Monomeric and Dimeric Forms and the Mechanism-Based Inactivation of 1-Aminocyclopropane-1-Carboxylate Synthase

The molecular mass of 1-aminocyclopropane-1-carboxylate (ACC)synthase from a variety of sources was examined by both high-performancegel-filtration chromatography and polyacryl-amide gel electrophoresisin the presence of sodium dodecylsulfate. Enzymes used wereprepared from wounded or non-wounded pericarp of ripe tomatofruits and wounded mesocarp of winter squash fruits, as wellas from cells of E. coli that had been transformed with cDNAsfor the wound-induced or ripening-induced ACC synthases of tomatoand the wound-induced or auxininduced enzymes from winter squash.The enzymes from tomato fruit tissues were isolated in a monomericform, whereas the enzymes synthesized in E. coli from cDNAsfor tomato ACC synthase were isolated in a dimeric form. ACCsynthases of winter squash obtained either from fruit tissuesor from transformed E. coli cells were isolated in dimeric forms.ACC synthase in the monomeric form was less sensitive to theinactivation that is associated with the catalytic reaction(the mechanism-based inactivation) than the enzyme in the dimericform. A plausible mechanism relating the difference in molecularform to sensitivity to the mechanism-based inactivation of tomatoACC synthase is discussed. (Received February 1, 1993; Accepted May 17, 1993)     

Molecular Size of Wound-Induced 1-Aminocyclopropane-1-carboxylate Synthase from Cucurbita maxima Duch. and Change of Translatable mRNA of the Enzyme after Wounding

Wound-induced 1-aminocyclopropane-1-carboxylate (ACC) synthasewas purified by an immunoaffinity column from wounded mesocarpof winter squash (Cucurbita maxima Duch. cv. Ebisu) fruit, anda specific antibody was raised in rabbit. Translatable mRNAcoding for ACC synthase was barely detectable in fresh tissuebut clearly increased after wounding. The apparent molecularsize of the purified enzyme as estimated by SDS-polyacrylamidegel electrophoresis (PAGE) was about 50 kDa. However, SDS-PAGEfluorograms of in vitro translation product of ACC synthasemRNA and the in vivo labeled enzyme as well as Western blotanalysis showed that the subunit size of the enzyme was 58 kDa.The enzyme was partially degraded or processed to a 50 kDa peptideboth in vivo and in vitro. (Received December 19, 1987; Accepted June 13, 1988)     

Molecular Cloning and Sequence of a Complementary DNA Encoding 1-Aminocyclopropane-l-carboxylate Synthase Induced by Tissue Wounding

1-Aminocyclopropane-l-carboxylate (ACC) synthase [EC 4.4.1.14 [EC] ]is the key enzyme regulating ethylene biosynthesis in higherplants. A complementary DNA encoding wound-induced ACC synthasefrom mesocarp of winter squash (Cucurbita maxima Duch.) fruitswas cloned, and its complete nucleotide sequence determined.The cloned cDNA contained an open reading frame of 1479 basepairs encoding a sequence of 493 amino acids. Identificationof the cDNA was accomplished by expression of active enzymein Escherichia coli harboring the cDNA and by the presence ofa partial amino acid sequence identical to that found in thepurified enzyme. A putative pyridoxal phosphate binding siteof the enzyme is suggested. Northern blot analysis showed thatthe ACC synthase gene was activated by tissue wounding, andits expression was repressed by ethylene. Genomic Southern analysisindicates the presence of at least another sequence which weaklyhybridizes with the cDNA. (Received June 26, 1990; Accepted August 7, 1990)     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Wound-Induced Ethylene Production and 1-Aminocyclopropane-1-carboxylic Acid Synthase in Mesocarp Tissue of Winter Squash Fruit

