Planta (1984) 160:276-280
Planta
9 Springer-Verlag 1984
Ethylene formation in Pisum sativum and Vicia faba protoplasts Micha Guy* and Hans Kende MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
Abstract. Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-l-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-l,1dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-14C]meth ionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation. Key words: 1-Aminocyclopropane-l-carboxylic acid - Aminoethoxyvinylglycine - Ethylene synthesis - Pisum (ethylene synthesis) - Vicia.
Introduction
Mattoo and Lieberman (1977) attempted to localize the site of ethylene biosynthesis in plant cells by using protoplasts isolated from apple fruits. They found that freshly prepared protoplasts had * Present address: Ben-Gurion University of the Negev, Blaus-
tein Institute for Desert Research, Sede Boqer, Israel Abbreviations: ACC = l-Aminocyclopropane-l-carboxylicacid; AVG=aminoethoxyvinylglycine; CCCP=carbonyl cyanide m-chlorophenylhydrazone; DCMU = 3-(3,4-dichlorophenyl)1,1-dimethylurea
lost their ability to produce ethylene. Ethylene synthesis resumed slowly as the protoplasts formed new cell walls, provided that methionine was added to the incubation medium. In later experiments, Anderson et al. (1979) found conditions under .which freshly isolated apple protoplasts did produce ethylene. Ethylene synthesis in apple protoplasts appeared to be anomalous in that it was relatively insensitive to aminoethoxyvinylglycine (AVG). Aminoethoxyvinylglycine is a potent inhibitor of ethylene synthesis in most plant tissues, including apple fruits, and acts by blocking the synthesis of 1-aminocyclopropane-l-carboxylic acid (ACC), the immediate precursor of ethylene (Adams and Yang 1979). Konze et al. (1980) reported that protoplasts prepared from flower tissue of Ipomoea tricolor Cav. produced ethylene, but only if ACC was added to the incubation mixture. We felt that protoplasts might be suitable to study the compartmentation of components of the ethylene-forming system and that the use of protoplasts might enable us to localize the intracellular site of ethylene synthesis. In the following, we describe conditions under which protoplasts isolated from Pisurn sativum and Viciafaba leaves synthesize both ACC and ethylene, and the effect of light on these processes. We also supply evidence that ACC and ethylene synthesis are compartmented in the plant cell. Materials and methods Plant material. Seeds of Pisum sativurn L., cv. Alaska (Ferry:
Morse Seed Co., Mountain View, Calif., USA), were imbibed overnight in aerated distilled water. They were sown in plastic trays either in vermiculite or a soil mixture consisting of three parts Sunshine Mix, Blend No. I (Fisons-Western Corp., Vancouver, B.C., Canada), one part sand, one part Michigan bog peat, one part loam soil, and 400 g of triple superphosphate
M. Guy and H. Kende : Ethylene formation in protoplasts per 50 1 of mixture. The plants were grown at 23 ~ C and a photoperiod of 16 h (cool-white fluorescent lamps; General Electric Co., Cleveland, O., USA; photon flux 125 p.mol m - 2 s-1). Seeds of Vicia faba L., cv. Long Pod (Burpee Co., Clinton, Ia., USA), were sown in the above soil mixture, two seeds per i-liter plastic container. The plants were grown in a growth chamber at 23 ~ C, a daily light period of 20 h (coolwhite fluorescent lamps, General Electric, and incandescent lamps; photon flux 350 g m o l m -2 s -a) and at 70% relative humidity.
277 30
80O
//~
A io
o
300
zo
o
) ~O0
10
--,a
Preparation of protoplasts. Fully expanded leaflets of 10- to 13-d-old pea plants were detached, surface-sterilized with 5% (w/v) sodium hypochlorite for approx. 30 s and then cut into 2-mm-wide strips. These were incubated in a filter-sterilized solution of 0.6 M mannitol and 1% (w/v) cellulysin at pH 5.6 in sterile Petri dishes at 30~ C for 2.5-3 h. In one experiment (Table 1), incubation of leaves in isolation medium was for 18 h at 22~ C. Protoplasts were isolated as described in Guy et al. (1978), and resuspended in a medium containing 0.2 mM KH~PO 4, ~ mM KNO~, i m M MgSO4, 10raM CaC12, 20 mM Mes [2-(N-morpholino)ethanesulfonic acid], 20 mM Hepes [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid] and 0.55 M mannitol, adjusted to pH 5.5 with NaOH. The number of protoplasts was determined under a microscope using a hemacytometer. Protoplasts were prepared from the youngest, fully expanded leaflets of V. faba plants as described for peas except that the leaves were not cut into 2-ram strips. The main veins were removed, and the epidermis from the abaxial side of the blade was peeled off with fine forceps before incubation in cellulysin.
