Novel inhibitors of ethylene production in higher plants

Of a number of O-substituted hydroxylamine derivatives, N-benzyloxycarbonyl-L-a-aminooxy-propionicacid and -aminooxyacetic acid inhibited ethylene productionby etiolated mung bean hypocotyls by 50% at 3 and 6 µmconcentrations, respectively. Their potency is thus similarto that of aminoethoxyvinylglycine (50% inhibition at 2 µM),the most potent inhibitor of ethylene production hitherto known.Methionine partially alleviated inhibition of ethylene productionby a-aminooxy-acetic acid. The results are in agreement withthe postulated involvement of pyridoxal phosphate in ethylenebiosynthesis. (Received August 31, 1979; )     

Oxidative stress, antioxidant capacity and ethylene production during ageing of cut carnation (Dianthus caryophyllus) petals

The objective of this work was to study the role of free radicalsin relation to ethylene production during ageing of cut carnationpetals. Ethylene production by freshly cut flowers was negligible,but 8 d after cutting ethylene production began to increaseand reached a peak by day 9, before beginning to decline again.The efflux of electrolytes (membrane damage index) increased101% and 2',7'-dichlorofluorescein oxidation rate (oxidativestress index) increased 53% from day 8 to day 11 after detachment.Ethylene peak was either (a) not affected significantly by thesupplementation of exogenous ethylene on the day of cutting,or (b) expanded between days 7–9 after ethylene supplementationon day 6 of cutting, or (c) was inhibited by amino-oxyaceticacid and paraquat treatments. After ethylene supplementation,conductivity and 2',7'-dichlorofluorescein oxidation increasedsignificantly as compared to control petals, and the activityof antioxidant enzymes was not affected. However, both -tocopheroland glutathione content decreased significantly after ethylenesupplementation on day 6 after detachment. Amino-oxyacetic acidtreatment prevented the increases in conductivity and 2',7'-dichlorofluoresceinoxidation, did not alter the activities of antioxidant enzymesand significantly increased the content of -tocopherol and glutathioneas compared to control carnation petals. Paraquat treatmentparalleled qualitatively ethylene supplementation after 6 dof cutting. Taken as a whole, the data presented here may be understoodas experimental evidence of a close association between ethyleneproduction and oxidative stress in ageing of cut carnations. Key words: Carnation petals, oxidative stress, ethylene, antioxidants     

Ethylene production by apple protoplasts

Freshly prepared protoplasts from apple tissue that produced ethylene were obtained. Ethylene production was inhibited by osmotic shock, 0.01% Triton X-100, and aminoethoxyvinyl glycine. Protoplasts as well as the ethylene system were not greatly affected by protease treatment.     

Modulation of ethylene synthesis in acotyledonous soybean and wheat seedlings

The characteristics of ethylene production and ACC conversion in 8-day-old soybean seedlings were examined and a relationship between cytochrome P-450 activity and ethylene-forming enzyme (EFE) activity was found. An atmosphere containing 10% carbon monoxide (CO) significantly inhibited ethylene production and ACC conversion in control soybean seedlings, but had only a slight effect on soybean seedlings treated with uniconazole. Foliar application of triclopyr, a pyridine analogue of the phenoxy herbicides, significantly increased ethylene production and ACC conversion in control, but not in uniconazoletreated seedlings. Triclopyr treatment also resulted in a three-fold increase in extractable cytochrome P-450 of 5-day-old etiolated soybeans. At equimolar concentrations tetcyclacis was more effective than uniconazole in reducing shoot elongation and endogenous ethylene production. Although uniconazole and tetcyclacis did not inhibit ACC conversion in nonherbicide-treated soybean seedlings, they did prevent the observed increase in ACC-dependent EFE activity following triclopyr application. However, the rate of ACC conversion in etiolated soybean segments was sensitive to uniconazole, and tetcyclacis inhibited the rate of ACC conversion by 2.6-fold in etiolated soybean segments within 4 h after treatment. Microsomal membranes were isolated from 5-day-old naphthalic anhydride-treated etiolated wheat shoots as this tissue contains much higher cytochrome P-450 levels than soybean shoots. Optical difference spectroscopy demonstrated that ACC generated binding spectrum characteristic of a reverse-type-I cytochrome P-450 substrate when combined with reduced microsomes. In vitro conversion of ACC to ethylene by microsomal membranes was NADPH-dependent, inhibited by CO, and had an apparent K_m and V_max of 45 M and 0.345 nl/mg protein/h, respectively. These results suggest that cytochrome P-450-mediated monooxygenase reactions may be intimately involved in the conversion of ACC to ethylene in young soybean and wheat seedlings.     

