Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit

We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green `Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO₂ evolution by 400 micrograms per gram fresh weight galactose. 1-Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol.     

Glucose Inhibits ACC Oxidase Activity and Ethylene Biosynthesis in Ripening Tomato Fruit

Experiments were carried out to evaluate the effect of glucose on ripening and ethylene biosynthesis in tomato fruit (Lycopersicon esculentum Mill.). Fruit at the light-red stage were vacuum infiltrated with glucose solutions post-harvest and changes in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, ACC oxidase, and ethylene production monitored over time. ACC oxidase activity was also measured in pericarp discs from the same fruits that were treated either with glucose, fructose, mannose, or galactose. While control fruit displayed a typical peak of ethylene production, fruit treated with glucose did not. Glucose appeared to exert its effect on ethylene biosynthesis by suppressing ACC oxidase activity. Fructose, mannose, and galactose did not inhibit ACC oxidase activity in tomato pericarp discs. Glucose treatment inhibited ripening-associated colour development in whole fruit. The extent of inhibition of colour development was dependent upon the concentration of glucose. These results indicate that glucose may play an important role in ethylene-associated regulation of fruit ripening.     

Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO₂ and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.     

Effects of aminoethoxyvinylglycine and countereffects of ethylene on ripening of bartlett pear fruits

Pear fruits (Pyrus communis L. var. Bartlett) were treated with solutions containing aminoethoxyvinylglycine (AVG) using a modified vacuum infiltration method that introduced 4.3 milliliters solution per 100 grams tissue. At concentrations of 1 millimolar, AVG strongly inhibited ethylene production and delayed for 5 days the respiratory climacteric and accompanying ripening changes in skin color and flesh firmness. AVG was less effective in inhibiting the ripening of more mature fruits. Fruit infiltrated with 5 millimolar AVG had not begun to ripen 12 days after initiation of ripening in the controls. When treated with ethylene the inhibited fruit exhibited a climacteric rise in respiration, softened, and became yellow. Treatment of the AVG infiltrated fruits with ethyelne for 24 hours resulted in no recovery in endogenous ethylene production, but in a stimulation of protein synthesis measured as a 200% increase in leucine incorporation by excised tissue and a 74% increase in the percentage of ribosomes present as polysomes.     

Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : II. EFFECT OF ETHYLENE

The effect of ethylene on cell wall metabolism in sections excised from etiolated pea stems was studied. Ethylene causes an inhibition of elongation and a pronounced radial expansion of pea internodes as shown by an increase in the fresh weight of excised, 1-cm sections. Cell wall metabolism was studied using centrifugation to remove the cell wall solution from sections. The principal neutral sugars in the cell wall solution extracted with H₂O are arabinose, xylose, galactose, and glucose. Both xylose and glucose decline relative to controls in air within 1 hour of exposure to ethylene. Arabinose and galactose levels are not altered by ethylene until 8 hours of treatment, whereupon they decline in controls in air relative to ethylene treatment. When alcohol-insoluble polymers are fractionated into neutral and acidic polysaccharides, xylose and glucose predominate in the neutral fraction and arabinose and galactose in the acidic fraction. Ethylene depresses the levels of xylose and glucose in the neutral fraction and elevates arabinose and galactose in the acidic fraction. Ethylene treatment does not affect the level of uronic acids extracted with H₂O; however, the level of hydroxyproline-rich proteins in this water-extracted cell wall solution is increased by ethylene. Extraction of sections with CaCl₂ results in an increase in the levels of neutral sugars particularly arabinose. Ethylene depresses the yield of arabinose in calcium-extracted solution relative to controls in air. Similarly, extraction with CaCl₂ increases the yield of extracted hydroxyproline in ethanol-insoluble polymers and ethylene depresses its level relative to controls. Metabolism of uronic acids and neutral sugars and growth in response to ethylene treatment contrast markedly with auxin-induced polysaccharide metabolism and growth. With auxin, sections increase mostly in length not radius, and this growth form is associated with an increase in the levels of xylose, glucose, and uronic acids. With ethylene, on the other hand, stem elongation is suppressed and expansion is promoted, and this growth pattern is associated with a decrease in xylose and glucose in the ethanol-insoluble polysaccharides.     

