Biosynthesis of ethylene. 4-Methylmercapto-2-oxobutyric acid, an intermediate in the formation from methionine

The enzyme responsible for the conversion of methionine into a precursor of ethylene in cauliflower florets is a transaminase. The formation of 4-methyl-mercapto-2-oxobutyric acid by this enzyme has been shown. The oxo acid stimulates the synthesis of ethylene when added to floret tissue, and tracer experiments have shown that (14)C is incorporated into ethylene from the labelled oxo acid. The evidence is consistent with the view that the oxo acid is an intermediate in the formation of ethylene from methionine.     

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.     

Biosynthesis of ethylene. Enzymes involved in its formation from methional

1. Two enzymes were shown to be necessary for the production of ethylene from methional; they were separated from extracts of cauliflower florets by fractionation on Sephadex and other methods. 2. The first enzyme, generating hydrogen peroxide, appears to be similar to the fungal glucose oxidase, for like the latter it is highly specific for its substrate d-glucose. 3. The second enzyme, in the presence of cofactors and peroxide generated by the first enzyme, cleaves methional to ethylene. 4. It was also found that hydrogen peroxide in these reactions may be replaced by hydroperoxide generated from linolenic acid by lipoxidase enzymes. 5. Dihydroxyphenols were shown to have a marked inhibitory effect on these reactions and to account for the initial phase of low activity that is always observed in aqueous extracts prepared from the floret tissue.     

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.     

Biosynthesis of ethylene: Formation of ethylene from methional by a cell-free enzyme system from cauliflower florets

1. The formation of ethylene from cauliflower florets is stimulated by the addition of either methionine or its hydroxy analogue. 2. Formation of ethylene from these compounds may also be demonstrated in cell-free extracts, but the most rapid formation is achieved by the addition of methional. 3. Fractionation of such extracts has shown that both particulate and non-particulate fractions are necessary for the formation of ethylene from methionine or its hydroxy analogues, but only the non-particulate fraction is necessary for its formation from methional. 4. A study of this system has shown that the conversion of methional into ethylene requires the presence of two enzyme systems, the first generating peroxide and the second catalysing the conversion of methional into ethylene in the presence of peroxide. 5. The presence of a heat-stable factor in cauliflower extracts that is necessary for the full activity of the enzyme converting methional into ethylene has also been shown. 6. The nature of this factor is at the present unknown; it is not a metal nor is it identifiable with many of the known coenzymes.     

Purification of an alcohol dehydrogenase involved in the conversion of methional to methionol in <Emphasis Type="Italic">Oenococcus oeni</Emphasis> IOEB 8406

Oenococcus oeni, the major lactic acid bacteria involved in malolactic fermentation (MLF) in wine, is able to produce volatile sulfur compounds from methionine. Methional reduction is the last enzymatic step of methionol synthesis in methionine catabolism. Alcohol dehydrogenase (ADH) activity was found to be present in the soluble fraction of O. oeni IOEB 8406. An NAD(P)H-dependent ADH involved in the reduction of methional was then purified to homogeneity. Sequencing of the purified enzyme and amino acid sequence comparison with the database revealed the presence of a conserved sequence motif specific to the medium-chain zinc-containing NAD(P)H-dependent ADHs. Despite the great importance of ADH activities in wine flavor modification, this is the first report of the purification of an ADH isolated from O. oeni. The purified ADH does not seem to be involved in the modification of buttery and lactic notes or to be involved in the specific formation of volatile alcohols during MLF. The enzyme was not strictly specific of methional reduction and the highest reducing activity was obtained with acetaldehyde as substrate. The function of the purified ADH remains unclear, although the role of the sulfur atom in methional molecules in the interaction between enzyme and substrate was evidenced.     

The Production of Ethylene by Whole Apple Fruits and by Tissue 1

It is shown that the production of ethylene by whole apples,discs of peel prepared from them, and extracts prepared fromthe discs and supplied with linolenic acid, increases simultaneouslyduring the development of the respiration climacteric in apples.As the climacteric peak is reached, the ability of the extractsto produce ethylene declines and this is associated with a rapidloss of ethylene-producing activity on short term (up to 24h) ageing of the peel discs from which the extracts were obtained. It is suggested that the gmn.ll ethylene production by extracts(with linolenic acid) from pre-climacteric fruits, which arenot themselves evolving sufficient ethylene for its detectionin the ambient atmosphere, may be explained on the grounds thateven before the climacterio the fruit is producing small amountsof ethylene within the tissue; the tissue, as the climactericphase is approached, becomes more sensitive to ethylene andautocatalysed production of the gas then stimulates the fruitinto the respiration climacteric.     

