Effects of inhibitors on the carbon dioxide-stimulation of ethylene-forming enzyme activity in fruit of Japanese pear and apple

Postharvest Biokxjyand Techr~)gy ELSEVIER

Postharvest Biologyand Technology4 (1994) 13-21

Effects of inhibitors on the carbon dioxide-stimulation of ethylene-forming enzyme activity in fruit of Japanese pear and apple M.S. Tian a,b, E.W. Hewett ,,a R.E. Lill b a Department of Plant Science, Massey University, Palmerston North, New Zealand b Crop and Food Research Institute, Levin, New Zealand

(Accepted 17 June 1993)

Abstract

Cobalt ions (Co2+) at 1 mM inhibited ethylene-forming enzyme (EFE) activity and ethylene production in apple (Granny Smith) discs. Low concentrations (0.01 mM) of Co 2+ ions stimulated, but higher concentrations (>0.02 mM) inhibited EFE activity in discs of both Japanese pear (Hosui) and apple. CO2 stimulated EFE activity but did not reverse the inhibition caused by Co 2+ ions. AgNO3 inhibited ethylene production of fruit discs in Granny Smith apple. Low concentrations (<0.25 mM) of Ag + stimulated, but higher levels (0.5 and 1 mM) inhibited EFE activity in discs of both fruits. CO2 did not reverse the inhibitory effect of Ag + (1 raM). 2,5-Norbornadiene (NDE) at 0.5% (v/v) inhibited EFE activity in discs from both fruits. CO2 partially reversed the NDE inhibition in discs of Hosui and Granny Smith fruit stored at 1 4- I°C for a short period.The possible mechanisms of the interactions between CO2 and the inhibitors on EFE activity and ethylene receptors are discussed. Key words: Carbon dioxide; Cobalt ion; Ethylene action; EFE; Ethylene production; Apple;

Japanese pear; 2,5-Norbornadiene; Silver ion

1. I n t r o d u c t i o n

Carbon dioxide is thought to be involved in the regulation of ethylene biosynthesis and action in different ways: (a) it regulates ethylene synthesis by affecting the enzymes in the ethylene biosynthetic pathway (Buffer, 1986; Chaves et al., 1984; Cheverry et al., 1988; Philosoph-Hadas et al., 1986) and, (b) it influences ethylene action by competing (Burg and Burg, 1967; Sisler, 1979) with ethylene for the * Corresponding author. 0925-5214/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved. SSDI 0925-5214(93)E0038-F

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M.S. Tian et aL / Postharvest Biology and Technology 4 (1994) 13-21

binding site on the ethylene receptor, or by promoting the binding of ethylene to its binding site(s) (Thomas et al., 1984). There have been very few reports on the interaction between CO2 and Co 2+, Ag +, or 2,5-norbornadiene (NDE) on ethylene-forming enzyme (EFE) in fruit tissues. Cheverry et al. (1988) showed that the maximal inhibition of EFE activity in avocado fruit at the autocatalytic stage of ethylene production occurred in the presence of NDE alone, while less inhibition was measured with a mixture of NDE and COz. In the presence of NDE, ethylene production in tissues excised from climacteric avocado fruits was no longer autocatalytic, and COz in the absence of any autocatalytic ethylene production stimulated conversion of ACC to ethylene. Carbon dioxide stimulated EFE activity in discs of both Japanese pear (cv. Hosui) and apple (cv. Granny Smith) fruit (Tian et al., 1994). CO2 also stimulated EFE biosynthesis in discs of preclimacteric Granny Smith fruit, but not in Hosui fruit discs. Inhibitors of EFE activity, such as Co z+ (McGarvey and Christoffersen, 1992; Smith, et al., 1992; Yu and Yang, 1979), and those of ethylene action, such as Ag + ion and NDE (Veen, 1985, 1986 and 1987; Wang, 1987) are useful tools for testing mechanisms of synthesis and activity of ethylene biosynthesis enzymes and the regulation of ethylene production by ethylene receptors (Veen, 1987; Yang, 1985). To investigate the mechanism of COz action on EFE activity, the interaction of Co 2+ and COz was studied on a climacteric (Granny Smith) and nonclimacteric (Hosui) fruit. In addition the interactions between CO2 and two ethylene action inhibitors, Ag + and NDE, and EFE were tested to elucidate the effect of CO2 on the ethylene receptor binding sites. 2. Materials and methods

