measured on alternate days so that the estimate of the time of onset for individual apples was less precise; also shortage of equipment led to the discarding of some fruit samples before ethylene production had commenced. In spite of these limitations it was clear that, from the earliest harvest date, Cox apples in air or oxygen at 3 C began to produce ethylene later than those at 18 C (Table 3). 10 1.0- hu <s o.oi- V 10 1.0T" 0.1 o 0001 10 20 10 20 10 20 Time (d) Fio. 2. Ethylene production (A) internal concentration (B) and ACC levels (c) in Golden Delicious apples harvested 13/10/81 and stored at 3 C or 18 C. A A apples at 3 C; apples at 18 C. Each point represents the mean for three apples.
Knee et al. Temperature and Ethylene Synthesis in Fruits 1211 TABLE 3. Mean time after harvest (d) for onset of ethylene production by Cox apples in relation to harvest date and storage conditions Ethylene production was estimated on alternate days. The results represent the mean time for 10 apples when a rate of production higher than 0-25 ft\ kg" 1 h~' was first recorded. 95% quartiles were calculated from data for all harvest dates except those where ethylene production was not observed. Harvest date 10 August 13 August 20 August 28 August 3 September 10 September 17 September (95% quartile) Oxygen concentration 21% Temperature >13 >16 >18 >14 13-7 10-1 14-7 (8-7) 18 C 9-0 14-5 10-2 121 9-6 12-9 8-0 (5-4) 100% Temperature >13 >16 15-3 10-9 10-7 10-1 12-2 (5-8) 18 C 7-3 9-7 90 7-6 7-2 6-7 4-9 (4-8) DISCUSSION The promotion of ethylene synthesis in Conference pears by exposure to low temperature corroborates earlier observations on the cultivars Bartlett (Looney, 1972), Bosc (Sfakiotakis and Dilley, 1974), and Anjour (Wang et al., 1971): this seems to be a general response of Pyrus communis L. cultivars. Of the apple cultivars tested only Golden Delicious showed a clearly analogous response. However, the greater synchrony of ripening, which was often observed at 3 C by comparison with higher temperatures, suggests a general response to low temperature. Presumably some other process, favoured by high temperature, regulates the development of ethylene production in most apple cultivars. The relative rates of the processes favoured by high and low temperature in different cultivars would determine whether the onset of rapid ethylene production occurred sooner at 3 C or 20 C. The temperature experience of the fruit prior to harvest would be expected to influence its subsequent progress to rapid ethylene production in store. Indeed it seemed possible that Cox and other 'unresponsive' varieties were predisposed to ethylene production by the time of their normal harvest date. But Cox harvested early were no more responsive to low temperature than at the usual date. This implies that the effects observed by Streif (1976) were not a direct fruit response but were mediated via a response of the tree to low night temperatures. Enhanced ethylene production is a common response of fruits and vegetative parts of plants to low temperatures, and it is often associated with chilling injury (Cooper, Rasmussen, and Waldon, 1969; Wright, 1974). Often a burst of ethylene synthesis is seen on transfer of chilled tissue to higher temperatures. In cucumber fruits ACC accumulates at 2-5 C and is rapidly metabolized to ethylene at 25 C (Wang and Adams, 1982). However, with Golden Delicious apple ACC levels remained low at 3 C until ethylene synthesis itself began to accelerate. The chilling response has also been associated with loss of fluidity of cell membranes (Wright, 1974). Although apples do not generally show symptoms of chilling injury even after prolonged periods at 3 C, the phase transition temperatures observed for mitochondrial lipids from apple cultivars are generally above 5 C (McGlasson and Raison, 1973). This
