received for publication accepted turnover 1-carboxylic ... · plant physiol. vol. 100, 1992 figure...

Plant Physiol. (1992) 100, 1126- 11310032-0889/92/100/11 26/06/$01 .00/0

Received for publication January 27, 1992Accepted June 6, 1992

Turnover of 1 -Aminocyclopropane- 1 -Carboxylic AcidSynthase Protein in Wounded Tomato Fruit Tissue1

Woo Taek Kim* and Shang Fa Yang

Mann Laboratory, Department of Vegetable Crops, University of California, Davis, California 95616

ABSTRACT

Ethylene production in plant tissues declines rapidly followinginduction, and this decline is due to a rapid decrease in the activityof 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a keyenzyme in ethylene biosynthesis. To study the nature of the rapidturnover of ACC synthase in vivo, proteins in wounded ripeningtomato (Lycopersicon esculentum) fruit discs were radiolabeledwith [5S]methionine, followed by a chase with nonradioactivemethionine. Periodically, the radioactive ACC synthase was iso-lated with an immunoaffinity gel and analyzed. ACC synthaseprotein decayed rapidly in vivo with an apparent half-life of about58 min. This value for protein turnover in vivo is similar to thatpreviously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlo-rophenylhydrazone and 2,4-dinitrophenol, potent uncouplers ofoxidative phosphorylation, strongly inhibited the rapid decay ofACC synthase protein in the tissue. Degradation of this enzymeprotein was moderately inhibited by the administration of ami-nooxyacetic acid, a competitive inhibitor of ACC synthase withrespect to its substrate S-adenosyl-L-methionine, a,a'-dipyridyl,and phenylmethanesulfonyl fluoride or leupeptin, serine proteaseinhibitors. These results support the notion that the substrate S-adenosyl-L-methionine participates in the rapid inactivation of theenzyme in vivo and suggest that some ATP-dependent processes,such as the ubiquitin-requiring pathway, are involved in the deg-radation of ACC synthase proteins.

The production of the phytohormone ethylene in planttissue is usually low but is greatly induced at certain stagesof plant development, such as seed germination, leaf absci-sion, and fruit ripening; by auxin treatment; or under variousenvironmental stresses, including wounding, drought, flood-ing, and pathogen invasion (1). It has been well establishedthat ethylene is biosynthesized via the following sequence:Met -- AdoMet -) ACC2 -- C2H4 (3). Because a promoted

ethylene synthesis is usually correlated with an increasedACC synthase activity, which catalyzes the conversion ofAdoMet to ACC, ACC synthase has been thought to be the

'This work was supported by grant No. DCB-9004129 from theNational Science Foundation.

2Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid;AdoMet, S-adenosyl-L-methionine; AOA, aminooxyacetic acid; AVG,aminoethoxyvinylglycine; CCCP, carbonylcyanide-m-chlorophenyl-hydrazone; CHI, cycloheximide; DNP, 2,4-dinitrophenol.

rate-limiting enzyme in the ethylene biosynthetic pathway(24).

Induction of ACC synthase activity by various stimuli isgreatly inhibited by CHI (10, 26). Kende and his associates(2, 5) showed that the wound-induced increase in ACCsynthase activity in tomato fruit tissue is due to de novosynthesis of ACC synthase. Recently, cDNA clones encodingACC synthases were isolated from various plant species.Northern blot analysis shows that the promoted ACC syn-thase activity is closely related to the enhanced level of itsmRNA (8, 11, 14, 15).The apparent half-life of ACC synthase activity in the

tissue, as measured in the presence of CHI, was estimated tobe about 40, 25, and 40 min in wounded tomato tissue (10),in auxin-treated mung bean hypocotyls (26), and in elicitor-treated tomato cell culture (21), respectively, indicating thatthe turnover of ACC synthase activity is rapid.Another important characteristic of ACC synthase is that