Japanese pear (Pyrus serotina cv. Hosui) and apple (Malus domestica Borkh. cv. Granny Smith) fruits were harvested from the Ruakura Research Orchard, Hamilton and the Fruit Crops Unit, Massey University, Palmerston North as previously reported (Tian et al., 1994). Harvest dates were 12 February, 1989 and 21 February, 1990 for pear and 25 April, 1989 and 1990 for apple. After harvest, Japanese pears were stored at 0°C for one night before air freighting to Palmerston North next morning. On arrival, fruit were sorted into various colour grades according to a colour chart (Suzuki, et al., 1981). Fruit of colour grade 2-3 and fresh weight (180 -4-5 g) were selected and then stored at 1 + I°C until ready for use. After harvest, apples in the range 178-192 g were selected and stored at 1-4-l°C until ready for use. Internal ethylene concentrations were not measurable at harvest in any experimental apples in the different seasons. The methods of preparation of discs, measurement of ethylene production, EFE activity and CO2 treatment (flushed with a gas mixture containing 20% CO2, 21% 02 and 59% N2) for both Hosui and Granny Smith fruit discs were the same as those used by Tian et al. (1994). Discs of both fruit were treated with Co 2+ (COC12) or Ag + (AgNO3) ions using the same method. To reduce possible variation of internal ion levels which might be caused by different tissue permeability to Co 2+ or Ag +, fruit discs were

M.S. Tian et al. / Postharvest Biology and Technology 4 (1994) 13-21

15

vacuum-infiltrated (a vacuum of 90 kPa was applied for 1 min then released; this procedure was carried out 3 times to ensure fuU penetration of the solution) with 3 ml of medium containing 0.4 M mannitol, 5 mM 1-aminocyclopropane-1carboxylic acid (ACC), 5 mM amino-oxyacetic acid (AOA) and various Co 2+ or Ag + concentrations. After vacuum-infiltration, fruit discs were dried on filter paper then six discs were sealed in each of 3 x 36 ml vials for 2 hours at 27°C before removal of 1 ml gas for C2H4 measurement by gas liquid chromatography. To test the effect of CO2 on EFE activity of discs in the presence of Co 2+ or Ag + only one concentration (1 mM) of both ions was used. After saturation with the medium, discs were treated with or without CO2 for 2 hours at 27°C before removal of I ml gas for C2H4 measurement. In the NDE experiment, discs were sampled from fruit that had been stored for 30 days (Hosui) and 60 and 102 days (Granny Smith) at 1 + 1°C, respectively. After discs were vacuum-infiltrated with the medium for EFE measurement for 3 minutes, CO2 was applied using the method described above. NDE was injected into each sealed vial with a syringe to give a concentration of 0.5% (v/v). After 2 hours incubation at room temperature (about 20°C) in the fume cabinet, 1 ml gas was sampled for ethylene analysis by GLC. All data was analysed using an SAS computing program which generated ANOVA, means, and standard errors, multiple comparisons, and determination of significant difference in the data (Steel and Torrie, 1981). 3. Results