Discs (9 mm in diameter and 2 mm in thickness) sliced from mesocarpof winter squash fruit (Cucurbita maxima Duch.) upon incubationat 24°C produced ethylene at an increasing rate after alag period of 3 h. 1-Aminocydopropane-l-carboxylic acid (ACC)synthase activity also increased at a rapid rate after lag periodof less than 3 h, reaching a peak 14 h after incubation andthen declining sharply. The rise in ACC synthase activity precededa rapid increase in ACC formation and ethylene production. Inductionof ACC synthase by wounding in sliced discs was strongly suppressedby the application of cycloheximide, actinomycin D and cordycepin,suggesting that the rise in ACC synthase activity may resultfrom de novo synthesis of protein. ACC synthase extracted from wounded tissue of winter squashmesocarp required pyridoxal phosphate for its maximum activity.The optimum pH of the reaction was 8.5. Km value for S-adenosylmethioninewas 120 µM. The reaction was markedly inhibited by aminoethoxyvinylglycinewith Ki value being 2.7 µM. (Received March 23, 1983; Accepted May 23, 1983)     

Characterization and Induction of the Activity of 1-Aminocyclopropane-1-Carboxylate Oxidase in the Wounded Mesocarp Tissue of Cucurbita maxima

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene-formingenzyme) was isolated from wounded mesocarp tissue of Cucurbitamaxima (winter squash) fruit, and its enzymatic properties wereinvestigated. The enzyme required Fe²⁺ and ascorbate for itsactivity as well as ACC and O₂ as substrates. The in vitro enzymeactivity was enhanced by CO₂. The apparent K_m value for ACCwas 175 µM under atmospheric conditions. The enzyme activitywas inhibited by sulfhydryl inhibitors and divalent cationssuch as Co²⁺, Cu²⁺, and Zn²⁺. ACC oxidase activity was induced at a rapid rate by woundingin parallel with an increase in the rate of ethylene production.The exposure of excised discs of mesocarp to 2,5-norbornadiene(NBD),an inhibitor of ethylene action, strongly suppressed inductionof the enzyme, and the application of ethylene significantlyaccelerated the induction of the activity of ACC oxidase inthe wounded mesocarp tissue. These results suggests that endogenousethylene produced in response to wounding may function in promotingthe induction of ACC oxidase. (Received January 13, 1993; Accepted April 15, 1993)     

Induction of 1-Aminocyclopropane-1-carboxylic Acid Synthase in Wounded Mesocarp Tissue of Winter Squash Fruit and the Effects of Ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase activityincreased rapidly after wounding of mesocarp tissue of wintersquash fruit (Cucurbita maxima Duch.) and reached a peak at16 h after excision and then declined sharply. The rise in ACCsynthase activity was followed by increases in the endogenousACC content and the rate of ethylene production. The activityof ethylene forming enzyme (EFE) also increased rapidly in theexcised discs of mesocarp of winter squash fruit. ACC synthase activity was strongly inhibited by aminoethoxyvinylglycinewith a Ki value of 2.1 µM. Michaelis-Menten constant ofACC synthase for S-adenosylmethionine was 13.3 µM. Ethylene suppressed the induction of ACC synthase in the woundedmesocarp tissue. The suppression by ethylene increased withthe increasing concentrations of applied ethylene and the maximumeffect was obtained at about 100 µl 1^–1 ethylene,at which point the induction was suppressed by 54%. Ethylenedid not inhibit ACC synthase activity, nor did it suppress theinduction of EFE, but rather it slightly enhanced the latter. (Received August 24, 1984; Accepted October 29, 1984)     

Inhibition of 1-Aminocyclopropane-l-carboxylic Acid Synthase Activity by Polyamines, Their Related Compounds and Metabolites of S-adenosylmethionine

