9 Springer-Verlag 1984
Conversion of 1-aminocyclopropane-l-carboxylic acid to ethylene by isolated vacuoles of Pisum sativum L. Micha Guy* and Hans Kende M S U - D O E Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
Abstract. We compared the distribution of 1-aminocyclopropane-l-carboxylic acid (ACC) between the vacuole of isolated pea (Pisum sativum L.) protoplasts and the remainder of the cell and found that over 80% of the ACC was localized in the vacuole. Isolated protoplasts and vacuoles evolved ethylene. Over 80% of the ethylene production by protoplasts could be accounted for as originating from the vacuole. Ethylene synthesis by isolated vacuoles was saturated at ACC concentrations above 1 raM, and the apparent K m for the conversion of ACC to ethylene was 61 gM. Ethylene production in isolated vacuoles was inhibited by Co 2+, n-propyl-gallate, in a N 2 atmosphere, and following lysis of the vacuoles. The ethylene-forming enzyme in pea vacuoles exhibited stereospecificity inasmuch as it catalyzed the conversion of (+)-allocoronamic acid to 1-butene but not that of (+)-coronamic acid. The same stereospecificity was also shown by leaf tissue. Based on competition studies with ACC and (+)-allocoronamic acid, we conclude that conversion of ACC to ethylene and (+)-allocoronamic acid to l-butene is mediated by the same enzyme in isolated vacuoles and in intact leaf tissue. Vacuoles isolated from Vicia faba L. leaves showed essentially the same characteristics with regard to ACC-dependent ethylene synthesis as did pea vacuoles. Key words: (+)-Allocoronamic acid - 1-Aminocyclopropane-l-carboxylic acid - Ethylene synthesis - Pisum (ethylene) - Protoplast - Vacuole. * Present address: Ben-Gurion University of the Negev, Blaustein Institute for Desert Research, Sede Boqer, Israel
ACC = 1-Aminocyclopropane-l-carboxylic acid; AEC=l-amino-2-ethylcyclopropane-l-carboxylic acid; AVG = aminoethoxyvinylglycine
In the preceding paper, we described conditions under which protoplasts isolated from leaves of pea and of Vicia faba produced ethylene and its immediate precursor, 1-aminocyclopropane-l-carboxylic acid (ACC) (Guy and Kende 1983). In protoplasts incubated on a medium containing [U-14C]methionine, the specific radioactivity of the C-2 and C-3 atoms of ACC was much higher than that of the newly formed ethylene. From these results, we concluded that ACC and ethylene were synthesized in different compartments of the ceil. We now provide evidence that vacuoles isolated from pea and V. faba protoplasts convert ACC to ethylene in a manner similar to that of intact plants. Most of the ethylene produced by pea protoplasts can be accounted for as originating from the vacuole. Materials and methods Growth of plants and preparation of protopIasts. Sources and culture of Pisum sativum L., cv. Alaska, and Vicia faba L., cv. Long Pod, were as described in Guy and Kende (1984). Protoplasts were prepared from fully expanded leaflets according to Guy et al. (1978) and Guy and Kende (1984). Leaf strips were usually incubated in protoplast-isolation medium at 30 ~ C for 3 h. When high levels of vacuolar ACC were required, this incubation period was extended to 18 h at 22 ~ C (see Table 2). Preparation of vacuoles. Vacuoles were released from protoplasts by the shearing force of ultracentrifugation through a step density gradient as described by Guy et al. (1979) with the following modifications: 7.5% and 10% (w/v) Ficoll were used for the step gradient, and centrifugation was performed at Rav 246,000 g (50,000 rpm) in a SW 56 Beckman rotor using a Model L-2 Ultracentrifuge (Beckman Instruments, Palo Alto, Calif., USA). Vacuoles were collected from both interphases of the gradient with a Pasteur pipette and checked for contamination under a light microscope. The vacuolar fraction at the 10% Ficoll interphase was discarded if it was found to be
282 contaminated with protoplasts or subprotoplast particles. The vacuoles were resuspended in the same medium as protoplasts (Guy and Kende 1984) except that the pH of the resuspension medium was adjusted to 7.2 with NaOH, and were counted under a microscope using a hemacytometer.