Ethylene and ACC determinations. For the measurement of ethylene formation, 1.106-2.106 protoplasts were incubated in 0.6-0.7 ml resuspension medium in 4-ml test tubes stoppered with serum vial caps. The test tubes were kept at a slant either in the dark or in front of fluorescent lights (cool white fluorescent lamps; General Electric; 70 gmol m -2 s-~) at 22 ~ C. Ethylene was sampled by first injecting 1 ml of air into each test tube with a tuberculin syringe, pumping the syringe several times, and then withdrawing 1 ml for analysis by gas chromatography (Kende and Hanson 1976). In calculating the amount of ethylene in each tube, a correction was applied for the successive injections of air and removals of gas samples. The specific radioactivity of ethylene was determined as in Konze et al. (1980).
For ACC determinations, protoplasts were lysed directly in the ACC-assay mixture of Lizada and Yang (1979); ACC was extracted from leaf tissue as described tbr tomato pericarp (Kende and Bolter 1981), and was measured in both instances according to Lizada and Yang (1979). The specific radioactivity of the C-2 and C-3 atoms of ACC was determined by first converting ACC to ethylene following the method of Lizada and Yang (1979) and then measuring the specific radioactivity of ethylene according to Konze et al. (1980).
Chemicals'. L-[U-~'~C]Methionine (l.06.J0r was purchased from Amersham, Arlington Heights, Ill., USA; ACC and cellulysin were from Calbiochem-Behring Corp., La Jolla, Calif., USA; Ficoll (molecular weight 400,000) from Pharmacia Fine Chemicals, Piscataway, N.J., USA; 3-(3,4-dichlorophenyl)-J,l-dimethylurea (DCMU) from Du Pont de Nemours & Co., Wilmington, Dela., USA; carbonyl cyanide m-chloropheny/hydrazone (CCCP) from Sigma Chemical Co., St. Louis, Mo., USA; Hepes from Research Organics, Cleveland, O., USA, and Mes from U.S. Biochemical Corp., Cleveland, O., USA. Aminoethoxyvinylglycinewas a gift of Dr. M. Lieberman (U.S. Department of Agriculture, Beltsville, Md., USA).
_~
r
-o
I
3o
If, ~ 240 45O
I
r5 TIME(rain)
Ill
~E o. v t)
Fig. 1. A Ethylene synthesis in protoplasts isolated from pea leaves that had been incubated in the dark (R), in the light (o), and in the light in the presence of 200 pM AVG (o), 0.J mM DCMU (n) and 10 pM CCCP (zx). B Levels of ACC in the same protoplasts at the beginning of the incubation (hatched bar) and after 450 min of incubation. Symbols as above. The vertical bars denote the spread of duplicate samples in the ACC assay; when no bar is given, the spread between duplicates has been smaller than the symbol used
Table 1. Induction of ACC synthesis in pea leaf tissue b) protoptast-isolation medium. Strips of leaf tissue were cut from the youngest fully expanded leaflets and were incubated in protoplast-isolation medium (1% cellulysin and 0.6 M mannitol) or on distilled water in darkness at 22 ~ C for 18 h. The ACC level of whole leaflets incubated on water for 18 h and of fresh leaflets was also measured Treatment
ACC content (nmol g - a FW)
Leaf strips on isolation medium Leaf strips on water Intact leaves on water Intact leaves, fresh
19.5 2.5 2.1 1.6
Results
Incubation of pea leaf tissue in protoplast isolation medium containing cellulysin and mannitol induced ACC synthesis (Table 1). The leaf tissue that remained intact for 18 h of incubation in the above medium contained 12 times more ACC than did fresh leaves. The increase in ACC content of leaves that were cut and incubated on water was, by comparison, small, as was the increase in the ACC content of whole leaves incubated on water. Protoplasts isolated from pea leaves evolved ethylene, even in the absence of added substrate (Fig. l A). Illuminated protoplasts produced up to 4.5-fold more ethylene than protoplasts kept in darkness. When 200 pM AVG was present in the medium in which the protoplasts were resuspended and incubated, ethylene production was only slightly inhibited. 3-(3,4-Dichlorophenyl)-l,l-di-
278
M. Guy and H. Kende: Ethylene formation in protoplasts 2o ,-//
800
fOl A
o
g
~15 o o
o
CorlfrOl AVG
"5
c'e"
%n \
4oo 3
w Z l.d
#
2::: I'-" LtJ
LU
~ __
20
50 TIME
85
Dark+ CoCl~ I
I
70
240
(min)
Fig. 2. A The effect of AVG on ethylene formation in pea protoplasts. Protoplasts were incubated in darkness in resuspension medium either without AVG (o) or with 200 gM AVG (e). In the third sample, 200 pM AVG was present in the isolation medium as well as in the resuspension medium (A). B Levels of ACC in the same protoplasts after 20 rain (hatched bars) and 240 min of incubation. Symbols as above. The vertical bars denote the spread of duplicate samples as in Fig. 1