Determination of an ethylene biosynthesis pathway in the unicellular green alga,Haematococcus pluvialis. Relationship between growth and ethylene production

In the freshwater ChlorophyceaeHaematococcus pluvialis, precursors of ethylene biosynthesis cycle are the same as those of higher plants: L-methionine S-adenosylmethionine 1-aminocyclopropane-1-carboxylic acid ethylene. However, the enzymatic complex of the last step of ethylene synthesis-ACCoxidase-differs from that of higher plants. It is stimulated by Co²⁺ (at least 10^-5 M), Mn²⁺ (at least 10^-6 M) and Ag²⁺ (at least 10^-4 M), inhibited by Cu²⁺ (at least 10^-5 M) and not affected by Zn²⁺, Fe²⁺ or Mg²⁺. ACCoxidase is also inhibited by salicylhydroxamic acid and by dark. Ethylene production is more important in young, mobile, green cells in active growth phase than in old, encysted and red cells in stationary growth phase. No peaks in ethylene production or respiration were observed during batch culture, as opposed to the situation with climacteric fruits.     

The relationships between ethylene production and germination ofCicer arietinum seeds

The germination percentage of chick-pea (Cicer arietinum) Seeds was greatly reduced by temperatures of 30°C and 35°C. This thermoinhibition was overcome by ethylene (ethrel). Both ABA and PEG diminished ethylene production and germination percentage in a parallel way. FC, MGBG and CHA stimulated both ethylene production and germination. AVG reduced ethylene production to some extent but did not inhibit germination. CoCl₂ and PG completely prevented both ethylene production and germination; this effect was reversed by ethylene but not by its immediate precursor ACC. NBE prevented both germination and ethylene production. Our results suggest that high ethylene production rates are not essential for germination of chick-pea seeds but that certain quantities of ethylene may be required.     

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. -DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and -aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.     

The mechanism involved in ethylene-enhanced ethylene synthesis in carnations

The plant hormone ethylene triggers and enhanced ethylene synthesis in certain ripening fruits and senescing flowers. Unlike most carnation (Dianthus caryophyllus L.) cultivars exhibiting climacteric rise in ethylene production at the onset of senescence, cv. Sandrosa does not show this phenomenon naturally. In order to understand the mechanism of autocatalytic ethylene production, we exposed carnation flowers cv. Sandrosa to ethylene which resulted in an enhanced capacity for ethylene synthesis in the petals. A short time response of one hour was measured for an increase in ACC oxidase activity, about five hours in advance of an increase in ACC synthase activity and ethylene production. The observed enhancement was dependent on the presence of exogeneous ethylene, and could be partially inhibited by prior treatment of the petals with -amanitin or cycloheximide. The results of the present study suggest that in response to ethylene, activation of an existing enzyme is taking place first. This is followed by an increase in expression of ACC oxidase and ACC synthase mRNAs.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DTT dithiothreitol - PMSF phenyl-methylsulfonyl fluoride - SAM S-adenosyl-L-methionine     

Carbon dioxide and 1-MCP inhibit ethylene production and respiration of pear fruit by different mechanisms

Ethylene production in relation to O2 partial pressure of whole pear fruit stored at 2C could be described by a Michaelis-Menten equation. This was indicated by the use of a gas exchange model. The maximum ethylene production rate was strongly inhibited while the KmO2 value (1.25 kPa) was not affected by elevated CO2. Ethylene production was also inhibited by 1-MCP, an inhibitor of ethylene perception. The reduction in ethylene production by CO2 was similar for 1-MCP treated and untreated pears. Elevated CO2, therefore, must have had an influence on ethylene production other than through ethylene perception. A possible site of inhibition by CO2 is the conversion of ACC to ethylene. The O2 uptake rate in relation to O2 partial pressure of whole pear fruit could be described by a Michaelis-Menten equation. The O2 uptake rate was inhibited by elevated CO2 at a level similar to the inhibition of ethylene production. Again the KmO2 value (0.68 kPa) was not affected by CO2. Using 1-MCP treatments it was shown that there was no direct effect of inhibited ethylene production on O2 uptake rate.     

