Ethylene-forming Enzyme Activity Research Articles

Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.

Overcoming substrate limitations for improved production of ethylene in E. coli.

BackgroundEthylene is an important industrial compound for the production of a wide variety of plastics and chemicals. At present, ethylene production involves steam cracking of a fossil-based feedstock, representing the highest CO2-emitting process in the chemical industry. Biological ethylene production can be achieved via expression of a single protein, the ethylene-forming enzyme (EFE), found in some bacteria and fungi; it has the potential to provide a sustainable alternative to steam cracking, provided that significant increases in productivity can be achieved. A key barrier is determining factors that influence the availability of substrates for the EFE reaction in potential microbial hosts. In the presence of O2, EFE catalyzes ethylene formation from the substrates α-ketoglutarate (AKG) and arginine. The concentrations of AKG, a key TCA cycle intermediate, and arginine are tightly controlled by an intricate regulatory system that coordinates carbon and nitrogen metabolism. Therefore, reliably predicting which genetic changes will ultimately lead to increased AKG and arginine availability is challenging.ResultsWe systematically explored the effects of media composition (rich versus defined), gene copy number, and the addition of exogenous substrates and other metabolites on the formation of ethylene in Escherichia coli expressing EFE. Guided by these results, we tested a number of genetic modifications predicted to improve substrate supply and ethylene production, including knockout of competing pathways and overexpression of key enzymes. Several such modifications led to higher AKG levels and higher ethylene productivity, with the best performing strain more than doubling ethylene productivity (from 81 ± 3 to 188 ± 13 nmol/OD600/mL).ConclusionsBoth EFE activity and substrate supply can be limiting factors in ethylene production. Targeted modifications in central carbon metabolism, such as overexpression of isocitrate dehydrogenase, and deletion of glutamate synthase or the transcription regulator ArgR, can effectively enhance substrate supply and ethylene productivity. These results not only provide insight into the intricate regulatory network of the TCA cycle, but also guide future pathway and genome-scale engineering efforts to further boost ethylene productivity.

REGULATION OF WATER-STRESS-INDUCED ETHYLENE IN CITRUS LEAVES

ABSTRACT Whole detached citrus leaves (Citrus sinensis [L] Osbeck) were subjected to water stress by allowing them to lose water until their initial fresh weight decreased by about 15%. Ethylene production in water-stressed leaves increased significantly reaching a peak 4 h after termination of the stress treatment and decreased rapidly to control level thereafter. The enhancement in ethylene production was the result of an increase both in the synthesis of 1-aminocyclopropane-l-carboxylic acid (ACC) and in the activity of ethylene-forming enzyme (EFE). The increase in ACC level peaked 2 h after termination of the stress treatment, whereas EFE activity increased gradually with time. Pretreatment with abscisic acid (ABA) had no effect on ethylene production in whole detached leaves subjected to water stress. In water-stressed leaf discs, ABA pretreatment stimulated ethylene production. Norbornadiene, a competitive inhibitor of ethylene, enhanced the ethylene production rate in water-stressed leaves and del...

Effect of calcium and calmodulin antagonists on ethylene biosynthesis in tomato fruits

The aim of this work was to elucidate the mode of action of calcium in calcium-inhibited ethylene biosynthesis. In one experiment, mature green tomatoes were treated with 1.5% (w/v) CaCl 2 + 60 μM N-6(aminohexyl)-1-naphthalene-sulphonamide (W5). 1.5% (w/v) CaCl 2 + 60 μM N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7), 1.5% (w/v) CaCl 2 alone or distilled water (control). The control and the W7 treatment had much higher ethylene production than either the CaCl 2 treatment alone or the CaCl 2 + W5 treatment. In another experiment, similar tomatoes were treated with 1.5% (w/v) CaCl 2 + 15 μM trifluoperazine (TFP), 1.5% (w/v) CaCl 2 only, or distilled water (control). The treatment with 1.5% CaCl 2 + 15 μM TFP gave higher ethylene production than 1.5% (w/v) CaCl 2 without TFP, and nearly the same level as the control towards the end of the experimental period. For enzyme and intermediate metabolite assays, mature green tomatoes were treated with 1.5% (w/v) CaCl 2 plus either W5 or W7. W5 treatment had a lower ethylene forming enzyme (EFE) activity, higher 1-aminocyclopropane-l-carboxylic acid (ACC) concentration, similar levels of ACC synthase activity and 1-(malonylamino)-cyclopropane-1-carboxylic acid concentration, compared to the control and W7 treatment. As W7 is a more potent calmodulin antagonist than W5, these results suggest a calcium-calmodulin inactivation of EFE activity.

