Inhibition of ethylene production in fruit slices by a rhizobitoxine analog and free radical scavengers

The rhizobitoxine analog, L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid (Ro), which effectively inhibits ethylene production in apple (Malus domestica Borkh.) and other tissues at concentrations at about 68 micromolar, inhibited ethylene production by about 50 to 70% in green tomato (Lycopersicon esculentum Mill.) fruit slices but only by about 15% in pink and ripe tomato tissue slices. Ethylene production in climacteric-rise and postclimacteric avocado slices was likewise relatively insensitive to 68 micromolar Ro. At 340 micromolar Ro, inhibition of ethylene production increased up to 50% in pink tomato slices, whereas 680 micromolar Ro was required to inhibit ethylene production by 30% in avocado slices. Incorporation of ¹⁴C from [¹⁴C]methionine into ethylene in green and pink tomato tissues was inhibited by Ro to about the same extent as inhibition of total ethylene production. Results thus far are inconclusive as to the mechanism of Ro resistance in tomato and avocado tissues. At 1 millimolar, free radical scavengers such as benzoate, propyl gallate, nordihydroguaiaretic acid, and to a lesser extent, eugenol, inhibited ethylene production in both Ro-sensitive (green tomato and apple) tissues and Ro-resistant (pink tomato and avocado) tissues. Therefore, free radical steps are suggested in the ethylene-forming systems.     

Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue

This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4-hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway.     

Influence of Plant Hormones on Ethylene Production in Apple, Tomato, and Avocado Slices during Maturation and Senescence

Ethylene production by tissue slices from preclimacteric, climacteric, and postclimacteric apples was significantly reduced by isopentenyl adenosine (IPA), and by mixtures of IPA and indoleacetic acid, and of IPA, indoleacetic acid, and gibberellic acid after 4 hours of incubation. Ethylene production by apple (Pyrus malus L.) slices in abscisic acid was increased in preclimacteric tissues, decreased in climacteric peak tissues, and little affected in postclimacteric tissues. Indoleacetic acid suppressed ethylene production in tissues from preclimacteric apples but stimulated ethylene production in late climacteric rise, climacteric, and postclimacteric tissue slices. Gibberellic acid had less influence in suppressing ethylene production in preclimacteric peak tissue, and little influenced the production in late climacteric rise, climacteric peak, and postclimacteric tissues. IPA also suppressed ethylene production in pre- and postclimacteric tissue of tomatoes (Lycopersicon esculentum) and avocados (Persea gratissima). If ethylene production in tissue slices of ripening fruits is an index of aging, then IPA would appear to retard aging in ripening fruit, just as other cytokinins appear to retard aging in senescent leaf tissue.     

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

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 mum canaline and almost complete inhibition with 300 mum 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 mum and by 90% at a concentration of 100 mum. Production of labeled carbon dioxide from acetate-1-(14)C 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 gamma-elimination.     

Localization of the Ethylene-synthesizing System in Apple Tissue

Apple (Malus sp.) slices gradually lost the ability to synthesize ethylene when incubated with a mixture of enzymes that digest cell walls. The released protoplasts did not produce ethylene. The release of protoplasts was faster from climacteric fruit slices than from preclimacteric tissue. In protoplast suspension culture, as new cell wall was deposited (as judged by the intensity of fluorescence of regenerating protoplasts stained with Calcofluor White and the incorporation of labeled myo-inositol into their ethanol-insoluble residue), ethylene synthesis was gradually regained. Restored ethylene synthesis reached a maximum after 80 hours in protoplasts from preclimacteric fruit and in 120 hours in those from climacteric tissue. Addition of methionine (1 mm) to the culture medium was essential for appreciable synthesis of ethylene; and this synthesis was inhibited by the aminoethoxy analogue of rhizobitoxine and by propyl gallate, inhibitors of ethylene synthesis in higher plants. We suggest that the ethylene-synthesizing enzyme system is highly structured in the apple cell and is localized in a cell wall-cell membrane complex.     

