Precursors of Ethylene

L-Canaline, a potent inhibitor of pyridoxal phosphate-mediated reactions, markedly inhibited the conversion of methionine to ethylene and carbon dioxide by apple tissue. A 50% inhibition of methionine conversion into ethylene was obtained with 50 jtM canaline and almost complete inhibition with 300 AM canaline. When 2, 4-dinitrophenol, an oxidative phosphorylation uncoupler, was fed to apple tissue, it inhibited the conversion of radioactive methionine to ethylene by 50% at a concentration of 60 AM and by 90% at a concentration of 100ItM. Production of labeled carbon dioxide from acetate-l-'4C was increased by 2,4-dinitrophenol, indicating that the inhibition of ethylene production was due to uncoupling of phosphorylation. Auxin-induced ethylene production by mungbean (Phaseolus mungo L.) hypocotyl sections was similarly inhibited by these inhibitors.These results support the proposal that pyridoxal phosphate is involved in the formation of ethylene from methionine, substantiate the requirement for ATP in ethylene production, and suggest that this ATP requirement occurs in the step(s) between methionine and ethylene. The biosynthetic mechanism probably involves activation of methionine by ATP followed by a pyridoxal phosphate-mediated y-elimination.Evidence from tracer studies has established methionine as the in vivo precursor of ethylene in fruit and vegetative tissues (1. 3,6,12,17,21). Conversion of methionine to ethylene in apple tissues represents the major pathway of methionine metabolism (6) and has an absolute requirement for 02 (3). A requirement for oxidative phosphorylation in the endogenous synthesis of ethylene has been demonstrated by inhibitor studies using respiratory uncouplers (8,11,20). Although DNP,2 a respiratory uncoupler, has been reported to inhibit the in vivo conversion of methionine to ethylene (4), the dependence of methionine conversion to ethylene on DNP concentration was not investigated.Recently it was found that rhizobitoxine, a phytotoxin produced by Rhizobium japonicum, markedly inhibited ethylene evolution by untreated and IAA-or kinetin-treated sorghum seedlings as well as inhibiting the formation of ethylene from 1 This work was supported by National Science Foundation Grant GB-33907X. (14) and spinach (10), Owens et al. (15) suggested that pyridoxal phosphate acted as a cofactor in the conversion of methionine to ethylene. Yang and Baur (21) proposed a concerted y-elimination reaction mechanism involving pyridoxal phosphate and 02 to account for the biological production of ethylene from methionine.We have found that L-canaline (2-amino-4-aminooxybutyric acid), a structural analog of L-ornithine, effectively inhibits the conversion of methionine to ethylene in apple tissue. L-Canaline has been shown to complex strongly with pyridoxal phosphate and inhibit activity of pyridoxal-dependent enzymes (16). This paper describes the effect of DNP and L-canaline on the conversion of L-methionine to ethylene in apple tissue and on the ethylene evolution by auxin-treated mu...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Inhibition of in Vivo Conversion of Methionine to Ethylene by l-Canaline and 2,4-Dinitrophenol

Murr

Yang²

1975

“…and otber tissues at concentratins at about 68 micromolar, inhibited ethylene productin by about 50 to 70% in green tomato (Lycopersicon esculentum Mill.) Since Lieberman et al (6) showed that ethylene was derived from carbon atoms 3 and 4 of methionine in apple slices, evidence has accumulated that methionine is the natural precursor of ethylene in several plant tissues (2,11). Despite this evidence, the exact reaction mechanism of the conversion of methionine to ethylene has not been established.…”

mentioning

confidence: 99%

Inhibition of Ethylene Production in Fruit Slices by a Rhizobitoxine Analog and Free Radical Scavengers

