Inhibited Ethylene Production Research Articles

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.

Inhibition of Ethylene Evolution in Papaya Pulp Tissue by Benzyl Isothiocyanate

Papaya (Carica papaya L.) pulp tissue disks in an incubation medium composed of 0.4 m sucrose evolve ethylene at an optimum pH of 5.25 at 30 C. Disks of young preclimacteric fruit evolve the gas linearly with fruit age until fruit age reaches 4 months. Disks from 5-month-old postclimacteric fruit produce approximately 5-fold more ethylene than disks from 4-month-old fruit. Ethylene evolution by unaged papaya disks is inhibited potently by benzyl isothiocyanate. The compound inhibits production of ethylene by approximately 60% at a concentration of 0.046 mm. However, in aged papaya disks benzyl isothiocyanate causes no inhibition of ethylene production indicating that the compound inhibits the induction of the ethylene-producing system rather than the evolution of the gas per se. Even at a 2-fold higher concentration benzyl isothiocyanate has no effect on respiration of unaged papaya disks. It is proposed that benzyl isothiocyanate may act as an endogenous regulator of ethylene evolution in papaya fruit.

The Generation of Hydrogen Peroxide, Superoxide Radical, and Hydroxyl Radical by 6-Hydroxydopamine, Dialuric Acid, and Related Cytotoxic Agents

Hydrogen peroxide, superoxide radical, and hydroxyl radical were all formed during the autoxidation of four cytotoxic agents, namely, 6-hydroxydopamine, 6-aminodopamine, 6,7-dihydroxytryptamine, and dialuric acid. Ascorbic acid and 5-hydroxydopamine, two autoxidizable agents of biological interest but without similar cytotoxic actions, were used as negative controls. The results tend to implicate O2- and ·OH, as well as H2O2, in the molecular mechanisms for cytotoxicity. Production of H2O2 was measured with an oxygen electrode. Accumulation of H2O2 was apparent from the return to solution of approximately one-half of the consumed oxygen after the addition of catalase. Superoxide radicals were detected by reduction of cytochrome c, with inhibition by superoxide dismutase. Although O2- radicals were observed previously by other means during the autoxidation of 6-hydroxydopamine, there was no clear effect in the cytochrome c system. This may have been due, in part, to a previously described scavenging of O2- by 6-hydroxydopamine itself. Hydroxyl radicals were detected by reaction with β-methylthiopropionaldehyde to form ethylene. Catalase or superoxide dismutase inhibited ethylene production. These results point to a reaction between H2O2 and O2- (Haber-Weiss reaction) as a major source of the ·OH radicals. Ethylene production was inhibited by known ·OH trappers, such as benzoate and ethanol, and by a variety of other agents. Catechol and the catecholamines, dopamine and norepinephrine, were very potent inhibitors of ethylene production. The effectiveness of these agents may have been due to a combined action of blocking ·OH formation as well as accelerating its removal. Superoxide dismutase had a biphasic effect in the 6-hydroxydopamine system, inhibitory at early times but causing increased accumulation of ethylene at later times. An explanation for this latter phenomenon may be based on a reaction between O2- and ·OH, as described by other investigators.

Effects of Cycloheximide on Indoleacetic Acid-induced Ethylene Production in Pea Root Tips

Cycloheximide inhibited ethylene production in excised pea root tips treated with high levels of indoleacetic acid (100 mum and 10 mum). In contrast, cycloheximide did not inhibit ethylene production induced by a lower concentration (1 mum) of indoleacetic acid unless it was added 2 hours before the indoleacetic acid treatment. These observations suggest that indoleacetic acid has two effects on the enzyme system involved in ethylene synthesis. At low concentrations (1 mum) indoleacetic acid increases ethylene production without protein synthesis, whereas at the higher concentrations, the synthesis of new protein is associated with increased ethylene production.