Use of a laser-driven photoacoustic detection system for measurement of ethylene production in cymbidium flowers

A laser-based photoacoustic method was used for determination of ethylene (C₂H₄) production of emasculated orchid (Cymbidium) flowers in a flow-through system. The laser photoacoustic equipment consisted of a line-tuneable CO₂ laser in conjunction with a single-pass resonant acoustic cell. The minimum detection limit of the system for C₂H₄ in air was 0.03 nanoliter per liter. C₂H₄ production of intact Cymbidium (cv Mary Pinchess `Del Rey') flowers was very low (0.015 nanoliter per gram per hour) and showed an increase within 3 hours following emasculation (removal of pollinia plus anthercap). Production peaked (0.14 nanoliter per gram per hour) 8 hours after emasculation and decreased thereafter. Production again increased 45 hours after emasculation. Coloration of the labellum appeared shortly after the first peak; wilting of the petals and sepals appeared during the second rise in ethylene production. The use of the laser photoacoustic technique in plant physiological studies is discussed.     

Ethylene production by auxin-deprived, suspension-cultured pear fruit cells in response to auxins, stress, or precursor

Auxin-deprived, mannitol-supplemented, suspension-cultured pear (Pyrus communis L. Passe Crassane) fruit cells produce large quantities (20-40 nanoliters ethylene per 10⁶ cells per hour) of ethylene in response to auxins, CuCl₂ or 1-amino-cyclopropane-1-carboxylic acid (ACC). Maximum rates of production are achieved about 12 hours after the addition of optimal amounts of indoleacetic acid (IAA), naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 4 to 5 hours after the addition of CuCl₂ and 1 to 2 hours after the addition of ACC. Supraoptimal concentrations of IAA result in a lag phase followed by a normal response. High concentrations of NAA and 2,4-D result in an early (4-5 hours) stress response and injury.

Continuous protein and RNA synthesis are essential for elaboration of the full IAA response; only protein synthesis is necessary for the response to CuCl₂ and ACC. Based on polysomal states and rates of amino acid incorporation, CuCl₂ partially inhibits protein synthesis while nonetheless stimulating ethylene production. In general, ethylene production by the pear cells resembles that of other plant systems. Some differences may reflect the sensitivity of the cells and are discussed. The relatively high levels of ethylene produced and the experimental convenience of the cultured cells should make them especially suitable for further investigations of ethylene production and physiology.

     

Effect of low oxygen and high carbon dioxide on the levels of ethylene and 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

The association of the level of ACC and the ethylene concentration in ripening apple fruit (Malus sylvestris Mill, var. Ben Davis) was studied. Preclimacteric apple contained small amounts of ACC and ethylene. With the onset of the climacteric and a concomitant decrease in flesh firmness, the level of ACC and ethylene concentration both increased markedly. During the postclimacteric period, ethylene concentration started to decline, but the level of ACC continued to increase. Ethylene production and loss of flesh firmness of fruits during ripening were greatly suppressed by treatments with low O₂ (O₂ 1–3%, CO₂ O%) or high CO₂ (CO₂ 20–30%, O₂ 15–20%) at the preclimacteric stage. However, after 4 weeks an accumulation of ACC was observed in treated fruits when control fruit was at the postclimacteric stage. Treatment of fruit with either low O₂ or high CO₂ at the climacteric stage resulted in a decrease of ethylene production. However, the ACC level in fruit treated with low O₂ was much higher than both control and high CO₂ treated fruit; it appears that low O₂ inhibits only the conversion of ACC to ethylene, resulting in an accumulation of ACC. Since CO₂ inhibits ethylene production but does not result in an accumulation of ACC, it appears that high CO₂ inhibits both the conversion of ACC to ethylene and the formation of ACC.     