Methionine metabolism in apple tissue in relation to ethylene biosynthesis

A comparison of the rate of ethylene production by apple fruit to the methionine content of the tissue suggests that the sulfur of methionine has to be recycled during its continuous synthesis of ethylene. The metabolism of the sulfur of methionine in apple tissue in relation to ethylene biosynthesis was investigated. The results showed that in the conversion of methionine to ethylene the CH₃S-group of methionine is first incorporated as a unit into S-methylcysteine. By demethylation, S-methylcysteine is metabolized to cysteine. Cysteine then donates its sulfur to form methionine, presumably through cystathionine and homocysteine. This view is consistent with the observation that cysteine, homoserine and homocysteine were all converted to methionine, in an order of efficiency from least to greatest. For the conversion to ethylene, methionine was the most efficient precursor, followed by homocysteine and homoserine. Based on these results, a methionine-sulfur cycle in relation to ethylene biosynthesis is presented.     

BIOGENESIS OF ETHYLENE

1. The main characteristics of the biosynthetic system forming ethylene in plant tissues have been reviewed. The dependence of synthesis on a liberal supply of oxygen is clearly indicated by the fact that atmospheres containing 3–5% oxygen prevent the synthesis in fruits. There is no close connexion between respiratory activity and synthesis. Ripening of fruits and the changes associated with it may be initiated by ethylene; under such conditions the progress of formation of the hydrocarbon is autocatalytic. 2. Synthesis appears to be dependent on some degree of cell organization, since it responds acutely to changes in toxcity, tissue wounding and tissue destruction. Homogenates of many plant tissues do not produce ethylene in vitro, and the inability to use such extracts has imposed serious restrictions on biochemical studies which have in the past been mainly concerned with tracer studies and the use of tissue slices. 3. The chief difficulty associated with tracer studies aimed at determining the nature of the precursor stems from the fact that the synthesis of ethylene is only a minor pathway on the general metabolism of the cell. Thus the ratio of CO₂ to ethylene production is of the order of 164 in the case of the apple and as high as 18,000 in the case of less vigorous producers of ethylene. The incorporation of label from labelled substrates which enter the general metabolism of the cell is thus usually very low, and this makes it difficult to determine whether the incorporation observed has any real physiological significance. In fact only where incorporation into ethylene relative to that into CO₂ is high, as is the case with methionine, can one conclude that the substance can be considered to be an immediate precursor. 4. Because of the difficulty of obtaining clear-cut results with tracer techniques, attention has been devoted to the production of ethylene by model systems from substances of physiological interest. The studies have revealed that many substances found in plant tissue can be decomposed to yield ethylene in model systems functioning under physiological conditions. Two such substances, which have received most attention, are methionine and linolenic acid, and conditions under which ethylene is formed from them have been described. 5. Such developments have stimulated research to obtain evidence for or against the operation of such model systems in vivo. Using tissue-slice techniques, methionine and linolenic acid have both been found to stimulate ethylene formation in tissue slices. 6. The first demonstration of the synthesis of ethylene in vitro by enzymes isolated from the florets of the cauliflower has now been reported. The system involves the intermediate formation of methional from methionine by enzymes contained in the mitochondria, and the subsequent enzymic decomposition of methional into ethylene by non-particulate enzymes. These latter consist of a glucose oxidase and a peroxidase. The glucose oxidase in the presence of its substrate generates hydrogen peroxide, and peroxidase, in the presence of two co-factors, ^-coumaric acid esters and methane sulphinic acid, utilizes the peroxide to produce ethylene from methional. Although all components of this system have been isolated from extracts of floret tissue, proof that this is the actual or only process in vivo for this or other plant tissue has not as yet been achieved. The more recent demonstration of the possible involvement of linolenic acid underlines the necessity for further work. 7. Whilst much work still remains to be done to establish the mechanism of synthesis, which may not be identical in different plants, the related question of the nature of the events which stimulate the tissue to produce ethylene remains to be answered. Recent work has suggested that these events, induced by ageing of the tissue, are associated with the synthesis of new enzyme proteins, which are themselves the cause of the rapid onset of synthesis of ethylene, observed in most fruits, at the climacteric. 8. Much more information on the nature of events leading to and changes associated with the ripening syndrome in fruits and onset of senescence in vegetable tissues is needed before authoritative answers can be given to any of the questions raised in this review.     