Ethylene in Plant Growth

Ethylene inhibits cell division, DNA synthesis, and growth in the meristems of roots, shoots, and axillary buds, without influencing RNA synthesis. Apical dominance often is broken when ethylene is removed, apparently because the gas inhibits polar auxin transport irreversibly, thereby reducing the shoot's auxin content just as if the apex had been removed. A similar mechanism may underly ethylene-induced release from dormancy of buds, tubers, root initials, and seeds. Often ethylene inhibits cell expansion within 15 min, but delays differentiation so that previously expanding cells eventually grow to enormous size. These cells grow isodiametrically rather than longitudinally because their newly deposited cellulose microfibrils are laid down longitudinally rather than radially. Tropistic responses are inhibited when ethylene reversibly and rapidly prevents lateral auxin transport. In most of these cases, as well as certain other instances, ethylene action is mimicked by application of an auxin, since auxins induce ethylene formation. Regulation by ethylene extends to abscission, to flower formation and fading, and to fruit growth and ripening. Production of ethylene is controlled by auxin and by red light, auxin acting to induce a labile enzyme needed for ethylene synthesis and red light to repress ethylene production. Numerous cases in which a response to red light requires an intervening step dependent upon inhibition of ethylene production have been identified. Ethylene action requires noncovalent binding of the gas to a metal-containing receptor having limited access, and produces no lasting product. The action is competitively inhibited by CO(2), and requires O(2). Ethylene is biosynthesized from carbons 3 and 4 of methionine, apparently by a copper-containing enzyme in a reaction dependent upon an oxygen-requiring step with a K(m) = 0.2% O(2). The oxidative step appears to be preceded by an energy-requiring step subsequent to methionine formation.

Interrelationships of Ethylene and Abscisic Acid in the Control of Rose Petal Senescence

The role of abscisic acid and ethylene in the senescence of rose petals cv. Golden-Wave was examined. A rise in ethylene evolution, followed by an increase in the level of abscisic acid was observed. The presence of abscisic acid in rose petals was established, using different chromatography systems, several bioassays, and immunoassay. External application of ethylene accelerated senescence and induced a rise in endogenous abscisic acid-like activity. Application of abscisic acid promoted senescence, but suppressed ethylene production. The data suggest that the participation of these two hormones in the control of senescence is via the same pathway. The possibility of interrelationship between abscisic acid and ethylene was tested and experimental evidence in favor of this hypothesis is presented. It was suggested that ethylene affects senescence in rose petals by inducing an increase in abscisic acid activity, which in turn may control ethylene evolution, via a feedback mechanism.

Relation of Phytochrome-enhanced Geotropic Sensitivity to Ethylene Production

Brief exposure of etiolated pea (Pisum sativum cv. Alaska) seedlings to red light enhances subsequent development of geotropic curvature of the stem. Both this response and inhibition of ethylene production by red light become maximal 8 hours after illumination. Very low concentrations of applied ethylene inhibit development of geotropic curvature, whereas hypobaric treatment enhances geotropic sensitivity by removing endogenous ethylene. Increased geotropic sensitivity after illumination is accompanied by increased lateral migration of (3)H-indoleacetic acid in response to gravity, and ethylene inhibits this lateral migration. It is suggested, therefore, that red light-enhanced geotropic sensitivity is caused by increased lateral auxin transport resulting from a reduction in ethylene production after illumination.

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, alpha-keto-gamma-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.Incorporation of (14)C from added methionine-(14)C 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 Penicillium digitatum, a fungus which produces ethylene via pathways not utilizing methionine as a precursor, was not affected by rhizobitoxine.

Physiology of Oil Seeds

Germination, ethylene production, and carbon dioxide production by dormant Virginia-type peanuts were determined during treatments with plant growth regulators. Kinetin, benzylaminopurine, and 2-chloroethylphosphonic acid induced extensive germination above the water controls. Benzylaminopurine and 2-chloroethylphosphonic acid increased the germination of the more dormant basal seeds to a larger extent above the controls than the less dormant apical seeds. Coumarin induced a slight stimulation of germination while abscisic acid, 2,4-dichlorophenoxyacetic acid, and succinic acid 2,2-dimethylhydrazide did not stimulate germination above the controls. In addition to stimulating germination, the cytokinins also stimulated ethylene production by the seeds. In the case of benzylaminopurine, where the more dormant basal seeds were stimulated to germinate above the control to a larger extent than the less dormant apical seeds, correspondingly more ethylene production was induced in the basal seeds. However, the opposite was true of kinetin for both germination and ethylene production. When germination was extensively stimulated by the cytokinins, maximal ethylene and carbon dioxide evolution occurred at 24 and 72 hours, respectively. Abscisic acid inhibited ethylene production and germinaton of the seeds while carbon dioxide evolution was comparatively high. The crucial physiological event for germination of dormant peanut seeds was enhancement of ethylene production by the seeds.