Basic amino acids, monoamines, diamines and polyamines inhibitedthe activity of 1-aminocyclopropane-1-carboxylic acid (ACC)synthase extracted from wounded mesocarp tissue of winter squashfruit (Cucurbita maxima Duch.). Among the amines tested, polyamineswere highly effective, while the synthetic triamine, 1,8-diamino-4-aminomethyloctane,was an even stronger inhibitor than the polyamine spermine.Polyamines inhibited ACC synthase activity in a non-competitivemanner, while metabolic inhibitors such as aminoethoxyvinylglycineand aminooxyacetic acid inhibited ACC synthase activity competitively,showing much lower Ki values than those of polyamines. ACC synthaseactivity was also inhibited by intermediates of the methionine-recyclingpathway, 5'-methylthioadenosine and -keto--methylthiobutyricacid and by S-adenosylhomocysteine, a product of transmethylationof S-adenosylmethionine. It appears that polyamines not only inhibit ACC synthase activitybut also suppress the induction of the enzyme. However, unlikeprevious reports, polyamines did not inhibit in vivo ethyleneforming enzyme activity in the wounded mesocarp tissue. (Received October 24, 1985; Accepted January 10, 1986)     

Properties and Partial Purification of 1-Aminocyclopropane-1-carboxylate Synthase

We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato (Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on ²H₂O as compared to ACC synthase from H₂O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of l-[³⁵S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding.     

The Effects of 2,5-Norbornadiene on the Induction of the Activity of 1-Aminocyclopropane-l-carboxylate Synthase and of Phenylalanine Ammonia-lyase in Wounded Mesocarp Tissue of Cucurbita maxima

Activities of both 1-aminocyclopropane-l-carboxylate (ACC) synthaseand phenylalanine ammonia-lyase (PAL) were rapidly induced inexcised mesocarp discs of Cucurbita maxima Duch. The increasein activity of ACC synthase preceded that of PAL. 2,5-Norbornadiene(NBD), an inhibitor of the action of ethylene [Sisler and Yang(1984) Phytochemistry 12: 2765-2768.], enhanced the level ofactivity of ACC synthase in excised mesocarp disc and overcamethe suppression by exogenous ethylene. NBD, by contrast, suppressedthe level of PAL activity induced in the wounded tissue. Theseresults suggest that endogenous ethylene produced in the woundedmesocarp tissue suppresses the induction of ACC synthase butpromotes the induction of PAL. (Received March 9, 1989; Accepted June 14, 1989)     

Monoclonal Antibodies against Apple 1-Aminocyclopropane-l-Carboxylate Synthase

1-Aminocyclopropane-l-carboxylate (ACC) synthase from applefruits was purified over 5,000-fold by conventional column chromatography.By immunizing mice with this partially purified enzyme preparation,8 hybridoma lines producing monoclonal antibodies against appleACC synthase were isolated. While all 8 clones immunoprecipitatednative ACC synthase, only two clones recognized the putative(48 kDa) ACC synthase on Western blots. When a partially purifiedACC synthase preparation was incubated with S-adenosyl-L-[carboxyl-¹⁴C]methionine(AdoMet), only one radioactive protein of 48 kDa was detectedon sodium dodecyl sulfate-poly-acrylamide gel electrophoresis.This radioactive protein was specifically immunoprecipitatedby the monoclonal antibodies, indicating that apple ACC synthaseis specifically radiolabeled by its substrate AdoMet, as istomato ACC synthase. Thus, the monoclonal antibodies recognizedboth native and AdoMet-inactivated forms of ACC synthase. Whilethese antibodies failed to im-munoprecipitate ACC synthase isolatedfrom ripe tomato fruits, ripe avocado fruits or auxin-treatedmungbean hypocotyls, they were effective in immunoprecipitatingthe enzyme isolated from ripe pear fruits. (Received August 11, 1990; Accepted October 17, 1990)     

Studies on the regulation of 1-aminocyclopropane-1-carboxylate synthase in tomato using monoclonal antibodies

