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 Auxin-Induced Ethylene Production by Lycoricidinol

Lycoricidinol, a natural growth inhibitor isolated from bulbsof Lycoris radiata Herb. strongly suppressed auxin-induced ethyleneproduction from the hypocotyl segments of etiolated mung bean(Vigna radiata Wilczek) seedlings. The inhibitor did not significantlyinhibit ethylene formation from its immediate precursor, 1-aminocyclopropane-1-carboxylicacid (ACG), during short-term (up to 4 h) incubation. The ACCcontent in tissue treated with IAA was reduced by lycoricidinolin close parallel with the inhibition of ethylene production.Examination of radioactive metabolites in tissues labeled with3,4-¹⁴C-methionine indicated that reduction of the ACC contentwas not due to any possible promotive effect of lycoricidinolon conjugation of ACC with malonate. Lycoricidinol showed noinhibitory effect on the activity of ACC synthase if appliedin vitro, but it almost completely abolished the increase inthe enzyme activity when applied in vivo during incubation ofthe tissue with IAA. Lycoricidinol also strongly inhibited incorporationof ¹⁴C-leucine into protein in the tissue. The suppression ofthe enzyme induction and, in turn, that, of ethylene productionby lycoricidinol were interpreted as being due to the inhibitionof protein synthesis. (Received September 30, 1983; Accepted December 8, 1983)     

The Synthesis of Ethylene in Melon Fruit during the Early Stage of Ripening

The levels of mRNA and polypeptide for a 1-aminocyclopropane-1-carboxylate(ACC) oxidase were studied to identify the tissues in whichthe synthesis of ethylene first occurs during the initial stageof ripening. RNA and immunoblot analysis showed that the levelsof the mRNA and polypeptide for ACC oxidase were very low inunripe fruit. They first became detectable in the placentaltissue at the pre-climacteric stage, and then their levels increasedin the mesocarp tissue during the climacteric increase in theproduction of ethylene. Two mRNAs for ACC synthase (transcribedfrom ME-ACS1 and ME-ACS2) were detected in the placental tissueand seeds at the pre-climacteric stage, but only the level ofME-ACS1 mRNA, which has been characterized as the mRNA for awound-inducible ACC synthase, increased in mesocarp, placentaltissues and seeds during ripening. The level of ME-ACS2 mRNAthat was isolated from etiolated seedlings of melon, did notchange markedly during ripening. These results suggest thatthe central region of melon fruit (placental tissue and seeds)plays a major role in the production of ethylene during theearly stage of ripening. ³These three authors made equal contribution to this study.     

Induction of chitinase and {beta}-1,3-glucanase activity in sunflower suspension cells in response to an elicitor from Phytophthora megasperma f. sp. glycinea (Pmg). Evidence for regulation by ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC)

In heterotrophic cell suspensions of sunflower (Helianthus annuusL. cv. Spanners Allzweck) the effect of Pmg elicitor, a fungalelicitor preparation from Phytophthora megasperma f. sp. glycinea,on the induction of chitinase and ß-1,3-glucanaseactivity was studied in relation to changes in ethylene biosynthesis.Dose-response experiments with Pmg elicitor showed that theonset of the induction of intracellular chitinase and ß-1,3-glucanaseactivity coincided or followed a transient rise in ethyleneand particularly endogenous 1-aminocyclopropane-1-carboxylicacid (ACC) levels within 5 h of application. Treatment with5 µg ml^–1 elicitor stimulated ethylene and ACC levels1.6-fold and 4-fold, relative to control, respectively. Themolar ratio of ACC to ethylene changed from approximately 3:1in controls to 9:1 in treated cells. During further incubation,ethylene formation and, to a lesser degree, ACC levels declinedand the ACC/ethylene ratio increased to 56:1 in elicitor-treatedcells. On a protein basis, the activities of ß-1,3-glucanaseand chitinase increased approximately 5-fold and 8-fold, respectively,48 h after elicitor application. Additional treatment with theACC synthesis inhibitor aminoethoxyvinyiglycine (AVG) decreasedelicitor-induced enzyme activities and the levels of both ethyleneand ACC. Elicitor effects on chitinase and ß-1,3-glucanaseactivities could be fully restored when ACC was additionallyapplied. Concomitantly, the ACC/ ethylene ratio increased. Neithertreatments with ACC alone, which simultaneously increased internalACC and ethylene levels, nor treatments with AVG alone, whichsimultaneously reduced ACC and ethylene levels, could generallystimulate chitinase or ß-1,3-glucanase activitiesin the cells. It is suggested that ACC functions as a promotingfactor in the induction of chitinase and ß-1,3-glucanaseactivity triggered by Pmg elicitor and appears to reverse aninhibiting influence of ethylene. Key words: 1-Aminocyclopropane-1-carboxylic acid, chitinase, ß-1,3-glucanase, ethylene, Helianthus cellsuspension cultures, Phytophthora megasperma-elicitor     