Effects of CoCl2 on EFE activity EFE activity in both Hosui and Granny Smith was stimulated, inhibited, or unaffected depending on the concentrations of cobalt ions applied (Fig. 1). In Hosui fruit, EFE activity in discs increased 16% (compared with control) at 0.01 mM COC12 (Fig. la), before declining to the control value at 0.05 mM. There was a rapid (58%) decrease in EFE activity as Co 2+ increased from 0.05 to 1 mM, followed by a slower (31%) decrease between 1 to 5 mM COC12 (Fig. la). In contrast no significant increase in EFE occurred in Granny Smith apple discs between control and 0.01 mM Co 2+ treatment (Fig. lb). Over the range of 0.1-3 mM, EFE activity decreased rapidly by 65% followed by a slower fall off (30%) between 3 and 10 mM CoCI2. Because 1 mM COC12 significantly reduced EFE activity, this concentration was selected for the experiment to test the interaction between Co 2+ and CO2 on EFE activity. In Hosui, ethylene production from discs was not measurable, thus the effect of Co 2+ on ethylene production could not be determined. CO2 increased EFE activity by 24.7% (Table 1) and Co 2+ significantly reduced EFE activity. Addition of CO2 did not overcome the inhibitory effect of Co 2+ alone. In Granny Smith fruit, CO2 did not affect ethylene production, but incubation with I mM Co 2+ decreased it by 65.4% (Table 1). The combination of CO2 and Co 2+ did not reverse the inhibitory effect of Co 2÷ on ethylene production. CO2 stimulated EFE activity, and Co 2÷ reduced it by 40.3%. CO2 did not reverse this inhibition.

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M.S. Tian et aL / Postharvest Biology and Technology 4 (1994) 13-21

20. 18.

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Fig. 1. EFE activity of Hosui (a) and Granny Smith (b) fruit discs treated with CoC12. EFE activity was measured by ethylene released from discs incubated at 20"C for 2 hours after being treated with 5 mM 1-aminocyclopropane-l-carboxylic acid (ACC), 5 mM aminooxyacetic acid (AOA) and 1 mM cyeloheximide (CHI) and various concentrations of COC12 in 0.4 M mannitol solution. Each point is the mean of 3 measurements. Vertical bars represent SE of the means. Inset: EFE activity at low CoO2 concentrations.

Effect of AgN03 on EFE activity and respiratory rate E F E activity was not affected when Hosui discs were treated with 0.02 to 0.1 m M Ag + (Fig. 2). A large decrease (66.8%) in EFE activity did occur at higher Ag + concentrations (1 and 3 mM) and at concentrations of 3 mM and above tissue

M.S. Tian et al. / Postharvest Biology and Technology 4 (1994) 13-21

17

Table 1 E F E activity in Hosui and Granny Smith fruit discs treated with 1 mM CoCI2 and 20% CO2 at 27°C

Treatments

Control + CO 2 + 1 m M CoCI 2 + CO2, + 1 m M COC12

Hosui

Granny Smith

EFE

C2H4

EFE

activity

production

activity

21.0 b 26.2 a 8.1 c 12.8 c

15.3 a 15.0 a 5.3 b 5.2 b

142.3 b 170.9 a 85.0 c 71.3 c

Data are given as means (n = 3). Values within a column followed by different letters represent significant differences at P < 0.05 (Duncan's multiple comparison). Value in nl C2H4 g - I h - l ) .

browning was found. Thus the decline in EFE activity above 3 mM AgNO3 can be attributed to toxicity and probable tissue death. Respiration rate of Hosui fruit discs was not affected by Ag + in the range of 0.02-0.1 mM (Fig. 2a). It increased at 0.5 and 1 mM, dropped significantly at 3 mM and then remained relatively constant between 3-10 mM AgNO3. In Granny Smith fruit discs, similar results were obtained (Fig. 2b). Compared with control, EFE activity increased marginally at 0.05 mM AgNO3 with concentrations greater than 0.5 mM markedly inhibiting EFE activity. There was no activity at 3 mM and higher, and tissue browning occurred at 3 and 5 mM. Respiration rate in Granny Smith fruit discs was not affected at 0.02 and 0.05 mM Ag +, but increased gradually over the range of 0.1-1 mM and reached a peak at 0.5 mM AgNO3 (Fig. 2b) before declining to a very low level at 3 and 5 mM concentration (Fig. 2b). This was probably caused by toxicity. Because 1 mM Ag + significantly decreased EFE activity without reducing respiration in each fruit type, this concentration was selected to test the interaction between Ag + and CO2 on EFE activity. Carbon dioxide increased EFE activity of Hosui fruit discs (Table 2). AgNO3 reduced EFE activity by 37.3% and this inhibition was not reversed by addition of CO2. Table 2 E F E activity of Hosui and Granny Smith fruit discs treated with 1 mM AgNO3 and 20% C O 2