the enzyme is inactivated in vitro by its substrate AdoMet (6,16). Satoh and Yang (17-19) demonstrated that incubationof a partially purified tomato ACC synthase preparation withAdoMet results in inactivation of the enzyme activity andthat this inactivation is due to a covalent linkage of theaminobutyrate portion of AdoMet to the enzyme. Becausethe half-life of ACC synthase inactivation by AdoMet in vitro(16, 17) is similar to that in vivo (10, 26), and because AVG,a competitive inhibitor of ACC synthase activity with respectto AdoMet, increases the enzyme half-life both in vitro (17)and in vivo (26), AdoMet-induced inactivation of the enzymeis suggested to be the cause of the rapid decrease in enzymeactivity observed in vivo (16, 17). On the contrary, Spanu etal. (21) recently reported that AVG does not alter the apparentturnover rate of ACC synthase activity in tomato leaves andin cell suspension cultures.To gain more insight into the nature of the ACC synthase

degradation, we examined the influence of various com-pounds on the degradation of ACC synthase protein inwounded tomato (Lycopersicon esculentum) fruit tissue. Fol-lowing pulse labeling with radioactive Met and chase withnonradioactive Met in the presence of various compounds,ACC synthase protein was purified with immunoaffinity gel.In this communication, we report that ACC synthase proteindegrades rapidly at a rate similar to that of the AdoMet-dependent enzyme activity and that AOA, a known compet-itive inhibitor of ACC synthase, and the uncouplers CCCPand 2,4-DNP are effective inhibitors of the degradation ofACC synthase in vivo.

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1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE TURNOVER IN VIVO

MATERIALS AND METHODS

Plant Material

Tomato (Lycopersicon esculentum Mill.) fruits at the pinkstage of ripeness were bought at a local market. For eachexperiment, 2.5 g of discs (1.2 cm in diameter and 5 mmthickness) prepared from pericarp tissue were used.

Determination of Ethylene

A 1-mL gas sample was withdrawn with a hypodermicsyringe from the head space of each flask containing tomatosamples, and ethylene was assayed on a gas chromatographequipped with an alumina column and a flame ionizationdetector.

In Vivo Labeling Experiments

After tomato discs were prepared and incubated at roomtemperature for 3 h, an aqueous solution (30 ,uL) containing100 ,uCi [35S]Met (>lOOOCi/mmol) was applied to the surfaceof the discs, and they were incubated for an additional 3 h.For the chase, radiolabeled tomato discs were infiltratedunder vacuum with 20 mL of 50 mm nonradioactive Met.After the discs were blotted dry and incubated for variousperiods (0-1.5 h), they were frozen with liquid nitrogen andstored at -800C until used. To examine the effect of varioustest solutions on the ACC synthase turnover, radiolabeleddiscs were similarly vacuum infiltrated with a test solutioncontaining 50 mM nonradioactive Met and chased for 2.5 h.Test solutions contained 2 mm AOA, 0.4 mm CCCP, 2 mM2,4-DNP, 30 mm dipyridyl, 5 mm PMSF, 100 ,ug/mL ofleupeptin, and 100 ,ug/mL of pepstatin. Radiolabeled discs(2.5 g) were homogenized in a 10-mL solution containing100 mm potassium phosphate (pH 7.5), 5 mm EDTA, and 5tLM pyridoxal phosphate (4). The supernatant was obtainedafter centrifugation at 15,000g for 20 min.

Immunoaffinity Purification of ACC Synthase Protein

A monoclonal antibody against tomato ACC synthase (4),a generous gift from Hans Kende, Michigan State University,was dialyzed against 0.1 M Mops, pH 7.5, for 6 h at 40C. Animmunoaffinity matrix was prepared by coupling 3 mg of thedialyzed antibody to 1 mL of activated Affi-Gel 10 (Bio-Rad).After incubation at 40C for 4 h with constant shaking, theremaining active ester in the gel was blocked by reacting with0.1 mL of 1 M ethanolamine-HCl (pH 8.0). A crude homog-enate (10 mL) of radiolabeled tomato discs (2.5 g) was incu-bated with 50 ML of immunoaffinity gel for 3 h. The gel wasseparated from the solution by centrifugation and washedfive times with 10 mL of PBS solution (10 mm Na-Pi [pH 7.5]and 0.9% NaCl). The gel-bound protein was eluted with 2%SDS, and the eluted protein was analyzed by SDS-PAGE(12). Radioactive bands were visualized by fluorography. Todetermine the radioactivity incorporated into ACC synthase,ACC synthase protein bands were cut out from the gel andsoaked in 200 ,tL of H20 for 1 h at room temperature. Afterthe addition of 1 mL of NCS tissue solubilizer (Amersham)/