A partially purified preparation of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) from tomato (Lycopersicon esculentum (Mill.) fruit tissue was used to generate monoclonal antibodies (MAb) specific for the two different MAbs yielded a 50-kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An enzyme-linked immunosorbent assay (ELISA) capable of detecting <1 ng of antigen was developed. The ELISA system was used to demonstrate that two of the MAbs recognized different epitopes on the ACC-synthase protein. Wound-induced increases in ACC-synthase activity in tomato fruit tissue were correlated with changes in ELISA-detectable protein. In-vivo labeling of wounded tissue with [³⁵S]methionine followed by extraction and immunopurification in the presence of various protease inhibitors yielded one major radioactive band of 50 kDa molecular mass. Pulse labeling with [³⁵S]methionine at various times after wounding indicated that the wound-induced increase in ACC-synthase activity involved de-novo synthesis of a rapidly turning over 50-kDa polypeptide.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - ELISA enzyme-linked immunosorbent assay - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Regulation of Auxin-induced Ethylene Biosynthesis. Repression of Inductive Formation of 1-Aminocyclopropane-1-carboxylate Synthase by Ethylene

Changes in the 1-aminocyclopropane-1-carboxylate (ACC) synthaseactivity which regulates auxin-induced ethylene production werestudied in etiolated mung bean hypocotyl segments. Increasesboth in ethylene production and ACC synthase activity in tissuetreated with IAA and BA were severely inhibited by cycloheximide(CHI), 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide,actinomycin D and -amanitin. Aminoethoxyvinylglycine (AVG),a potent inhibitor of the ACC synthase reaction, increased theactivity of the enzyme in the tissue 3- to 4-fold. This stimulationalso was severely inhibited by the above inhibitors. Stimulationof the increase in the enzyme content by AVG was partially suppressedby an exogenous supply of ACC or ethylene. Suppression of theincrease in the enzyme took place with 0.3 µl/liter ethylene,and inhibition was increased to 10 µl/liter, which caused65% suppression. Air-flow incubation of the AVG-treated tissue,which greatly decreased the ethylene concentration surroundingthe tissue, further increased the amount of enzyme. Thus, oneeffect of AVG is to decrease the ethylene concentration insidethe tissue. The apparent half life of ACC synthase activity,measured by the administration of CHI, was estimated as about25 min. AVG lengthened the half life of the activity about 2-fold.Feedback repression by ethylene in the biosynthetic pathwayof auxin-induced ethylene is discussed in relation to the effectof AVG. (Received January 22, 1982; Accepted March 26, 1982)     

Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits

Biggs, M. S., Woodson, W. R. and Handa, A. K. 1988. Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits. Physiol. Plant. 72: 572578
Incubation of fruits of tomato ( Lycopersicon esculentum Mill. cv. Rutgers) at 34°C or above resulted in a marked decrease in ripening-associated ethylene production. High temperature inhibition of ethylene biosynthesis was not associated with permanent tissue damage, since ethylene production recovered following transfer of fruits to a permissive temperature. Determination of pericarp enzyme activities involved in ethylene biosynthesis following transfer of fruits from 25°C to 35 or 40°C revealed that 1-aminocyclopropane-l-carboxylic acid (ACC) synthase (EC 4.4.1.14) activity declined rapidly while ethylene forming enzyme (EFE) activity declined slowly. Removal of high temperature stress resulted in more rapid recovery of ACC synthase activity relative to EFE activity. Levels of ACC in pericarp tissue reflected the activity of ACC synthase before, during, and after heat stress. Recovery of ethylene production following transfer of pericarp discs from high to permissive temperature was inhibited in the presence of cycloheximide, indicating the necessity for protein synthesis. Ethylene production by wounded tomato pericarp tissue was not as inhibited by high temperature as ripening-associated ethylene production by whole fruits.     

Turnover of 1-aminocyclopropane-1-carboxylic Acid synthase protein in wounded tomato fruit tissue

Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [³⁵S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins.     

Radiolabeling of a wound-inducible pyridoxal phosphate-utilizing enzyme: evidence for its identification as ACC synthase

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase, a pyridoxal phosphate-utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate-limiting step in the biosynthesis of the plant hormone ethylene. We report the partial purification (400-fold) of ACC synthase from wounded pink tomato pericarp. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase-enriched fraction was achieved by reduction using tritiated sodium borohydride. Evidence that this radiolabeled protein is ACC synthase is presented.