Ethylene biosynthesis in fruit tissues

Tracer studies with avocado tissues indicate that methionine is converted to ethylene at stages of the climacteric rise and the climacteric peak, but not at the preclimacteric stage. The results suggest that the control of ethylene biosynthesis is at a step after methionine is synthesized. The endogenous content of methionine was found to be so low that methionine must be actively turned over for ethylene biosynthesis during the stages when the rate of ethylene production is high. Oxygen was found to be essential for this conversion, indicating that at least one of the steps in conversion of methionine to ethylene is oxygen-dependent. The ability of methionine and its keto analogue (α-keto-γ-methylthiobutyric acid) to serve as ethylene precursors by apple tissues was compared. Chemical and kinetic evidence support the view that methionine is a closer precursor of ethylene than its keto analogue.     

An evaluation of 4-s-methyl-2-keto-butyric Acid as an intermediate in the biosynthesis of ethylene

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 synthesis from 3-methylthioribose in apple tissue

The primary fate of 5-methylthioribose in apple tissue is the formation of methionine. Using dual labeled 5-methylthioribose, it was shown that both the CH₃S- group and the ribose portion of 5-methylthioribose were equally incorporated into methionine. Thus, the pathway involves modification of the ribose portion of 5-methylthioribose into the 2-aminobutyrate portion of methionine. This pathway functions to recycle methionine for continued synthesis of ethylene in fruit tissues. The methionine cycle in relation to ethylene biosynthesis is presented.     

Inhibition of ethylene production by rhizobitoxine

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 sorghum was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, α-keto-γ-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.     

Chemical reactivity of metallic copper in a model system containing biological metabolites

Chemical reactivity of metallic copper in a model system containing biological metabolites is described. Methionine, methional, and propanal produced ethylene when exposed to metallic copper in the presence of oxygen. It may be that metallic copper in this system serves as the '1 electron reducing agent' in the proposed chemical model system (Kumamoto et al). The requirement for oxygen was verified by removing this electron acceptor and observing the reduced ethylene production. Preliminary studies have shown that other reaction products of the reaction of copper metal with methionine include dimethyl sulfide and dimethyl disulfide or methyl mercaptan or both. These data further suggest that these chemicals are liberated from methionine when copper comes in contact with methionine-containing biological fluids.     

Stimulation of ethylene production in tomato tissue by propionic Acid

Propionic acid (10⁻³m) increases ethylene production by about 30 to 60% in tissue from green and half-ripe tomatoes (Lycopersicon esculentum Mill. var. Homestead) but does not increase ethylene production in tissue from ripe fruit. Stimulation is not due to the conversion of propionic acid to ethylene but appears to be secondary in nature and to operate on the endogenous ethylene-forming system. Thus conversion of methionine to ethylene in green and half-ripe tomato tissue is increased in the presence of propionic acid. Inhibitors which affect the normal endogenous ethylene-forming system similarly affect the propionic acid-stimulated system. Endogenous propionic acid may play a role in the regulation of ethylene production in tomato tissues.     

A comparative study of the ability of methionine or linolenic acid to act as precursors of ethylene in plant tissues

A comparative study has been made of the ability of three plant tissues to incorporate (14)C into ethylene from (14)C-labelled methionine and derivatives and from (14)C-labelled linolenic acid. Incorporation of label occurs readily from methionine and its derivative 4-methylmercapto-2-oxobutyric acid with apple, tomato or cauliflower floret tissue. No incorporation of label occurred, however, from uniformly (14)C-labelled linolenate.     

Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts

Ethylene production in apple fruit and protoplasts and in leaf tissue was inhibited by spermidine or spermine. These polyamines, as well as putrescine, inhibited auxin-induced ethylene production and the conversion of methionine and 1-aminocyclopropane-1-carboxylic acid to ethylene. Polyamines were more effective as inhibitors of ethylene synthesis at the early, rather than at the late, stages of fruit ripening. Ca²⁺ in the incubation medium reduced the inhibitory effect caused by the amines. A possible mode of action by which polyamines inhibit ethylene production is discussed.     

Free radical and free radical scavenger effects on indole-3-acetic acid levels and ethylene production

Stable free radicals, together with horseradish peroxidase, promoted degradation of indole-3-acetic acid (IAA). These reactions were retarded by the free radical scavengers Bromoxynil, Na-benzoate and kinetin. Certain free radicals promoted, but the free radical scavenger Bromoxynil retarded ethylene production in apple slices and mung bean stem tissues. The interdependency of free radicals and free radical scavengers in systems controlling IAA levels and ethylene production is discussed.     