H20 (9:1, v/v), the mixture was further incubated at 500Cfor 6 h and then neutralized with 50 ,uL of acetic acid. Theradioactivity was assayed in a liquid scintillation counter aftermixing with 5 mL of scintillation cocktail.

Extraction of AdoMet

Extraction and determination of AdoMet were performedas described previously (28) with modifications. Discs infil-trated under vacuum with 2 mm DNP, 0.4 mM CCCP, orwater were homogenized and extracted with 1 M HC104 atroom temperature for 1 h. The extract was then centrifuged,and the pH of the supernatant was adjusted to 4.5 by slowlyadding solid KHCO3. The precipitated salt was discarded,and the supernatant was passed through an ion exchangeresin (Bio-Rex 70, H' form) column. After the column waswashed until no absorbance at 260 nm was detected, AdoMetwas eluted with 0.1 N HCl. The concentration of AdoMetwas determined spectrophotometrically at 260 nm, assuminga molar absorption coefficient of 15,000 cm-' M-1.

RESULTS AND DISCUSSION

Ripening tomato fruit tissue is a good source of ACCsynthase, and its activity can be further increased by wound-ing (7, 10, 27, 29). After the wounded ripening tomato fruittissue was radiolabeled with [35S]Met, ACC synthase proteinin the homogenate was purified with an immunoaffinity gel.

A

M 1 2kDa688

43.

I B*14.

B

M 1 2

4

Figure 1. Fluorograph of [35S]Met-labeled, immunopurified pro-teins analyzed by SDS-PAGE. A, Tomato discs (2.5 g) labeled for 3h with 100 ACi [35S]Met were homogenized in 10 mL of homoge-nization buffer; 50 ,L of the homogenate was subjected to SDS-PAGE analysis (lane 2), and the remaining solution was subjectedto immunoaffinity purification, followed by SDS-PAGE (lane 1). B,Lane 1, SDS-PAGE analysis of the fraction eluted from the immu-noaffinity gel with 2% SDS; lane 2, the fraction eluted from a secondaliquot of immunoaffinity gel added to the preparation after theproteins had been removed by the first aliquot of the immunoaffin-ity gel as used in lane 1. Lane M in both panels are "4C-labeledmolecular size markers (Bethesda Research Laboratories): BSA (68kD), ovalbumin (43 kD), carbonic anhydrase (29 kD), j3-lactoglobulin(18 kD), and lysozyme (14 kD).

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Plant Physiol. Vol. 100, 1992

Figure 1 shows a typical result of our immunoaffinity puri-fication. One major radioactive band of about 48 kD wasdetected (Fig. 1A, lane 1). This band was judged to be ACCsynthase on the basis that its apparent molecular sizematched that previously reported for tomato ACC synthase(4, 5, 17, 18, 23) and it was specifically bound to an anti-ACC synthase monoclonal antibody (4, 5, 17). To ascertainthat under the present experimental conditions our immu-noaffinity gel was effective in removing all the ACC synthaseprotein, a repetitive purification procedure was carried out.To the supernatant from which the gel-bound proteins hadbeen removed after the first-round purification, a secondaliquot (50 AL) of immunoaffinity gel was added, and anyproteins bound to that gel were similarly analyzed. As shownin Figure 1B, no radioactive bands were detected from thegel after the second-round purification step. These resultsindicate that 50 ML of immunoaffinity gel is enough to bindmost, if not all, ACC synthase protein in the extract preparedfrom 2.5 g of tomato tissue. Because only one radioactiveband was observed, our results confirmed that the presentimmunoaffinity purification is highly specific for ACC syn-thase (4, 5, 17). Hence, this one-step purification method wasused for our subsequent experiments. To ascertain the repro-ducibility of our immunoaffinity purification, experimentsdescribed in this paper were repeated three or four times,and in all cases similar results were obtained.