Treatments

Control + CO2 + 1 mM AgNO3 + CO2, + 1 mM AgNO3

Hosui

Granny Smith

EFE

C2H4

activity

production

activity

35.5 b 54.9 a 13.3 e 13.9 c

18.7 a 19.9 a 1.0 b 0.9 b

42.6 b 55.2 a 13.5 c 12.1 e

EFE

Data are given as means (n = 4). Different letters within the same column represent significant differences at P < 0.05 (Duncan's multiple comparison). Initial EFE activity was 5.7 nl C2H4 g-1 h - l . Ethylene production of discs were not measurable.

18

M.S. Tian et aL / Postharvest Biology and Technology 4 (1994) 13-21

~,1oo3

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Fig. 2. EFE activity and respiration rates of Hosui (a) and Granny Smith (b) fruit discs treated with AgNO3. EFE activity was measured using the same method as for the cobalt treatment. For measurement of ethylene production, ACC, AOA and CHI were not added to the mannitol solutions. Each point is the mean of 4 measurements. Vertical bars represent SE of the means. Inset: EFE activity at low AgNO3 concentrations.

E t h y l e n e p r o d u c t i o n of G r a n n y Smith fruit discs was unaffected by CO2 (Table 2), b u t almost completely inhibited by A g +. CO2 did not reverse this inhibitory effect. E F E activity in G r a n n y Smith fruit discs was stimulated 29.5% by CO2. A g + r e d u c e d E F E activity by 68.2% and again CO2 was not able to reverse the inhibitory effect o f A g N O 3 .

M.S. Tian et al. / Postharvest Biology and Technology 4 (1994) 13-21

19

Table 3 E F E activity in discs of Hosui and Granny Smith fruit, maintained at 1 + I°C for various times before assay, treated with 0.5% norbornadiene (NDE) and 20% CO2 Storage time

Control + COz + NDE + COz, + N D E

Hosui

Granny Smith

30 days

60 days

102 days

37.4 b 58.4 a 19.2 d 31.7 b

36.5 b 48.7 a 10.4 a 15.2 c

85.8 b 116.8 a 33.8 c 40.9 c

Data are given as means (n = 4). Different letters within the same column represent significant differences at P < 0.05 (Duncan's multiple comparison). E F E activity in nl C2H4 g-I h-1.

Effects of NDE and carbon dioxide on EFE activity In Hosui discs, C O 2 enhanced EFE activity by 56% and NDE treatment decreased it by 48.8% (Table 3). When discs were treated with a mixture of NDE and CO2, the inhibitory effect of NDE was reversed, although the full stimulatory effect of COz was not achieved. In Granny Smith fruit which had been stored for 60 days, CO2 increased EFE activity in discs by 33.5%, while NDE inhibited it by 71.6% (Table 3). CO2 slightly reversed the inhibition caused by NDE. EFE activity increased 102% during the period from 60 to 102 days in coolstore. In fruit stored for 102 days CO2 increased EFE activity by 36.3%. NDE reduced it by 60.6% and COz did not reverse the inhibitory effect of NDE (Table 3). 4. Discussion

