Ethylene Production by Albedo Tissue of Satsuma Mandarin (Citrus Unshiu Marc.) Fruit Received for Publication April 27, 1976 and in Revised Form June 16, 1976

Plant Physiol. (1977) 59, 111-113

Short Communication Ethylene Production by Albedo Tissue of Satsuma Mandarin (Citrus unshiu Marc.) Fruit Received for publication April 27, 1976 and in revised form June 16, 1976

HIROSHI HYODO Department of Horticulture, Faculty ofAgriculture, Shizuoka University, Ohya, Shizuoka 422, Japan

ABSTRACT Erlenmeyer flask and the flask was sealed with a rubber serum cap. Discs were incubated in the dark at 26 C. After closure for Isolated albedo tissue of Satsuma mandarin (Citrus unshiu Marcov- 1 hr, the atmosphere in the flasks was sampled through the itch, cv. Owari) fruit produced a large quantity of ethylene during serum caps at regular intervals with a plastic syringe, and ethyl- incubation at 26 C in the dark. When sliced, albedo tissue began produc- ene concentration was determined. After each sampling, the ing ethylene at an increasing rate until a maximum was reached after flasks were flushed with air and then resealed. Ethylene was incubation for about 30 hours. Aged albedo discs which were capable of measured on a Hitachi gas chromatograph equipped with a producing ethylene, actively converted L-IU-14CJmethionine into both hydrogen flame ionization detector and an activated alumina ethylene and carbon dioxide. In fresh tissue, almost no measurable column. Ethylene was produced at an increasing rate, reaching a conversion of radioactive methionine into ethylene took place. Conver- maximum about 30 hr after the start of incubation. sion of labeled L-methionine into ethylene was totally inhibited by the Similar aged (preincubated) discs, known to be capable of addition of nonradioactive L-methionine or L-ethionine. It appears possi- vigorous ethylene production, were used for the following incor- ble, from these finding, that methionine is a precursor of ethylene in the poration studies. Twelve albedo discs (1.1-1.6 g) were placed in aged albedo discs. Ethylene synthesis in the aged albedo tissnue was a 55-ml Erlenmeyer flask on 3 ml of medium containing 2.5 uCi markedly reduced in the presence of cycloheximide, suggesting that (0.01 ,umol) L-[U-_4C]methionine obtained from New England there may be a rapid turnover ofthe ethylene-producing system, and that Nuclear. The flask was sealed with a rubber serum cap. In the its formation involves protein synthesis. Actinomycin D exerted no flask were suspended two polypropylene center wells (Kontes effect. Glass Co.), one containing 0.1 ml of 0.25 M mercuric perchlo- rate solution and the other 0.1 ml of 20% KOH solution, and fitted with filter paper wicks, to absorb evolved ethylene and CO2. The flasks were placed on a shaker at 23 C with a shaking speed of 110 strokes/min. The ethylene and CO2 produced were trapped with an efficiency greater than 80 and 90%, respec- It has been demonstrated by Aharoni (1) that young, unripe tively, by this procedure. After an appropriate period of incuba- oranges evolve a large amount of ethylene after harvest, paral- tion, the filter paper wicks were taken out and put in vials with leling a marked increase in respiration. Similar findings have 10 ml of Bray's solution. The radioactivity was measured in a been made by Eaks (3) for young fruits of grapefruit and or- Horiba liquid scintillation spectrometer. In some experiments, anges. Recently, I have also reported that young, immature fruit actinomycin D, cycloheximide, L-methionine, D-methionine, or of Satsuma mandarin produced a great amount of ethylene after L-ethionine was included in the test solution to examine the harvest (5). The rate of ethylene production by Satsuma man- effects of these compounds on ethylene production. All chemi- darin fruit markedly decreased as the fruit developed and in- cals used were obtained commercially except for actinomycin D, creased in weight. When young fruits were cut into small pieces which was a generous gift of Merck Sharp & Dohme. (e.g. quarters or eighths) ethylene was produced at a much greater rate than by the intact fruit (5). Similar results were RESULTS obtained with isolated albedo tissue. Albedo discs vigorously evolved ethylene, even though the tissue was taken from fruit The rate of ethylene production by freshly prepared albedo which did not produce ethylene when intact. Subsequently, al- discs was very low, but increased markedly during incubation in bedo has been used for studying the mode of ethylene produc- the dark at 26 C (Fig. 1). It reached a maximum after about 30 tion in the Satsuma mandarin. Methionine has been shown to be hr, then declined gradually or remained steady. The rise in the a precursor of ethylene in plants (2, 7). In the present experi- rate of ethylene production seemed faster in young fruit than in ments L-[I4C]methionine was used to test the efficiency of methi- more mature fruit. In albedo discs aged (preincubated) for 18 hr, onine as a possible precursor of ethylene in the albedo tissue. radioactive label from methionine was extensively incorporated into both ethylene and CO2. The incorporation of radioactivity MATERIALS AND METHODS into ethylene, starting after a 40-min lag period, continued linearly for about 3 hr (Fig. 2). No incorporation of '4C from Satsuma mandarin fruit (Citrus unshiu Marcovitch, cv. Owari) uniformly labeled L-leucine into ethylene was observed, al- were harvested from the orchard during the months from July to though it was converted substantially into CO2. In contrast to November 1975. Ethylene production, respiration, increase in aged tissue, freshly prepared albedo discs produced very little fresh weight, and some properties of the postharvest fruit were ethylene and incorporated very little label from methionine into described elsewhere (5). Discs of the peel of the fruit were ethylene (Fig. 3). Incorporation of label from methionine into excised with a cork borer 9 mm in diameter. Albedo discs (2-4 ethylene in the aged albedo discs was prevented by 5 mm unla- mm thick), separated from the flavedo, were placed in a 130-ml beled L-methionine or by 5 mm L-ethionine (Fig. 4). ii-Methio- 111 Downloaded from on January 22, 2020 - Published by www.plantphysiol.org Copyright © 1977 American Society of Plant Biologists. All rights reserved. 112 HYODO Plant Physiol. Vol. 59, 1977 September or later produced no measurable quantities of ethylene (5). However, excised albedo tissue evolved a great amount 60- Oct. 6 of ethylene during incubation, even if the fruit from which it was excised was incapable of producing ethylene. This may be due to wound-induced ethylene production. Imaseki et al. (6) reported 50\ that sweet potato root tissue produced ethylene in response to / cutting injury. Riov et al. (10) found that flavedo discs evinced increasing ethylene production upon incubation. In the case of Sept.18 Au Satsuma mandarin fruit, aged flavedo tissue showed a similar l w increase in ethylene production, but albedo tissue had a far greater productive capacity. It is of interest to ask what kind of ssNov.2 substance or condition is responsible for the induction of ethyl- c 30- / 1 l 1ene evolution in the albedo tissue in response to wounding. It is

