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Lf U-14CjMethionine fed to apple tissue was efficiently converted to ethylene when the tissue was incubated in air. In nitrogen, however, it was not metabolized to ethylene but was instead converted to 1-aminocyclopropane-1-carboxylic acid (ACC). When apple tissues were fed with L[methylI4Cjmethionine or L{35S~methionine and incubated in nitrogen, radioactivity was found subsequently in methylthioribose. This suggests that methionine is first converted to S-adenosylmethionine which is in turn fragmented to ACC and methylthioadenosine. Methylthioadenosine is then hydrolyzed to methylthioribose. The conclusion that ACC is an intermediate in the conversion of methionine to ethylene is based on the following observations: Labeled ACC was efficiently converted to ethylene by apple tissue incubated in air; the conversion of labeled methionine to ethylene was greatly decreased in the presence of unlabeled ACC, but the conversion of labeled ACC to ethylene was little affected by the presence of unlabeled methionine; and 2-amino-4(2'-aminoethoxy)trans-3-butenoic acid, a potent inhibitor of pyridoxal phosphate-mediated enzyme reactions, greatly inhibited the conversion of methionine to ethylene but did not inhibit conversion of ACC to ethylene. These data indicate the following sequence for the pathway of ethylene biosynthesis in apple tissue: methionine -_ S-adenosylmethionine -ACC -ethylene. A possible mechanism accounting for these reactions is presented. Ethylene is a plant hormone that initiates fruit ripening and regulates many aspects of plant growth and development (1). It is generally thought that methionine is the common precursor of ethylene throughout diverse higher plant tissues in which the hormone occurs and exerts its many effects (2-4). In apple tissue the conversion of methionine to ethylene represents the major metabolism of methionine (5). In this conversion, C-1 of methionine is converted to CO2, C-2 to formic acid, and C-3,4 to ethylene. The sulfur atom, however, is retained in the tissue (2). Because the conversion of methionine to ethylene is greatly inhibited by uncouplers of oxidative phosphorylation, Burg (6) and Murr and Yang (7) proposed that S-adenosylmethionine (SAdoMet), formed from methionine and ATP, is an intermediate between methionine and ethylene. Adams Feeding Experiments. Plugs, 1 cm in diameter and 2 cm long, were cut from apple fruit with a cork borer and scalpel. They were quickly rinsed in 2% (wt/vol) KC1 and blotted with a paper towel. Indicated substrates were infused by a vacuum infiltration technique as described (11). For incubation in a nitrogen or air atmosphere, a tissue plug was placed in a 12-ml plastic syringe fitted with a three-way stopcock. Two holes (3 mm diameter) were made near the end of the syringe to allow flushing with a stream of nitrogen or air from the stopcock inlet.After flushing, the syringe was sealed for incubation. Gas samples were withdrawn periodically from the incubation syringe with a sampling syringe and assayed for total and rad...

A characteristic response of tomato to waterlogging is epinasty of the petioles (10). Recent studies have demonstrated that epinastic growth of the petioles is a response to accelerated rates of ethylene synthesis (2,8). Anaerobic conditions in the root zone are sufficient to cause elevated ethylene synthesis and epinasty in the shoot regardless of whether the anaerobiosis is imposed by waterlogging (8,11), or by flushing with N2 (2,8,9,14). Indirect evidence suggested that a signal from the anaerobic roots is transported to the shoot where it stimulates ethylene synthesis (2,8,9). Using plants with a divided root system, Jackson and Campbell (8) demonstrated that the transport of such a signal was probably through the xylem.A burst of ethylene is often observed when plant tissues are transferred from anaerobic to aerobic conditions (4). Such a surge of ethylene evolution following an anaerobic incubation has been observed in excised tomato roots (9). Recently, Adams and Yang (1) Ethylene Measurements. Ethylene production by excised petioles was estimated by the procedure of Jackson and Campbell (7). Proximal sections (5 cm) of the second, third, and fourth oldest petioles were enclosed in a 4.1-ml test tube, flushed with ethylene-free air, and capped with a serum stopper. After 30 min, a 0.5-ml gas sample was taken with a gas-tight syringe. The ethylene content was determined by gas chromatography with a flame ionization detector. All data are corrected for any ethylene present in blank tubes and are expressed on a fresh weight basis.