Recently EFE has been partially purified and characterized from climacteric fruit such as melon (Ververidis and John, 1991; Smith, et al., 1992), apple (FernandezMaculet and Yang, 1992; Kuai and Dilley, 1992) and avocado (McGarvey and Christoffersen, 1992). No work has been reported on the purification and characteristics in vitro of EFE from nonclimacteric fruit. It has been suggested that EFE in preclimacteric apple and avocado is localized in the cytosol, but loosely binds to particulate fractions during the cell disruption (Fernandez-Maculet and Yang, 1992). Two EFE fractions were found in avocado fruit (McGarvey and Christoffersen, 1992) and divalent metals, especially Co a+, inhibited EFE activity in vitro (Smith, et al., 1992). Inhibition of EFE activity occurred in both Hosui and Granny Smith fruit discs treated with 1 mM COC12. Addition of CO2 was not able to reverse the inhibitory effect of Co 2+ on EFE activity. Yu and Yang (1979) showed that Co 2+ affected ethylene production by inhibiting conversion of ACC to ethylene (i.e. EFE activity). Co 2+ showed similar effects on EFE activity in discs from both Hosui and Granny Smith fruit. This indicates that the inhibitory effect of Co 2+ on EFE activity may be through the same mechanism in climacteric and nonclimacteric fruit. As CO2 stimulated EFE activity but did not interact with Co 2+, this suggests that CO2 and Co 2+ are attached to different binding sites on EFE.

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M.S. Tian et aL / Postharvest Biology and Technology 4 (1994) 13-21

To investigate whether CO2 stimulated EFE activity by affecting the ethylene receptor, the interaction between CO2 and inhibitors of ethylene action, Ag + and NDE, were tested. Silver nitrate and NDE inhibited EFE activity in both Hosui and Granny Smith apple. Because the inhibitory patterns of Ag + and NDE on EFE activity in discs of Hosui and Granny Smith fruit at preclimacteric stage were similar, it is suggested that both of these chemicals inhibited EFE activity in nonand climacteric fruits through the same mechanism. It has been suggested that ethylene biosynthesis is regulated by the System I receptor in nonclimacteric and immature climacteric fruit tissues, and by System I and System II receptors in mature climacteric fruit tissues (McGlasson, 1985). It is logical to assume that EFE is regulated by the System I ethylene receptor in vivo (Yang, 1987a). Addition of CO2 did not reverse the inhibitory effect of Ag + on EFE activity indicating that CO2 did not compete with Ag + for the same binding site on the ethylene receptors. As ethylene production was inhibited more by Ag + than was EFE activity in discs of Granny Smith fruit at the early climacteric stage, this may indicate that Ag + inhibited both ACC synthesis and conversion of ACC to ethylene by inactivating both ethylene receptors. Although Yang (1987b) suggested that NDE competitively inhibits ethylene action by reversibly binding to the same binding site of the receptor as does ethylene, no information is available indicating whether it binds to the System I or System II ethylene receptor. The present work indicates that NDE competes with ethylene on the System I receptor in Hosui and preclimacteric Granny Smith fruit. CO2 reversed, at least partially, the inhibitory effect of NDE on EFE activity and this result indicates that CO2 can compete with NDE at the same binding site on the ethylene receptor. It is likely that EFE activity in climacteric (apple) and nonclimacteric (Japanese pear) fruits is regulated by the System I ethylene receptor through the same mechanism. Ag + and NDE probably inhibited EFE activity by inhibiting ethylene action through binding to different binding sites on the ethylene receptors. CO2 may stimulate ACC-dependent ethylene production (ie. EFE activity) by binding to the same site as ethylene and NDE on the System I receptor forming an active CO2-receptor complex. The nature of these different binding sites have not been determined.

Acknowledgement Thanks to the New Zealand Ministry of Agriculture and Fisheries (MAF), Trigon Packaging System (NZ) Ltd and a Helen E. Akers Scholarship from Massey University, New Zealand, for financial support and to Drs M.E. Hopping and D.J. Klinae from the Ruakura Research Orchard, MAF for supplying experimental fruit.

References Buffer, G., 1986. Ethylene-promoted conversion of 1-aminocyclopropane-l-carboxylic acid to ethylene in peel of apple at various stages of fruit development. Plant Physiol., 80: 539-543.