STUDIES IN THE PHYSIOLOGY OF FUSARIA. THE RESPIRATORY AND FERMENTATIVE MECHANISMS

1.Fusarium tricothecoides was selected for a study of the respiratory and fermentative activities of Fusaria. "Resting cell" suspensions were investigated by the Barcroft manometric technique. 2. The results of the investigation indicate clearly that the mechanism of endogenous metabolism (respiration) is distinct from the exogenous mechanism (fermentation). Anaerobically no significant CO₂ production is apparent without added substrate. In the presence of glucose the anaerobic CO₂ evolution is practically equal to the added CO₂ evolved aerobically in the presence of added glucose. Low concentrations of iodoacetate or fluoride selectively poison the exogenous mechanism but do not affect the endogenous mechanism. Alcohol is not produced in the course of endogenous metabolism, but is produced in the presence of added glucose. 3. A study of the metabolism of the organism throughout its entire growth phase from 1 to 7 days has been made. 4. The ability of suspensions of Fusarium sp. H., obtained by growth on a variety of common substrates, to attack a large number of carbon sources with the production of exogenous CO₂ was determined. It is found that organisms grown on glucose will attack only glucose, mannose, and fructose, but none of the common intermediary metabolites except pyruvic acid. Organisms grown on galactose attack galactose, as well as the other hexoses, indicating an adaptive mechanism. 5. An identical mechanism for the dissimilation of glucose, mannose, and galactose is indicated since no additive effects with these substrates were observed. Growths on non-hexose carbon sources attack glucose slightly under the experimental conditions with the evolution of CO₂, but do not attack any other substrate. This would indicate a residual glucose-dissimilating mechanism in all growths investigated. 6. Striking similarities between the general metabolism of resting suspensions of Fusarium sp. H. and resting suspensions of yeast cells are apparent.     

Galactose inhibits the conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in aged tobacco leaf discs

d-Galactose has been shown to have toxic and growth inhibitory effects in plants. When applied at levels of 50 millimolar to tobacco (Nicotiana tabacum L. cv Xanthi) leaf discs galactose caused a rapid increase in ethylene production during the first 2 days of incubation, followed by a rapid return to the basal level on the third day. This pattern of galactose-stimulated ethylene production was accompanied by increased formation of 1-aminocyclopropane-1-carboxylic acid (ACC), which accumulated without being metabolized to ethylene or to the ACC-conjugate. The inhibitory effect of galactose (50 millimolar) on the conversion of ACC of ethylene was relieved partially by d-glucose or sucrose (50 millimolar), and completely by CO₂ (10%), which were shown to enhance this conversion by themselves. Consequently, application of galactose plus any one of these compounds increased ethylene production and decreased free ACC levels. The data suggest that galactose toxicity may result in both an increased ethylene production as well as in accumulation of free ACC in aged discs. The increased ethylene production rates and ACC levels may, in turn, play a role in the development of symptoms associated with galactose toxicity.     

Deferral of senescence and abscission by chemical inhibition of ethylene synthesis and action in bean explants

Three compounds known to inhibit ethylene synthesis and/or action were compared for their ability to delay senescence and abscission of bean explants (Phaseolus vulgaris L. cv Contender). Aminoethoxyvinyl-glycine (AVG), AgNO₃, and sodium benzoate were infiltrated into the petiole explants. Their effect on abscission was monitored by measuring the force required to break the abscission zone, and their effect on senescence was followed by measuring chlorophyll and soluble protein in the distal (pulvinus) sections. AVG at concentrations between 1 and 100 micromolar inhibited ethylene synthesis by about 80 to 90% compared to the control during sampling periods of 24 and 48 hours after treatment. This compound also delayed the development of abscission and senescence. Treatment with AgNO₃ at concentrations between 1 and 100 micromolar progressively reduced ethylene production, but to a lesser extent than AVG. The effects of AgNO₃ on senescence and abscission were quite similar to those of AVG. Sodium benzoate at 50 micromolar to 5 millimolar did not inhibit ethylene synthesis during the first 24 hours, but appreciably inhibited ethylene synthesis 48 hours after treatment. It also delayed the development of abscission and senescence. The effects of AVG, Ag⁺, and sodium benzoate suggest that ethylene could play a major role in both the senescence induction phase and the separation phase in bean explants.     

