J. Japan. Soc. Hort. Sci. 61 (4) : 805-812. 1993. Behaviors of 1-Aminocyclopropane-1-carboxylic Acid (ACC) and ACC Synthase Responsible for Ethylene Production in Normal and Mutant (nor and rin) Tomato Fruits at Various Ripening Stages irofumi Terai Faculty of Agriculture, Kobe University, Nada-ku, Kobe 657 H S ummary The behavior of 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC synthase responsible for ethylene production was studied using three types of tomato fruits; ebeiju' as a ripening type, nor mutant as an intermediate ripening type and rin mutant as a non-ripening type. In ebeiju' tomatoes, the rate of ethylene production, ACC synthase activity.and the amounts of ACC and 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) increased with ripening. In nor tomatoes, a slight increase in ethylene production and ACC synthase activity was observed about 50 days after anthesis. ACC and MACC accumulated markedly during the late stage of fruit development. In rin tomatoes, on the other hand, the activity of ACC synthase and the amounts of ACC and MACC hardly changed at any stage and the rate of ethylene production also remained nearly constant. Wounding the pericarp tissue in Yin tomatoes induced ACC synthase activity, whereas the activity was scarcely observed in intact fruits. Wounding of ebeiju' and nor tomatoes during fruit development accelerated ACC synthase activity markedly and the seasonal patterns of the activity in the wounded fruits were similar to that in intact fruits. Introduction Mutant tomato fruits are frequently used to study the difference in gene expression between ripening and non-ripening fruits (Biggs and Handa, 1989; DellaPenna et al., 1987; Giovannoni et al., 1989; Lincoln and Fischer, 1988). However, the difference in the ripening mechanism between normal and mutant tomato fruits has not been clearly elucidated yet. To clarify the difference in the ripening mechanism of these tomatoes, the regulation mechanism of ethylene production between ripening and non-ripening types, focusing on the ethylene production pathway in tomato fruits was studied. In a previous paper, Terai (1990) reported on the behavior of ethylene forming enzyme (EFE) and ACC during fruit development, 1) in 'Rutgers', a ripening type, the EFE activity was stimulated which paralleled the accumulation of ACC in the tissue during the ripening period, 2) in nor mutant, Received for publication 5 November 1990. a slight ripening type, the increase in ACC content was observed at a late stage of fruit development while EFE activity had already decreased by this time, and 3) in rin mutant, a non-ripening type, the ACC content in the tissue was very low and the EFE activity did not increase at any stage of fruit development. To understand the regulating mechanism of ethylene production in the three types of tomatoes, information with respect to the changes in the levels of ACC and MACC, the end product formed from ACC, and in the activity of ACC synthase during fruit development, is needed. This paper deals with the regulating mechanism of ACC content and ACC synthase activity in intact fruits and in wounded fruits using three tomato types, 'Beiju', nor and rin at various stages of development. Materials and Methods Three types of tomato plants (Lycopersicon esculentum Mill.) ebeiju' as a ripening type, nor mutant as a slight ripening type and rin mutant as a 805
806 H. TERAI In the wounding experiments, discs with the peel (10 mm diam. and 3~4 mm thick) were excised from the pericarp with a cork-borer and quartered with a razor. For the measurement of ethylene production, 20g of the quartered discs were kept at 30 C with air flowing at 800 ml Ehr -1 (Fig. 3), and 3g of the quartered discs were kept at 30 C in a 70 ml Erlenmeyer flask with a serum cap (Fig. 4). For analyses of ACC and ACC synthase in both experiments, comparable quartered discs were incubated in beakers with loose covers at 30 for a given period of time. After incubation the discs were frozen in liquid nitrogen. To determine ACC and MACC contents, 5g of pericarp tissues were heated in 50 ml of 80% ethanol for 30 min at 70 C and then homogenized in a blender. The homogenate was centrifuged at 10,000 x g for 15 min at 1 C. The supernatant (11 ml) was concentrated to dryness in vacuo at 40 C. The residue was dissolved in 4 ml of water and the aqueous solution was filtered through a membrane with 1µm pore size. An aliquot (0.8 ml) was assayed for the ACC. Another aliquot (2 ml) of extract was passed through a cation exchange resin column (Dowex 50W, 3 ml of bed-volume) to