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Effect of Ethephon on the Activity of the Ethylene-forming Enzyme and the Biosynthesis of Ethylene in Winter Rye Seedlings
Biochem. Physiol. Ptlan/cn 186, 221 - 22R ( 19l)() I Gustav Fischer Verlag .kna
Effect of Ethephon on the Activity of the Ethylene-forming Enzyme and the Biosynthesis of Ethylene in Winter Rye Seedlings GEDERTS IEVINSH, YJACHESLAV IUIN, OLGERTS KREICBERGS and OLGA ROMANOVSKAYA Institute of Biology, Latvian Academy of Sciences, Salaspils, Latvian SSR, USSR Key Term Index: ACC, ACC-oxidase, EFE, ethephon, ethylene biosynthesis; Secale cereale
Summary The effect of ethylene-releaser ethephon on the intensity of ethylene production, ACC content, and EFE activity in situ and in vitro (activity of ACC-oxidase) in primary leaves of intact winter rye seedlings was investigated. There were a sharp increase of ethylene production immediately after the treatment and an increase in ACC-oxidase activity in vitro and EFE activity in situ after a lag period of at least 15 min. Inversely, the level of ACC decreased after an equal lag period. Such an effect of ethephon on ethylene biosynthesis was most remarkable 1 to 24 h after the treatment. An additional treatment with 0.1 mM CoS0 4 before the application of ethephon inhibited the ethylene production. It is suggested that a major part of the increase in the ethylene production is related to the decomposition of ethephon having penetrated into leaf tissues, while the other part is related to an increased formation of endogenous ethylene from the accessible pool of ACC due to enhanced EFE activity. Treatment with gaseous ethylene (0.8 ppm) caused an increase in ethylene production and a decrease in ACC content. The results reveal an autocatalytic action of exogenous ethylene in intact juvenile tissues and emphasize the probable importance of EFE in a positive regulation of the ethylene formation.
Introduction The autocatalytic effect of ethylene can appear both at the synthesis of the ethylene precursor, ACC, and at the conversion of ACC to ethylene (YANG and HOFFMAN 1984). Many data concern the effect of exogenous ethylene on the last step of ethylene biosynthesis in fruits and on the aging or injured vegetative tissues (YANG and HOFFMAN 1984), but there is little information about its effect in intact juvenile plants. The last stage of ethylene biosynthesis is catalysed by a so-called ethylene-forming enzyme, EFE, which is labile and related to the integrity of cell membranes and tissues (APELBAUM et at. 1981; PORTER et at. 1986). Therefore, the activity of the enzyme cannot be determined in vitro, but a probable activity of EFE is estimated according to the capacity or tissues to convert the exogenous ACC into ethylene in situ. In homogenates and membrane fractions of plant material the ability to convert the exogenous ACC to ethylene can be estimated in vitro (activity of ACC-oxidase) (KONZE and KENDE 1979; MAYAK et al. 1981). Though some investigations show a certain physiological significance of this parameter (CROUZILLAT et al. 1985; CREVECOEUR et al. 1986; BOYER et al. 1987; KEVERS et al. 1989), in general the functioning of ACC-oxidase as a native EFE is doubtful. The Abbreviations: ACC, l-aminocyclopropane-l-carboxylic acid; EFE, ethylene-forming enzyme
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main evidence in this case is a lack of the basic characteristics of EFE in ACC-oxidase - high affinity towards the substrate and selectivity for stereoisomers of ethyl-substituted ACC (YANG et al. 1985). In a previous work we demonstrated that the activity of ACC-oxidase in the homogenate of winter rye primary leaves had some physiological significance, being partially dependent upon the intensity of ethylene production (IEVINSH et al. 1988). The aim of the present paper is to study the effect of the ethylene-producer ethephon on the activity of EFE in situ and in vitro (activity of ACC-oxidase) and on the ACC level as a function of the intensity of ethylene production in the primary leaf of winter rye seedlings.
Material and Methods Plant material and treatment Winter rye (Secale cereale L. cv. Priekulu) seeds were des infected in 0.1 M KMn04, soaked in water for 2 h and planted on plastic trays (32 x 25 cm) with wet cotton-wool. The trays were kept in darkness at 23°C for 72 h, then transferred to a growth chamber with day/night temperature 20/1 rc, irradiance of 25 W . m- 2 for 16 h. Seedlings were watered with tap water or with 0.1 mM CoS04 beginning with the 3rd day. On the 4th day when the seedlings were 50 ± 5 mm high and the primary leaf had exceeded the coleoptile by 5-10 mm, they were sprayed with 0.01 M ethephon solution (10 ml per tray). Controls were sprayed with water. Another part of seedlings were exposed in 61 glas boxes flushed with a 100 I . h -1 flow of humidified air with or without 0.8 ppm ethylene for 72 h. After certain intervals of time seedlings were cut, rinsed with water and dried with filter paper, then the coleoptile and the second leaf were removed, but the primary leaf was used for analyses.