Ethylene and carbon dioxide production by developing strawberries show a correlative pattern that is indicative of ripening climacteric fruit

Laser photoacoustic spectroscopy continuously quantified the ethylene (C₂H₄) produced by strawberry flowers and fruits developing in planta. C₂H₄ was first detected as flower buds opened and exhibited diurnal oscillations (to approximately 200 pl flower^?1 h^?1) before petal abscission. Exogenous application of silver thiosulphate (STS) to detached flowers inhibited petal abscission and flower senescence. In fruit, C₂H₄ production was maintained at a ‘low level’ (10–60 pl fruit^?1 h^?1) until fruit expanded when levels increased in a diurnal pattern (to 200 pl fruit^?1 h^?1). After expansion, C₂H₄ production declined to a low level until fruit attained the red‐ripe stage for at least 24 h. After this time, C₂H₄ levels increased linearly (no diurnal fluctuation) to approximately 1 nL fruit^?1 h^?1. Twenty‐four hours after the re‐initiation of C₂H₄ production by red fruit, CO₂ levels increased approximately three‐fold, indicative of a respiratory climacteric. STS applied to fruits developing in planta and dissected fruit parts ex situ established that C₂H₄ production is regulated by negative feedback until fruits had expanded. The C₂H₄ produced by red‐ripe fruit was regulated by positive feedback. Anti‐1‐amino‐cyclopropane‐1‐carboxylic acid oxidase IgG localization identified immunoreactive antigens of 40 and 30 kDa (M_r) within the fruit achenes of expanding and red‐ripe fruit. Analysis of dissected fruit showed that seed C₂H₄ accounts for 50% the C₂H₄ that is detectable from ripe fruit.     

Mannosyl- and Xylosyl-Containing Glycans Promote Tomato (Lycopersicon esculentum Mill.) Fruit Ripening

The oligosaccharide glycans mannosylα1-6(mannosylα1-3)mannosylα1-6(mannosylα1-3) mannosylβ1-4-N-acetylglucosamine and mannosylα1-6(mannosylα1-3)(xylosylβ1-2) mannosylβ1-4-N-acetylglucosaminyl(fucosylα1-3) N-acetylglucosamine were infiltrated into mature green tomato fruit (Lycopersicon esculentum Mill., cv Rutgers). Coinfiltration of 1 nanogram per gram fresh weight of the glycans with 40 micrograms per gram fresh weight galactose, a level of galactose insufficient to promote ripening, stimulated ripening as measured by red coloration and ethylene production.     

钙对香蕉采后乙烯释放的抑制

本实验用CaCl_2溶液对香蕉(Musa acuminata cf. 'Dwarf Davendish')组织进行真空浸透处理,研究Ca~(2 )对香蕉采后乙烯释放、EFE活性、ACC水平以及ACC/MACC比值的影响。结果表明,Ca~(2 )处理可抑制香蕉果皮和果肉组织乙烯生成,对抑制果皮的乙烯生成尤为明显。Ca~(2 )处理还可降低内源ACC水平,抑制EFE活性。结果还显示,Ca~(2 )处理对组织中ACC/MACC比值有一定影响。     

Ethylene-Induced Chlorosis in the Pathogenesis of Bipolaris sorokiniana Leaf Spot of Poa pratensis

Endogenous ethylene of Poa pratensis leaves infected by Bipolaris sorokiniana was evaluated as a factor in leaf chlorosis during pathogenesis. Detectable increases in endogenous ethylene of leaves of intact plants under normal ambient pressure occurred 12 hours after inoculation and was maximum at 48 hours; from 48 to 96 hours the ethylene progressively decreased. Necrotic lesions surrounded by chlorotic halos occurred on infected leaves between 24 and 48 hours. Midvein chlorosis interconnecting individual lesions and complete chlorosis of all tissues not directly affected by the lesions occurred between 72 and 96 hours, after maximum production of ethylene at 48 hours. The chlorophyll loss in infected leaves by 96 hours was 44% compared with controls.