methylurea, an inhibitor of photosynthetic electron transport, reduced light-stimulated ethylene synthesis by 60% (Fig. 1A). In other experiments, D C M U reduced ethylene production in illuminated protoplasts to that of protoplasts in darkness. Carbonyl cyanide m-chlorophenylhydrazone, a proton conductor and uncoupler of phosphoryladon, proved to be a very potent inhibitor of ethylene synthesis in pea protoplasts (Fig. 1 A). The level of ACC doubled during the 450 rain of incubation in darkness and increased fourfold in the light (Fig. 1 B); AVG, D C M U and CCCP all inhibited the increase in the level of ACC. When AVG was present in the medium in which protoplasts were isolated and then again in the resuspension medium, ethylene synthesis was inhibited by more than 90% (Fig. 2A). The inhibitory effect of AVG on ethylene synthesis was fully reversible with added ACC (results not shown). Aminoethoxyvinylglycine again had only a minor effect on ethylene synthesis when it was added, after isolation of the protoplasts, to the medium in which the protoplasts were resuspended. 1Aminocyclopropane-l-carboxylic-acid levels increased in the control protoplasts during the 4 h of the experiment (Fig. 2 B). Aminoethoxyvinylglycine inhibited ACC formation, both in the protoplasts that had been treated with AVG in the resuspension medium and in those that were also isolated in the presence of AVG. Protoplasts isolated from V. faba leaves also
135 TIME(rain)
I 210
270
Fig. 3. Ethylene synthesis in protoplasts isolated from V. faba leaves and incubated in the light with 1 mM COC12 0x) and without CoC12 (o) or in the dark with I mM CoCI~ (A) and without CoCI 2 (e). CoC12 was not present during isolation of the protoplasts
20C
-2
g~
v >b-
~IOC O fls h
n CO
o ----r--'-T~ ~o 4o
L 8o
i~o
'
l~o
~io
3" I ME (rain)
Fig. 4. The specific radioactivity of ACC (o) and ethylene (o) during incubation of pea protoplasts in the light in ~ ml of incubation medium containing 188.5kBq (=18nmol) of L-[U-14C]methionine
evolved ethylene, and light enhanced ethylene production just as in pea protoplasts (Fig. 3). Cobalt (II), a potent inhibitor of ACC-dependent ethylene synthesis (Yu and Yang 1979), inhibited ethylene production in the light and in the dark. In the experiment of Fig. 3, i mM CoC1 z reduced ethylene synthesis in illuminated V. faba protoplasts by 80% after 210 min of incubation. In pea protoplasts, ethylene formation was also inhibited by CoC12. In one experiment very similar to the one shown in Fig. 3, light-stimulated ethylene formation in pea protoplasts was inhibited by 75% after 185min of incubation in resuspension medium containing 1 mM CoClz.
M. Guy and H. Kende: Ethylene formation in protoplasts
We examined the incorporation of radioactivity from L-[U-14C]methionine into ACC and ethylene in illuminated pea protoplasts (Fig. 4). The specific radioactivity of the C-2 and C-3 atoms of ACC increased up to the third hour of incubation and declined thereafter. This decline was observed in other experiments as well and might have been caused by depletion of the tracer levels of [lr from the medium. The specific radioactivity of ethylene increased linearly but at a much slower rate than that of ACC. After 180 min of incubation, the specific radioactivity of ethylene was less than one-sixth that of ACC. Discussion
We described conditions under which protoplasts isolated from pea and V. faba leaves synthesized ethylene from endogenous substrate (Figs. 1-3). Neither methionine nor ACC had to be added to the incubation medium for sustained ethylene production to occur. Therefore, pea and V. faba protoplasts appear to be well suited for studies on ethylene biosynthesis and for the cellular localization of components of the ethylene-synthesizing system. Synthesis of ACC was induced during isolation of protoplasts because the cells were exposed to cellulysin and mannitol and it continued during incubation in resuspension medium (Table 1, Figs. 1 B, 2B). Anderson et al. (1982) found that cellulysin stimulated ethylene production in tobacco leaf discs by promoting synthesis of ACC, and Riov and Yang (1982) reported that formation of ACC and conversion of ACC to ethylene were stimulated by mannitol in leaf tissue of citrus. Light enhanced both ACC and ethylene synthesis (Figs. l, 3). This was probably due, at least in part, to increased availability of energy-rich compounds in the photosynthesizing cells. The inhibition of ACC and ethylene synthesis by D C M U and CCCP, inhibitors of electron transport and phosphorylation, respectively, was consistent with this notion. The stimulation of ethylene synthesis by light was not only based on increased availability of ACC but also on increased conversion of ACC to ethylene. Protoplasts in darkness and AVG-treated protoplasts in light had closely similar ACC contents, yet AVG-treated protoplasts in light produced almost four times more ethylene than did protoplasts in darkness (Fig. 1). Stimulation of ACC-dependent ethylene formation in pea and V. faba protoplasts by light might have been analogous to that observed by Grodzinski et al. (1982) in corn leaf discs and by Kao and Yang
279