2: 20 well known that wounding causes an activation of plant metabo- (3 il /lism, particularly an increase in some enzyme activities. The 20 [lpossible formation of a triggering substance in the wound tissue, or by a wound reaction, remains obscure (4). Methionine has been shown to be a precursor of ethylene in 0 1 plants (2, 7, 11, 12). From the results obtained above it is most

0 10 20 30 40 50 Incubation time (hr) 4 FIG. 1. Effect of incubation time on the rate of ethylene production by albedo tissues prepared from fruit harvested from August to Novem- t / ber. 0Aged tissue x 3

8- & Leu-C02 E

26~~~~~~~~~~~~~~~~~~~~~~~~~ 0 ~~~~~~~~~~~~~~~0

E Met-CO2 4 0 Fresh tissue 0 0 Met-C214 1 2 3 4 2-//Incubation time (hr) a /2 FIG. 3. Incorporation of "4C from methionine into ethylene by fresh albedo tissue or aged (18 hr) albedo tissue. For methods see Fig. 2.

C AiA ^ ^A Leu-C2H4 2 3 4 Incubation time (hr) 0 FIG. 2. Incorporation of '4C from methionine or leucine into ethyl- x ene and CO2. Albedo discs aged for 18 hr were incubated in a medium 2- containing 2.5 I±Ciof L-[U-14CJmethionine or 2.5 ACi of L-[U- , o c .'4C]leucine. Ethylene and CO2 evolved were trapped and their radioac- C. c o tivities were measured in a liquid scintillation spectrometer. e o c E c CL 0 J C 1 nine (5 mM) also inhibited incorporation from L-methionine into Q + + + ethylene, although to a far less extent. The application of 72 AM cycloheximide significantly reduced incorporation into ethylene, suggesting that the formation of the ethylene-producing system 0 may involve de novo synthesis of protein (Fig. 5). Actinomycin D (80 Mm), however, exerted no effect. l- DISCUSSION FIG. 4. Effects of unlabeled L-methionine, L-ethionine, and D-methionine on the incorporation of [(4C]methionine into ethylene. Albedo Young immature fruit of Satsuma mandarin produced a great discs aged for 18 hr were incubated in a medium containing 2.5 ,uCi of L- amount of ethylene after harvest. The rate of ethylene produc- [U-_4C]methionine for 2.5 hr in the absence (control) or in the presence tion decreased as fruit growth progressed. The fruit harvested in of 5 mm L-methionine, 5 mm L-ethionine, or 5 mm o-methionine.