Baker¹,

Lieberman²,

Anderson³

1978

The rhizobitoxine analog, L-2-amino-4-(2-aminoethoxy)-traas-3-butenoic acid (Ro), wich effectively Iits ethylene productio in apple (Mahus domestca Brkh.) and otber tissues at concentratins at about 68 micromolar, inhibited ethylene productin by about 50 to 70% in green tomato (Lycopersicon esculentum Mill.) Since Lieberman et al. (6) showed that ethylene was derived from carbon atoms 3 and 4 of methionine in apple slices, evidence has accumulated that methionine is the natural precursor of ethylene in several plant tissues (2,11). Despite this evidence, the exact reaction mechanism of the conversion of methionine to ethylene has not been established. With the discovery that rhizobitoxine inhibits ethylene production from methionine (1 1), a tool was provided for probing the pathway(s) of ethylene biosynthesis in plant tissues. Rhizobitoxine was found to inhibit effectively ethylene production of apple and banana slices, pea seedlings, tobacco leaves, sorghum seedlings (11), and flower tissues (5); the assumption was that methionine was the major precursor of ethylene in those tissues. Rhizobitoxine and certain of its analogs (Ro)' inhibit ethylene production irreversibly, presumably by binding to an enzyme involved in the first step of the methionine to ethylene pathway (7). One purpose of the present investigation was to extend study of the rhizobitoxine effect of other fruits and to determine whether sensitivity of the ethylene-producing system to rhizobitoxine varies with the different stages of maturation and senescence. A second purpose was to determine whether free radical scavengers inhibit ethylene production in vivo, as they have been shown to do in a model system which converts methional to ethylene following hydroxyl radical attack (4). A preliminary report of this work was published earlier (1).' Abbreviations: Ro: ethoxy analog of rhizobitoxine; Inhib.: inhibition. MATERIALS AND METHODSPlant Material and Procedure. Tomato (Lycopersicon esculentum Mill., cvs. Rutgers, Cal Ace, and Walter) and apple (Malus domestica Borkh., Red Delicious) fruit were grown at the Beltsville Agricultural Research Center, and unknown varieties of tomato were purchased from a local wholesale market. The responses of tomato fruit slices to inhibitors of ethylene production appeared to be independent of variety. Avocado (Persea gratissima Gaertn.) fruit were from a local market or provided by Calavo Growers of California. Tomato pericarp tissue was cut into about 0.5-cm cubes; avocado and apple tissues were cut into discs about 0.3 cm thick and I cm in diameter. Tissue samples (2 g) were incubated in 25-ml stoppered Erlenmeyer flasks containing 3 ml of 0.4 M sucrose in 0.027 M citrate-0.047 M Na-phosphate buffer (pH 4.6).The rhizobitoxine analog, sodium benzoate, gailic acid, and propyl gallate stock solutions were made up in that buffer. Nordihydroguaiaretic acid and eugenol solutions were prepared with ethanol, and the final concentration of ethanol in the incubation flasks was 1.5%. The flasks were incubated ...

“…We assume that approximately 20% was absorbed by the tissue. Why then is there no increase in ethylene production in the "no solution system" preincubated 1 6.5 ("buffer solution system"). In "buffer solution system", 3 g of florets were incubated 4 hr in 3 ml of 0.1 M phosphate buffer, pH 6.5, at 25 C with or without SMKB.…”

Section: Methodsmentioning

confidence: 99%

An Evaluation of 4-S-Methyl-2-Keto-Butyric Acid as an Intermediate in the Biosynthesis of Ethylene

Lieberman¹,

Kunishi²

1971

Stimulation of ethylene production by cauliflower (Brassica oleracea var. botrytis L.) tissue in buffer solution containing 4-S-methyl-2-keto-butyric acid is not due to activation of the natural in vivo system. Increased ethylene production derives from an extra-cellular ethylene-forming system, catalyzed by peroxidase and other factors, which leak from the cauliflower tissue and cause the degradation of 4-S-methyl-2-keto-butyric acid. This exogenous ethylene-forming system is similar to the ethylene-forming horseradish peroxidase system which utilizes methional or 4-S-methyl-2-keto-butyric acid as substrate. We conclude that 4-S-methyl-2-keto-butyric acid is probably not an intermediate in the biosynthetic pathway between methionine and ethylene.Methionine is now established as a precursor of ethylene in higher plants (2, 5). There is, however, no unequivocal information concerning the enzymes and intermediates involved in the pathway from methionine to ethylene. Peroxidase was suggested as an enzyme system which could be involved in ethylene biosynthesis (5). Yang (14) subsequently described a model system for ethylene production in which horseradish peroxidase, catalyzed the conversion of methional or the a-keto analogue of methionine (4-S-methyl-2-keto-butyric acid) to ethylene. This system required a combination of monophenol, sulfite and Mn2+ or hydrogen peroxide as cofactors.Mapson and colleagues, in a series of papers, demonstrated a peroxidase in cauliflower florets which catalyzed production of ethylene from methional in the presence of p-coumaric acid or its methyl ester, hydrogen peroxide, and methane sulfinic acid (7-10). They identified all these accessory factors and a hydrogen peroxide-forming system in cauliflower extracts. Takeo and Lieberman (13) MATERIALS AND METHODSTissue and Tissue Treatment. Cauliflower (Brassica oleracea var. botrytis L.) heads were purchased in the wholesale market and stored at 0 C to be used as needed. The heads were separated into florets 5 mm in diameter and 7 mm long. In most experiments 3 g of florets were placed in 3 ml of 0.1 M phosphate buffer solution, pH 6.5, with or without SMKB, in a 25-ml Erlenmeyer flask, which was stoppered with a serum cap and incubated 4 hr at 30 C. This was designated a "buffer solution system" in contrast to a "no solution system" which was incubated in absence of buffer solution after a 1-hr preincubation soaking in SMKB.In the experiments with apple (Malus sylvestris Mill. cv. Rome) tissue, 4 apple plugs (about 1 g), prepared as described (5), were incubated 4 hr at 30 C in 5 ml of 0.4 M sucrose-0.1 M bicarbonate buffer, pH 8.7, in 25-ml flasks stoppered with serum caps. In all the closed systems a vial containing 0.5 ml of 10% KOH was included in the flask to absorb CO2.Tomato (Lycopersicon esculentum Mill. cv. Homestead) pericarp tissue (4 g) from half-ripe fruit was cut into 0.5-cm squares, as previously described (6)