It has long been recognized that ethylene production inplant tissues declines rapidly following induction, and thisdecline is accompanied by a decline in ACC synthase activity(10, 21, 26). The apparent half-life of tomato ACC synthaseactivity from wounded ripening fruit was previously esti-mated to be about 40 min (10) in experiments in which CHIwas used to block the new synthesis of the enzyme. BecauseCHI is also known to inhibit other metabolic processes, e.g.energy transfer (10, 13), it is conceivable that CHI mayinterfere with not only the new synthesis of ACC synthasebut also the inactivation/degradation of ACC synthase.With the present pulse-chase-labeling technique, wounded

tomato discs were first radiolabeled with [35S]Met for 3 h andthen chased with nonradioactive Met for various time periodsin the absence of CHI, before the radioactive ACC synthaseprotein was determined by the immunoaffinity assay. Be-cause the newly synthesized proteins during the chase periodwould not be radiolabeled, they would not be detected bythe present method. Whereas the enzyme activity assay re-lates only to the amount of native ACC synthase, the presentimmunoassay measures the summation of both native andinactive ACC synthase proteins. To measure the decrease inradioactive ACC synthase protein, ACC synthase bands werecut out from the gel and their radioactivities were assayed byscintillation counting. During the course of chasing, ACCsynthase protein decayed rapidly following first-order kinet-ics as indicated by the linear decay curve in a semilogarithmicplot (Fig. 2). The apparent half-life was determined to be 58min (Fig. 2B). In another experiment, a half-life of 48 minwas observed (data not shown). In wounded tomato fruitdiscs treated with CHI, Kende and Boller (10) observed thatACC synthase activity decreased rapidly with a half-life of40 min. These results indicate that the turnovers of ACC

A0 0.5 1,5 M

68

7_1~3 18

B

Figure 2. Time course for the degradation of ACC synthase proteinin vivo. A, Tomato discs labeled with [35S]Met for 3 h and chasedwith 50 mmv nonradioactive.Met for various time periods as indi-cated were homogenized, and the radioactive ACC synthase pro-tein was purified by the immunoaffinity gel. The ACC synthaseband was cut out from the gel after SDS-PAGE analysis and fluoro-graphy and assayed with a liquid scintillation counter. The amountof radioactive ACC synthase at 0, 0.5, and 1.5 h of chasingwere 1020, 653, and 373 dpm, respectively. B, The decay of radio-active ACC synthase protein during the chasing is plottedsemilogarithmically.

synthase protein and ACC synthase activity are rapid withcomparable half-lives.

Boller (6) and Satoh and Esashi (16) made the initialobservation that extracted ACC synthase was inactivated invitro by its substrate AdoMet during its catalytic reaction.Satoh and Yang (17) showed that the rate of ACC synthaseinactivation in vitro depends upon the concentration ofAdoMet and that in the presence of AVG, a competitiveinhibitor of ACC synthase with respect to AdoMet (7), thehalf-life of the enzyme activity in vitro increases from 54 to108 mmn. Later work showed that this AdoMet-dependentinactivation results from an irreversible covalent linkageof the 2-aminobutyrate moiety of AdoMet to the enzyme(18, 25).

1128 KIM AND YANG




Because the half-life of ACC synthase actithe presence of AdoMet is similar to the half-libecause the competitive inhibitor AVG increasof the enzyme activity in vitro as well as in vbeen suggested that the AdoMet-induced ina(first step leading to the rapid inactivation cfound in the tissue (16, 17). We have, therefo:effect of AOA, a well-known competitive inhsynthase with respect to AdoMet (27), on tiACC synthase protein in vivo. To ascertain theof AOA required to inhibit ethylene synthesiswere vacuum infiltrated with various concentrateand ethylene production was measured peric

A 200

en0

(n

c_

1-

a)

a)

0-

B

kDa

-a-

-9.-

M

control0.3 mM AOA

1 mMAOA5 mM AOA

2 4 6

incubation (h)

1 2 3

68

43.