Inactivation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase,which catalyses the final step in the biosynthesis of ethylene,showed a non-linear time-course in vitro and activity decayedwith a half-life of around 14 min. This loss of activity wasstudied using tomato ACC oxidase purified from Escherichia coiltransformed with the cDNA clone pTOM13. Inactivation was notdue to end-product inhibition by dehydroascorbic acid or cyanide.Preincubatlon of enzyme in the combined presence of Fe²⁺ ascorbateand ACC, which together allowed catalytic turnover, resultedin almost total loss of ACC oxidase activity. Enzyme Inactivatedby catalysis could not be reactivated by passage through SephadexG-25 or by treating with combina tions of DTT and CO₂ A non-lineartime-course and inactivation in the presence of all substratesand cofactors was also shown for the enzyme assayed in vivowith melon fruit discs. Using the purified tomato enzyme a distinctascorbate-dependent inactivation was also observed, which occurredIn the absence of catalysis and was prevented, although notreversed, by catalase. This ascorbate-dependent inactivationmay thus be due to H₂O₂ attack on ACC oxidase. Key words: 1-aminocyclopropane-1-carboxylate (ACC) oxidase, catalase, catalytic inactivation, ethylene     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Development of Ethylene Biosynthesis in Pear Fruits at --1 {degrees}C

Knee, M. 1987. Development of ethylene biosynthesis in pearfruits at — 1 °C.—J. exp. Bot. 38: 1724–1733. The regulation of ethylene synthesis in pear fruits was investigated.During storage for 60 d at — 1 °C the rate of ethylenesynthesis increased 100-fold but the concentration of 1-aminocyclopropane-l-carboxylicacid (ACC) increased only 2-fold and ACC synthase activity waslow. On transfer to 15 °C after storage at — 1 °Cethylene synthesis increased 10-fold within 10 h but ACC synthaseactivity only increased rapidly after 24 h; the decline in ACClevels during the first 16 h at 15 °C was insufficient tosustain ethylene synthesis. Ethylene synthesis was further investigatedusing discs cut from the mid cortex of pear fruits. Synthesiswas inhibited by aminoethoxyvinylglycine (AVG) and amino-oxyaceticacid at all stages of ripening. The rate of synthesis and ACCsynthase activity increased rapidly after slicing of pears heldat — 1 °C but more slowly in discs cut from pearsimmediately after harvest. Cycloheximide (CHI) inhibited theseincreases and reversed increases resulting from pre-incubationof discs. A combination of CHI and AVG abolished the capacityof discs to synthesize ACC and ethylene production was curtailed.Cordycepin and actinomycin-D were less effective as inhibitorsof the development of ethylene synthesis and ACC synthase activitythan as inhibitors of incorporation of 5-[³H] uridine into totalRNA or poly A rich RNA. The ability of discs to develop ethylenesynthesis and ACC synthase activity in the presence and absenceof cordycepin increased concurrently during storage of wholefruits at — 1 °C. This suggested that mRNA for ACCsynthase was formed at — 1 °C. Key words: 1-Aminocyclopropane-l-carboxylic acid, ethylene, fruit ripening, Pyrus communis L. (fruit ripening)     