ACC-oxidase extraction and assay Tissues of primary leaves were ground in a mortar in liquid nitrogen, then extracted twice by cooled acetone (-20°C,S m1 per g FW). Acetone powder was extracted with 0.1 M sodium phosphate buffer, pH 7.8, at 4 °cfor 15 min (3 ml per 0.1 g of powder). The homogenate was filtered through nylon cloth. After centrifugation (15,000 x g for 15 min, 0-4 0c) the supernatant was immediately used for the assay of ACC-oxidase activity. The reaction mixture consisted of 300 flmol sodium phosphate buffer (PH 6.8), 5 flmol ACC, 5 !!mol MnCI 2 , 1 !!mol pyridoxal-5'-phosphate, the total volume being 5 ml with 0.1 m1 of supernatant. The mixture was incubated in 15 ml serum bottles sealed with a rubber septum for 3 h at 28°C in darkness, then the produced ethylene was analysed. Determination of ethylene production The primary leaves were weighed and placed in 15 ml serum bottles containing 0.1 m1 water to prevent dehydration, sealed with a rubber septum and incubated for 3 h at 28°C in darkness. The 1 ml samples were then withdrawn with a hypodermic syringe from the gas phase. Ethylene was assayed on a gas chromatograph Chrom-5 (Czechoslovakia) equipped with a glass column (Ah03, 80°C) and a flame ionization detector, with helium as carrier gas. EFE activity After vacuum infiltration with I mM ACC, leaves were placed in 15 ml serum bottles and sealed with a rubber septum. Incubation conditions and ethylene analysis were the same as described above. The activity of EFE was calculated by substracting the amount of ethylene produced without ACC from that produced with ACC. ACC extraction and determination A 1 g aliquot of ground leaf tissue was extracted in 30 m1 of 80 % acetone at 60°C for I h. The mixture was filtered through filter paper. The acetone was evaporated from the filtrate under vacuum at 45°C. The dry residue was dissolved in 3 ml of water and 2 ml of chloroform. ACC was determined in water phase
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according to LIZAl)!\ and 'I \ N(, \ IY79), 4 f,lmol of HgCI2 were added to 0,7 ml of extract in a 15 ml serum bottle, The bottle was sealed wi th a rubber septum and kept in water with ice, About 0,6 ml of cooled mixture of NaOCI and ION NaO H (2: I, v/v) was injected into the bottle through the septum, The bottle was shaken before and after the 4 min incubation in water with ice , and I ml gas sample was withdrawn for ethylene determination, The effi cienc y of conversion of ACC into ethylene was 60-70 %, Chemicals; replication
ACC was purchased from Sigma Chemical Co , and pyridoxal-5' -phosphate from Merck, Ethephon was obtained from Bitterfeld , G,D,R, All experiments were repeated at least three times with 2-5 replicates, The data presented are of a typical experiment.
Results Early Effect of Ethephon on the Activity of ACC-oxidase, EFE and Ethylene Biosynthesis
The activity of ACC-oxidase in vitro in the control and ethephon-treated leaves was measured at different intervals after treatment. Figure 1 shows that ACC-oxidase activity in the control leaves remained constant during the experiment. In treated plants a lag-period of at 'I.e
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Fig, I , Effect of ethephon on the activity oj ACC-oxidase in primary leaves o.fwinrer rye seedlings, (0), Control ; (e ), treatment with 0,01 M ethephon on the 4th day. ACC-oxidase activity was assayed in tissue homogenate as described in Material and Methods. Values are the means of 3 measurements at each time point , S.E. are smaller th an symbols. Fig. 2. Effect (~f ethephon 011 the activity ofEFE in primary leaves oJ winter rye seedlings. (0) , Control; ee), treatment with 0.0 I Methephon on the 4th day. EFE activity was assayed in situ by incubation of 10 leaves after infiltration with I 111M ACe. Values are the means of 3 measurements at each time point ±S.E" where S.E, bars are larger than symbols.
least 15 min was observed, after which the activity of ACC-oxidase rose sharply, After 45 min ACC-oxidase activity exceeded that of the controls by 60 %, The acti vity of EFE was measured at the same time intervals as that of ACC-oxidase, The activity of EFE in situ in the leaves of control and ethephon-treated seedlings was identical to ACC-oxidase activity (Fig. 2). The rate of ethylene production from the leaves of control seedlings also remained constant during the experiment (Fig. 3). In contrast, the rate of ethylene production from leaves of BPP 186 (1990) 4
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Fig. 3. Effect of ethephon on the intensity of ethylene production in primary leaves of winter rye seedlings. (0), Control; (e), treatment with 0.01 M ethephon on the 4th day. Ethylene production was measured after incubation of 10 leaves. Values are the means of 5 measurements at each time point. Vertical bars indicate S.E. Fig. 4. Effect of ethephon on the content of ACC in primary leaves of winter rye seedlings. (0), Control; (e), treatment with 0.01 M ethephon on the 4th day. ACC content was measured as described in Material and Methods. Values are the means of 3 measurements at each time point. Vertical bars indicate S.E., where S.E. bars are larger than symbols.
ethephon-treated seedlings increased sharply immediately after the treatment. This increase was linear for the period of the experiment and after 1 h was 4.4 times the control rate. The initial level of ACC in untreated leaves was 1.7 nmol g -1, remaining constant during the experiment (Fig. 4). In leaves of ethephon-treated seedlings after a lag-period of at least 15 min there was a considerable decrease in the level of ACC. At the end of the experimental period the ACC content in treated leaves was 1.2 nmol g -1, about 70 % of that in the leaves of control seedlings. The pattern of change in ACC content was inverse to that of EFE and of ACC-oxidase.
Continuous Effect of Ethephon on ACC-oxidase Activity, EFE and Ethylene Biosynthesis Table 1 shows that the most remarkable effect of ethephon on the activity of ACC-oxidase and EFE was 1 to 24 h after the treatment, coinciding with the period of growth inhibition caused by ethephon (Table 2). The activity of ACC-oxidase in ethephon-treated seedlings 24 h after the treatment was 1.6-fold the control and the activity ofEFE was 1. 7-fold. Further on (72 and 168 h after treatment) the effect of ethephon diminished. In the same way, enhancement of ethylene production caused by ethephon reached its maximum in the period 1 to 24 h after the treatment, and this parameter was 6.4 and 6 times greater, respectively, than in the control. The level of ACC in leaves of untreated seedlings 168 h after the treatment was 75 % of that in the control.