29S

18-14*

Figure 3. A, Effect of AOA on ethylene synthe!tomato discs. Tomato discs were infiltrated under i

mm Met or 50 mm Met containing 0.3, 1, or 5 mMproduced during the 7-h incubation was assayedEffect of AOA and CCCP on the turnover of ACC s

in vivo. Tomato discs labeled for 3 h with [35S]Met0 h following vacuum infiltration with 50 mM MEdpm) or chased for 2.5 h following vacuum infiltrateMet (lane 2, 383 dpm) or 50 mm Met + 2 mm A(dpm) or 50 mm Met + 0.4 mm CCCP (lane 4, 1145Molecular size markers.

vity in vitro in the incubation. Wound-ethylene production increased mark-ife in vivo, and edly during the incubation and reached about 140 nL/g afteres the half-life 7 h incubation (Fig. 3A). Ethylene production was inhibitedivo (26), it has about 50% with 300 Mm AOA, and the inhibition was nearlyactivation is the complete at 1 or 5 mm AOA (Fig. 3A). Therefore, we chose)f the enzyme to use 2 mm AOA in the present experiment.tre, studied the After tomato discs were labeled with [35S]Met, they wereLibitor of ACC chased with 50 mm nonradioactive Met containing 0 or 2 mMie turnover of AOA for 2.5 h. Analysis of ACC synthase protein revealedM concentration that infiltration of AOA significantly inhibited the decay of5, tomato discs ACC synthase (Fig. 3B, lanes 2 and 3), indicating that AOAnations of AOA, stabilizes ACC synthase in vivo. These results are in agree-dically during ment with the previous report that AVG increases the enzyme

half-life in vivo by about 2-fold in mung bean hypocotyls(26) and are consistent with the hypothesis that AdoMetparticipates in the rapid inactivation of ACC synthase invivo. At variance with these results is the recent report ofSpanu et al. (21), who observed that AVG did not signifi-cantly alter the apparent turnover of stress-induced ACCsynthase activity in tomato leaf tissue and cell suspension

3 cultures.It should be noted that the level of ACC synthase activity

in the tissue is determined by both enzyme synthesis andenzyme inactivation. Thus, measurements of ACC synthaseactivity may reflect the decay of ACC synthase only whenthe enzyme synthesis is completely blocked during the incu-bation period. Spanu et al. (21), however, examined the effectof AVG on the decrease in ACC synthase activity in vacuum-infiltrated tomato leaf discs in the absence of CHI. Thus,

*l their observed changes in ACC synthase activity may not8 reflect the decay of ACC synthase. In another experiment,

Spanu et al. (21) investigated the effect of AVG on the decayof ACC synthase in tomato cell cultures treated with a fungalelicitor in the presence of CHI (fig. 3 in ref. 21). When we

4 replotted their data semilogarithmically, AVG resulted in anincrease in enzyme half-life from 75 to 150 min, a conclusionin agreement with our present data (Fig. 3B) and otherprevious data (26).

It should be noted that both native and AdoMet-inacti-_4 vated ACC synthase proteins are recognized by the mono-

clonal antibody (17, 25). Thus, if the inactivation of ACCsynthase by AdoMet represents the first step in its turnover,this inactivated enzyme must be further degraded by a rapidproteolytic process (17). In the case of phytochrome, it hasbeen shown that Pr is first converted into Pfr, which is thendegraded by a specific proteolytic pathway involving ATP-dependent conjugation of ubiquitin (9, 20). To examine apossible involvement of proteolysis in the turnover of ACCsynthase in vivo, we examined the effects of various proteaseinhibitors and uncouplers of oxidative phosphorylation on