Inhibition of Ethylene Production in Banana Fruit Tissue by Ethylene Treatment

A temporary ethylene treatment, sufficient to stimulate ripening in banana fruit tissue, partly suppresses endogenous ethylene production and the evolution of ethylene from methionine. The production of endogenous ethylene does not return to rates normal for naturally ripening fruit after the exogenous ethylene is removed. The extent of inhibition is related to the concentration of applied ethylene up to 5-10 p.p.m., and to the duration of treatment within the period 12 hI' to 3 days. Other characteristics of ripening appear to develop normally, except in the shorter treatments, where respiration shows a lower climacteric peak and chlorophyll breakdown is delayed.

Hormonal regulation of cell elongation in the hypocotyl of rootless soybean: an evaluation of the role of DNA synthesis.

A method was developed where soybean seedlings were grown without roots to study the influence of hormones of root origin on shoot growth. Excision of the root resulted in inhibition of apical section growth and DNA synthesis and inhibited elongating section growth. A synthetic cytokinin restored DNA synthesis in the apical section, but did not influence growth in either the apical or elongating sections. Low concentrations of gibberellin with the cytokinin restored growth in the apical section. Gibberellin alone was sufficient to restore growth in the elongating section. An inhibitor of DNA synthesis, 5-fluorodeoxyuridine, inhibited the increase in apical section DNA without inhibiting control or gibberellin-induced growth in the elongating section. Experiments with (14)C-thymidine resulted in no DNA labeling differences in the elongating section under conditions where gibberellin-induced elongation varied from 50% to 73% above controls. It was concluded that gibberellin-induced elongation in soybean hypocotyl occurred in the absence of DNA synthesis. Gibberellin does stimulate DNA synthesis in the apical tissue apart from its effect on cell elongation. Excised soybean hypocotyl elongated maximally at 10(-6)m auxin. At higher auxin concentrations, fresh weight and ethylene production increased, but elongation was reduced. Addition of GA to the higher auxin concentrations resulted in a 50% inhibition in auxin-induced ethylene production and resumption in maximal elongation. Added ethylene inhibited elongation 30% at 2 mul/l. Addition of up to 100 mul/l ethylene did not inhibit elongation with GA present in the incubation medium. Thus GA may counteract ehtylene inhibition of cell elongation in addition to inhibiting ethylene production in auxin-treated tissues.

Control of Apple Ripening by Succinic Acid 2,2-Dimethyl Hydrazide, 2-Chloroethyltrimethylammonium Chloride, and Ethylene

Ripening of ;Tydeman's Early' apples (Malus sylvestris L.) assessed by the occurrence of the respiratory climacteric was delayed by succinic acid 2,2-dimethyl hydrazide (B-9) but not by 2-chloroethyltrimethylammonium chloride (CCC), each applied 14 days after bloom. B-9 also inhibited ethylene production by fruits exhibiting the climacteric rise. Inhibition of the climacteric by B-9 was overcome by exogenous ethylene, but the latter treatment failed to completely reverse the B-9 inhibition of endogenous ethylene production.In apple fruitlets harvested within 6 weeks of bloom, ethylene stimulated respiration but did not initiate a climacteric respiratory pattern, did not induce a color change, and did not give rise to any measurable increase in endogenous ethylene production. However, all of these changes did occur in more mature fruit. These data suggest that apples have 2 physiological stages with regard to their reaction to exogenous ethylene.

Stimulation of Ethylene Production in Apple Tissue Slices by Methionine

Methionine can induce more than a 100% increase in ethylene production by apple tissue slices. The increased amount of ethylene derives from carbons 3 and 4 of methionine. Only post-climacteric fruit tissues are stimulated by methionine, and stimulation is optimum after 8 months' storage. Copper chelators such as sodium diethyl dithiocarbamate and cuprizone very markedly inhibit ethylene production by tissue slices. Carbon monoxide does not effect ethylene production by the slices. These data suggest that the mechanism for the conversion of methionine to ethylene, in apple tissues, is similar to the previously described model system for producing ethylene from methionine and reduced copper. Therefore, it is suggested that one of the ethylene-forming systems in tissues derives from methionine and proceeds to ethylene via a copper enzyme system which may be a peroxidase.