Gibberellin-induced ethylene production and its effect on cowpea epicotyl elongation

The effect of gibberellin A₁ (GA₁) on production of ethylene by cowpea (Vigna sinensis cv Blackeye pea no. 5) epicotyl explants and its relationship to epicotyl elongation was investigated. The explants were placed upright in water and incubated in sealed culture tubes or in large jars. GA, and IAA in ethanol solution were injected into the subapical tissues of the decapitated epicotyls. Cowpea epicotyl explants elongated after GA but not after IAA treatment, and they were very sensitive to exogenous ethylene. As little as 0.14 1/1 ethylene reduced significantly GA₁-induced epicotyl elongation.Treatment with GA₁ induced the production of ethylene which began 10 h after GA application, showed a peak at about 22 h and then declined. The yield of ethylene was proportional to the amount of GA, injected. The inhibition of epicotyl elongation in closed tubes was avoided by absorbing ethylene released with Hg(Cl0₄)₂ , or by adding AVG to the incubation solution to inhibit ethylene production. Treatment with IAA elicited a rapid production of ethylene which ceased about 10 h after application. The effects of IAA and GA₁ on ethylene production were additive.Abbreviations AVG aminoethoxyvinylglycine 2-amino-4-(2-aminoethoxy)-trans-3butenoic acid - ACC 1-aminocyclopropane-1-carboxylic acid - GA gibberellin - IAA indole-3-acetic acid     

Ethylene-promoted malonylation of 1-aminocyclopropane-1-carboxylic acid participates in autoinhibition of ethylene synthesis in grapefruit flavedo discs

The increase in ethylene formation and in 1-aminocyclopropane-1-carboxylic acid (ACC) content in flavedo tissue of grapefruit (Citrus paradisi Macfad. cv. Ruby Red) in response to excision was markedly inhibited by exogenous ethylene. Ethylene treatment inhibited the synthesis of ACC, but increased the tissue's capability to malonylate ACC to N-malonyl-ACC, resulting in further reduction in the endogenous ACC content. The development of extractable ACC-malonyl-transferase activity in the tissue was markedly promoted by treatment with exogenous ethylene. These results indicate that the autoinhibition of ethylene production in this tissue results not only from suppression of ACC synthesis, but also from promotion of ACC malonylation; both processes reduce the availability of ACC for ethylene synthesis.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethyoxyvinylglycine (2-amino-4-(2-aminoexthoxy)-trans-3-butenoic acid) - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid     

Coleoptile removal-induced ethylene production in winter rye seedlings

Coleoptile removal-induced ethylene production was investigated in light-grown winter rye seedlings. Removal of the coleoptile induced 1-aminocyclopropane-l-carboxylic acid (ACC) synthesis and ethylene production by primary leaves and caused an inhibition of elongation growth of the leaves. The activity of ethylene-forming enzyme (EFE) was associated with the increase in ethylene evolution. Both rise in ethylene and ACC production, as well as EFE activity were inhibited by cycloheximide. Wounding the tissue 40 min after the initial treatment resulted in the second increase in ethylene evolution. Derooting of the seedlings without coleoptile removal did not induce ethylene production. It is suggested that the coleoptile represents a barrier for wound-induced ethylene production from actively growing leaf tissue.     

Studies on octylphenoxy surfactants: X. Effect of oxyethylene chain length on ethylene production by cowpea and mung bean and comparison with linear alcohol hydrophobes