(1982) in rice leaf segments. In both instances, conversion of ACC to ethylene was enhanced by light when the level of CO 2 in the ambient atmosphere was increased. Because diffusion of gases from cells into water is much slower than diffusion of gases into air, it is very likely that the CO 2 level in protoplasts increases as a result of incubation in liquid medium. This would create the proper conditions for the stimulation of ethylene synthesis by light. Ethylene synthesis was saturated at ACC concentrations that were about one-third to one-half those of illuminated protoplasts. This followed from a comparison of ACC levels and ethylene synthesis in protoplasts that had been resuspended in media with and without AVG (Figs. 1, 2). Ethylene synthesis was reduced by 10-15% in the presence of 200 gM AVG while the final ACC levels were 60-75% lower. However, when the ACC level was reduced by more than 90% as a result of treatment with AVG in the isolation and resuspension media, ethylene production was inhibited by over 90% (Fig. 2). Our results may explain why Anderson et al. (1979) have not found consistent inhibition of ethylene synthesis by AVG in apple protoplasts even though ethylene production in apples is very effectively blocked by AVG. The level of ACC in apple protoplasts probably also rises during isolation, and treatment of the cells with AVG in the resuspension medium does not lower the ACC level enough for ethylene synthesis to be markedly reduced. Radioactivity from [~C]methionine was incorporated into both ACC and ethylene (Fig. 4). However, the specific radioactivity of C-2 and C-3 of ACC, the carbon atoms which are incorporated into ethylene, rose much faster than the specific radioactivity of ethylene. Therefore, ethylene was synthesized primarily from pre-existing, non-radioactive ACC rather than from newly formed, radioactive ACC. These data indicated the existence of two ACC pools in pea protoplasts. It appeared that ACC was synthesized from methionine in one compartment of the cell and was converted to ethylene in another compartment which already contained previously formed ACC. We found indeed that 85% of the ACC in pea and V. faba protoplasts was localized in the vacuole and that isolated vacuoles synthesized ethylene (Guy and Kende 1984). We therefore suggest that ACC synthesis is localized in the cytoplasm while ethylene is formed primarily in the vacuole of pea and V. faba protoplasts. This work was supported by the U.S. Department of Energy under Contract No. DE-AC02-76-ER01338.
280
References Adams, D.O., Yang, S.F. (1979) Ethylene biosynthesis: identification of l-aminocyClopropane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76, 170-174 Anderson, J.D., Lieberman, M., Stewart, R.N. (1979) Ethylene production by apple protoplasts. Plant Physiol. 63, 931-935 Anderson, J.D., Mattoo, A.K., Lieberman, M. (1982) Induction of ethylene biosynthesis in tobacco leaf discs by cell wall digesting enzymes. Biochem. Biophys. Res. Commun. 10/, 588-596 Grodzinski, B., Boeset, I., Horton, R.F. (1982) Ethylene release from leaves of Xanthium strumarium L. and Zea mays L. J. Exp. Bot. 33, 344-354 Guy, M., Kende, H. (1984) Conversion of 1-aminocyclopropane-l-carboxylic acid to ethylene by isolated vacuoles of Pisum sativum L. Planta 160, 281-287 Guy, M., Reinhold, L., Laties, G.G. (1978) Membrane transport of sugars and amino acids in isolated protoplasts. Plant Physiol. 61, 281-287 Kao, C.H., Yang, S.F. (1982) Light inhibition of the conversion of l-aminocyclopropane-l-carboxylic acid to ethylene in leaves is mediated through carbon dioxide. Planta 155, 261-266
M. Guy and H. Kende: Ethylene formation in protoplasts Kende, H., Boller, T. (1981) Wound ethylene and 1-aminocyclopropane-l-carboxylate synthase in ripening tomato fruit. Planta 151,476-481 Kende, H., Hanson, A.D. (1976) Relationship between ethylene evolution and senescence in morning-glory flower tissue. Plant Physiol. 57, 523-527 Konze, J.R., Jones, J.F., Boller, T., Kende, H. (1980) Effect of 1-aminocyclopropane-l-carboxylic acid on the production of ethylene in senescing flowers of Ipomoea tricolor Cav. Plant Physiol. 66, 566-571 Lizada, M.C.C., Yang, S.F. (1979) A simple and sensitive assay for 1-aminocyclopropane-l-carboxylic acid. Anal. Biochem. 100, 140 145 Mattoo, A.K., Lieberman, M. (1977) Localization of the ethylene-synthesizing system in apple tissue. Plant Physiol. 60, 794-799 Riov, J., Yang, S.F. (1982) Stimulation of ethylene production in citrus leaf discs by mannitol. Plant Physiol. 70, 142-146 Yu, Y.-B., Yang, S.F. (1979) Auxin-induced ethylene production and its inhibition by aminoethoxyvinylglycine and cobalt ion. Plant Physiol. 64, 1074-1077
Received 21 September; accepted 18 November 1983
Planta
9 Springer-Verlag 1984
Ethylene formation in Pisum sativum and Vicia faba protoplasts Micha Guy* and Hans Kende MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
Abstract. Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-l-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-l,1dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-14C]meth ionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation. Key words: 1-Aminocyclopropane-l-carboxylic acid - Aminoethoxyvinylglycine - Ethylene synthesis - Pisum (ethylene synthesis) - Vicia.
