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

Abstract

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)

Methionine-induced Ethylene Production by Penicillium digitatum

“…Results with the chelators indicate that a heavy metal ion, possibly an oxidized Cu, may be involved in the reactions; and the inhibition by catalase suggests the involvement of H202. SMKB stimulation of ethylene production in filtrates from both the methionine-induced and the water control shake cultures (Table III) suggests that constituents of a model system may leak out of the fungal cells into the incubation solution, as they have been shown to do from higher plants (13).…”

“…Vol (Table II). Addition of SMKB (10-4 M), previously indicated as a substrate for ethylene (13), to both the nonboiled and boiled filtrates resulted in a markedly increased ethylene evolution, which was further stimulated by the addition of horseradish peroxidase (Table III). Similarly, filtrates from the 24-hr shake-incubated fungal cells in water released ethylene when SMKB was added.…”

“…Similarly, filtrates from the 24-hr shake-incubated fungal cells in water released ethylene when SMKB was added. SMKB is known (13) to be readily converted to ethylene nonenzymically especially at low pH, such as that of the filtrate and incubation solution (Table II). However, we were unable to detect SMKB, by chromatography or by the ethylene-peroxidase assay (13) Inhibitor was applied in a methanolic solution (6 IL/ml), and final concentration was 50 uLM.…”

Ethylene and Agriculture: the Role of the Microbe

“…(2) Catalase failed to inhibit ethylene production by cauliflower florets (Lieberman & Kunishi 1971). The significance of these results is open to question.…”

Ethylene in Plant Growth

“…Moreover in apples, [14C]2-keto-4-methylthiobutyrate is converted less efficiently than [14C]methionine to ethylene and then only after conversion to methionine (64). Objection also has been raised to the proposed role of 2-keto-4-methylthiobutyrate in ethylene production by other tissues (70).…”