Sutwumary. AMethionine can iniduce more thani a 100 % inlcrease in ethylene prodluction by apple tissue slices. The increased amounit of ethvlenie derives from carbolns 3 and 4 of methionine. On4l post-climacteric fruit tissues are stimulated bv iimethionine, and stimulatioin is optimum after 8 months' storage. Copper chelators such as sodium diethvl (lithiocarbam-nate and cuprizone very markedly inhibit ethylene production by tissuie slices. Carbon monoxide does not effect ethylene produiction by the slices. These (lata sluggest that the mechanism for the coniversion of mnethionine to ethylene, in apple tissues, is similar to the previously descriibed model system for pro(duicing ethylene fromii imiethionine and reduced copper. Therefore, it is suggested that one of the ethylene-forming systemis in tissues derives fromii miiethionine and proceeds to ethylene via a copper enzyme system which may be a peroxidase. over inoninfiltrated treatmiienits, of rem,oving the preformiied ethylenie fromii the slices. Inl somiie later experiments air was replacedI by 0,. since it w-as found( that ethylene production by apple slices immiiiiersed in liquid media was stimulated b) O.-Gas Anialy-sis. Gases evolved by the tissue slices were sample(d by syringe anid determined by gas chromatography with a flamiie-ionization detector in a system using either alumiinia or silicolle (30% silicolne oil on celite) colulmniiis (3).T'racer EIxperinients. Tracer studies were carrie(d out with 14C. CH, labeled r-methionine, I)J.-methionine carboxy 4C, DL wlethioninle 2, 14C. or I)! n'ethionine 3, 4, 14C, added to the sucrose-bicarbonate inicubation mlixture. A 2 mil aliquiot of the gases evolved by the apple tissue slices xvas first assaye(l for ethylene onl the gas chromlatograph, and(I then 50 % of the gaseous atmosphere in the incuibation flask was remioved with a 50-nl gas-tight syringe for 14C analy sis.
Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorgh-um was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, a-keto-ymethylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.Incorporation of "4C from added methionine-'4C into ethylene was curtailed by rhizobitoxine to about the same extent as was ethylene production. These results suggest that rhizobitoxine interferes with ethylene biosynthesis by blocking the conversion of methionine to ethylene and not indirectly by inhibiting the biosynthesis of methionine. Ethylene production by Pemicillium digitatum, a fungus which produces ethylene via pathways not utilizing methionine as a precursor, was not affected by rhizobitoxine.Two model systems for the generation of ethylene in plant tissues have been described by Lieberman and co-workers, one utilizing methionine as a substrate (8), and the other utilizing linolenate (1 1). In addition, methionine can serve as a precursor of ethylene in plant tissues (2, 7). To help assess the physiological importance of methionine as an ethylene precursor, a specific inhibitor of methionine biosynthesis was sought. Rhizobitoxine appeared to offer that potential.Rhizobitoxine is a phytotoxin produced by certain strains of the soybean root nodule bacterium Rhizobium japonicum (15).It inhibits greening of new leaf tissue of many plants and causes the main visual symptom of the disease in soybean known as rihizobial-induced chlorosis (14). The precise structure of rhizobitoxine remains to be elucidated; however, it is known to be a basic sulfur-containing amino acid which yields an ether derivative of homoserine upon desulfurization (13). Rhizobitoxine inhibits the growth of Salmonella typhimurium by inhibiting /3-cystathionase, an enzyme in the methionine biosynthetic pathway (12). It also irreversibly inactivates /Bcystathionase isolated from spinach leaves (4); however, the physiological effect of this lesion on the biosynthesis of methionine in spinach has yet to be assessed. We report here that rhizobitoxine inhibits ethylene biosynthesis in sorghum seedlings and in senescent apple tissue by the unexpected mechanism of blocking the conversion of methionine to ethylene. Hegari were surface-sterilized by wetting with ethanol and then immersing in an aqueous solution of 0.2% HgCl2 + 1% HCl for 2 min. After rinsing well, the seeds were germinated on moist filter paper in a Petri dish at 27 C in the dark. Two days after imbibition, the seedlings were transplanted to 50-ml Erlenmeyer flasks constructed with a side arm to collect CO2. Six seedlings per flask (about 300 mg fresh wt) were supported on a nylon mesh screen held 1.0 cm above the flask bottom...
1. A new reaction is described in which ethylene is formed from the Cu+-catalysed breakdown of methionine in phosphate buffer at 300 in air. Some of the other products of the reaction are methionine sulphone, methionine sulphoxide, homocysteic acid, homocystine, acrolein, dimethyl disulphide, methanethiol, ethyl methyl sulphide, methane and ethane. These are considered to be produced in different reaction pathways. The formation of ethylene in a model system in which Cu+ catalyses the breakdown of peroxidated linolenic acid has been described (Lieberman & Mapson, 1964). It was proposed that the biosynthesis ofethylene may proceed through an analogous system catalysed by a copper enzyme. Recent experiments, however, suggest that ethylene in tissues may arise from more than one source. For example, an analysis of particulate fractions, isolated from apples, shows that at least two types of compounds are present which can yield ethylene in the Cu+-catalysed system. The first is associated with the fat-soluble constituents of the cell and the second with the water-soluble constituents.Search for other precursors of ethylene has led to the finding that L-methionine, in reaction with the cuprous generating system, also forms ethylene. It is shown below that methionine and related substances are unique in their ability to form relatively large quantities of ethylene in a model system. The present paper describes characteristics of this ethylene-forming system, demonstrates that ethylene is derived from C-3 and C-4 of methionine, and suggests a mechanism for the reaction. MATERIALS AND METHODSModel 8y8tem. The model system consisted of L-methionine (1 mM), CU2+ (1.0 or 0-1 mm), ascorbate (10mm) and phosphate buffer, pH6.8 (60mm). Components of the 15 model system were contained in 25 ml. flasks sealed with one-hole rubber stoppers containing clamped capillary tubes. The flasks were incubated at 300 in a water bathshaker and internal atmospheres above the reaction mixture were sampled periodically with gas-tight syringes. Corrections were made to account for the increase in gas volume resulting from this sampling technique.Gas analysis. Gases produced in the reaction were determined by gas chromatography. Alumina or silicone (30% silicone oil on Celite) columns and a flame-ionization detector were used to determine ethylene. Details of the complete system have been described by Meigh, Norris, Craft & Lieberman (1960). Acrolein, ethyl methyl sulphide, dimethyl sulphide, dimethyl disulphide and methanethiol were detected on a capillary column (80ft. x 0-015in. diam.) at 150 with di-(2-cyanoethyl) ether as stationary phase, with an enrichment trap cooled in liquid 02 to first concentrate the gases. The vapours were subsequently released from the trap by passing an electric current through the trap.Methional. This compound was prepared from acrolein and methanethiol by the method described by Pierson, Giella & Tishler (1948
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