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Burg, S.P. and Burg, E.A., 1967. Molecular requirements for the biological activity for ethylene, Plant Physiol., 42: 144-152. Chaves, A.R. and Tomas, J.O., 1984. Effect of a brief CO2 exposure on ethylene production. Plant Physiol., 76: 88-91. Cheverry, J.L., Sy, M.O., J. Pouliqueen, J. and Marcellin, E, 1988. Regulation by CO2 of 1aminocyclopropane-l-carboxylicacid conversion to ethylene in climacteric fruits. Physiol. Plant., 72: 535-540. Fernandez-Maculet, J.C. and Yang ES., 1992. Extraction and partial characterization of the ethyleneforming enzyme from apple fruit. Plant Physiol., 99: 751-754. Kual, J. and Dilley, D.R., 1992. Extraction, partial purification and characterization of 1-aminocyclopropane-l-carboxylic acid oxidase from apple fruit. Postharvest Biol. Technol., 1: 203-211. McGarvey, D.J. and Christofferson, R.E., 1992. Characterization and kinetic parameters of ethyleneforming enzyme from avocado fruit. J. Biol. Chem.. 267: 5964--5967. McGlasson, W.B., 1985. Ethylene and fruit ripening. HortScience, 20: 51-54. Philosoph-Hadas, S., Aharoni, N. and Yang, S.E, 1986. Carbon dioxide enhances the development of ethylene forming enzyme in tobacco leaf discs. Plant Physiol., 82: 925-929. Sisler, E.C., 1979. Measurement of ethylene binding in plant tissue. Plant Physiol., 64: 538-542. Smith, J.J., Ververidis, E and John, E, 1992. Characterization of the ethylene-forming enzyme partially purified from melon. Phytochemistry, 31: 1485-1494. Steel, R.G.D. and Torrie, J.H., 1981. Principles and Procedures of Statistics: A Biometrical Approach. McGraw-Hill, London. Suzuki, K., Uamazaki, R., Murase, S., Miyagawa, H., Nogata, T., Mitobe, M. and Morita, A., 1981. Colour charts: useful guide to evaluate the fruit maturity, III. Fruit skin colour in relation to the fruit maturity. Bull. Fruit Tree Res. Stn., Yatabe, Ibaraki, Japan, Ser. 8, pp. 85-100. Thomas, C.J.R., Smith, A.R. and Hall, M.A., 1984. The effect of solubilisation on the character of an ethylene-binding site from Phaseolus vulgaris L. cotyledons. Planta, 160: 474-479. Tian, M.S., Hewett, E.W. and Lill, R.E., 1994. Effects of carbon dioxide on ethylene-forming enzyme in Japanese pear and apple. Postharvest Biol. Technol., 4:1-12 (this issue). Veen, H., 1985. Antagonistic effect of silver thiosulphate or 2,5-norbornadiene on 1-aminocyclopropane1-carboxylic acid-stimulated growth of pistils in carnation buds. Physiol. Plant., 65: 2-8. Veen, H., 1986. A theoretical model for anti-ethylene effects of silver thiosulphate and 2,5norbornadiene. Acta Hortic., 181: 129-134. Veen, H., 1987. Use of inhibitors of ethylene action. Acta Hortic., 210: 213-222. Ververidis, E, and John, P., 1991. Complete recovery in vitro of ethylene-forming enzyme activity. Phytochemistry, 30: 725-727. Wang, C.Y., 1987. Use of ethylene biosynthesis inhibitors in horticulture. Acta Hortic., 201: 187-194. Yang, S.E, 1985. Biosynthesis and action of ethylene. HortScience, 20(1): 41-45. Yang, S.E, 1987a. The role of ethylene and ethylene synthesis in fruit ripening. In: W.W. Thomson, E.A. Nothnagel and R.C. Huffaker (Editors), Plant Senescence: Its Biochemistry and Physiology. American Society of Plant Physiologist, Rockville, Md., pp. 156-166. Yang, S.E, 1987b. Regulation of biosynthesis and action of ethylene. Acta Hortic., 201: 53-59. Yu, Y.B. and Yang, S.E, 1979. Auxin-induced ethylene production and its inhibition by aminoethyoxyvinylglycine and cobalt ion. Plant Physiol., 64: 1074-1077.