Subjecting inoculated leaves of intact plants to a controlled atmospheric-environmental system with an atmospheric pressure of 233 millibars and O₂ and CO₂ partial pressures adjusted to approximately that of normal ambient pressure during infection and disease development prevented most midvein chlorosis and complete chlorosis, but did not prevent necrotic lesion or chlorotic halo development. Under the hypobaric conditions, chlorophyll loss during disease development was reduced to 22% compared with controls at 96 hours. The observations suggest that ethylene may function late in pathogenesis of this host-pathogen interaction and is responsible for much of the chlorophyll loss after its maximum production at 48 hours.

     

Effect of carbon dioxide and light on ethylene production in intact sunflower plants

High CO₂ concentration (0.5%) increased the rate of ethylene production, measured in a continuous flow system, in intact sunflower (Helianthus annuus L.) plants. However, the rate of ethylene production subsided to near control levels after approximately 24 hours. The effect of high CO₂ could only be observed in light. Although high CO₂ concentration had no effect on the rate of ethylene production in darkness, prolonged exposure (approximately 16 hours) of plants to high CO₂ in the dark prevented the increase in ethylene production when the plants were exposed to light and high CO₂.     

Involvement of wound and climacteric ethylene in ripening avocado discs

Avocado (Persea americana Mill. cv Hass) discs (3 mm thick) ripened in approximately 72 hours when maintained in a flow of moist air and resembled ripe fruit in texture and taste. Ethylene evolution by discs of early and midseason fruit was characterized by two distinct components, viz. wound ethylene, peaking at approximately 18 hours, and climacteric ethylene, rising to a peak at approximately 72 hours. A commensurate respiratory stimulation accompanied each ethylene peak. Aminoethoxyvinyl glycine (AVG) given consecutively, at once and at 24 hours following disc preparation, prevented wound and climacteric respiration peaks, virtually all ethylene production, and ripening. When AVG was administered for the first 24 hours only, respiratory stimulation and softening (ripening) were retarded by at least a day. When AVG was added solely after the first 24 hours, ripening proceeded as in untreated discs, although climacteric ethylene and respiration were diminished. Propylene given together with AVG led to ripening under all circumstances. 2,5-Norbornadiene given continuously stimulated wound ethylene production, and it inhibited climacteric ethylene evolution, the augmentation of ethylene-forming enzyme activity normally associated with climacteric ethylene, and ripening. 2,5-Norbornadiene given at 24 hours fully inhibited ripening. When intact fruit were pulsed with ethylene for 24 hours before discs were prepared therefrom, the respiration rate, ethylene-forming enzyme activity buildup, and rate of ethylene production were all subsequently enhanced. The evidence suggests that ethylene is involved in all phases of disc ripening. In this view, wound ethylene in discs accelerates events that normally take place over an extended period throughout the lag phase in intact fruit, and climacteric ethylene serves the same ripening function in discs and intact fruit alike.     

Induction by Electric Currents of Ethylene Biosynthesis in Cucumber (Cucumis sativus L.) Fruit

The effects of an electric current on ethylene biosynthesis were investigated in cucumber (Cucumis sativus L.) fruit that were producing almost no ethylene. Direct currents at 0.5 to 3.0 milliamperes induced much ethylene synthesis, with a rapid continuous increase in the rate, which reached a peak within 5 to 6 hours and then decreased. The rate of production was greater with a stronger current. Ethylene production was not observed after the use of a sine-wave alternating current (60 hertz) at 3 milliamperes, the magnitude at which a direct current had the greatest effect. The activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ethylene forming enzyme (EFE) increased before the rise in ethylene production. ACC synthase and EFE were activated sixfold and fourfold, respectively, by 2 hours. The concentration of ACC increased linearly up to 6 hours and then decreased. Ethylene induction by an electric current was suppressed almost completely by the infiltration of the cucumbers with 5 millimolar aminooxyacetic acid, an inhibitor of ACC synthase, and was also suppressed 70% by 5 millimolar salicylic acid, an inhibitor of EFE. The results indicate that the ethylene induced by the direct current was synthesized via the ACC-ethylene pathway as a result of electrical stress, a new kind of stress to be identified.     