Auxin is known to stimulate greatly both C2H4 production and the conversion of methionine to ethylene in vegetative tissues, while aminoethoxyvinylglycine (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, 13-14Clmethionine was administered to IAA or IAA-plus-AVG-treated mung bean hypocotyls, and the conversion of methionine to S-adenosylmethionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), and C211 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 --C2H) 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.Auxins are known to stimulate ethylene production in a wide variety of plant tissues (1,4,7,8,13,18,25). Many of the effects of auxin on growth and on other processes, such as epinasty, hook opening, inhibition of growth, root induction, and geotropism, are now attributed to its ability to induce ethylene production (1,4,7). In vegetative tissues the rate of ethylene production is thought to be regulated by the internal level of free auxin (1). Cytokinins stimulate ethylene production only slightly, but a remarkable synergistic effect of cytokinins on IAA-induced ethylene production has been observed in etiolated seedlings of several species (4,8,12,13). Auxin-induced ethylene production, as well as other ethylene production systems, are known to be inhibited by AVG2 (2,10,15) and by Co2+ (9,11,14 were germinated and grown in Vermiculite for 3.5 days in darkness at 25 C. Twenty 2-cm-long hypocotyl segments, 1 to 3 cm below the hook, were incubated in 5 ml of a medium consisting of 2% sucrose, 50 ,ug/ml chloramphenicol, and 50 mm Mes buffer (pH 6.1) in a 50-ml Erlenmeyer flask. Where indicated, addenda were 50 ,UM IAA plus 20 ,UM TZ (Tables I, II, and III), 50 ,LM IAA plus 25 ,UM IPA (Table IV, Figs. 1 and 2), 10 ,UM AVG, and 0.2 mm ACC. The flasks were flushed with air...

ABSTRACT1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene. Following conventional column fractionation, the enzyme was purified 180-fold to near homogeneity with a specific activity of 20 nmol/(mg min). This purified enzyme preparation migrated as a single protein band with an apparent molecular mass of 35 kDa on SDS/PAGE and 39 kDa on gel filtration. As in vivo, the purified enzyme required CO2 for activity. Removal of CO2 from the reaction mixture completely abolished the enzyme activity, while 0.5% atmospheric CO2 (0.15 mM in the medium) gave half-maximal activity. The purified enzyme agreed well with that deduced from the pAE12 sequence. When the protein was cleaved with CNBr and one of the peptide fragments was isolated and sequenced for 20 cycles, its sequence (KE-FAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115-134). When preclimacteric apple fruit was treated with ethylene, a parallel increase in in vivo and in vitro ACC oxidase activities was observed, and this increase was accompanied by a concomitant increase in the level of pAE12 transcript. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.The simplest olefin, ethylene, is a gaseous plant hormone which regulates many physiological processes of plant growth and development. The biosynthetic pathway for ethylene in higher plants has been elucidated as follows:. Two enzymes that are unique to this pathway are ACC synthase and ACC oxidase (also known as ethylene-forming enzyme). ACC synthase converts S-adenosylmethionine to ACC, and ACC oxidase catalyzes the oxidation of ACC to ethylene. In fruits, both ACC synthase and ACC oxidase are induced during ripening and contribute to the regulation of ethylene biosynthesis.While much progress has been achieved on the characterization of ACC synthase at the biochemical and molecular levels (1-5), progress on the isolation of in vitro ACC oxidase has been slow. ACC oxidase activity is readily demonstrated in vivo by supplying tissues with ACC. These in vivo studies have established that the enzyme has a high affinity for its substrate ACC, exhibits stereospecificity toward the stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid for the synthesis of 1-butene, and specifically converts ACC to ethylene, CO2, and cyanide (6). Although a number of plant enzyme preparations were shown to be capable of oxidizing ACC to ethylene, these preparations lacked these characteristics. Based on these criteria it was concluded that these reported in vitro systems were not the authentic ACC oxidase that functions in vivo (6).Recent advances in the molecular biology of this enzyme have led to the successful isolation ofauthentic ACC oxidase. Based on the observations that ACC oxidase activity in tomato fruit was greatly reduced by an antisense gene of a ripening-related cDNA, pTOM13, Hamilton et al. (7) suggested that the pTOM13 gene was related to ACC oxidase. Later work confirmed that pTOM13 and relat...

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It has been shown that 1-aminocyclopropane-1-carboxylic acid (ACC) is the immediate precursor of ethylene, which is derived from C-2 and C-3 of ACC. When [1-14C]ACC was administered to etiolated mungbean (Vigna radiata) hypocotyls, -16% of the ACC was converted to ethylene and about 10% of the radioactivity was converted to[14C]asparagine in 7 hr. In etiolated epicotyls of common vetch (Vicia saliva), after 7 hr about 14% of the ACC was converted to ethylene and 16% of the radioactivity was converted to 1 cyanoalanine plus -glutamyl--cyanoalanine. It is known that in most plants cyanide is metabolized to asparagine via the intermediate (3cyanoalanine, whereas in a few plants such as V. saliva, 1-cyanoalanine is converted to the conjugate y-glutamyl-f8-cyanoalanine. We