Introduction
Mattoo and Lieberman (1977) attempted to localize the site of ethylene biosynthesis in plant cells by using protoplasts isolated from apple fruits. They found that freshly prepared protoplasts had * Present address: Ben-Gurion University of the Negev, Blaus-
tein Institute for Desert Research, Sede Boqer, Israel Abbreviations: ACC = l-Aminocyclopropane-l-carboxylicacid; AVG=aminoethoxyvinylglycine; CCCP=carbonyl cyanide m-chlorophenylhydrazone; DCMU = 3-(3,4-dichlorophenyl)1,1-dimethylurea
lost their ability to produce ethylene. Ethylene synthesis resumed slowly as the protoplasts formed new cell walls, provided that methionine was added to the incubation medium. In later experiments, Anderson et al. (1979) found conditions under .which freshly isolated apple protoplasts did produce ethylene. Ethylene synthesis in apple protoplasts appeared to be anomalous in that it was relatively insensitive to aminoethoxyvinylglycine (AVG). Aminoethoxyvinylglycine is a potent inhibitor of ethylene synthesis in most plant tissues, including apple fruits, and acts by blocking the synthesis of 1-aminocyclopropane-l-carboxylic acid (ACC), the immediate precursor of ethylene (Adams and Yang 1979). Konze et al. (1980) reported that protoplasts prepared from flower tissue of Ipomoea tricolor Cav. produced ethylene, but only if ACC was added to the incubation mixture. We felt that protoplasts might be suitable to study the compartmentation of components of the ethylene-forming system and that the use of protoplasts might enable us to localize the intracellular site of ethylene synthesis. In the following, we describe conditions under which protoplasts isolated from Pisurn sativum and Viciafaba leaves synthesize both ACC and ethylene, and the effect of light on these processes. We also supply evidence that ACC and ethylene synthesis are compartmented in the plant cell. Materials and methods Plant material. Seeds of Pisum sativurn L., cv. Alaska (Ferry:
Morse Seed Co., Mountain View, Calif., USA), were imbibed overnight in aerated distilled water. They were sown in plastic trays either in vermiculite or a soil mixture consisting of three parts Sunshine Mix, Blend No. I (Fisons-Western Corp., Vancouver, B.C., Canada), one part sand, one part Michigan bog peat, one part loam soil, and 400 g of triple superphosphate
M. Guy and H. Kende : Ethylene formation in protoplasts per 50 1 of mixture. The plants were grown at 23 ~ C and a photoperiod of 16 h (cool-white fluorescent lamps; General Electric Co., Cleveland, O., USA; photon flux 125 p.mol m - 2 s-1). Seeds of Vicia faba L., cv. Long Pod (Burpee Co., Clinton, Ia., USA), were sown in the above soil mixture, two seeds per i-liter plastic container. The plants were grown in a growth chamber at 23 ~ C, a daily light period of 20 h (coolwhite fluorescent lamps, General Electric, and incandescent lamps; photon flux 350 g m o l m -2 s -a) and at 70% relative humidity.
277 30
80O
//~
A io
o
300
zo
o
) ~O0
10
--,a
Preparation of protoplasts. Fully expanded leaflets of 10- to 13-d-old pea plants were detached, surface-sterilized with 5% (w/v) sodium hypochlorite for approx. 30 s and then cut into 2-mm-wide strips. These were incubated in a filter-sterilized solution of 0.6 M mannitol and 1% (w/v) cellulysin at pH 5.6 in sterile Petri dishes at 30~ C for 2.5-3 h. In one experiment (Table 1), incubation of leaves in isolation medium was for 18 h at 22~ C. Protoplasts were isolated as described in Guy et al. (1978), and resuspended in a medium containing 0.2 mM KH~PO 4, ~ mM KNO~, i m M MgSO4, 10raM CaC12, 20 mM Mes [2-(N-morpholino)ethanesulfonic acid], 20 mM Hepes [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid] and 0.55 M mannitol, adjusted to pH 5.5 with NaOH. The number of protoplasts was determined under a microscope using a hemacytometer. Protoplasts were prepared from the youngest, fully expanded leaflets of V. faba plants as described for peas except that the leaves were not cut into 2-ram strips. The main veins were removed, and the epidermis from the abaxial side of the blade was peeled off with fine forceps before incubation in cellulysin.