Limitations in the Use of Cycloheximide in Studies of Apple Ripening

In order to discover whethor the production of aroma volatilesby apple fruits is dependent on the synthesis of appropriateenzymes during ripening, excised peel, excised cortical tissue,and whole apples were treated with cycloheximide (CH). Volatilerelease, ethylene production, respiration, flesh softening,and peel chlorophyll degradation were measured. The ethylene and volatile compounds produced by excised peelapparently resulted from wounding rather than processes analogousto fruit ripening. Excised cortical tissue was capable of autonomousripening with ethylene production, respiration, and softeningcomparable to that in intact fruits. After infiltration withsucrose solution the same changes occurred, but they were delayedby up to 4 d. Cycloheximide inhibited respiration although theextent of this inhibition decreased after 3 d. Cycloheximideprevented the onset of rapid ethylene production but stimulatedproduction of ethanol, ethyl acetate, and other volatiles. Softeningof CH-treated cortical discs was associated with progressivenecrosis. When whole apples were infiltrated with CH through hypodermicneedles inserted into the core, [¹⁴C]valine incorporation wasinhibited from the core to the mid-cortex but not in the peeland outer cortex. Infiltration with sucrose solution delayedmany ripening changes although the time of maximum [¹⁴C]valineincorporation was unaffected. Early effects of CH on respirationwere masked by the effects of infiltration, but after 5 d CH-infiltratedfruit contained higher CO₂ concentrations and respired morerapidly than controls. Internal ethylene concentrations wereusually lower in CH-treated apples than in controls. CH stimulated release of ethanol and ethyl acetate but inhibitedrelease of higher molecular weight esters such as propyl andbutyl acetates. Cycloheximide-treated fruit softened, but thiswas apparently due to internal necrosis. Peel chlorophyll degradationwas inhibited by CH treatment of whole apples although the tissuehad apparently received no inhibitor.     

Auxin-induced Ethylene Production and Its Inhibition by Aminoethyoxyvinylglycine and Cobalt Ion

Auxin is known to stimulate greatly both C₂H₄ production and the conversion of methionine to ethylene in vegetative tissues, while amino-ethoxyvinylglycine (AVG) or Co²⁺ ion effectively block these processes. To identify the step in the ethylene biosynthetic pathway at which indoleacetic acid (IAA) and AVG exert their effects, [3-¹⁴C]methionine was administered to IAA or IAA-plus-AVG-treated mung bean hypocotyls, and the conversion of methionine to S-adenosylmethionine (SAM), 1-amino-cyclopropane-1-carboxylic acid (ACC), and C₂H₄ was studied. The conversion of methionine to SAM was unaffected by treatment with IAA or IAA plus AVG, but active conversion of methionine to ACC was found only in tissues which were treated with IAA and which were actively producing ethylene. AVG treatment abolished both the conversion of methionine to ACC and ethylene production. These results suggest that in the ethylene biosynthetic pathway (methionine → SAM → ACC → C₂H₄) IAA stimulates C₂H₄ production by inducing the synthesis or activation of ACC synthase, which catalyzes the conversion of SAM to ACC. Indeed, ACC synthase activity was detected only in IAA-treated tissues and its activity was completely inhibited by AVG. This conclusion was supported by the observation that endogenous ACC accumulated after IAA treatment, and that this accumulation was completely eliminated by AVG treatment. The characteristics of Co²⁺ inhibition of IAA-dependent and ACC-dependent ethylene production were similar. The data indicate that Co²⁺ exerts its effect by inhibiting the conversion of ACC to ethylene. This conclusion was further supported by the observation that when Co²⁺ was administered to IAA-treated tissues, endogenous ACC accumulated while ethylene production declined.