M. Guy and H. Kende: Ethylene formation by vacuoles Table 1. The level of marker enzymes, chlorophyll and protein in the protoplast and vacuolar fractions of pea leaves Marker
Determination of ACC, AEC, ethylene and 1-butene. Protoplasts and vacuoles were lysed directly in the ACC-assay mixture of Lizada and Yang (1979). The concentration of ACC and of 1-amino-2-ethylcyclopropane-l-carboxylicacid (AEC) was determined by converting ACC to ethylene and AEC to 1-butene according to the method of Lizada and Yang (1979). For measurement of ethylene or 1-butene production, 1.106- 3.106 protoplasts or vacuoles were incubated at 22~ C in 0.6-0.7 ml resuspension medium in 4-rot test tubes that were stoppered with serum vial caps and were kept at a slant. For gas sampling, I ml air (or N 2 in the case of experiments where vacuoles were incubated in an atmosphere of N2) was injected into each tube with a tuberculin syringe. The syringe was pumped several times, and a l-ml sample was withdrawn for analysis by gas chromatography according to Kende and Hanson (1976). For the calculation of ethylene and 1-butene production, a correction was applied for the repeated injections of air (or N2) into the tubes and the removal of gas samples. For the determination of ethylene and 1-butene formation in intact leaf tissue, five leaf discs (diameter 11 ram) cut from fully expanded leaflets of P. sativum were incubated in the dark at 27~ C in a 25-mi Erlenmeyer flask on a Whatman No. 1 filter-paper disc wetted with 0.5 ml test solution. The flasks were stoppered with serum vial caps, and gas samples were withdrawn and analyzed as described above. For measurement of AEC uptake into leaf tissue, leaf discs were extracted and analyzed as described for ACC determinations in tomato pericarp discs (Kende and Boiler 1981).
Enzyme, chlorophyll and protein determinations. Glucose-6phosphate dehydrogenase, hexosephosphate isomerase, a-mannosidase and acid phosphatase were measured according to Boller and Kende (1979), cytochrome-c oxidase according to Hanks et al. (1981), and glucan synthase II according to Ray (1977). Conversion of ACC to ethylene in a homogenate of etiolated pea epicotyls was assayed following the procedure of Konze and Kende (1979). Chlorophyll was determined according to Arnon (1949) and protein by the method of Bradford (1976).
Binding assays. Binding of [3H]naphthylphthalamic acid (NPA) was assayed as described by Ray (1977) and of fiuoresceinisothiocyanate-labeledconcanavalin A according to Thom et al. (1982).
Purity of the vacuolar fraction. The purity of the vacuolar fraction was monitored routinely by staining the vacuoles with neutral red and examining the vacuolar fraction under the light microscope. In addition, contamination of the vacuolar fraction was assessed by assaying for cytoplasmic and organellar marker enzymes and for chlorophyll and protein content (Table 1). Essentially all of the acid-phosphatase and ~-mannosidase activities of the protoplast were found in the vacuolar fraction. The contamination of the vacuolar fi'action with mitochondria, chloroplasts and, implicitly, with protoplasts was low as judged by the low levels of cytochrome-c oxidase activity (5-I 5%) and chlorophyll (5-9%) in the vacuolar fraction. The levels of the cytoplasmic marker enzymes glucose-6-phosphate dehydrogenase and hexosephosphate isomerase in the vacuolar fraction were 14% and 27%, respectively. We employed three tests that are thought to be specific for the detection of the plasma mem-
~-Mannosidase (nmol min- 1)
Level per 106 protoplasts
Level per 106 vacuoles
Relative level in vacuolar fraction
Acid phosphatase (nmol min- 1)
Glucose-6-phosphate dehydrogenase (nmol min- 1) Hexosephosphate isomerase (nmol min- 1) Cytochrome-c oxidase (nmol rain- 1)
Chlorophyll (pg) Protein (lag)
brane. First, we assayed the activity of glucan synthase II in membrane fractions of pea protoplasts and found that it was very low. No reliable data concerning its intracellular distribution could be obtained. Second, we tried unsuccessfully to detect binding of fluorescein-isothiocyanate-labeled concanavalin A to the plasma membrane of intact protoplasts at concentrations that did label the cell wall of yeast (up to 1.6 mg ml- 1). Third, we assayed for specific binding of [3H]naphthylphthalamic acid to the membrane fraction ofprotoplast homogenates but could detect none. Thorn et al. (1982) found an 18% contamination of their vacuolar preparation with concanavalin-Abinding membrane material, presumably plasmalemma. Since the vacuolar isolation method and the marker-enzyme data of Thorn et al. (1982) were very similar to ours, our vacuolar preparation might have been contaminated with plasma membrane to a similar extent.