The effects of ethoxy (EO) chain length on surfactant-induced ethylene production for selected octylphenoxy (OP) and linear alcohol (LA) surfactants were established using primary leaves of cowpea (Vigna unguiculata (L.) Walp. subsp. unguiculata Dixielee) seedlings. OP surfactant-induced ethylene production was concentration dependent and decreased log linearly with increasing EO content. C_12–15 LA-induced ethylene production decreased log linearly with increasing EO content at 0.1%; however, at 1.0% the relationship was curvilinear with maximum response at 7 EO. Relationships for the C_9–11 and C₉ LA series were nonlinear with greatest biological activity at intermediate (8–12) EO content. Short EO chain length OP surfactants were only slightly water soluble, and induced low levels of ethylene production and phytotoxicity. Addition of OP+1EO to a long chain, water soluble, non-ethylene inducing surfactant (OP+40EO) solution significantly increased ethylene production by OP+1EO in cowpea. A similar response was found for surfactant-induced phytotoxicity and EO chain length as between ethylene production and EO content. Similar EO chain length and ethylene production relationships were found for germinating mung bean (Vigna radiata (L.) R. Wilcz) seeds as for ethylene production and phytotoxicity in cowpea. Radicle growth was markedly inhibited by OP surfactants with an EO chain length of 10 or less and, in some cases, radicles were irreversibly damaged by ethylene inducing surfactants.     

Involvement of cellular membrane in regulation of ethylene production

Effects of various substances which would change membrane structureson auxin-induced ethylene production in etiolated mung beanhypocotyl segments were investigated. Auxin-induced ethyleneproduction was not affected by treatment of tissue segmentswith pronase, trypsin, Con A and amphotericin B. PhospholipaseD inhibited ethylene production and its action was reversible,suggesting that the inhibitory action may not be due to enzymaticaction. Lecithin, Tween 20, Triton X 100 and SDS also significantlyinhibited ethylene production. Inhibition by the former twosubstances was completely, and that of the latter two was partiallyreversed by removing them from the tissue segments. All thesubstances which inhibited ethylene production also suppressedIAAsp formation by the tissue. It was concluded that inhibitionresulted from structural changes of cell membranes caused byreversible interaction with the lipophilic substances and thatethylene production and IAAsp formation were under the controlof membrane function. ¹ Supported in part by grants from the Ministry of Educationand the Ministry of Agriculture and Forestry, Japan. (Received December 24, 1976; )     

{alpha}-Aminoisobutyric acid : A probable competitive inhibitor of conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

Of 16 compounds related to 1-aminocyclopropane-1-carboxylicacid (ACC), aminoisobutyric acid (AIB) inhibited the productionof endogenous ethylene in the cotyledonary segments of cocklebur(Xanthium pennsylvanicum Wallr.) seeds most strongly. AIB at4 mM inhibited the formation of ethylene by about 50%, althoughthe O₂ uptake of the segments was not affected even at 20 mM.AIB also inhibited ethylene formation in the stem segments ofetiolated pea (Pisum sativum L. cv. Alaska) seedlings. Kineticanalysis with cell free extracts from etiolated pea shoots revealedthat AIB competitively inhibits the conversion of ACC into ethylene. (Received May 26, 1980; )     

Cytokinin-induced ethylene biosynthesis in nonsenescing cotton leaves

The influence of cytokinins on ethylene production was examined using cotton leaf tissues. Treatment of intact cotton (Gossypium hirsutum L. cv LG 102) seedlings with both natural and synthetic cytokinins resulted in an increase in ethylene production by excised leaves. The effectiveness of the cytokinins tested was as follows: thidiazuron BA isopentyladenine ≥ zeatin kinetin. Using 100 micromolar thidiazuron (TDZ), an initial increase in ethylene production was observed 7 to 8 hours post-treatment, reached a maximum by 24 hours and then declined. Inhibitors of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis and its oxidation to ethylene reduced ethylene production 24 hours post-treatment; however, by 48 hours only inhibitors of ACC oxidation were effective. The increase in ethylene production was accompanied by a massive accumulation of ACC and its acid-labile conjugate. TDZ treatment resulted in a significant increase in the capacity of tissues to oxidize ACC to ethylene. Endogenous levels of methionine remained constant following TDZ treatment. It was concluded that the stimulation of ethylene production in cotton leaves following cytokinin treatment was the result of an increase in both the formation and oxidation of ACC.     