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Table I. Continuous
production and
Ace
~tk,1
0/ elilephol1 on the activit',' Iff ACe-oxidase, EFE, intensity of ethylene
coll/el1l
ill primary leaves of winter rye seedlings. Seedlings were treated with
0.01 M ethephon on the 4th day. Values are the means ± S.E. of 3 measurements at each time point. Treatment
Time after treatment (h)
Control
Ethephon
ACC-oxidase
EFE activity
C 2H4
ACC content
activity
(nmol C 2H 4 • gFW- 1 h-')
production
(nmo\'gFW-')
(nmol C1 H4 ' gFW- 1 h- 1) 6.42 ± 0.05
(nmol· gFW- 1 h- 1)
6.0 ± 0.5
0.54 ± 0.10
l.83 ± 0.04
24
2.83 ± 0.Q3
5.1±0.2
0.31 ± 0.08
72
1.65 ± 0.05
4.7 ± 0.2
0.22 ± 0.03
168
0.70 ± 0.04
2.7 ± 0.1
0.20 ± 0.03
1.64 ± 0.10
1
10.27 ± 0.10
10.0 ± 0.4
3.24 ± 0.10
1.31 ± 0.20
24
4.50 ± 0.04
8.7 ± 0.3
1.80 ± 0.20
72
2.21 ± 0.05
6.0 ± 0.2
0.60 ± 0.05
168
0.92 ± 0.Q3
3.3 ± 0.1
0.31 ± 0.06
1.23 ± 0.10
Table 2. Effect of ethephon on the height of primary leaves of winter rye seedlings (mm). Seedlings were treated with 0.01 M ethephon on the 4th day. Values are the means of 25 leaves ± S.E. at each time point. Treatment
Time after treatment (h)
o Control Ethephon
50 ± 2 51
±2
24
72
168
73 ± 1 67 ± 3
94 ± 3 82 ± 2
93 ± 2 83 ± 4
Changes in the Activity of ACC-oxidase, EFE and Ethylene Production during the Leaf Growth It is obvious from the Tables I and 2 that ACC-oxidase activity was partially dependent upon leaf age and extension growth. During the growth of the primary leaf the activity of ACCoxidase diminished. Age-dependent changes in EFE activity in situ and intensity of ethylene production had the same character (Table I). However, the level of ACC changed little during leaf growth.
Effect of Cobalt on Ethylene Production Table 3 shows that an additional treatment of winter rye seedlings with 0.1 mM cobalt, the inhibitor of EFE, before the application of ethephon, inhibited ethylene production by 48 % 24 h after the treatment with ethephon. There was a significant inhibition of ethylene production under the effect of cobalt also in the control seedlings. BPP 186 (1990) 4
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Table 3. Effect of cobalt on ethylene production intensity in primary leaves of winter rye seedlings. Seedlings were preliminary treated with 0.1 M CoS04 on the 3rd day and treated with 0.01 M ethephon on the 4th day. Values are the means ± S.E. of 3 measurements. Time (days)
CZH4 production (nmol· gFW- 1 h -I) Control
4 5 6 7
0.65 0.49 0.45 0.36
± ± ± ±
Cobalt 0.10 0.06 0.05 0.10
0.47 0.35 0.13 0.08
± ± ± ±
0.05 0.04 0.06 0.05
+ Cobalt
Ethephon
Ethephon
1.33 ± 0.20 1.11 ± 0.05 0.82 ± 0.10
0.69 ± 0.10 0.56 ± 0.05 0.35 ± 0.05
Changes in Ethylene Production and ACC Content in Ethylene-treated Seedlings An additional experiment was provided in order to investigate the effect of gaseous ethylene on ethylene production intensity and ACC level. It is obvious from the Table 4 that Table 4. Effect of ethylene on ethylene production intensity and ACC content in primary leaves of winter rye seedlings. Seedlings were exposed in 61 glas boxes flushed with a 100 I· h -I flow of humidified air with or without 0.8 ppm ethylene beginning with the 4th day. Values are the means ± S.E. of 2 measurements. Treatment
Time (days)
CZH4 production (nmol·gFW- 1 h- I )
ACC content (nmol·gFW- 1)
Control
5 7 5 7
0.78 ± 0.08 0.48 ± 0.10 1.52 ± 0.20 0.76±0.14
2.06 ± 0.10 2.05 ± 0.10 1.45 ± 0.08 1.31±0.1O
Ethylene
there was an increase in ethylene production paralleled by a decrease in ACC content in ethylene-treated winter rye seedlings. Discussion It is generally recognized that synthesis of ACC is the main regulating step in ethylene biosynthesis (YANG and HOFFMAN 1984). It has been suggested that ACC conversion to ethylene may be a limiting step in ethylene formation in aging tissues (EVENSEN 1984). In such cases there is increased ACC accumulation. Our investigations show that under the effect of ethephon the activities of ACC-oxidase and EFE in situ increase in parallel with the increase of ethylene production in the primary leaves of winter rye seedlings. The lag-period (15 min) before such an increase induced by ethephon, possibly indicates a synthesis of specific protein(s) as a part of the induction process. Approximately the same lag-period has been shown in the case of induction of stress ethylene production and activity of other enzymes (YANG and HOFFMAN 1984; CHAPELL et al. 1984). These results reveal autocatalytic action of exogenous ethylene in intact juvenile tissues and support the suggestion that EFE is a positive regulator of ethylene formation. Such a
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possibility is strengtheneJ 0) lower ACe content due to ethephon treatment accompanying increased EFE activity with equal lag-periods. As the intensity of ethephon-stimulated ethylene production increased by about 6 times during I h , and the activity of EFE by 1.5 times , it is possible that the major part of increased ethylene production is related to the decomposition of ethephon having penetrated into plant tissues . The other part is related to increased formation of endogenous ethylene from the accessible pool of ACC due to enhanced EFE activity. Besides, the decrease in ethylene production in ethephon-treated plants preliminary exposed with 0.1 mM cobalt, the inhibitor ofEFE (Y ANG and HOFFMAN 1984), as well as an enhancement of ethylene production intensity in ethylene-treated seedlings are in agreement with the suggestion of a possible autocatalytic effect of ethylene in winter rye. Our additional experiments with gaseous ethylene also demonstrate that the effect of increased endogenous ethylene production is not related to enhanced synthesis of ACe. Indeed, a similar effect of ethephon was observed in sunflower cell suspension culture, where an ethylene-releaser caused an increase of ethylene production with a simultaneous decrease in ACC content (SAUERBREY et al. 1988). Changes of ACC-oxidase activity in vitro in primary leaves of winter rye seedlings under the effect of ethephon coincided with the changes in EFE activity in situ. In addition, the activity of ACC-oxidase in control plants shows the same dependence upon the stage of leaf development as the activity of EFE and the intensity of ethylene production. With increased leaf age and ceased extension growth these indicators sharply decreased. These results correlate with the data of high rate of ethylene production in actively growing tissues (HUXTER et a\. 1979). The mechanisms of ACC-oxidase reaction in vitro and of native EFE are probably not identical, the specific features of EFE being related to the localization of this enzyme in the cell membrane (KACPERSKA and KUBACKA-ZEBALSKA 1985). After tissue homogenization and extraction, separate parts of the splitting complex of EFE may produce a certain amount of ethylene due to oxidation of exogenous ACC. Such a capability increases sharply in the presence of various cofactors. Thus, the measurement of ACC-oxidase activity can reflect the activity of native EFE in intact tissues. Similarly , changes in microsomal activity of ACC conversion into ethylene coincided with the changes in EFE activity in situ and the intensity of ethylene production in internodes of Bryonia dioica due to mechanical rubbing (CROUZILLAT et al. \985). The physiological character of ACC-oxidase, i.e., its assimilation to the native EFE activity, will vary according to the plant species, tissues, and their physiological condition. Tn such a way, the presence or absence of various features of ACC-oxidase activity in vitro, in comparison with the native EFE, will depend on manipulations carried out with the plant material in the process of isolation. The oxidase activity activity
present investigations confirm our previous data (lEVINSH et al. 1988) that ACCactivity has certain physiological meaning and can serve as an indicator of the EFE in vivo . Therefore , ACC-oxidase activity provides an estimation of changes in EFE where the assay of this activity in situ is impossible. Acknowledgements
The authors wish to thank Dr. E. correction of this article.