isolate MACC from contaminations. The resin was rinsed 3 times with 3 ml of water and effluent was concentrated to dryness in vacuo at 40 C. After the residue was re-dissolved in 2 ml of water, an aliquot (0.8 ml) containing MACC was hydrolyzed with 2 N HC1 for 3 hr at 100 C. Following neutralization with 2 N NaOH, the hydrolysate was assayed for ACC according to the method of Lizada and Yang (1979). ACC synthase activity in the tissue was determined by the method described in a previous paper (Terai, 1990), and protein content was measured according to the method of Bradford (1976). Results and discussion The pattern of changes in the rates of carbon dioxide and ethylene production and the amounts of ACC and MACC in the three types of tomato fruits were shown in Fig. 1. In ebeiju' tomatoes, the rate of carbon dioxide production was minimum at the mature green stage and maximum at the pink stage; ethylene production began after mature green stage. In both nor and rin tomatoes, the rates of carbon dioxide production were highest 16 non-ripening type, were grown in a field at Kobe University. Fruits of these types were picked at various stages. Immature fruits were picked and maintained at 20 C for 1 day and analyzed. Mature green fruits were picked and stored at 20 C until a given stage and analyzed. Several intact fruits were put in a jar (4,000 to 4,500 ml) which was sealed for 0.5 to 1 hr at 20 or 30 C for carbon dioxide and ethylene determinations. The rates of carbon dioxide and ethylene production of the fruits were measured by gas chromatography. After the measurement, the pericarp tissue was frozen in liquid nitrogen until ready for ACC, MACC and ACC synthase activity assays. days after anthesis, but after 26 days the rate hardly changed. The rate of ethylene production in nor tomatoes increased from 37 to 61 days after anthesis (DAA), but slower than that in 'Beiju' tomatoes; it then decreased 79 DAA. In rin tomatoes, ethylene production was scarcely detectable at any stage. Although the tomato has been classified as a climacteric fruit, these data reveal that nor and rin tomatoes have the respiratory pattern of a non-climacteric fruit (McGlasson 1985; Biale and Young 1981). However, nor tomatoes produced a small amount of ethylene and developed some pink coloring 50 to 60 DAA (data not shown). The nor tomatoes seemed to be an intermediate between climacteric and non-climacteric types and also seemed to be a tomato that ripens very slowly. The ACC content in ebeiju' tomatoes tended to increase through the ripening stages 26 DAA and increased significantly at the overripe stage. In nor tomatoes, a marked change in the ACC content was not observed for the period between 26 and 50 DAA, but at day 61 the level increased, attaining a maximum level 79 DAA. In spite of the large accumulation of ACC, ethylene production decreased 79 DAA. In nor tomatoes, the decrease in ethylene production at the final stage coincided with a decrease in EFE activity, as reported previously (Terai, 1990). In rin tomatoes, the ACC content did not change significantly, unlike ebeiju' and nor tomatoes. MACC content in ebeiju' tomatoes did not change until the breaker stage and then increased after the pink stage; in nor tomatoes MACC increased notably 61 to 79 DAA. Unlike Beiju' and nor tomatoes, MACC content ein rin tomatoes remained nearly constant. MACC con-
J. Japan. Soc. Hort. Sci. 61 (4): 805-812. 1993. 807
J. Japan. Soc. Hort. Sci. 61 (4) : 805-812. 1993. 809 tents increased with the development of ebeiju' and nor tomatoes, paralleling that of ACC content. Hoffman et al. (1983a, 1983b) reported that MACC was irreversibly formed from ACC, and furthermore, Liu et al. (1985a, 1985b) and Yang et al. (1985) described that the activity of this pathway was accelerated by ethylene. The pattern of changes in the ethylene production rate, ACC content and ACC synthase activity in the three types of tomatoes during their development stage is shown in Fig. 2. The changes in ethylene production and ACC content were similar to those shown in Fig. 1. ACC synthase activity in `Beiju' tomatoes showed an initial peak at the pink stage, decreased temporarily at the table ripe stage, then increased again at the overripe stage. In nor tomatoes, ACC synthase activity increased continuously until 68 DAA and then decreased at day 78. ACC synthase activities in ebeiju' and nor tomatoes followed a similar seasonal pattern in the both ethylene production rate and the ACC content; exception was an increase in the ACC content in nor tomatoes at day 78. The decrease in ACC synthase