Biosynthesis of ethylene. Methanesulphinic acid as cofactor in the enzymic formation of ethylene from methional

The second cofactor in the peroxidase-catalysed formation of ethylene from methional in cauliflower extracts was identified as methanesulphinic acid. The progress of the reaction is described and the activities of related sulphinic acids were determined.     

Ethylene inhibitors enhance in vitro root formation from apple shoot cultures

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and three ethylene inhibitors, AgNO₃, aminoethoxyvinyglycine (AVG) and CoCl₂, on root formation were tested in vitro using shoot cultures of the apple (Malus×domestica Borkh.) cultivar Royal Gala. ACC inhibited root formation by delaying root emergence and increasing callus formation at the bases of shoots. In contrast, ethylene inhibitors promoted root formation. Both AgNO₃ and AVG at the appropriate concentrations increased the percentage of shoots producing roots and reduced callus formation at the base of these shoots. AgNO₃ stimulated root emergence and enhanced root growth, while AVG increased the number of roots per shoot. CoCl₂ slightly increased root number and rooting efficiency. These promotive effects may result from a reduction in ethylene concentration or inhibition of ethylene action. The results found in this study may be used to improve the rooting efficiency of other apple cultivars and rootstocks, and possibly of other plant species. Received: 2 March 1997 / Revision received: 1 July 1997 / Accepted: 18 July 1997     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid by microsomal membranes from senescing carnation flowers

Isolated membranes from the petals of senescing carnation flowers (Dianthus caryophyllus L. cv. White-Sim) catalyze the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. A microsomal membrane fraction obtained by centrifugation at 131,000 g for 1 h proved to be more active than the membrane pellet isolated by centrifugation at 10,000 g for 20 min. The ethylene-producing activity of the microsomal membranes is oxygen-dependent, heat-denaturable, sensitive to n-propyl gallate, and saturable with ACC. Corresponding cytosol fractions from the petals are incapable of converting ACC to ethylene. Moreover, the addition of soluble fraction back to the membrane fraction strongly inhibits the ACC to ethylene conversion activity of the membranes. The efficiency with which isolated membranes convert ACC to ethylene is lower than that exhibited by intact flowers based on the relative yield of membranes per flower. This may be due to the presence of the endogenous soluble inhibitor of the reaction, for residual soluble fraction inevitably remains trapped in membrane vesicles isolated from a homogenate.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA aminoxyacetic acid - AVG aminoethoxyvinylglycine - EPPS N-2-hydroxyethylpiperazine propane sulfonic acid     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

Ethylene formation in Pisum sativum and Vicia faba protoplasts

Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-1-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-¹⁴C]methionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Evaluation of the role of methional, 2-keto-4-methylthiobutyric acid and peroxidase in ethylene formation by Escherichia coli.

During growth of Escherichia coli strain SPA O in the presence of methionine, an intermediate accumulates in the medium. This intermediate reacts with 2,4-dinitrophenylhydrazine, and can be degraded to ethylene either enzymically or photochemically, the latter being stimulated by the addition of a flavin. The pH optimum for the photochemical degradation of this intermediate and 2-keto-4-methylthiobutyric acid (KMBA) is pH 3 whereas the optimum for methional is pH 6. The enzyme which converts the intermediate to ethylene also converts KMBA to ethylene and has many of the properties of a peroxidase including inhibition by catalase, cyanide, azide and anaerobiosis. The enzyme which synthesizes the intermediate is not known but requires oxygen and pyridoxal phosphate. A pathway for ethylene biosynthesis is proposed in which methionine is converted to KMBA which can be degraded either by peroxidase or in a flavin-mediated photochemical reaction. Its relevance to the properties of other ethylene-producing bacteria and to the proposed pathway of ethylene release by higher plants is discussed.     

Ethylene involvement in vegetative bud formation in tobacco thin layers

Summary The role of ethylene in vegetative bud regeneration was studied in cultured tobacco (Nicotiana tabacum L. cvSamsun) thinlayer expiants. The experimental approach consisted in supplementing the bud-inducing medium with an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), an ethylene antagonist, silver thiosulphate (STS), or an ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA), at various concentrations. The organogenic response was assessed both macroscopically (percentage of bud-forming expiants, final number of buds per expiant) and cytohistologically (number, characteristics, and localisation of meristemoids and bud primordia). The time course of ethylene production during culture was also evaluated. At the end of culture (day 27) all the expiants treated with these compounds had a lower number of buds compared to controls. STS was detrimental to meristemoid initiation at all the concentrations tested. In contrast, 0.5 M AVG, which strongly inhibited ethylene production, provoked a large increase in the formation of meristemoids early in culture and the appearance of anomalous (twin) buds. CEPA reduced meristemoid formation but, at the lower concentrations (1 and 10 M) speeded up bud emergence. On the whole it mainly favoured disorganised growth and xylogenesis. The results of this work highlight the contrasting effects of ethylene in relation to the two critical stages of the organogenic process, i.e., meristemoid formation and bud primordium development.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - STS silver thiosulphate - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - BA benzyladenine - HF hormone-free     

Ethylene accumulation in waterlogged Rumex plants promotes formation of adventitious roots