Manipulation of ethylene biosynthesis

The plant hormone ethylene is involved in many plant processes ranging from seed germination to leaf and flower senescence and fruit ripening. Ethylene is synthesized from methionine, via S-adenosyl-L-methionine (SAM) and 1-amino-cyclopropane-1-carboxylic acid (ACC). The key ethylene biosynthetic enzymes are ACC synthase (ACS) and ACC oxidase (ACO). Manipulation of ethylene biosynthesis by chemicals and gene technology is discussed. Biotechnological modification of ethylene synthesis is a promising method to prevent spoilage of agricultural and horticultural products.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.     

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     

Methionine metabolism and ethylene formation in etiolated pea stem sections

Stem sections of etiolated pea seedlings (Pisum sativum L. cv. Alaska) were incubated overnight on tracer amounts of l-[U-(14)C]methionine and, on the following morning, on 0.1 millimolar indoleacetic acid to induce ethylene formation. Following the overnight incubation, over 70% of the radioactivity in the soluble fraction was shown to be associated with S-methylmethionine (SMM). The specific radioactivity of the ethylene evolved closely paralleled that of carbon atoms 3 and 4 of methionine extracted from the tissue and was always higher than that determined for carbon atoms 3 and 4 of extracted SMM.Overnight incubation of pea stem sections on 1 millimolar methionine enhanced indoleacetic acid-induced ethylene formation by 5 to 10%. Under the same conditions, 1 millimolar homocysteine thiolactone increased ethylene synthesis by 20 to 25%, while SMM within a concentration range of 0.1 to 10 millimolar did not influence ethylene production. When unlabeled methionine or homocysteine thiolactone was applied to stem sections which had been incubated overnight in l-[U-(14)C]methionine, the specific radioactivity of the ethylene evolved was considerably lowered. Application of unlabeled SMM reduced the specific radioactivity of ethylene only slightly.     

Rapidly induced ethylene formation after wounding is controlled by the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis

Bean leaves from Phaseolus vulgaris L. var. Pinto 111 react to mechanical wounding with the formation of ethylene. The substrate for wound ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC). It is not set free by decompartmentation but is newly synthesized. ACC synthesis starts 8 to 10 min after wounding at 28°C, and 15 to 20 min after wounding at 20°C. Aminoethoxyvinylglycine (AVG), a potent inhibitor of ethylene formation from methionine via ACC, inhibits wound ethylene synthesis by about 95% when applied directly after wounding (incubations at 20°C). AVG also inhibits the accumulation of ACC in wounded tissue. AVG does not inhibit conversion of ACC to ethylene. Wound ethylene production is also inhibited by cycloheximide, n-propyl gallate, and ethylenediaminetetraacetic acid.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine - EDTA ethylenediaminetetraacetic acid     

Stimulation of Fruit Ethylene Production by Wounding and by Botrytis cinerea and Geotrichum candidum Infection in Normal and Non-Ripening Tomatoes

Inoculations with both Botrytis cinerea and Geotrichum candidum stimulated ethylene evolution in the pre-climacteric normal tomato fruit and the non-ripening nor mutant which did not show any rise in ethylene when uninfected. In the post-climacteric normal fruits, new peaks in ethylene production were formed. The rise in ethylene evolution in all types of infected fruits has already been detected during the incubation period of the disease. Ethylene peaks were detected earlier and were higher in fruits infected with B. cinerea than with G. candidum, coinciding with the faster rate of growth of the former. Mechanical wounding also stimulated ethylene synthesis by the non-ripening fruits, production being directly proportional to wound dimension. Considerably higher rates of ethylene were recorded for infected fruits than for mechanically-injured fruits in which wound dimensions were similar to those of lesion development. Applying aminoxyacetic acid at the site of inoculation inhibited ethylene production by 55–60 % in the normal fruits and by about 80 % in the nor mutant fruits. A similar pathway of ethylene synthesis was suggested for normally ripening tomato fruit and non-ripening infected tissues.     