DUMPE
for critical review and G.
SURINA
for the English
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References APELBAUM, A., WANG, S. Y., BURGOON, A. C., BAKER, J. G., and LIEBERMAN, M.: Inhibition of the conversion of I-aminocyclopropane-I-carboxylic acid to ethylene by structural analogs, inhibitors of electron transfer, uncouplers of oxidative phosphorylation and free radical scavengers. Plant Physiol. 67,74-79 (1981). BOYER, N., DEJAEGHER, G., BON, M. c., and GASPAR, T.: Cobalt inhibition ofthigmomorphogenesis in Bryonia dioica: possible role and mechanism of ethylene production. Physiol. Plant. 67, 552-556 (1986). CHAPELL, J., HAHLBROCK, K., and BOLLER, T.: Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase. Planta 161, 475-480 (1984). CREVECOEUR, M., PENEL, c., GREPPIN, H., and GASPAR, T.: Ethylene production in spinach leaves during floral induction. J. Exp. Bot. 37, 1218-1224 (1985). CROUZILLAT, D., DESBIEZ, M. 0., PENEL, C., and GASPAR, T.: Lithium, aminoethoxyvinylglycine and cobalt reversal of the cotyledonary pricking-induced growth inhibition in the hypocotyl of Bidens pilosus in relation to ethylene and peroxidases. Plant Sci. 40, 7-11 (1985). DE JAEGHER, G., BOYER, N., BON, M. C., and GASPAR, T.: Thigmomorphogenesis in Bryonia dioica: early events in ethylene biosynthesis pathway. Biochem. Physiol. Pflanzen 182, 49-56 (1987). EVENSEN, K. B.: Calcium effects on ethylene and ethane production and I-aminocyclopropane-lcarboxylic acid content in potato disks. Physiol. Plant. 60, 125-128 (1984). HUXTER, T. J., REID, D. M., and THORPE, T. A.: Ethylene production by tobacco (Nicotiana tabacum) callus. Physiol. Plant. 46, 374-380 (1979). IEVINSH, G., ROMANOVSKAYA, O. I., and ILJIN, V. V.: Activity of I-amino cyclopropane-I-carboxylic acid oxidase in winter rye seedlings. Latv. PSR Zinat. Akad. Vestis 12, 80-86 (1988) (In Russian). KACPERSKA, A., and KUBACKA-ZEBALSKA, M.: Is lipoxygenase involved in the formation of ethylene from ACC? Physiol. Plant. 64, 333-338 (1985). KEVERS, C., GOLDBERG, R., and GASPAR, T.: Gradients in ethylene metabolism along the growing mung bean hypocotyl. Saussurea 19, 129~134 (1989). KONZE, J. R., and KENDE, H.: Ethylene formation from l-aminocyclopropane-l-carboxylic acid in homogenates of etiolated pea seedlings. Planta 146, 293- 301 (1979). LIZADA, M. C. c., and YANG, S. F.: A simple and sensitive assay for I-aminocyclopropane-l-carboxylic acid. Anal. Biochem. 100, 140-145 (1979). MAYAK, S., LEGGE, R. L., and THOMPSON, J. E.: Ethylene formation from l-aminocyclopropane-Icarboxylic acid by microsomal membranes from senescing carnation flowers. Planta 153, 49-55 (1981). PORTER, A. J. R., BORLAKOGLU, J. T., and JOHN, P.: Activity of the ethylene-forming enzyme in relation to plant cell structure and organization. J. Plant Physiol. 125, 207-216 (1986). SAUERBREY, E., GROSSMAN, K., and JUNG, J.: Ethylene production by sunflower cell suspensions. Effects of plant growth retardants. Plant Physiol. 87, 510-513 (1988). YANG, S. F., and HOFFMAN, N. E.: Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155-189 (1984). YANG, S. F., Lm, Y., Su, L., PEISER, G. D., HOFFMAN, N. E., and McKEON, T.: Metabolism of 1amino cyclopropane-I-carboxylic acid. In: Ethylene and Plant Development (Eds. J. A. ROBERTS and G. A. TUCKER) pp. 9-21. Butterworths, London 1985.
Received June 19, 1989; revised form accepted January 15, 1990 Authors' address: Dr. G. IEVINSH, Dr. V. ILJIN, Dr. O. KREICBERGS and Dr. O. ROMANOVSKAYA; Institute of Biology, Latvian Academy of Sciences, 229021 Salaspils, 3 Miera street, Latvian SSR, USSR.