Accumulation of the gaseous plant hormone ethylene is very importantfor the induction of several responses of plants to flooding.However, little is known about the role of this gas in the formationof flooding-induced adventitious roots. Formation of adventitiousroots in Rumex species is an adaptation of these plants to floodedsoil conditions. The large air-spaces in these roots enablesdiffusion of gases between shoot and roots. Application of ethylene to non-flooded Rumex plants resultedin the formation of adventitious roots. In R. palustris Sm.shoot elongation and epinasty were also observed. The numberof roots in R. thyrsiflorus Fingerh. was much lower than inR. palustris, which corresponds with the inherent differencein root forming capacity between these two species. Ethyleneconcentrations of 1.5–2µI I_{– 1} induced a maximumnumber of roots in both species. Quantification of ethylene escaping from root systems of Rumexplants that were de-submerged after a 24 h submergence periodshowed that average ethylene concentrations in submerged rootsreached 1.8 and 9.1 µl I_–1 in R. palustris and R.thyrsiflorus, respectively. Inhibition of ethylene productionin R. palustris by L--(2-aminoethoxyvinyl)-glycine (AVG) or-aminobutyric acid (AIB) decreased the number of adventitiousroots induced by flooding, indicating that high ethylene concentrationsmay be a prerequisite for the flooding-induced formation ofadventitious roots in Rumex species. Key words: Adventitious roots, epinasty, ethylene, flooding, Rumex, shoot elongation     

Ethylene in relation to compression wood formation in Abies balsamea shoots

 The terminal (1-year-old) shoot of dormant, 2-year-old balsam fir [Abies balsamea (L.) Mill.] seedlings was either left vertically oriented or tilted to an angle of 60° from the vertical (tilting experiment), or was ringed with N-1-naphthylphthalamic acid (NPA), an inhibitor of indole-3-acetic acid transport, at a concentration of 0, 1 or 10 mg g⁻¹ lanolin (NPA experiment). After 6 weeks of growth, ethylene evolution from the cambial region was measured by gas chromatography – flame ionization detection, and tracheid production and compression wood formation were determined by microscopy. In vertical seedlings of the tilting experiment and in 0 mg g⁻¹-treated seedlings of the NPA experiment, compression wood was not formed and neither ethylene evolution nor tracheid production varied longitudinally or circumferentially within the stem. Tilting induced compression wood formation and increased ethylene evolution and tracheid production on the lower side of the stem, while decreasing tracheid production on the upper side. Compression wood formation was induced and tracheid production and ethylene evolution were stimulated at and above the point where 1 or 10 mg NPA g⁻¹ was applied, whereas below this point compression wood was not formed and tracheid production was inhibited. In both tilting and NPA experiments, there was a positive correlation between ethylene evolution and tracheid production when data from all seedlings were analyzed, but not when data from seedlings forming compression wood were excluded. The results indicate that cambial region ethylene evolution is enhanced when compression wood is being formed, and that the enhancement is related to compression wood formation per se rather than the associated increase in tracheid production. Received: 19 February 1998 / Accepted: 26 October 1998     

Effect of low and high methional concentrations on prostaglandin biosynthesis in microsomes from bovine and sheep vesicular glands.

The effect of methional on prostaglandin biosynthesis from 5,8,11,14-eicosatetraenoic acid was studied with microsomes from both bovine vesicular glands (BVG) and sheep vesicular glands (SVG). Ethylene was identified when methional was added to the fatty acid-microsome incubation systems showing that oxygen centered radicals such as hydroxyl radical were generated during incubation. A low methional level, 1 mM, enhanced the rate of prostaglandin biosynthesis in both BVG and SVG. A high methional level, 10 mM, inhibited prostaglandin biosynthesis in both BVG alone and SVG solubilized with 1% Tween 20. The inhibitory effect of 10 mM methional was reversed by lyophilization. These data suggest that oxygen centered radicals are used in prostaglandin biosynthesis even though they inactivate the enzyme complex.     

Effect of low and high methional concentrations on prostaglandin biosynthesis in microsomes from bovine and sheep vesicular glands

The effect of methional on prostaglandin biosynthesis from 5,8,11, 14-eicosatetraenoic acid was studied with microsomes from both bovine vesicular glands (BVG) and sheep vesicular glands (SVG). Ethylene was identified when methional was added to the fatty acid-microsome incubation systems showing that oxygen centered radicals such as hydroxyl radical were generated during incubation. A low methional level, 1 mM, enhanced the rate of prostaglandin biosynthesis in both BVG and SVG. A high methional level, 10 mM, inhibited prostaglandin biosynthesis in both BVG alone and SVG solubilized with 1% Tween 20. The inhibitory effect of 10 mM methional was reversed by lyophilization. These data suggest that oxygen centered radicals are used in prostaglandin biosynthesis even though they inactivate the enzyme complex.     

Ethylene influences green plant regeneration from barley callus

The plant hormone ethylene is involved in numerous plant processes including in vitro growth and regeneration. Manipulating ethylene in vitro may be useful for increasing plant regeneration from cultured cells. As part of ongoing efforts to improve plant regeneration from barley (Hordeum vulgare L.), we investigated ethylene emanation using our improved system and investigated methods of manipulating ethylene to increase regeneration. In vitro assays of regeneration from six cultivars, involving 10 weeks of callus initiation and proliferation followed by 8 weeks of plant regeneration, showed a correlation between regeneration and ethylene production: ethylene production was highest from ‘Golden Promise’, the best regenerator, and lowest from ‘Morex’ and ‘DH-20’, the poorest regenerators. Increasing ethylene production by addition of 1-aminocyclopropane 1-carboxylic acid (ACC) during weeks 8–10 increased regeneration from Morex. In contrast, adding ACC to Golden Promise cultures during any of the tissue culture steps reduced regeneration, suggesting that Golden Promise may produce more ethylene than needed for maximum regeneration rates. Blocking ethylene action with silver nitrate during weeks 5–10 almost doubled the regeneration from Morex and increased the Golden Promise regeneration 1.5-fold. Silver nitrate treatment of Golden Promise cultures during weeks 8–14 more than doubled the green plant regeneration. These results indicate that differential ethylene production is related to regeneration in the improved barley tissue culture system. Specific manipulations of ethylene were identified that can be used to increase the green plant regeneration from barley cultivars. The timing of ethylene action appears to be critical for maximum regeneration.