Chemicals. Cellulysin and ACC were purchased from Calbiochem-Behring Corp., La Jolla, Calif., USA; Ficoll (molecular weight 400,000) from Pharmacia Fine Chemicals, Piscataway, N.J., USA; digitonin from United States Biochemical Corp., Cleveland, O., USA, and all other chemicals from Sigma Chemical Company, St. Louis, Mo., USA. (+)-Coronamic acid and (_+)-allocoronamic acid were a gift of Dr. M. Venis, Sittingbourne Research Centre, Sittingbourne, UK; naphthylphthalamic acid was a gift of Dr. M. Jacobs, Swarthmore College, Swarthmore, Pa., USA, and AVG a gift of Dr. M. Lieberman, U.S. Department of Agriculture, Beltsville, Md., USA.
Compartmentation of ACC. We investigated the distribution of ACC between the vacuole and the cytoplasm of freshly isolated pea protoplasts and found that 88% of the ACC was localized in the vacuole (average of seven experiments +_12% standard deviation). The level of ACC in protoplasts
M. Guy and H. Kende: Ethylene formation by vacuoles //
m 60C o
< -6 50C
9 I 2.5
hl Z hl
150 185 T I ME (min)
Fig. I . A Levels of ACC in isolated pea protoplasts and vacuoles. Protoplasts were isolated from pea leaves, resuspended and incubated in the light (70 pmol m - 2 s-1). The ACC level of protoplasts (o) was determined immediately after isolation (time zero) and after 2.5, 5 and 18 h of incubation. At these times, vacuoles were isolated from the protoplasts, and their ACC content was determined as well (e). B Protoplasts (PpI) were isolated and incubated in media with and without 200 gM AVG. Their ACC level was determined immediately after isolation (time zero) and after 6 h of incubation in darkness. Vacuoles (Vat) were isolated from control and AVG-treated protoplasts, and their ACC content was determined immediately upon isolation (time zero) and after 6 h of incubation in darkness
incubated in resuspension medium increased with time (Fig. 1 A). Vacuoles isolated from these protoplasts at different time points contained between 80% and 85% of the total cellular ACC, and the ACC level in vacuoles mirrored the increase in the ACC level of protoplasts (Fig. 1 A). When vacuoles were isolated from freshly prepared protoplasts at time zero and incubated for 6 h, the ACC level increased very little, if significantly at all, while that of the corresponding protoplasts rose almost fivefold (Fig. 1 B). In other experiments, the ACC content of isolated vacuoles actually dropped by 10-28% during 6-7 h of incubation. The ACC level in protoplasts that had been isolated in the presence of 200 pM AVG and then incubated for 6 h in resuspension medium containing 200 gM AVG was very low as was the ACC content of vacuoles isolated from AVG-treated protoplasts (Fig. ~ B).
Ethylene formation in isolated vacuoles. Vacuoles isolated from freshly prepared protoplasts and incubated in resuspension medium without any further additions produced ethylene (Fig. 2). An aliquot of the original protoplasts was incubated under identical conditions to compare the level of ethylene formation in isolated vacuoles and protoplasts. The amount of ethylene produced per vacuole was 75-86% of that formed per protoplast.
Fig. 2. Ethylene production in pea protoplasts and vacuoles. Protoplasts (o) and vacuoles (a) isolated from them at time zero were incubated in darkness, and ethylene production was determined. Ethylene synthesis by vacuoles incubated in a medium containing 1 mM CoC12 was also measured (rT) 7
0.1, UA Z lJ ,_.J >-
r : 0.985
ACC (y.M)-~ Fig. 3. Lineweaver-Burk plot of ethylene production by pea leaf vacuoles. Vacuoles were prepared from protoplasts that had been treated with 200 gM AVG during their isolation. The reciprocal rate of ethylene production at the linear phase of ethylene synthesis is plotted against the reciprocal external ACC concentration
Vacuoles obtained from protoplasts that had been isolated in the presence of 200 ~tM AVG produced only trace amounts of ethylene from the low levels of endogenous ACC but converted exogenously supplied ACC very effectively to ethylene. Such vacuoles were used to study the kinetic parameters of ACC-dependent ethylene synthesis. Ethylene synthesis was saturated at 1 mM ACC (Fig. 3), as was ethylene synthesis in pea leaf discs incubated on increasing concentrations of ACC (results not shown). The apparent Km for ACCdependent ethylene synthesis in isolated vacuoles was 61 gM and the Vm.x 32 pmol h-1 per 106 v a c u o l e s (Fig. 3).
Ethylene formation from endogenous ACC was greatly inhibited in isolated vacuoles by C o 2 +, n-propyl-gallate and in an atmosphere of N 2 (Fig. 2, Table 2). Ethylene synthesis also ceased when the vacuoles were lysed by passage through a syringe and a hypodermic needle (Table 2). Vacuoles that were lysed in a medium containing 5 mM ACC produced at most 3% of the ethylene evolved by intact vacuoles incubated without ACC. Inhibition of ACC-dependent ethylene synthesis by lysis of the vacuoles was observed in the usual resuspension medium at pH 7.2 and also in the same medium at pH 5.5.