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Effect of Ethephon on the Activity of the Ethylene-forming Enzyme and the Biosynthesis of Ethylene in Winter Rye Seedlings GEDERTS IEVINSH, YJACHESLAV IUIN, OLGERTS KREICBERGS and OLGA ROMANOVSKAYA Institute of Biology, Latvian Academy of Sciences, Salaspils, Latvian SSR, USSR Key Term Index: ACC, ACC-oxidase, EFE, ethephon, ethylene biosynthesis; Secale cereale
Summary The effect of ethylene-releaser ethephon on the intensity of ethylene production, ACC content, and EFE activity in situ and in vitro (activity of ACC-oxidase) in primary leaves of intact winter rye seedlings was investigated. There were a sharp increase of ethylene production immediately after the treatment and an increase in ACC-oxidase activity in vitro and EFE activity in situ after a lag period of at least 15 min. Inversely, the level of ACC decreased after an equal lag period. Such an effect of ethephon on ethylene biosynthesis was most remarkable 1 to 24 h after the treatment. An additional treatment with 0.1 mM CoS0 4 before the application of ethephon inhibited the ethylene production. It is suggested that a major part of the increase in the ethylene production is related to the decomposition of ethephon having penetrated into leaf tissues, while the other part is related to an increased formation of endogenous ethylene from the accessible pool of ACC due to enhanced EFE activity. Treatment with gaseous ethylene (0.8 ppm) caused an increase in ethylene production and a decrease in ACC content. The results reveal an autocatalytic action of exogenous ethylene in intact juvenile tissues and emphasize the probable importance of EFE in a positive regulation of the ethylene formation.
Introduction The autocatalytic effect of ethylene can appear both at the synthesis of the ethylene precursor, ACC, and at the conversion of ACC to ethylene (YANG and HOFFMAN 1984). Many data concern the effect of exogenous ethylene on the last step of ethylene biosynthesis in fruits and on the aging or injured vegetative tissues (YANG and HOFFMAN 1984), but there is little information about its effect in intact juvenile plants. The last stage of ethylene biosynthesis is catalysed by a so-called ethylene-forming enzyme, EFE, which is labile and related to the integrity of cell membranes and tissues (APELBAUM et at. 1981; PORTER et at. 1986). Therefore, the activity of the enzyme cannot be determined in vitro, but a probable activity of EFE is estimated according to the capacity or tissues to convert the exogenous ACC into ethylene in situ. In homogenates and membrane fractions of plant material the ability to convert the exogenous ACC to ethylene can be estimated in vitro (activity of ACC-oxidase) (KONZE and KENDE 1979; MAYAK et al. 1981). Though some investigations show a certain physiological significance of this parameter (CROUZILLAT et al. 1985; CREVECOEUR et al. 1986; BOYER et al. 1987; KEVERS et al. 1989), in general the functioning of ACC-oxidase as a native EFE is doubtful. The Abbreviations: ACC, l-aminocyclopropane-l-carboxylic acid; EFE, ethylene-forming enzyme
BPP 186
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4
221
main evidence in this case is a lack of the basic characteristics of EFE in ACC-oxidase - high affinity towards the substrate and selectivity for stereoisomers of ethyl-substituted ACC (YANG et al. 1985). In a previous work we demonstrated that the activity of ACC-oxidase in the homogenate of winter rye primary leaves had some physiological significance, being partially dependent upon the intensity of ethylene production (IEVINSH et al. 1988). The aim of the present paper is to study the effect of the ethylene-producer ethephon on the activity of EFE in situ and in vitro (activity of ACC-oxidase) and on the ACC level as a function of the intensity of ethylene production in the primary leaf of winter rye seedlings.
Material and Methods Plant material and treatment Winter rye (Secale cereale L. cv. Priekulu) seeds were des infected in 0.1 M KMn04, soaked in water for 2 h and planted on plastic trays (32 x 25 cm) with wet cotton-wool. The trays were kept in darkness at 23°C for 72 h, then transferred to a growth chamber with day/night temperature 20/1 rc, irradiance of 25 W . m- 2 for 16 h. Seedlings were watered with tap water or with 0.1 mM CoS04 beginning with the 3rd day. On the 4th day when the seedlings were 50 ± 5 mm high and the primary leaf had exceeded the coleoptile by 5-10 mm, they were sprayed with 0.01 M ethephon solution (10 ml per tray). Controls were sprayed with water. Another part of seedlings were exposed in 61 glas boxes flushed with a 100 I . h -1 flow of humidified air with or without 0.8 ppm ethylene for 72 h. After certain intervals of time seedlings were cut, rinsed with water and dried with filter paper, then the coleoptile and the second leaf were removed, but the primary leaf was used for analyses.