activity in nor tomatoes 78 DAA did not affect the ACC content, inferring that one of the factors related to the accumulation of ACC was a decline in ACC conversion to ethylene, which can be attributed to a decrease in EFE activity. In rin tomatoes, however, ACC synthase activity like ethylene production was hardly detectable at any stage. Furthermore, it is noted that the ACC content of rin tomatoes remained nearly constant. A possible explanation for the regulating mechanism of ACC and MACC contents in the intact tomato fruits is as follows. In ebeiju' tomatoes, the stimulation of ACC synthase activity with the onset of ripening results in an increase of ACC content, and increased ACC content also brings about an accumulation of MACC. In nor tomatoes, increased ACC content results from the stimulation of ACC synthase activity. But the accumulation of ACC at the final stage in spite of low level of ACC synthase is attributed to the reduced EFE activity. This accumulation of ACC is due to an increase in MACC content. In rin tomatoes, ACC synthase activity is very low, and furthermore the conversion of ACC to ethylene or MACC hardly occurs during the fruit development, and consequently, there is not significant change in ACC content. This behavior of ethylene synthesis pathway in rin tomatoes may be a reason to classify them as a non-climacteric type. The ethylene production rate, ACC content and ACC synthase activity in the wounded pericarp tissue (quartered discs) of ebeiju' tomatoes at mature green stage increased slightly for 3 hr after the beginning of incubation as shown in Fig. 3. After 6 hr, ACC synthase activity increased appreciably and after 14 hr, both the ethylene production rate and ACC content increased notably, but ACC synthase activity decreased. It is well-known that Fig. 3. Changes in the rate of ethylene production, the amount of ACC and the activity of ACC synthase in pericarp tissue of 'Beiju' tomato fruits at mature green stage after wounding at 30 C.
810 H. TERAI Fig. 4. Changes in the rate of ethylene production, the amount of ACC and the activity of ACC synthase in the wounded pericarp tissues of ebeiju', nor and rin tomato fruits picked at various stages. Ethylene production for a 6-hr wounding at 30 C was measured. After incubation, pericarp tissues were frozen in liquid nitrogen prior to analyses for ACC and ACC synthase. 'Beiju' tomato fruits (SG, 52% weight of MG tomato, and MG stages), nor tomatoes (21, 33 and 41 days after anthesis) and rin tomatoes (21, 33 and 41 days after anthesis) were picked 1 day before analysis. Each type of tomato fruits at mature stage ('Beiju', MG; nor, 37-39 days after anthesis; rin, 38-42 days after anthesis) was picked and stored at 20 C until the given stages ( ebeiju', B, P and TR; nor, 63 and 84 days; rin, 45, 49, 62 and 70 days). The tomato fruits that reached a given stage were analyzed for the experiment.
J. Japan. Soc. Hort. Sci. 61 (4): 805-812. 1993. 811 ethylene production is stimulated by wounding the plant tissue (Hyodo and Nishino, 1981; Kende and Boller, 1981; Konze and Kwiatkowski, 1981; Yu and Yang, 1980). Hyodo et al. (1985) who examined the changes in ethylene production, ACC content and ACC synthase activity in excised squash tissue during incubation reported that initially ACC synthase activity was induced followed by an increase in ACC content, and subsequently by an acceleration in ethylene production. In this experiment, the influence of wounding by excising tomato tissue appeared 6 hr after incubation. ACC content and ACC synthase activity in the excised pericarp of several developmental stages were measured 6 hr after incubation to obtain further information about the stimulation of ethylene production by wounding, as shown in Fig. 4. The rate of ethylene production in `Beiju' tissues was very low at the small-green and mature green stages, increased markedly at the breaker stage and reached the highest level at the pink stage. In nor and rin tomatoes, the ethylene production rate decreased as development progressed, except an increase 84 DAA in the nor type. ACC content in nor tomatoes increased as the fruit continued to develop, whereas in rin type decreased. ACC synthase activity in nor and Yin tomatoes increased as the fruit developed; the highest activities were 63 and 41 DAA, respectively, after which the activities decreased. Thus, in wounded fruit tissue, the patterns of changes in ACC synthase activity at different developmental stages were similar among the three types of tomatoes. In ebeiju' tomatoes, the patterns of seasonal trend in