sis in wounded the turnover of ACC synthase. Figure 4 shows that the rapidvacuum with 50 breakdown of ACC synthase was inhibited by leupeptin (laneAOA. Ethylene 4) and a,a'-dipyridyl (lane 8), and slightly inhibited by PMSFperiodically. B, (lane 3), but was little affected by pepstatin (lane 5). These;ynthase protein results suggest that serine and/or metal-dependent proteasewere chased for result est tha segad/or m - nden poteaeset (lane 1, 1206 are involved in the degradation of ACC synthase. However,tion with 50 m6 we could not rule out the possibility that the differentialDA (lane 3, 776 effects of these protease inhibitors are due to their different5 dpm). Lane M, permeability into the cell.

DNP and CCCP have long been known to inhibit C2H4

1129



Plant Physiol. Vol. 100, 1992

6.s

k:.

a. D !<

.s A. I'd I'.,

14IN. -., -m "lm o -

4

Figure 4. Effect of various protease inhibitors, CCCP, DNP, anddipyridyl, on the turnover of ACC synthase protein in vivo. Tomatodiscs labeled for 3 h with [35S]Met were chased for 0 h followingvacuum infiltration with 50 mm Met (lane 1, 1401 dpm) or chasedfor 2.5 h following vacuum infiltration with 50 mm Met (lane 2, 657dpm), 50 mm Met + 5 mm PMSF (lane 3, 881 dpm), 50 mm Met +100 ,ug/mL of leupeptin (lane 4, 1113 dpm), 50 mm Met + 100 Mg/mL of pepstatin (lane 5, 675 dpm), 50 mm Met + 0.4 mm CCCP(lane 6, 1394 dpm), 50 mm Met + 2 mm DNP (lane 7, 1427 dpm),or 50 mm Met + 30 mm dipyridyl (lane 8, 1031 dpm). Purificationand analysis of ACC synthase were carried out as in Figure 2.

production in various plant tissues (28). The effect of DNP(2 mM) or CCCP (0.4 mM) on the decay of ACC synthaseprotein in vivo was examined, and the results are shown inFigures 3B and 4. Both CCCP and DNP markedly inhibitedthe rapid degradation of ACC synthase protein (Fig. 4, lanes6 and 7); their effectiveness was greater than that exerted byAOA (Fig. 3B, lanes 3 and 4).

There are two possible explanations that could account forthe stabilization of ACC synthase protein by CCCP or DNP.Because they are potent uncouplers of oxidative phosphoryl-ation, it is reasonable to assume that they block ATP synthesisand thereby inhibit the ATP-dependent formation of AdoMetfrom Met. This will lead to a lower concentration of AdoMetin the tissue and consequently to'a reduced AdoMet-depend-ent inactivation of ACC synthase. The second possibility isthat the rapid turnover of ACC synthase in vivo is caused byan ATP-dependent proteolytic process. To'examine the effectof CCCP or DNP on the level of AdoMet in the tomatotissue, AdoMet was extracted from the tomato discs that hadbeen vacuum infiltrated with 2 mm DNP or 0.4 mm CCCP as

shown in Figure 4, and their concentrations were determined.The concentrations of AdoMet in control, DNP-, or CCCP-treated tissue were 15.5, 15.4, and 13.0 nmol/g, respectively.These results indicate that these uncouplers under the presentexperimental conditions exerted little influence on the levelof AdoMet. Such a conclusion is consistent with our previousfindings that the concentrations of AdoMet from mung bean

hypocotyls incubated with or without 50 gM DNP for 9 hwere similar (28). Thus, we can conclude that the stabilizationof ACC synthase protein caused by DNP or CCCP is not dueto the decrease in AdoMet concentration but to some ATP-dependent proteolytic processes.

Ubiquitin is a protein involved in the selective degradationof cytoplasmic proteins in both animal and plant cells throughits ATP-dependent covalent ligation to target proteins des-tined for catabolism (22). Our finding that inhibitors of ATP-generating systems effectively inhibit the rapid breakdownof ACC synthase in vivo leads us to suggest that the ubiqui-tin-dependent proteolytic pathway may be involved in thedegradation of ACC synthase.