ACC-oxidase extraction and assay Tissues of primary leaves were ground in a mortar in liquid nitrogen, then extracted twice by cooled acetone (-20°C,S m1 per g FW). Acetone powder was extracted with 0.1 M sodium phosphate buffer, pH 7.8, at 4 °cfor 15 min (3 ml per 0.1 g of powder). The homogenate was filtered through nylon cloth. After centrifugation (15,000 x g for 15 min, 0-4 0c) the supernatant was immediately used for the assay of ACC-oxidase activity. The reaction mixture consisted of 300 flmol sodium phosphate buffer (PH 6.8), 5 flmol ACC, 5 !!mol MnCI 2 , 1 !!mol pyridoxal-5'-phosphate, the total volume being 5 ml with 0.1 m1 of supernatant. The mixture was incubated in 15 ml serum bottles sealed with a rubber septum for 3 h at 28°C in darkness, then the produced ethylene was analysed. Determination of ethylene production The primary leaves were weighed and placed in 15 ml serum bottles containing 0.1 m1 water to prevent dehydration, sealed with a rubber septum and incubated for 3 h at 28°C in darkness. The 1 ml samples were then withdrawn with a hypodermic syringe from the gas phase. Ethylene was assayed on a gas chromatograph Chrom-5 (Czechoslovakia) equipped with a glass column (Ah03, 80°C) and a flame ionization detector, with helium as carrier gas. EFE activity After vacuum infiltration with I mM ACC, leaves were placed in 15 ml serum bottles and sealed with a rubber septum. Incubation conditions and ethylene analysis were the same as described above. The activity of EFE was calculated by substracting the amount of ethylene produced without ACC from that produced with ACC. ACC extraction and determination A 1 g aliquot of ground leaf tissue was extracted in 30 m1 of 80 % acetone at 60°C for I h. The mixture was filtered through filter paper. The acetone was evaporated from the filtrate under vacuum at 45°C. The dry residue was dissolved in 3 ml of water and 2 ml of chloroform. ACC was determined in water phase
222
BPP 186 (1990) 4
according to LIZAl)!\ and 'I \ N(, \ IY79), 4 f,lmol of HgCI2 were added to 0,7 ml of extract in a 15 ml serum bottle, The bottle was sealed wi th a rubber septum and kept in water with ice, About 0,6 ml of cooled mixture of NaOCI and ION NaO H (2: I, v/v) was injected into the bottle through the septum, The bottle was shaken before and after the 4 min incubation in water with ice , and I ml gas sample was withdrawn for ethylene determination, The effi cienc y of conversion of ACC into ethylene was 60-70 %, Chemicals; replication
ACC was purchased from Sigma Chemical Co , and pyridoxal-5' -phosphate from Merck, Ethephon was obtained from Bitterfeld , G,D,R, All experiments were repeated at least three times with 2-5 replicates, The data presented are of a typical experiment.
Results Early Effect of Ethephon on the Activity of ACC-oxidase, EFE and Ethylene Biosynthesis
The activity of ACC-oxidase in vitro in the control and ethephon-treated leaves was measured at different intervals after treatment. Figure 1 shows that ACC-oxidase activity in the control leaves remained constant during the experiment. In treated plants a lag-period of at 'I.e
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Fig, I , Effect of ethephon on the activity oj ACC-oxidase in primary leaves o.fwinrer rye seedlings, (0), Control ; (e ), treatment with 0,01 M ethephon on the 4th day. ACC-oxidase activity was assayed in tissue homogenate as described in Material and Methods. Values are the means of 3 measurements at each time point , S.E. are smaller th an symbols. Fig. 2. Effect (~f ethephon 011 the activity ofEFE in primary leaves oJ winter rye seedlings. (0) , Control; ee), treatment with 0.0 I Methephon on the 4th day. EFE activity was assayed in situ by incubation of 10 leaves after infiltration with I 111M ACe. Values are the means of 3 measurements at each time point ±S.E" where S.E, bars are larger than symbols.
least 15 min was observed, after which the activity of ACC-oxidase rose sharply, After 45 min ACC-oxidase activity exceeded that of the controls by 60 %, The acti vity of EFE was measured at the same time intervals as that of ACC-oxidase, The activity of EFE in situ in the leaves of control and ethephon-treated seedlings was identical to ACC-oxidase activity (Fig. 2). The rate of ethylene production from the leaves of control seedlings also remained constant during the experiment (Fig. 3). In contrast, the rate of ethylene production from leaves of BPP 186 (1990) 4
223
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Fig. 3. Effect of ethephon on the intensity of ethylene production in primary leaves of winter rye seedlings. (0), Control; (e), treatment with 0.01 M ethephon on the 4th day. Ethylene production was measured after incubation of 10 leaves. Values are the means of 5 measurements at each time point. Vertical bars indicate S.E. Fig. 4. Effect of ethephon on the content of ACC in primary leaves of winter rye seedlings. (0), Control; (e), treatment with 0.01 M ethephon on the 4th day. ACC content was measured as described in Material and Methods. Values are the means of 3 measurements at each time point. Vertical bars indicate S.E., where S.E. bars are larger than symbols.
ethephon-treated seedlings increased sharply immediately after the treatment. This increase was linear for the period of the experiment and after 1 h was 4.4 times the control rate. The initial level of ACC in untreated leaves was 1.7 nmol g -1, remaining constant during the experiment (Fig. 4). In leaves of ethephon-treated seedlings after a lag-period of at least 15 min there was a considerable decrease in the level of ACC. At the end of the experimental period the ACC content in treated leaves was 1.2 nmol g -1, about 70 % of that in the leaves of control seedlings. The pattern of change in ACC content was inverse to that of EFE and of ACC-oxidase.
Continuous Effect of Ethephon on ACC-oxidase Activity, EFE and Ethylene Biosynthesis Table 1 shows that the most remarkable effect of ethephon on the activity of ACC-oxidase and EFE was 1 to 24 h after the treatment, coinciding with the period of growth inhibition caused by ethephon (Table 2). The activity of ACC-oxidase in ethephon-treated seedlings 24 h after the treatment was 1.6-fold the control and the activity ofEFE was 1. 7-fold. Further on (72 and 168 h after treatment) the effect of ethephon diminished. In the same way, enhancement of ethylene production caused by ethephon reached its maximum in the period 1 to 24 h after the treatment, and this parameter was 6.4 and 6 times greater, respectively, than in the control. The level of ACC in leaves of untreated seedlings 168 h after the treatment was 75 % of that in the control.