Effect of antagonists of ethylene action on binding of ethylene in cut carnations

Five hours after cut carnations had been treated with a pulse of 1 or 4 mM silver thiosulfate (STS), in vivo ethylene binding in petals was inhibited by 22 and 29%, respectively. When binding was measured 4 days after the 4-mM STS treatment, binding was inhibited by 81%. 2,5-Norbornadiene, which substantially delays carnation senescence, inhibited ethylene binding by 41% at a concentration of 1000 l/l. The Kd for ethylene binding in carnations was estimated to be 0.1 l/l in petals and 0.09 l/l in leaves. The concentration of binding sites was estimated to be 6.0×10^–9 mol/kg of petals and 2.0×10^–9 mol/kg of leaves     

Role of ethylene action in ethylene production and poststorage leaf senescence and survival of pelargonium cuttings

This study investigated the role of ethylene action in ethylene production and in poststorage performance of pelargonium cuttings. Cuttings of zonal pelargonium (Pelargonium x hortorum L.H. Bailey) of the cultivars Isabell and Mitzou were treated with ethylene and with the ethylene action inhibitors 1-methylcyclopropene (MCP), silver-thiosulfate (STS) and silver nitrate (SN) and were stored in the dark at different temperatures (5, 12, and 20 °C) for 48 h. Ethylene concentrations in the storage boxes were monitored and poststorage leaf senescence, survival and root formation of cuttings were determined. Applications of MCP resulted in a significant increase of ethylene evolution by cuttings of both cultivars which was more pronounced with increasing storage temperature. After 48 h of storage at 20 °C, ethylene concentrations were more than 20-fold higher for the MCP-treated cuttings as compared to those of the untreated controls. Also preharvest applications of STS and SN increased ethylene evolution by cuttings, even though these effects were less pronounced. However, application of these inhibitors caused severe poststorage leaf injury. Application of ethylene during storage had no effect on subsequent leaf damage. Leaf senescence during rooting and decay of cuttings, which raised with increasing storage temperature, could only partially been reduced by MCP. The results strongly support the conclusion, that in zonal pelargonium cuttings, ethylene production is controlled by autoinhibition, and clearly indicate, that temperature dependent processes other than ethylene action are substantially involved in storage-induced leaf senescence and decay.     

冷激诱导高温胁迫下番茄幼苗矮化机理

为了探明冷激诱导高温胁迫下番茄幼苗矮化的机理,育苗期间,每天8:00对幼苗分别进行5、10、15 ℃持续时间依次为10、20、30 min的冷激处理,测试了不同冷激强度下番茄幼苗乙烯释放速率,研究了冷激处理T10 ℃ D10 min(10 ℃持续10 min)结合不同生长调节物质对番茄幼苗乙烯释放速率、赤霉素(GA₃)含量和幼苗生长特性的影响.结果表明： 冷激处理刺激了番茄幼苗乙烯的产生,随着冷激温度的降低和冷激时间的延长,冷激诱导乙烯释放的效应显著增强.5 ℃持续30 min的冷激处理番茄幼苗乙烯产生速率最大,达到60.3 nL·h^-1·g^-1,为对照的6.5倍;乙烯利(ETH)、硫代硫酸银(STS)、GA₃和多效唑(PP333)均不能完全阻止冷激处理T10 ℃D10 min诱发的高乙烯产生率.冷激处理T10 ℃D10 min番茄幼苗茎叶GA₃含量为80.8 μg·g^-1,与对照(130.6 μg·g^-1)相比降低了38.1%.喷施ETH、STS对冷激诱发的幼苗矮化效应无显著影响,而GA₃显著减弱了冷激的矮化效应,PP333显著增强了冷激的矮化效应.以株高作为衡量指标,浓度为4.0 mg·L^-1的PP333处理,相当于10 ℃冷激处理.冷激诱导的番茄幼苗矮化效应主要原因在于冷激降低了番茄幼苗茎叶GA₃的含量.T10 ℃ D10 min可以在降低幼苗株高的同时不降低幼苗干物质的积累.     

Some characteristics of ethylene production in peach (Prunus persica L.) seeds

Summary The seed of peach fruits develop the capacity to produce ethylene with a lag phase of about 1 h after excision. The site of ethylene synthesis is in the seed coat and rates as high as 6,000 l kg^–1 h^–1 were recorded. Ethylene production was reduced to less than 1% of the control by 10 g/ml cycloheximide. Although the tissue had only a small methionine pool, supplying the seed with exogenous methionine did not influence ethylene production at any stage of seed development. Label from [U-¹⁴C]methionine was readily incorporated into ethylene.