ACKNOWLEDGMENT

We thank Dr. Shigeru Satoh for his helpful discussion.

LITERATURE CITED

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10. Kende H, Boller T (1981) Wound ethylene and 1-aminocyclo-propane-1-carboxylate synthase in ripening tomato fruit.Planta 151: 476-481

11. Kim WT, Silverstone A, Yip WK, Dong JG, Yang SF (1992)Induction of ACC synthase mRNA by auxin in mungbeanhypocotyls and apple cultured shoots. Plant Physiol 98:465-471

12. Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227:680-685

13. MacDonald IR, Ellis RJ (1969) Does cycloheximide inhibitprotein synthesis specifically in plant tissues? Nature 222:791-792

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15. Olson DC, White JA, Edelman L, Harkins RN, Kende H (1991)Differential expression of two genes for 1 -aminocyclopropane-1-carboxylate synthase in tomato fruits. Proc Natl Acad SciUSA 88: 5340-5344

16. Satoh S, Esashi Y (1986) Inactivation of 1-aminocyclopropane-1-carboxylate synthase of etiolated mungbean hypocotyl seg-ments by its substrate, S-adenosyl-L-methionine. Plant CellPhysiol 27: 285-291

17. Satoh S, Yang SF (1988) S-Adenosylmethionine-dependent in-activation and radiolabeling of 1-aminocyclopropane-1-car-boxylic acid synthase isolated from tomato fruits. Plant Physiol88: 109-114

18. Satoh S, Yang SF (1989) Specificity of S-adenosyl-L-methioninein the inactivation and the labeling of 1-aminocyclopropane-1-carboxylic acid synthase in tomato fruits. Arch BiochemBiophys 271: 107-113

19. Satoh S, Yang SF (1989) Inactivation of 1-aminocyclopropane-1-carboxylate synthase by L-vinylglycine as related to themechanism-based inactivation of the enzyme by S-adenosyl-L-methionine. Plant Physiol 91: 1036-1039

20. Shanklin J, Jabben M, Vierstra RD (1987) Red light-inducedformation of ubiquitin-phytochrome conjugates. Identificationof possible intermediates of phytochrome degradation. ProcNatl Acad Sci USA 84: 359-363

21. Spanu P, Felix G, Boller T (1990) Inactivation of stressedinduced 1-aminocyclopropane-1-carboxylate synthase in vivo

differs from substrate-dependent inactivation in vitro. PlantPhysiol 93: 1482-1485

22. Vierstra RD (1987) Ubiquitin, a key component in the degra-dation of plant proteins. Plant Physiol 70: 103-106

23. White JA, Kende H (1990) 1-Aminocyclopropane-1-carboxylatesynthase: are there charge and size variants in tomatos? J PlantPhysiol 136: 646-652

24. Yang SF, Hoffman NE (1984) Ethylene biosynthesis and itsregulation in higher plants. Annu Rev Plant Physiol 35:155-189

25. Yip WK, Dong JG, Kenny JW, Thompson GA, Yang SF (1990)Characterization and sequencing of the active site of 1-ami-nocyclopropane-1-carboxylate synthase. Proc Natl Acad SciUSA 87: 7930-7934

26. Yoshii H, Imaseki H (1982) Regulation of auxin-induced eth-ylene biosynthesis. Repression of inductive formation of 1-aminocyclopropane-1 -carboxylate synthase by ethylene. PlantCell Physiol 23: 639-649

27. Yu Y-B, Adams DO, Yang SF (1979) 1-Aminocyclopropane-1-carboxylate synthase, a key enzyme in ethylene biosynthesis.Arch Biochem Biophys 198: 280-286

28. Yu Y-B, Adams DO, Yang SF (1980) Inhibition of ethyleneproduction by 2,4-dinitrophenol and high temperature. PlantPhysiol 66: 286-290

29. Yu Y-B, Yang SF (1980) Biosynthesis of wound ethylene. PlantPhysiol 66: 281-285

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