ethylene production rate, ACC content and ACC synthase activity with the progress of ripening stage are similar. In nor tomatoes, the trends between ethylene production and ACC content differed. The decrease of ethylene production may be due to the decline of EFE activity as the fruit developed. The reason for the increase of ethylene production rate 84 DAA is not clear. In yin tomatoes, ethylene production was barely detectable in intact fruits (Figs. 1 and 2), whereas the excised pericarp tissue produced significant amounts of ethylene, although the rate became weaker with time. Based on the difference between wounded tissues and intact fruits for induction of ethylene production, it was demonstrated that ethylene production in three types of tomatoes can be induced as the results of accelerating ACC synthase activity and inducing an accumulation of ACC by wounding, whereas the system of autocatalytic ethylene synthesis in intact rin fruit is lacking during fruit development but is present in vbeiju' and nor tomatoes. Acknowledgments The author obtained the seeds of rin and nor mutant tomatoes from Professor Dr. W.B. McGlasson, University of Western Sydney in Australia, through the effort of Dr. Y. Ueda, Department of Agriculture, Osaka Prefecture University. The author is grateful to them. Literature Cited Biale, J.B. and R.E. Young. 1981. Respiration and ripening in fruits-retrospect and prospect (Ch.1). p.1-39. In : J. Friend and M.J.C. Rhodes (eds.). Recent advances in the biochemistry of fruits and vegetables. Academic press, London. Biggs, M.S. and A. K. Handa. 1989. Temporal regulation of polygalacturonase gene expression in fruits of normal, mutant, and heterozygous tomato genotypes. Plant Physiol. 89 : 117-125. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 : 248-254. DellaPenna, D., D.S. Kates and A.B. Bennett. 1987. Polygalacturonase gene expression in Rutgers, rin, nor, and Nr tomato fruits. Plant physiol. 85 : 502-507. Giovannoni, J. J., D. DellaPenna, A.B. Bennett and R. L. Fischer. 1989. Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening. The Plant Cell 1 : 53-63. Hoffman, N.E., J. R. Fu and S.F. Yang. 1983a. Identification and metabolism of 1-(malonylamino)cyclopropane-1-carboxylic acid in germinating peanut seeds. Plant Physiol. 71 : 197-199. Hoffman, N.E., Y. Liu and S.F. Yang. 1983b. Changes in 1-(malonylamino)cyclopropane-l-carboxylic acid content in wilted wheat leaves in relation to their ethylene production rates and 1-aminocyclopropane- 1-carboxylic acid content. Planta 157: 518-523. Hyodo, H. and T. Nishino. 1981. Wound-induced ethylene formation in albedo tissue of citrus fruit. Plant Physiol. 67 : 421-423. Hyodo, H., K. Tanaka and J. Yoshisaka. 1985. Induction of 1-aminocyclopropane-1-carboxylic acid synthase in wounded mesocarp tissue of winter squash
812 H. TERAI fruit and the effects of ethylene. Plant Cell Physiol. 26 : 161-167. Kende, H. and T. Boller. 1981. Wound ethylene and 1- aminocyclopropane-l-carboxylate synthase in ripening tomato fruit. Planta 151 : 476-481. Konze, J.R. and G.M.K. Kwiatkowski. 1981. Rapidly induced ethylene formation after wounding is controlled by the regulation of 1-aminocyclopropane- 1-carboxylic acid synthesis. Planta 151 : 327-330. Lincoln, J.E. and R.L. Fischer. 1988. Regulation of gene expression by ethylene in wild-type and rin tomato (Lycopersicon esculentum) fruit. Plant Physiol. 88 : 370-374. Liu, Y., N.E. Hoffman and S.F. Yang. 1985a. Ethylene-promoted malonylation of 1-aminocyclopropane-1-carboxylic acid participates in autoinhibition of ethylene synthesis in grapefruit flavedo discs. Planta 164: 565-568. Liu, Y., L. Y. Su and S.F. Yang. 1985b. Ethylene promotes the capability to malonylate 1-aminocyclopropane-1-carboxylic acid and D-amino acids in preclimacteric tomato fruits. Plant Physiol. 77 : 891-895. Lizada, M. C. C. and S. F. Yang. 1979. A simple and sensitive assay for 1-aminocyclopropane-l-carboxylic acid. Anal. Biochem. 100: 140-145. McGlasson, W.B. 1985. Ethylene in postharvest biology and technology of horticultural crops: Ethylene and fruit ripening. HortScience 20 : 51-54. Terai, H. 1990. Regulation mechanism of ethylene production in normal 'Rutgers', and mutant nor and rin tomato fruits. J. Japan. Soc. Hort. Sci. 59 : 121-128. Yang, S.F., Y. Liu, L. Su, G.D. Peiser, N.E. Hoffman and T. McKeon. 1985. Metabolism of 1-aminocyclopropane-l-carboxylic acid. p.9-21 In : J.A. Roberts and G.A. Tucker (eds.). Ethylene and plant development. Butterworths, London. Yu, Y. and S.F. Yang. 1980. Biosynthesis of wound ethylene. Plant Physiol. 66 : 281-285.