Changes of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Activity in Stressed Pinus Sylvestris Needles

Stimulation of ethylene biosynthesis in pine needles by hydrogen peroxide and sodium bisulfite coincided with the activation of ACC oxidase at the level of protein synthesis. Decrease in ethylene production at high concentrations of sodium bisulfite (above 7 mM) was apparently due to inhibition of ACC oxidase activity. Treatment of pine needles with aminotriazole caused an inhibition of both ethylene production and ACC oxidase activity. Both methylviologen and methyl jasmonate stimulated ACC oxidase activity in a concentration-dependent manner with no parallel changes in ethylene production. The presented results suggest that ACC oxidase plays an important role in regulation of ethylene formation in pine needles in response to different stimuli.     

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: II. GRADIENT-PURIFIED MITOCHONDRIA LACK CYANIDE-INSENSITIVE RESPIRATION

Wheat leaves normally produced very little ethylene, but following a water deficit stress which caused a loss of 9% initial fresh weight, ethylene production increased more than 30-fold within 4 hours and declined rapidly thereafter. The changes in ethylene production were paralleled by an increase and subsequent decrease in 1-aminocyclopropanecarboxylic acid (ACC) content. The level of S-adenosylmethionine was unaffected, suggesting that the conversion of S-adenosylmethionine to ACC is a key reaction in the production of water stress-induced ethylene. This view was further supported by the observation that application of ACC to nonstressed leaf tissue caused a 70-fold increase in ethylene production, while aminoethoxyvinylglycine, a known inhibitor of the conversion of S-adenosylmethionine to ACC, inhibited ACC accumulation as well as the surge in ethylene production if the inhibitor was applied prior to the stress treatment. Cycloheximide, an inhibitor of protein synthesis, effectively blocked both ethylene production and ACC formation, suggesting that water stress induces de novo synthesis of ACC synthase, which is the rate-controlling enzyme in the pathway of ethylene biosynthesis.     

Exogenous Polyamines Stimulate Ethylene Synthesis by Soybean Leaf Tissues

Exogenous supply of spermine (Spm) markedly stimulated ethyleneevolution from intact soybean leaves of leaf discs, stronglyincreased the level of free 1-aminocyclopropane-1-carboxylicacid (ACC), and slightly stimulated ethylene forming-enzyme(EFE) activity Spm treatment also resulted in leaf epinastyand accelerated leaf senescence Ethylene stimulation was depressed,but not abolished, by light, and was suppressed by inhibitorsof ACC synthase and EFE activity Spermidine had a less pronouncedstimulatory effect on ethylene production whereas the diaminesputrescine and diaminopropane were without effect These resultscontrast with other reports indicating that di- and polyaminesinhibit ethylene biosynthesis in plants, and extend our previousresults on detached tobacco leaves exogenously treated withpolyamines Glycine max, ethylene, polyamines     

The Role of 1-Aminocyclopropane-1-carboxylic acid in the Control of Low Temperature Induced Ethylene Production in Leaf Tissue of Phaseolus vulgaris L .

Ethylene production from leaf discs of dwarf bean (Phaseolausvulgaris L.) was less than 02 nl g^–1 h^–1 at 5 Cbut rapidly increased tenfold on transfer to 25 C. The lowethylene production at 5 C and the potential for overshootproduction on transfer to 25C were not associated with accumulationof the ethylene synthesis intermediate 1-aminocyclopropane-1-carboxylicacid (ACC). Addition of exogenous ACC to leaf discs incubatedat 5C increased ethylene production, while similarly incubatedleaf discs did not synthesize increasing amounts of endogenousACC until they were transferred to 25 C. The basis for theovershoot in ethylene production when leafdiscs were transferredfrom 5 to 25 C appears to reside in changes to the pathwayleading to the synthesis of ACC or an earlier intermediate inthe pathway of ethylene biosynthesis. Ethylene, 1-aminocyclopropane-l-carboxylic acid, Phuseolru vulgaris L., dwarf bean, temperature     