1212 Knee et al. Temperature and Ethylene Synthesis in Fruits phase transition should be an immediate effect of chilling whereas rapid ethylene synthesis in apples began after several days at 3 C. This examination of apple cultivars has shown that at least one commercially important cultivar, Golden Delicious, shows a response which had previously been known only among pears. Other apple cultivars should be tested for their response; this could be critically important for successful application of recent developments in storage technology. The removal of ethylene from storage chambers by chemical means (Liu, 1979) or by use of hypobaric conditions (Lougheed, Murr, and Berard, 1978) and the rapid establishment of controlled atmosphere conditions (Lau and Looney, 1982) are all intended to delay the onset or minimize the effects of rapid ethylene production. Furthermore, this aspect of fruit physiology should be considered in attempts to relate fruit development to weather parameters. However, the complexity of the response of fruit and tree to temperature suggests that equally complex models will have to be considered. ACKNOWLEDGEMENTS We acknowledge the statistical advice given by D. A. Holland and technical assistance by Mrs. J. Perkins and the late Mrs. J. T. Cockburn. LITERATURE CITED COOPER, W. C, RASMUSSEN, G. K., and WALDON, E. S., 1969. Ethylene evolution stimulated by chilling in Citrus and Persea spp. Plant Physiology, 44, 1194-6. KIDD, F., and WEST, C, 1934. The influence of the composition of the atmosphere upon the incidence of the climacteric in apples. Report of the Food Investigation Board for 1933, 51-7. KNEE, M., SMITH, S. M., and JOHNSON, D. S., 1983. Comparison of methods for estimating the onset of the respiration climacteric in unpicked apples. Journal of Horticultural Science (in press). LAU, O. L., and LOONEY, N. E., 1982. Improvement of fruit firmness and acidity in controlledatmosphere-stored 'Golden Delicious' apples by a rapid O 2 reduction procedure. Journal of the American Society of Horticultural Science, 107,531 4. Liu, F. W., 1979. Interaction of daminozide. harvesting date and ethylene in CA storage on Mclntosh apple quality. Ibid. 104, 599-601. LIZADA, M. C. C, and YANG, S. F., 1979. A simple and sensitive assay for 1-amino cyclopropane- 1-carboxylic acid. Analytical Biochemistry, 100, 140-5. LOONEY, N. E., 1972. Interaction of harvest maturity, cold storage and two growth regulators on ripening of Bartlett pears. Journal of the American Society of Horticultural Science, 97,81-3. LOUGHEED, E. C, MURR, D. P., and BERARD, L., 1978. Low pressure storage for horticultural crops. Horticultural Science, 13, 21-7. MCGLASSON, W. B., and RAISON, J. K., 1973. Occurrence of a temperature-induced phase transition in mitochondria isolated from apple fruit. Plant Physiology, 52,390-2. REID, M. S., RHODES, M. J. C, and HULME, A. C, 1973. Changes in ethylene and CO 2 during the ripening of apples. Journalofthe Science of Food and Agriculture, 24,971-9. SFAKIOTAKIS, E. M., and DILLEY, D. R., 1973. Induction of autocatalytic ethylene production in apple fruits by propylene in relation to maturity and oxygen. Journal of the American Society of Horticultural Science, 98, 504-8. 1974. Induction of ethylene production in 'Bosc' pears by postharvest cold stress. Horticultural Science, 9, 336-8. STREIF, J., 1976. Einfluss der Temperature auf verschiedene Reifemerkmale von apfeln. Erwerbsobstbau, 18, 168-71. ULRICH, R., 1961. Temperature and maturation: pears require preliminary cold treatment. Recent Advances in Botany, 1961, 11 72-6. WANG, C. Y., and ADAMS, D. O., 1982. Chilling-induced ethylene production in cucumbers (Cucumis sativus L.). Plant Physiology, 69,424-7. MELLENTHTN, W. M., and HANSEN, E., 1971. Effect of temperature on development of premature ripening in Bartlett pears. Journal of the American Society of Horticultural Science, 96, 122-5. WRIGHT, M., 1974. The effect of chilling on ethylene production, membrane permeability and water loss of leaves ofphaseolus vulgaris. Planta, 120,63-9.