Ethylene and ACC determinations. For the measurement of ethylene formation, 1.106-2.106 protoplasts were incubated in 0.6-0.7 ml resuspension medium in 4-ml test tubes stoppered with serum vial caps. The test tubes were kept at a slant either in the dark or in front of fluorescent lights (cool white fluorescent lamps; General Electric; 70 gmol m -2 s-~) at 22 ~ C. Ethylene was sampled by first injecting 1 ml of air into each test tube with a tuberculin syringe, pumping the syringe several times, and then withdrawing 1 ml for analysis by gas chromatography (Kende and Hanson 1976). In calculating the amount of ethylene in each tube, a correction was applied for the successive injections of air and removals of gas samples. The specific radioactivity of ethylene was determined as in Konze et al. (1980).
For ACC determinations, protoplasts were lysed directly in the ACC-assay mixture of Lizada and Yang (1979); ACC was extracted from leaf tissue as described tbr tomato pericarp (Kende and Bolter 1981), and was measured in both instances according to Lizada and Yang (1979). The specific radioactivity of the C-2 and C-3 atoms of ACC was determined by first converting ACC to ethylene following the method of Lizada and Yang (1979) and then measuring the specific radioactivity of ethylene according to Konze et al. (1980).
Chemicals'. L-[U-~'~C]Methionine (l.06.J0r was purchased from Amersham, Arlington Heights, Ill., USA; ACC and cellulysin were from Calbiochem-Behring Corp., La Jolla, Calif., USA; Ficoll (molecular weight 400,000) from Pharmacia Fine Chemicals, Piscataway, N.J., USA; 3-(3,4-dichlorophenyl)-J,l-dimethylurea (DCMU) from Du Pont de Nemours & Co., Wilmington, Dela., USA; carbonyl cyanide m-chloropheny/hydrazone (CCCP) from Sigma Chemical Co., St. Louis, Mo., USA; Hepes from Research Organics, Cleveland, O., USA, and Mes from U.S. Biochemical Corp., Cleveland, O., USA. Aminoethoxyvinylglycinewas a gift of Dr. M. Lieberman (U.S. Department of Agriculture, Beltsville, Md., USA).
_~
r
-o
I
3o
If, ~ 240 45O
I
r5 TIME(rain)
Ill
~E o. v t)
Fig. 1. A Ethylene synthesis in protoplasts isolated from pea leaves that had been incubated in the dark (R), in the light (o), and in the light in the presence of 200 pM AVG (o), 0.J mM DCMU (n) and 10 pM CCCP (zx). B Levels of ACC in the same protoplasts at the beginning of the incubation (hatched bar) and after 450 min of incubation. Symbols as above. The vertical bars denote the spread of duplicate samples in the ACC assay; when no bar is given, the spread between duplicates has been smaller than the symbol used
Table 1. Induction of ACC synthesis in pea leaf tissue b) protoptast-isolation medium. Strips of leaf tissue were cut from the youngest fully expanded leaflets and were incubated in protoplast-isolation medium (1% cellulysin and 0.6 M mannitol) or on distilled water in darkness at 22 ~ C for 18 h. The ACC level of whole leaflets incubated on water for 18 h and of fresh leaflets was also measured Treatment
ACC content (nmol g - a FW)
Leaf strips on isolation medium Leaf strips on water Intact leaves on water Intact leaves, fresh
19.5 2.5 2.1 1.6
Results
Incubation of pea leaf tissue in protoplast isolation medium containing cellulysin and mannitol induced ACC synthesis (Table 1). The leaf tissue that remained intact for 18 h of incubation in the above medium contained 12 times more ACC than did fresh leaves. The increase in ACC content of leaves that were cut and incubated on water was, by comparison, small, as was the increase in the ACC content of whole leaves incubated on water. Protoplasts isolated from pea leaves evolved ethylene, even in the absence of added substrate (Fig. l A). Illuminated protoplasts produced up to 4.5-fold more ethylene than protoplasts kept in darkness. When 200 pM AVG was present in the medium in which the protoplasts were resuspended and incubated, ethylene production was only slightly inhibited. 3-(3,4-Dichlorophenyl)-l,l-di-
278
M. Guy and H. Kende: Ethylene formation in protoplasts 2o ,-//
800
fOl A
o
g
~15 o o
o
CorlfrOl AVG
"5
c'e"
%n \
4oo 3
w Z l.d
#
2::: I'-" LtJ
LU
~ __
20
50 TIME
85
Dark+ CoCl~ I
I
70
240
(min)
Fig. 2. A The effect of AVG on ethylene formation in pea protoplasts. Protoplasts were incubated in darkness in resuspension medium either without AVG (o) or with 200 gM AVG (e). In the third sample, 200 pM AVG was present in the isolation medium as well as in the resuspension medium (A). B Levels of ACC in the same protoplasts after 20 rain (hatched bars) and 240 min of incubation. Symbols as above. The vertical bars denote the spread of duplicate samples as in Fig. 1