Changes of Ethylene Evolution, ACC Content, Ethylene Forming Enzyme Activity and Respiration in Fruits of Highbush Blueberry.

Summary We measured ethylene evolution, ACC content, ethylene forming enzyme (EFE) activity, and respiration in highbush blueberry (Vaccinium corymbosum L.) from flowering to harvest to assess the role of ethylene in the processes. Cultivars used were 'Collins' (early maturing), 'Berkeley' (mid-season), and 'Dixi' (late maturing). Ethylene evolution increased at both petal-fall and maturation stages, and was higher in the early stage than in the late stage. Ethylene at petal fall was produced by the calyx and ovary parts, but not the corolla. This indicates that ethylene evolution in flowering is correlated with pollination or fertilization or both. Ethylene evolution rate in the maturation stage was high between the blue pink (BP) peel color in 'Collins', and mature green (MG) stage in 'Berkeley', and green pink (GP) stage in 'Dixi'. Ethylene evolution was higher for 'Collins' than for the others in both years and higher in 1995 than in 1994. The pattern of EFE activity was not similar to that of ethylene evolution except in 'Collins'. Therefore, the correlation between ethylene evolution and EFE activity in the maturation stage was different with each cultivar. Berry ACC content was high at GP stage in three cultivars before ethylene evolution increased.

Photosynthetic conversion of carbon dioxide to ethylene by the recombinant cyanobacterium, Synechococcus sp. PCC 7942, which harbors a gene for the ethylene-forming enzyme of Pseudomonas syringae

Photosynthetic conversion of carbon dioxide to ethylene was studied using the recombinant cyanobacterium, Synechococcus sp. strain PCC 7942 R2-SPc which expresses the ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding the EFE ( efe gene) from P. syringae was introduced into the cyanobacterium utilizing the pUC303 shuttle vector into which the efe gene was placed under the control of various transcriptional signals, i.e., the native promoter and terminator of the efe gene (pUC303-EFE03), the Escherichia coli lacZ promoter and the efe gene terminator (pUC303-EFE10 and pUC303-EFE30), or the promoter and terminator of the psbAI gene from Synechococcus sp. PCC7942 which codes for the D1 protein in photosystem II (pEXE3). Among these configurations, EFE activity measured in the cell-free extracts of transformants that harbored the pEXE3 was highest. However, ethylene production in vivo of the transformants carrying pEXE3 declined with the number of generations, because homologous recombination of DNA sequences on the pEXE3 plasmid and host chromosomal psbAI locus took place. Deletion of the 5′-upstream region of the psbAI promoter and the 3′-downstream region of the psbAI terminator in pEXE3 resulted in pEXE3Δ8 which showed the highest level of ethylene-forming activity, although the latter plasmid was still unstable with a half-life of only 12 generations. The amount of carbon incorporated into ethylene was calculated as a percentage of the total carbon fixed, the maximum value of which was 5.84% in the recombinant cyanobacterium harboring pUC303-EFE03.

