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Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl3, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.

Bean leaves from Phaseolus vulgaris L. var. Pinto 111 react to mechanical wounding with the formation of ethylene. The substrate for wound ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC). It is not set free by decompartmentation but is newly synthesized. ACC synthesis starts 8 to 10 min after wounding at 28°C, and 15 to 20 min after wounding at 20°C. Aminoethoxyvinylglycine (AVG), a potent inhibitor of ethylene formation from methionine via ACC, inhibits wound ethylene synthesis by about 95% when applied directly after wounding (incubations at 20°C). AVG also inhibits the accumulation of ACC in wounded tissue. AVG does not inhibit conversion of ACC to ethylene. Wound ethylene production is also inhibited by cycloheximide, n-propyl gallate, and ethylenediaminetetraacetic acid.

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Cell wail preparations (elicitors) from Phytophthora megasperma var. sojae increase C2H4 formation, phenylalanine ammonia lyase activity, and glyceollin accumulation in soybean cotyledons within about 1.5, 3, and 6 hours after treatment, respectively. The immediate precursor of C2H4, 1-aminocyclopropane-1-carboxylic acid, stimulates C2H4 formation like the elicitor within 1.5 hours after administration, whereas phenylalanine ammonia lyase activity and glyceollin concentration remain unchanged. Aminoethoxyvinylglycine, a specific inhibitor of C2H4 formation in higher plants, inhibits elicitor-induced C2H4 formation by about 95% but has no effects on phenylalanine ammonia lyase or glyceollin accumulation. It was concluded that C2H4 is a signal accompanying the specific recognition process which finally leads to the induction of phytoalexin formation, but it is not functioning as a link or messenger in the induction sequence of glyceollin accumulation.

Several in vitro model systems have been described in which ethylene is formed from methionine through a reaction involving free radicals (6, 9, 13). In our attempts to elucidate the pathway of ethylene biosynthesis, we investigated whether compounds which are known to quench free radicals inhibited ethylene generation in plant tissues. Among the substances tested were selenoamino acids which have been reported to act as protectants against freeradical attack (12). We found that selenomethionine and selenoethionine greatly enhanced ethylene formation in senescing flower tissue and in pea stem sections treated with IAA. Our results indicate that in these tissues both selenoamino acids are better precursors of ethylene than is methionine. MATERIALS AND METHODSPlant Material. Morning glory plants (Ipomoea tricolor Cav., cv. Heavenly Blue) were grown as described before (2). Rib segments were prepared from flower buds according to Kende and Hanson (5). The following designations are used to describe the age of the flower tissue: day 0, day of opening and fading of the flower; day -1, 1 day before flower opening; day -2, 2 days before flower opening.Pea seeds, Pisum sativum L., cv. Alaska (Vaughan's Seed Co., Downers Grove, Ill.), were imbibed for 5 hr or overnight in aerated tap water. They were sown in Vermiculite and grown in the dark at 25 C for 6 to 9 days. Pea stem sections were isolated between 4:00 and 6:00 PM, and batches of 12 sections were floated in 6-cm Petri dishes on 2 ml of distilled H20 or a solution of the compound to be tested. The dishes were kept overnight in the dark at 25 C. The stem sections were removed from the Petri dishes between 8:00 and 9:00 AM the next morning. They were rinsed with distilled H20, blotted dry, and transferred to 25-ml Erlenmeyer flasks containing 1 ml of distilled H20 or the appropriate test solution with or without 0.1 mM IAA. Each flask was flushed with ethylene-free air for 2 min, closed with a serum-vial cap and incubated in darkness at 27 C.The pH of all test solutions was adjusted with 0.1 N NaOH to 6.5 to 7.0. Each experiment was repeated at least three times with similar results.Determination of Ethylene Formation. Ethylene production was measured by gas chromatography as described earlier (5).Determination of Specific Radioactivity of Ethylene. The specific radioactivity ofethylene was determined according to Hanson and Kende (3) RESULTS Effect of Selenoamino Acids on Ethylene Synthesis in FlowerTissue. When rib segments excised from flower buds of I. tricolor were incubated continuously from the afternoon of day -I through the evening of day 0 on solutions containing selenomethionine, ethylene production on day 0 commenced earlier and proceeded at a higher rate than in the control tissue (Fig. 1). The total amount of ethylene produced on day 0 by rib segments treated with selenomethionine was usually three times higher than that produced by rib segments incubated on water. This result was surprising since methionine, the precursor of ethylene in mor...