methylurea, an inhibitor of photosynthetic electron transport, reduced light-stimulated ethylene synthesis by 60% (Fig. 1A). In other experiments, D C M U reduced ethylene production in illuminated protoplasts to that of protoplasts in darkness. Carbonyl cyanide m-chlorophenylhydrazone, a proton conductor and uncoupler of phosphoryladon, proved to be a very potent inhibitor of ethylene synthesis in pea protoplasts (Fig. 1 A). The level of ACC doubled during the 450 rain of incubation in darkness and increased fourfold in the light (Fig. 1 B); AVG, D C M U and CCCP all inhibited the increase in the level of ACC. When AVG was present in the medium in which protoplasts were isolated and then again in the resuspension medium, ethylene synthesis was inhibited by more than 90% (Fig. 2A). The inhibitory effect of AVG on ethylene synthesis was fully reversible with added ACC (results not shown). Aminoethoxyvinylglycine again had only a minor effect on ethylene synthesis when it was added, after isolation of the protoplasts, to the medium in which the protoplasts were resuspended. 1Aminocyclopropane-l-carboxylic-acid levels increased in the control protoplasts during the 4 h of the experiment (Fig. 2 B). Aminoethoxyvinylglycine inhibited ACC formation, both in the protoplasts that had been treated with AVG in the resuspension medium and in those that were also isolated in the presence of AVG. Protoplasts isolated from V. faba leaves also
135 TIME(rain)
I 210
270
Fig. 3. Ethylene synthesis in protoplasts isolated from V. faba leaves and incubated in the light with 1 mM COC12 0x) and without CoC12 (o) or in the dark with I mM CoCI~ (A) and without CoCI 2 (e). CoC12 was not present during isolation of the protoplasts
20C
-2
g~
v >b-
~IOC O fls h
n CO
o ----r--'-T~ ~o 4o
L 8o
i~o
'
l~o
~io
3" I ME (rain)
Fig. 4. The specific radioactivity of ACC (o) and ethylene (o) during incubation of pea protoplasts in the light in ~ ml of incubation medium containing 188.5kBq (=18nmol) of L-[U-14C]methionine
evolved ethylene, and light enhanced ethylene production just as in pea protoplasts (Fig. 3). Cobalt (II), a potent inhibitor of ACC-dependent ethylene synthesis (Yu and Yang 1979), inhibited ethylene production in the light and in the dark. In the experiment of Fig. 3, i mM CoC1 z reduced ethylene synthesis in illuminated V. faba protoplasts by 80% after 210 min of incubation. In pea protoplasts, ethylene formation was also inhibited by CoC12. In one experiment very similar to the one shown in Fig. 3, light-stimulated ethylene formation in pea protoplasts was inhibited by 75% after 185min of incubation in resuspension medium containing 1 mM CoClz.
M. Guy and H. Kende: Ethylene formation in protoplasts
We examined the incorporation of radioactivity from L-[U-14C]methionine into ACC and ethylene in illuminated pea protoplasts (Fig. 4). The specific radioactivity of the C-2 and C-3 atoms of ACC increased up to the third hour of incubation and declined thereafter. This decline was observed in other experiments as well and might have been caused by depletion of the tracer levels of [lr from the medium. The specific radioactivity of ethylene increased linearly but at a much slower rate than that of ACC. After 180 min of incubation, the specific radioactivity of ethylene was less than one-sixth that of ACC. Discussion
We described conditions under which protoplasts isolated from pea and V. faba leaves synthesized ethylene from endogenous substrate (Figs. 1-3). Neither methionine nor ACC had to be added to the incubation medium for sustained ethylene production to occur. Therefore, pea and V. faba protoplasts appear to be well suited for studies on ethylene biosynthesis and for the cellular localization of components of the ethylene-synthesizing system. Synthesis of ACC was induced during isolation of protoplasts because the cells were exposed to cellulysin and mannitol and it continued during incubation in resuspension medium (Table 1, Figs. 1 B, 2B). Anderson et al. (1982) found that cellulysin stimulated ethylene production in tobacco leaf discs by promoting synthesis of ACC, and Riov and Yang (1982) reported that formation of ACC and conversion of ACC to ethylene were stimulated by mannitol in leaf tissue of citrus. Light enhanced both ACC and ethylene synthesis (Figs. l, 3). This was probably due, at least in part, to increased availability of energy-rich compounds in the photosynthesizing cells. The inhibition of ACC and ethylene synthesis by D C M U and CCCP, inhibitors of electron transport and phosphorylation, respectively, was consistent with this notion. The stimulation of ethylene synthesis by light was not only based on increased availability of ACC but also on increased conversion of ACC to ethylene. Protoplasts in darkness and AVG-treated protoplasts in light had closely similar ACC contents, yet AVG-treated protoplasts in light produced almost four times more ethylene than did protoplasts in darkness (Fig. 1). Stimulation of ACC-dependent ethylene formation in pea and V. faba protoplasts by light might have been analogous to that observed by Grodzinski et al. (1982) in corn leaf discs and by Kao and Yang