Engineering of cysteine and methionine biosynthesis in potato

Summary. Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato. Received December 19, 2001 Accepted January 7, 2002     

Efficiency of indoleacetic acid,gibberellic acid and ethylene synthesized in vitro by Fusarium culmorum strains with different effects on cereal growth

Three different Fusarium culmorum strains having a pathogenic, a deleterious (deleterious rhizosphere microorganism), or a promoting (plant growth promoting fungus) effect on plant growth were studied for their ability to synthesize in vitro the phytohormones indoleacetic acid (IAA), gibberellic acid (GA), and ethylene. All the phytohormones tested were synthesized in cultures supplemented with wide concentration ranges of glucose and tryptophan or methionine (precursors of phytohormone synthesis). The amounts of these secondary metabolites synthesized by the particular strains were found to be significantly different. The non-pathogenic PGPF strain (DEMFc2) synthesized the highest amounts of IAA and GA, a fact that could be responsible for the growth-promoting properties of this strain. A pathogenic strain synthesized the highest amount of ethylene, which could be responsible for the negative effect of this strain on plant growth. F. culmorum isolates with a high capacity for IAA synthesis also have a high capacity for GA synthesis and irrespective of the growth conditions, a high positive correlation (R > 0.9) between the concentrations of synthesized IAA and GA in F. culmorum cultures was found. It is worth mentioning that the optimal conditions for the growth of F. culmorum isolates and the synthesis of the individual phytohormones differed from one another. The optimal growth conditions were 1.0% of glucose and 9.9 mM of methionine or 6.0 mM of tryptophan. The optimal conditions for ethylene synthesis were 0.5% of glucose and 6.6 mM of methionine, whereas 1.0% of glucose and 9.0 mM of tryptophan were optimal for IAA and GA synthesis.     

Ethylene formation from methional

The biosynthetic precursor of ethylene is 3-methylthiopropanal (methional). It has been claimed that hydroxyl (HO·) radicals are involved in this biosynthetic sequence, and that the production of ethylene from methional can be used as a specific probe for the presence of the HO· radical. We have now shown, however, that a variety of organic radicals lead to the production of ethylene from methional. Clearly this reaction cannot be used to test for the presence of HO· radicals, and the mechanism for the conversion of methional to ethylene will have to be reexamined.     

Ethylene production by wounded tissue of citrus fruit

Segments cut from young immature fruits and albedo discs excisedfrom both immature and mature fruits of Satsuma mandarin ormature fruits of Natsudaidai produced much ethylene during incubationat 26?C in the dark. Ethylene formation was markedly acceleratedby the application of abscisic acid but markedly delayed by3,5-dibromo-4-hydroxybenzoic acid. Both the stimulation andretardation decreased greatly during the course of incubation.Both compounds seem to be associated with the early stages ofethylene formation by wounded citrus fruit tissues. Albedo discs were fed ¹⁴C methionine labeled at one of threedifferent positions. Of the three radioanalogs (carbon-2, carbon-3and methyl carbon), the label at the 3 position was preferentiallyincorporated into ethylene. This agrees with the former observationthat ethylene is derived from carbon-3 and -4 of methionine.Incorporation of label into ethylene from L-[3-¹⁴C] methioninewas strongly inhibited by L-canaline, L-ethionine, 2,4-dinitrophenoland cycloheximide. Ethylene evolution was also strongly inhibitedby 2,4-dinitrophenol, KCN, NaN₃ and cycloheximide, but lesscompletely by L-canaline and L-ethionine. These results supportthe view that ATP and pyridoxal phosphate are utilized in activationof methionine to form ethylene. (Received October 25, 1977; )     

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.     

Galactose-induced Ethylene Evolution in Mung Bean Hypocotyls: A Possible Mechanism for Galactose Retardation of Plant Growth

Galactose has long been known to inhibit growth in certain plant systems and more recently to promote abscission. These same systems are similarly affected by ethylene. The mung bean (Phaseolus aureus Roxb.) hypocotyl system was employed to ascertain whether the inhibitory effects of galactose might be regulated through ethylene. Galactose alone (at 10 and 100 mM) of the many carbohydrates tested elicited high rates of ethylene evolution (1.5–4.0 nl/g fresh weight x h) as determined by gas chroma-tography. Hook opening, pigment formation, and hypocotyl elongation were inhibited by this resultant ethylene. Galactose and auxin were found to act synergistically with respect to ethylene induction. Use of an auxin antagonist and auxin transport inhibitor revealed that galactose-induced ethylene formation is auxin dependent. Time course studies indicate that this effect may be auxin-sparing. Methionine appears to be the substrate of galactose-induced ethylene. since a methionine antagonist [L-2-amino-4-(2′-amino ethoxy)-trans-3-butenoic acid] abolished the induction. Potential interrelationships between galactose and ethylene synthesis are discussed.