Ethylene biosynthesis in Amaranthus caudatus seeds in response to methyl jasmonate

Methyl jasmonate (JA-Me) at 10^–3 M completely inhibited Amaranthus caudatus seed germination. Exogenous ethylene could totally reverse this inhibition. The inhibitor of ethylene action, 2,5-norbornadiene (NBD), increased the sensitivity of seeds to JA-Me. Methyl jasmonate inhibited ethylene production and also decreased both 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl ACC (MACC) content. Likewise, ACC oxidase activity in vivo was decreased by jasmonate. Similarly ACC oxidase activity in vitro isolated from seeds incubated in the presence of JA-Me was lower than that isolated from untreated seeds.The inhibitory JA-Me action on seed germination seems to be mainly associated with the inhibition of ethylene biosynthesis. Both inhibition of ACC synthase and ACC oxidase activity and/or synthesis can be involved.     

Differential Expression of Genes Involved in the Biosynthesis and Perception of Ethylene during Ripening of Passion Fruit (Passiflora edulis Sims)

An increase in the enzyme activity of 1-aminocyclopropane-1-carboxylicacid (ACC) synthase and ACC oxidase induces the evolution ofethylene during the ripening of passion fruit. A much higherlevel of ethylene is produced in arils than in seeds or peelsduring ripening. The pattern of expression of two ACC synthasegenes (PE-ACS1 and PE-ACS2), one ACC oxidase gene (PE-ACO1),and two ethylene receptor genes (PE-ETR1 and PE-ERS1) revealedthat the expression of these genes is differentially regulated.Expression of PE-ACS1 and PE-ACO1 was enhanced during ripeningand after ethylene treatment. However, prominent expressionof PE-ACS1 was delayed compared to that of PE-ACO1. Much largerquantities of PE-ACS1 mRNA and PE-ACO1 mRNA were seen in arilsthan in seeds; this corresponds well with an increase in theamount of ethylene produced by the plant tissue itself. Thelevel of PE-ACS2 mRNA was detectable in arils of the preclimactericfruit, although it decreased during ripening. These resultssuggest that expression of PE-ACS1 and PE-ACO1 is required toincrease the activity of ethylene biosynthetic enzymes duringripening. The level of expression of PE-ETR1 and PE-ERS1 didnot significantly change over the course of ripening; however,the mRNA levels of PE-ETR1 and PE-ERS1 were much higher in arilsthan in seeds. ⁴Present address: Center forMolecular Genetics Research, Shizuoka University, Shizuoka, 422-8529 Japan.     

No difference in the regulation pattern of calcium on ethylene biosynthesis between wild-type and never-ripe tomato fruit at mature green stage

This work investigated how calcium regulates the ethylene biosynthesis in the fruits of wild-type tomato (Lycopersicon esculentum L.) and their ethylene receptor never-ripe (Nr) mutants. In Nr tomato, the ethylene perception was blocked. When both materials were treated with calcium, the content of 1-aminocyclopropane-1-carboxylic acid (ACC)/malonyl-ACC and the activity of ACC oxidase (ACO) in tomato fruit discs increased, whereas the production of ethylene, content of malondialdehyde, and membrane permeability decreased. Calcium treatment did not affect the activity of ACC synthase, which is the first committed step in the ethylene biosynthesis pathway. The expression of LeACO1 in mature green fruit was inhibited significantly by calcium treatment in wild-type and Nr tomatoes, but the expression of LeACS2, the key ACC synthase gene in ethylene synthesis during tomato fruit maturing, was not affected. These results revealed that the effect of calcium on ethylene biosynthesis in tomato mature green fruit was independent of ethylene perception. The results also revealed that the targeting step of calcium preventing ethylene production was located at the ACC conversion to ethylene, by means of inhibiting ACC availability for ACO through enhancing cell membrane integrity and by means of preventing LeACO1 gene expression. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 60–67. The text was submitted by the authors in English.