Influence of calcium and magnesium on ethylene production by apple tissue slices

The decline in ethylene production in apple (Pyrus malus L. cv. Golden Delicious) tissue slices during 24 hours incubation in 600 millimolar sorbitol and 10 millimolar 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) is recognized as a senescent phenomenon. The inclusion of very high concentrations (100 millimolar) of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ severely inhibited ethylene production during the first 6 hours of incubation. However, after 6 hours and up to 24 hours the ethylene-forming system was stablized. These high concentrations of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ virtually eliminated lipid peroxidation and protein leakage from these slices. Also conversion of 1-aminocyclopropane-1-carboxylic-1-acid to ethylene and the influence of indoleacetic acid on ethylene production was stabilized after 24 hours of incubation by these high concentrations of Ca²⁺, Mg²⁺, and Ca²⁺ plus Mg²⁺. Addition of divalent ionophores severely inhibited ethylene production, but this inhibition was prevented by Ca²⁺ in concentrations greater than the ionophore. These data suggest that the loss of ethylene production by aging tissue slices results from degradation of membranes. They support previous work that indicates that the ethylene-forming system, perhaps the segment of the pathway from 1-aminocyclo-propane-1-carboxylic-1-acid to ethylene, resides in the plasma membrane.     

Ethylene production from propanal

Tracer studies using a model system consisting of Cu²⁺ and ascorbate indicate that carbons 2 and 3 of propanal are converted to ethylene, and carbon 1 is converted to formic acid and CO₂. A mechanism accounting for this reaction is described. In apple tissue, methionine but not propanal is readily incorporated into ethylene. It is therefore concluded that propanal is not a precursor of ethylene in this fruit.     

Cyanide metabolism in relation to ethylene production in plant tissues

HCN is the putative product of C-1 and amino moieties of 1-aminocyclopropane-1-carboxylic acid (ACC) during its conversion to ethylene. In apple (Malus sylvestrus Mill.) slices or auxin-treated mungbean (Vigna radiata L.) hypocotyls, which produced ethylene at high rates, the steady state concentration of HCN was found to be no higher than 0.2 micromolar, which was too low to inhibit respiration (reported Ki for HCN to inhibit respiration was 10-20 micromolar). However, these tissues became cyanogenic when treated with ACC, the precursor of ethylene, and with 2-aminoxyacetic acid, which inhibits β-cyanoalanine synthase, the main enzyme to detoxify HCN; the HCN levels in these tissues went up to 1.7 and 8.1 micromolar, respectively. Although ethylene production by avocado (Persea gratissima) and apple fruits increased several hundred-fold during ripening, β-cyanoalanine synthase activity increased only one- to two-fold. These findings support the notion that HCN is a co-product of ethylene biosynthesis and that the plant tissues possess ample capacity to detoxify HCN formed during ethylene biosynthesis so that the concentration of HCN in plant tissues is kept at a low level.     

Methionine metabolism in apple tissue: implication of s-adenosylmethionine as an intermediate in the conversion of methionine to ethylene

If S-adenosylmethionine (SAM) is the direct precursor of ethylene as previously proposed, it is expected that 5′-S-methyl-5′-thioadenosine (MTA) would be the fragment nucleoside. When [Me-¹⁴C] or [³⁵S]methionine was fed to climacteric apple (Malus sylvestris Mill) tissue, radioactive 5-S-methyl-5-thioribose (MTR) was identified as the predominant product and MTA as a minor one. When the conversion of methionine into ethylene was inhibited by _l-2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid, the conversion of [³⁵S] or [Me¹⁴C]methionine into MTR was similarly inhibited. Furthermore, the formation of MTA and MTR from [³⁵S]methionine was observed only in climacteric tissue which produced ethylene and actively converted methionine to ethylene but not in preclimacteric tissue which did not produce ethylene or convert methionine to ethylene. These observations suggest that the conversion of methionine into MTA and MTR is closely related to ethylene biosynthesis and provide indirect evidence that SAM may be an intermediate in the conversion of methionine to ethylene.