Ethylene formation from α-keto-γ-methylthiobutyrate by tomato fruit extracts

Ku, Han; Yang, Shang Fa; Pratt, Harlan K.

doi:10.1016/s0031-9422(00)85401-1

Cited by 25 publications

(6 citation statements)

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“…Yang (30) speculated that the pea system may be a peroxidase and later Mapson and Wardale (24) further characterized the cauliflower enzyme system and concluded that the enzyme may indeed be a peroxidase. Although methionine appears to be a substrate for the production of ethylene in pea tissue treated with IAA (8,22), peroxidase only produces ethylene from methional or a-keto-y-methylthiobutyric acid (21), but not from S-adenosyl methionine or methionine. Furthermore, there is evidence that methional and the a-keto-analogue are not converted to ethylene as readily as methionine in vivo, and in addition C-2 of methionine is not converted to CO, in vivo (8), whereas peroxidase converts C-2 of the a-ketoanalogue to COa in vitro (31).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Auxin-induced Ethylene Production

1971

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Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.It has been suggested that auxin may stimulate ethylene production by inducing the enzymes involved in the formation of the gas (1). This theory is based primarily upon the finding that inhibitors of protein and RNA synthesis prevent auxininduced ethylene production, and also on the observation that stimulation of ethylene production by auxin has a substantial lag period (1,5,7,18,19). Extracts of pea seedlings contain an enzyme that converts methional to ethylene in a cell-free system (20). The enzyme has been identified as a peroxidase (30), a protein which also catalyzes a number of other reactions including oxidation of IAA (13,14,26). The present study presents evidence which shows the peroxidase content not to play a role in the regulation of ethylene biosynthesis in pea seedlings. Growth, ethylene production, the endogenous IAA content, exogenous IAA level, and rate of metabolism of growth hormone were continuously determined and compared in an attempt to determine the mechanism by which auxin induces ethylene production. (29) which is approximately 100% efficient. The radioactivity remaining in the tissue after alcohol extraction was determined after squashing the sections and drying them on a planchet, under which condition self-absorption was negligible. Enzyme Assay for Ethylene Synthesis. Sections of subapical internodes or plumular hooks were homogenized in 50 mm potassium phosphate buffer at pH 7.8 (10 sections/ml), strained through cheesecloth, and centrifuged at lOOOg for 15 min. The preparation could be further purified by dialysis and ammonium sulfate precipitation as described by Ku et al. for the pea enzyme (20), but a variable loss in activity occurred at each step, making the crude extract preferable for quantitative studies. The resulting supernatant (enzyme preparation) was assayed in 5 ml of reaction mixture described by Yang (30), consisting of 10 mM potassium phosphate buffer (pH 7.8), 0.6 mm methional, 3 em manganese sulfate, 80 mm resorcinol, 2 ytM ethylenediaminetetraacetic acid, 80 fM sodium hydrogen sulfite, with 1 ml of enzyme extract. The mixture was incubated at 25 C in a 25-ml Erlenmeyer flask sealed with a vaccine cap, and typically 1-ml samples of air were withdrawn from the flask at 30-min intervals, and ethylene was ...

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Section: Discussionmentioning

confidence: 99%

Mechanism of Auxin-induced Ethylene Production

1971

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“…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.…”

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

Plant Physiol.

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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)

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“…Dithiothreitol is oxidized in the presence of tomato or horseradish peroxidase with the enzymes accumulating in their oxyferroperoxidase forms during the oxidation reaction. Whereas HRP returns to its free ferric form at the end of the reaction, the tomato enzyme is converted into a form that absorbs at 442 nanometers.A single peroxidase which has been shown to exist in tomato fruit extracts and to exhibit some IAA oxidase activity (5, 8) has been implicated both in the production of ethylene (12,17,18) and in the destruction of the plant growth hormone IAA (8). Although it is not unusual to relate the action of the enzyme peroxidase with the control of the above hormones, in the case of tomato fruit it is premature to assume such a relationship because of insufficient information about the physical and catalytic properties of this peroxidase and the lack of a purification method which would allow a quantitative estimation and a complete isozyme composition of the noncovalently bound peroxidases of the fruit (22, 23).…”

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confidence: 99%

Tomato Peroxidase

Kokkinakis¹,

Brooks²

1979

Plant Physiol.

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A major peroxidase has been found in the tomato pericarp (Lycopersicon esculentum var. Tropic) of the ripe and green fruit. A purification scheme yielding this enzyme approximately 85% pure has been developed. The tomato enzyme resembles horseradish peroxidase (HRP) in a standard peroxidase assay and in its ability to be reduced to ferroperoxidase, to be converted to oxyferroperoxidase (compound III), and to form peroxidase complexes with hydrogen peroxide (compounds I and II). In contrast to the HRP, the tomato peroxidase fails to catalyze the aerobic oxidation of indole-3-acetic acid in the presence of 2,4-dichlorophenol and manganese. The tomato peroxidase can be resolved into two nonidentical subunits in the presence of dithiothreitol while HRP remains as a single polypeptide chain after such treatment. Dithiothreitol is oxidized in the presence of tomato or horseradish peroxidase with the enzymes accumulating in their oxyferroperoxidase forms during the oxidation reaction. Whereas HRP returns to its free ferric form at the end of the reaction, the tomato enzyme is converted into a form that absorbs at 442 nanometers.A single peroxidase which has been shown to exist in tomato fruit extracts and to exhibit some IAA oxidase activity (5, 8) has been implicated both in the production of ethylene (12,17,18) and in the destruction of the plant growth hormone IAA (8). Although it is not unusual to relate the action of the enzyme peroxidase with the control of the above hormones, in the case of tomato fruit it is premature to assume such a relationship because of insufficient information about the physical and catalytic properties of this peroxidase and the lack of a purification method which would allow a quantitative estimation and a complete isozyme composition of the noncovalently bound peroxidases of the fruit (22, 23).Spectral properties and pH optima of the tomato fruit peroxidase in catalyzing the oxidation of redogenic substrates in the presence of H202 have been previously reported by Evans (6). The present report deals with additional properties of the purified enzyme including its capability to be converted to complexes of higher oxidation states (compounds I, II, III) which are thought to be important for the IAA oxidase activity of peroxidases (13,26). The enzyme HRP,3 a well studied peroxidase, was employed in order to compare some of its properties with similar properties of the tomato enzyme. ' cheesecloth, and the residue was washed twice with 100 ml of 0.1 M phosphate buffer (pH 6.5). The filtrates were combined and centrifuged at 2,000 g for 10 min. The supernatant, to be referred to as crude soluble peroxidase fraction, was stored at 0 C for further use. The pellet and the solid material on the cheesecloth were suspended in 200 ml of 0.2 M sodium maleate-0.2 M calcium chloride adjusted to pH 6.5. The suspension was then sonicated for 5 min and centrifuged at 2,000g for 10 min. Under this treatment peroxidases that were ionically bound to the tomato pulp were solubilized (17). These per...

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Ethylene formation from α-keto-γ-methylthiobutyrate by tomato fruit extracts

Cited by 25 publications

References 14 publications

Mechanism of Auxin-induced Ethylene Production

Mechanism of Auxin-induced Ethylene Production

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

Tomato Peroxidase

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