224
BPP 186 (1990) 4
Table I. Continuous
production and
Ace
~tk,1
0/ elilephol1 on the activit',' Iff ACe-oxidase, EFE, intensity of ethylene
coll/el1l
ill primary leaves of winter rye seedlings. Seedlings were treated with
0.01 M ethephon on the 4th day. Values are the means ± S.E. of 3 measurements at each time point. Treatment
Time after treatment (h)
Control
Ethephon
ACC-oxidase
EFE activity
C 2H4
ACC content
activity
(nmol C 2H 4 • gFW- 1 h-')
production
(nmo\'gFW-')
(nmol C1 H4 ' gFW- 1 h- 1) 6.42 ± 0.05
(nmol· gFW- 1 h- 1)
6.0 ± 0.5
0.54 ± 0.10
l.83 ± 0.04
24
2.83 ± 0.Q3
5.1±0.2
0.31 ± 0.08
72
1.65 ± 0.05
4.7 ± 0.2
0.22 ± 0.03
168
0.70 ± 0.04
2.7 ± 0.1
0.20 ± 0.03
1.64 ± 0.10
1
10.27 ± 0.10
10.0 ± 0.4
3.24 ± 0.10
1.31 ± 0.20
24
4.50 ± 0.04
8.7 ± 0.3
1.80 ± 0.20
72
2.21 ± 0.05
6.0 ± 0.2
0.60 ± 0.05
168
0.92 ± 0.Q3
3.3 ± 0.1
0.31 ± 0.06
1.23 ± 0.10
Table 2. Effect of ethephon on the height of primary leaves of winter rye seedlings (mm). Seedlings were treated with 0.01 M ethephon on the 4th day. Values are the means of 25 leaves ± S.E. at each time point. Treatment
Time after treatment (h)
o Control Ethephon
50 ± 2 51
±2
24
72
168
73 ± 1 67 ± 3
94 ± 3 82 ± 2
93 ± 2 83 ± 4
Changes in the Activity of ACC-oxidase, EFE and Ethylene Production during the Leaf Growth It is obvious from the Tables I and 2 that ACC-oxidase activity was partially dependent upon leaf age and extension growth. During the growth of the primary leaf the activity of ACCoxidase diminished. Age-dependent changes in EFE activity in situ and intensity of ethylene production had the same character (Table I). However, the level of ACC changed little during leaf growth.
Effect of Cobalt on Ethylene Production Table 3 shows that an additional treatment of winter rye seedlings with 0.1 mM cobalt, the inhibitor of EFE, before the application of ethephon, inhibited ethylene production by 48 % 24 h after the treatment with ethephon. There was a significant inhibition of ethylene production under the effect of cobalt also in the control seedlings. BPP 186 (1990) 4
225
Table 3. Effect of cobalt on ethylene production intensity in primary leaves of winter rye seedlings. Seedlings were preliminary treated with 0.1 M CoS04 on the 3rd day and treated with 0.01 M ethephon on the 4th day. Values are the means ± S.E. of 3 measurements. Time (days)
CZH4 production (nmol· gFW- 1 h -I) Control
4 5 6 7
0.65 0.49 0.45 0.36
± ± ± ±
Cobalt 0.10 0.06 0.05 0.10
0.47 0.35 0.13 0.08
± ± ± ±
0.05 0.04 0.06 0.05
+ Cobalt
Ethephon
Ethephon
1.33 ± 0.20 1.11 ± 0.05 0.82 ± 0.10
0.69 ± 0.10 0.56 ± 0.05 0.35 ± 0.05
Changes in Ethylene Production and ACC Content in Ethylene-treated Seedlings An additional experiment was provided in order to investigate the effect of gaseous ethylene on ethylene production intensity and ACC level. It is obvious from the Table 4 that Table 4. Effect of ethylene on ethylene production intensity and ACC content in primary leaves of winter rye seedlings. Seedlings were exposed in 61 glas boxes flushed with a 100 I· h -I flow of humidified air with or without 0.8 ppm ethylene beginning with the 4th day. Values are the means ± S.E. of 2 measurements. Treatment
Time (days)
CZH4 production (nmol·gFW- 1 h- I )
ACC content (nmol·gFW- 1)
Control
5 7 5 7
0.78 ± 0.08 0.48 ± 0.10 1.52 ± 0.20 0.76±0.14
2.06 ± 0.10 2.05 ± 0.10 1.45 ± 0.08 1.31±0.1O
Ethylene
there was an increase in ethylene production paralleled by a decrease in ACC content in ethylene-treated winter rye seedlings. Discussion It is generally recognized that synthesis of ACC is the main regulating step in ethylene biosynthesis (YANG and HOFFMAN 1984). It has been suggested that ACC conversion to ethylene may be a limiting step in ethylene formation in aging tissues (EVENSEN 1984). In such cases there is increased ACC accumulation. Our investigations show that under the effect of ethephon the activities of ACC-oxidase and EFE in situ increase in parallel with the increase of ethylene production in the primary leaves of winter rye seedlings. The lag-period (15 min) before such an increase induced by ethephon, possibly indicates a synthesis of specific protein(s) as a part of the induction process. Approximately the same lag-period has been shown in the case of induction of stress ethylene production and activity of other enzymes (YANG and HOFFMAN 1984; CHAPELL et al. 1984). These results reveal autocatalytic action of exogenous ethylene in intact juvenile tissues and support the suggestion that EFE is a positive regulator of ethylene formation. Such a
226
BPP 186 (1990) 4
possibility is strengtheneJ 0) lower ACe content due to ethephon treatment accompanying increased EFE activity with equal lag-periods. As the intensity of ethephon-stimulated ethylene production increased by about 6 times during I h , and the activity of EFE by 1.5 times , it is possible that the major part of increased ethylene production is related to the decomposition of ethephon having penetrated into plant tissues . The other part is related to increased formation of endogenous ethylene from the accessible pool of ACC due to enhanced EFE activity. Besides, the decrease in ethylene production in ethephon-treated plants preliminary exposed with 0.1 mM cobalt, the inhibitor ofEFE (Y ANG and HOFFMAN 1984), as well as an enhancement of ethylene production intensity in ethylene-treated seedlings are in agreement with the suggestion of a possible autocatalytic effect of ethylene in winter rye. Our additional experiments with gaseous ethylene also demonstrate that the effect of increased endogenous ethylene production is not related to enhanced synthesis of ACe. Indeed, a similar effect of ethephon was observed in sunflower cell suspension culture, where an ethylene-releaser caused an increase of ethylene production with a simultaneous decrease in ACC content (SAUERBREY et al. 1988). Changes of ACC-oxidase activity in vitro in primary leaves of winter rye seedlings under the effect of ethephon coincided with the changes in EFE activity in situ. In addition, the activity of ACC-oxidase in control plants shows the same dependence upon the stage of leaf development as the activity of EFE and the intensity of ethylene production. With increased leaf age and ceased extension growth these indicators sharply decreased. These results correlate with the data of high rate of ethylene production in actively growing tissues (HUXTER et a\. 1979). The mechanisms of ACC-oxidase reaction in vitro and of native EFE are probably not identical, the specific features of EFE being related to the localization of this enzyme in the cell membrane (KACPERSKA and KUBACKA-ZEBALSKA 1985). After tissue homogenization and extraction, separate parts of the splitting complex of EFE may produce a certain amount of ethylene due to oxidation of exogenous ACC. Such a capability increases sharply in the presence of various cofactors. Thus, the measurement of ACC-oxidase activity can reflect the activity of native EFE in intact tissues. Similarly , changes in microsomal activity of ACC conversion into ethylene coincided with the changes in EFE activity in situ and the intensity of ethylene production in internodes of Bryonia dioica due to mechanical rubbing (CROUZILLAT et al. \985). The physiological character of ACC-oxidase, i.e., its assimilation to the native EFE activity, will vary according to the plant species, tissues, and their physiological condition. Tn such a way, the presence or absence of various features of ACC-oxidase activity in vitro, in comparison with the native EFE, will depend on manipulations carried out with the plant material in the process of isolation. The oxidase activity activity
present investigations confirm our previous data (lEVINSH et al. 1988) that ACCactivity has certain physiological meaning and can serve as an indicator of the EFE in vivo . Therefore , ACC-oxidase activity provides an estimation of changes in EFE where the assay of this activity in situ is impossible. Acknowledgements
The authors wish to thank Dr. E. correction of this article.