Downloaded from on January 22, 2020 - Published by www.plantphysiol.org Copyright © 1977 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 59, 1977 ETHYLENE PRODUCTION BY ALBEDO TISSUE 113 reduced in the presence of cycloheximide, but not actinomycin D, which suggests that there may be a rapid turnover of the ethylene-producing system, and that its formation requires pro- 3 tein synthesis. The lack of effect of actinomycin D may indicate that mRNA, once formed, is stable in the aged albedo tissue. 10 x Acknowledgments-I thank S. F. Yang and G. Von Abrams, Department of Vegetable Crops, University of California, Davis, for reading the manuscript and for helpful suggestions.

E C) LITERATURE CITED 1. AHARONI, Y. 1968. Respiration of oranges and grapefruits harvested at different stages of development. Plant Physiol. 43: 99-102. 2- 2. BAUR, A. H., S. F. YANG, H. K. PRATT, AND J. BIALE. 1971. Ethylene biosynthesis in fruit t + Cycloheximide tissues. Plant Physiol. 47: 696-699. 3. EAKS, I. L. 1970. Respiratory response, ethylene production, and response to ethylene of citrus fruit during ontogeny. Plant Physiol. 45: 334-338. 4. HYoWo, H. 1976. Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. 0 Development of enzyme activity and effects of antibiotics. J. Biochem. 79: 277-282. 1 2 3 4 5. HYoWo, H. 1976. Ethylene production and respiration of Satsuma mandarin (Citrus unshiu Incubation time (hr) Marc.) fruit harvested at different stages of development. J. Jap. Soc. Hortic. Sci. In press. 6. IMASEKI, H., I. URITANI, AND M. A. STAHMANN. 1968. Production of ethylene by injured FIG. 5. Effects of cycloheximide and actinomycin D on the incorpo- sweet potato root tissue. Plant Cell Physiol. 9: 757-768. ration of 14C of methionine into ethylene. Albedo discs aged for 17 hr 7. LIEBERMAN, M., A. KUNISHI, L. W. MAPSON, AND D. W. WARDALE. 1966. Stimulation of were incubated with labeled methionine in the absence (control) or in ethylene production in apple tissue slices by methionine. Plant Physiol. 41: 376-382. the presence of 72 gmM cycloheximide or 80 jmM actinomycin D. 8. MURR, D. P. AND S. F. YANG. 1975. Inhibition of in vivo conversion of methionine to ethylene by L-canaline and 2,4-dinitrophenol. Plant Physiol. 55: 79-82. 9. OWENS, L. D., M. LIEBERMAN, AND A. KUNISHI. 1971. Inhibition of ethylene production likely that the precursor of ethylene in the aged albedo tissue is by rhizobitoxine. Plant Physiol. 48: 1-4. also methionine. In the present experiments, uniformly labeled 10. Riov, J., S. P. MONSELISE, AND R. S. KAHAN. 1969. Ethylene-controlled induction of ['4C]methionine was used. In future studies, specifically labeled phenylalanine ammonia-lyase in citrus fruit peel. Plant Physiol. 44: 631-635. 1'4C]methionine will be applied and inhibitors specific for methi- 11. SAKAI, S. AND H. IMASEKI. 1972. Ethylene biosynthesis: methionine as an in vivo precursor of ethylene in auxin-treated mung bean hypocotyl segments. Planta 105: 165-173. onine metabolism (8, 9) will be used. 12. YANG, S. F. AND A. H. BAUR. 1969. Pathways of ethylene biosynthesis. Qual. Plant. Mat. Ethylene evolution in the aged albedo discs was markedly Veg. 19: 201-220.