279
(1982) in rice leaf segments. In both instances, conversion of ACC to ethylene was enhanced by light when the level of CO 2 in the ambient atmosphere was increased. Because diffusion of gases from cells into water is much slower than diffusion of gases into air, it is very likely that the CO 2 level in protoplasts increases as a result of incubation in liquid medium. This would create the proper conditions for the stimulation of ethylene synthesis by light. Ethylene synthesis was saturated at ACC concentrations that were about one-third to one-half those of illuminated protoplasts. This followed from a comparison of ACC levels and ethylene synthesis in protoplasts that had been resuspended in media with and without AVG (Figs. 1, 2). Ethylene synthesis was reduced by 10-15% in the presence of 200 gM AVG while the final ACC levels were 60-75% lower. However, when the ACC level was reduced by more than 90% as a result of treatment with AVG in the isolation and resuspension media, ethylene production was inhibited by over 90% (Fig. 2). Our results may explain why Anderson et al. (1979) have not found consistent inhibition of ethylene synthesis by AVG in apple protoplasts even though ethylene production in apples is very effectively blocked by AVG. The level of ACC in apple protoplasts probably also rises during isolation, and treatment of the cells with AVG in the resuspension medium does not lower the ACC level enough for ethylene synthesis to be markedly reduced. Radioactivity from [~C]methionine was incorporated into both ACC and ethylene (Fig. 4). However, the specific radioactivity of C-2 and C-3 of ACC, the carbon atoms which are incorporated into ethylene, rose much faster than the specific radioactivity of ethylene. Therefore, ethylene was synthesized primarily from pre-existing, non-radioactive ACC rather than from newly formed, radioactive ACC. These data indicated the existence of two ACC pools in pea protoplasts. It appeared that ACC was synthesized from methionine in one compartment of the cell and was converted to ethylene in another compartment which already contained previously formed ACC. We found indeed that 85% of the ACC in pea and V. faba protoplasts was localized in the vacuole and that isolated vacuoles synthesized ethylene (Guy and Kende 1984). We therefore suggest that ACC synthesis is localized in the cytoplasm while ethylene is formed primarily in the vacuole of pea and V. faba protoplasts. This work was supported by the U.S. Department of Energy under Contract No. DE-AC02-76-ER01338.
280
References Adams, D.O., Yang, S.F. (1979) Ethylene biosynthesis: identification of l-aminocyClopropane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76, 170-174 Anderson, J.D., Lieberman, M., Stewart, R.N. (1979) Ethylene production by apple protoplasts. Plant Physiol. 63, 931-935 Anderson, J.D., Mattoo, A.K., Lieberman, M. (1982) Induction of ethylene biosynthesis in tobacco leaf discs by cell wall digesting enzymes. Biochem. Biophys. Res. Commun. 10/, 588-596 Grodzinski, B., Boeset, I., Horton, R.F. (1982) Ethylene release from leaves of Xanthium strumarium L. and Zea mays L. J. Exp. Bot. 33, 344-354 Guy, M., Kende, H. (1984) Conversion of 1-aminocyclopropane-l-carboxylic acid to ethylene by isolated vacuoles of Pisum sativum L. Planta 160, 281-287 Guy, M., Reinhold, L., Laties, G.G. (1978) Membrane transport of sugars and amino acids in isolated protoplasts. Plant Physiol. 61, 281-287 Kao, C.H., Yang, S.F. (1982) Light inhibition of the conversion of l-aminocyclopropane-l-carboxylic acid to ethylene in leaves is mediated through carbon dioxide. Planta 155, 261-266
M. Guy and H. Kende: Ethylene formation in protoplasts Kende, H., Boller, T. (1981) Wound ethylene and 1-aminocyclopropane-l-carboxylate synthase in ripening tomato fruit. Planta 151,476-481 Kende, H., Hanson, A.D. (1976) Relationship between ethylene evolution and senescence in morning-glory flower tissue. Plant Physiol. 57, 523-527 Konze, J.R., Jones, J.F., Boller, T., Kende, H. (1980) Effect of 1-aminocyclopropane-l-carboxylic acid on the production of ethylene in senescing flowers of Ipomoea tricolor Cav. Plant Physiol. 66, 566-571 Lizada, M.C.C., Yang, S.F. (1979) A simple and sensitive assay for 1-aminocyclopropane-l-carboxylic acid. Anal. Biochem. 100, 140 145 Mattoo, A.K., Lieberman, M. (1977) Localization of the ethylene-synthesizing system in apple tissue. Plant Physiol. 60, 794-799 Riov, J., Yang, S.F. (1982) Stimulation of ethylene production in citrus leaf discs by mannitol. Plant Physiol. 70, 142-146 Yu, Y.-B., Yang, S.F. (1979) Auxin-induced ethylene production and its inhibition by aminoethoxyvinylglycine and cobalt ion. Plant Physiol. 64, 1074-1077
Received 21 September; accepted 18 November 1983