DUMPE
for critical review and G.
SURINA
for the English
BPP 186 (1990) 4
227
References APELBAUM, A., WANG, S. Y., BURGOON, A. C., BAKER, J. G., and LIEBERMAN, M.: Inhibition of the conversion of I-aminocyclopropane-I-carboxylic acid to ethylene by structural analogs, inhibitors of electron transfer, uncouplers of oxidative phosphorylation and free radical scavengers. Plant Physiol. 67,74-79 (1981). BOYER, N., DEJAEGHER, G., BON, M. c., and GASPAR, T.: Cobalt inhibition ofthigmomorphogenesis in Bryonia dioica: possible role and mechanism of ethylene production. Physiol. Plant. 67, 552-556 (1986). CHAPELL, J., HAHLBROCK, K., and BOLLER, T.: Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase. Planta 161, 475-480 (1984). CREVECOEUR, M., PENEL, c., GREPPIN, H., and GASPAR, T.: Ethylene production in spinach leaves during floral induction. J. Exp. Bot. 37, 1218-1224 (1985). CROUZILLAT, D., DESBIEZ, M. 0., PENEL, C., and GASPAR, T.: Lithium, aminoethoxyvinylglycine and cobalt reversal of the cotyledonary pricking-induced growth inhibition in the hypocotyl of Bidens pilosus in relation to ethylene and peroxidases. Plant Sci. 40, 7-11 (1985). DE JAEGHER, G., BOYER, N., BON, M. C., and GASPAR, T.: Thigmomorphogenesis in Bryonia dioica: early events in ethylene biosynthesis pathway. Biochem. Physiol. Pflanzen 182, 49-56 (1987). EVENSEN, K. B.: Calcium effects on ethylene and ethane production and I-aminocyclopropane-lcarboxylic acid content in potato disks. Physiol. Plant. 60, 125-128 (1984). HUXTER, T. J., REID, D. M., and THORPE, T. A.: Ethylene production by tobacco (Nicotiana tabacum) callus. Physiol. Plant. 46, 374-380 (1979). IEVINSH, G., ROMANOVSKAYA, O. I., and ILJIN, V. V.: Activity of I-amino cyclopropane-I-carboxylic acid oxidase in winter rye seedlings. Latv. PSR Zinat. Akad. Vestis 12, 80-86 (1988) (In Russian). KACPERSKA, A., and KUBACKA-ZEBALSKA, M.: Is lipoxygenase involved in the formation of ethylene from ACC? Physiol. Plant. 64, 333-338 (1985). KEVERS, C., GOLDBERG, R., and GASPAR, T.: Gradients in ethylene metabolism along the growing mung bean hypocotyl. Saussurea 19, 129~134 (1989). KONZE, J. R., and KENDE, H.: Ethylene formation from l-aminocyclopropane-l-carboxylic acid in homogenates of etiolated pea seedlings. Planta 146, 293- 301 (1979). LIZADA, M. C. c., and YANG, S. F.: A simple and sensitive assay for I-aminocyclopropane-l-carboxylic acid. Anal. Biochem. 100, 140-145 (1979). MAYAK, S., LEGGE, R. L., and THOMPSON, J. E.: Ethylene formation from l-aminocyclopropane-Icarboxylic acid by microsomal membranes from senescing carnation flowers. Planta 153, 49-55 (1981). PORTER, A. J. R., BORLAKOGLU, J. T., and JOHN, P.: Activity of the ethylene-forming enzyme in relation to plant cell structure and organization. J. Plant Physiol. 125, 207-216 (1986). SAUERBREY, E., GROSSMAN, K., and JUNG, J.: Ethylene production by sunflower cell suspensions. Effects of plant growth retardants. Plant Physiol. 87, 510-513 (1988). YANG, S. F., and HOFFMAN, N. E.: Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155-189 (1984). YANG, S. F., Lm, Y., Su, L., PEISER, G. D., HOFFMAN, N. E., and McKEON, T.: Metabolism of 1amino cyclopropane-I-carboxylic acid. In: Ethylene and Plant Development (Eds. J. A. ROBERTS and G. A. TUCKER) pp. 9-21. Butterworths, London 1985.
Received June 19, 1989; revised form accepted January 15, 1990 Authors' address: Dr. G. IEVINSH, Dr. V. ILJIN, Dr. O. KREICBERGS and Dr. O. ROMANOVSKAYA; Institute of Biology, Latvian Academy of Sciences, 229021 Salaspils, 3 Miera street, Latvian SSR, USSR.
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