Stereospec~'city of the ethylene-forming system in leaf discs and isolated vacuoles. Hoffman et al. (1982) reported that one of the four stereoisomers of AEC, namely (1 R, 2 S)-AEC, was preferentially converted to 1-butene by apple and mung-bean hypocotyl tissue. They suggested that conversion of (1 R, 2 S)-AEC to l-butene was mediated by the same enzyme as conversion of ACC to ethylene was. We investigated whether isolated vacuoles converted AEC to 1-butene and whether the vacuolar system discriminated between the stereoisomers of AEC. We did not have all four stereoisomers of AEC at our disposal but the racemic mixtures of (1 R, 2S)- and (1 S, 2R)-AEC, called (_+)-allocoronamic acid, and (1 S, 2S)- and (1 R, 2R)-AEC, called (_+)-coronamic acid (see insert, Fig. 5 A). It is important to note that (+)-allocoronamic acid contains the isomer that was preferentially converted to 1-butene (Hoffman et al. 1982) while (+_)-coronamic acid does not. Leaf discs cut from fully expanded pea leaves converted ACC to ethylene and (_+)-allocoronamic acid to 1-butene, but no detectable amounts of l-butene were formed from 0.1 mM (+)-coronamic acid (Fig. 4). 1-Butene production from 0.1 mM (+_)-allocoronamic acid was inhibited by more than 80% when 1 mM ACC was present in the incubation medium. Vacuoles that had been isolated from AVG-treated protoplasts also produced 1-butene from 1 m M (+)-allocoronamic acid (Fig. 5A, B) but not in detectable amounts from t mM (_+)-coronamic acid (Fig. 5 A). Ethylene synthesis from 0.1 mM ACC was reduced by 70% in the presence of 1 m M (_+)-allocoronamic acid (Fig. 5A) and, conversely, synthesis of 1-butene from I mM (_+)-allocoronamic acid was inhibited by 60% in the presence of 4 mM ACC (Fig. 5 B). Figure 5 demonstrates another important point that was seen in all experiments where ethylene synthesis in vacuoles from AVG-treated proto-
M. Guy and H. Kende: Ethylene formation by vacuoles Table 2. Inhibition of ethylene synthesis in isolated pea vacuoles by COC12, n-propyl-gallate, lack of 0 2 (N z atmosphere) and lysis. The vacuoles were prepared from protoplasts that had been isolated from pea leaves following incubation of the leaf tissue on protoplast-isolation medium at 22 ~ C for 18 h Treatment
Ethylene produced (pmol h -1 per 106 vacuoles)
Control CoC1 z (500 gM) n-Propyl-gallate (300 gM) N 2 atmosphere
1] .3 4.2 4.4 0.5
0 63 61 96
Control (pH 7,2) i 1.5 Lysed (pH 7,2) 99 97
Control (pH 5.5) 10.0 Lysed (pH 5.5) 99 97
TI M E (h)
Fig. 4. Stereospecificity of the ethylene-forming system in pea leaf discs. Ethylene evolution from leaf discs incubated in darkness on I mM ACC (o) was monitored. 1-Butene synthesis from leaf discs incubated in darkness on 0.1 mM (_)-allocoronamic acid (zx), 0.1 mM (+)-allocoronamic acid + i mM ACC (A), or on 0.1 mM (_+)-coronamic acid (D) was measured
plasts was measured. In such vacuoles, which produced very little ethylene from the low amounts of endogenous substrate but converted exogenous ACC to ethylene, there was always a time lag of 1-2 h between the start of the incubation in ACC and the onset of ethylene production. No such lag was observed when vacuoles produced ethylene from endogenous ACC (see Fig. 2), even though the eventual rate of ethylene production from endogenous ACC was much lower than that from high concentrations of exogenous substrate (compare Figs. 2 and 5). Konze and Kende (1979) described a cell-free system obtained from etiolated pea epicotyls that converted ACC to ethylene. We compared the capacity of this in-vitro system to form 1-butene
M. Guy and H. Kende: Ethylene formation by vacuoles
R4 =-C2H5 : (+)-allocoronamic acid R2 =-C2H5 (-)-allocoronarnic acid RI =-CtH5 ( + ) - c o r o n o m i c acid
R~' =-C2H5 (-) . . . . . . . .
,)~'@e'~ 6c~, a~ . - ' ~