Inhibitors of Ethylene Biosynthesis and Action Do Not Prevent Injury to Wheat Seedlings Infested with Schizaphis graminum (Homoptera: Aphididae)

Experiments were conducted to determine whether inhibitors of ethylene biosynthesis and action can prevent symptoms of aphid damage to wheat seedlings. ‘TAM 107’ wheat, Triticum aestivum L., seedlings infested with biotype G greenbugs, Schizaphis graminum (Rondani), for 6 h developed chlorosis and necrotic spots within 4 d after the aphids were removed. AVG (1 mM 2-aminoethoxyvinylglycine), an ethylene biosynthesis inhibitor, reduced ethylene production but not development of lesions or loss of chlorophyll from infested seedlings. STS [silver thiosulfate complex (0.5 mM AgN03, 2.0 mM Na thiosulfate)], an inhibitor of ethylene action, did not prevent a reduction in chlorophyll concentration or the development of lesions. A secondary objective was to determine how the ethylene biosynthetic pathway was differentially affected by aphid biotypes previously shown to stimulate high or low levels of ethylene. ‘Largo’ and TAM 107 wheat seedlings were infested with biotype E or G greenbugs, then harvested after 1, 6, or 48 h. Both wheat genotypes are susceptible to biotype G. TAM 107 is susceptible to biotype E and Largo is resistant. Increased ethylene production 6 h after seedlings were exposed to biotype G was associated with an increase in ACC (1-aminocyclopropane-l-carboxylic acid, the immediate precursor of ethylene) and EFE (ethylene-forming-enzyme) activity in both wheat genotypes. Ethylene production, ACC content, and EFE activity declined by 48 h, concomitant with an increase in MACC (N-malonyl-ACC, an inactive conjugate of ACC). Biotype E triggered a smaller increase in ethylene production than biotype G. ACC levels were not affected by biotype E, but EFE activity increased by 33% in Largo and 151% in TAM 107 after 6 h. Ethylene production from greenbug-infested wheat seedlings was controlled primarily by ACC content. Ethylene appeared to be a symptom, not a mediator of green bug-induced injury.

Non-involvement of ACC and ACC oxidase activity in pepper fruit ripening

Abstract Pepper of the variety California shows sigmoid-type growth curves and changes of colour, which are characteristic of the fruit reaching maturity, when it has practically reached full size. Respiratory activity is high a few days after fruit-set and then falls progressively. Ethylene production and 1-aminocyclopropane-1-carboxylic acid (ACC) levels remain low throughout fruit development and maturation, while ethylene-forming enzyme (EFE) activity gradually falls throughout the development. We show that treatment with propylene has no effect on the post-harvest maturation of pepper, although treatment with silver thiosulphate (STS) increases respiratory intensity and ethylene production due to the stress caused which, in turn, causes an increase in ACC levels after treatment. All these results lead to the conclusion that pepper behaves like a non-climacteric fruit during maturation, and that maturation is not regulated by ethylene.

Production of ethylene, its precursors and implied enzyme activities in isolated chickpea embryonic axes during the onset of growth

The embryonic axes of chickpea (Cicer arietinum) seeds were used to quantify 1-(malonyl)aminocyclopropane-1-carboxylic acid (MACC), 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene and some related enzymes during the initial 18 h imbibition period (anaerobic growth phase). Longer cold storage (stratification) of seeds produced higher levels of MACC and ACC. Maximum accumulation of MACC and malonyl-transferase activity occurred after 5 h of growth but MACC levels later became insignificant. ACC-synthase activity and endogenous ACC seem to reach a maximum 2 h after MACC accumulation. MACC-hydrolase activity was measured“in-vitro” and reached a maximum after 5.5 h of growth. These results suggest that endogenous MACC did not seem to be an end product; it may be involved in ACC production and the regulation of ethylene production before the emergence of the radicle. Ethylene-forming enzyme (EFE) activity reached a maximum after 12 h and ethylene production after 18 h of growth. The physiological implications of this temporal separation of MACC, ACC, ethylene and related enzymes is discussed.

Effects of carbon dioxide on ethylene-forming enzyme in Japanese pear and apple

Ethylene-forming enzyme (EFE) activity was stimulated by CO 2 in both of Hosui and Granny Smith fruit discs. In Hosui fruit, EFE activity remained unchanged up to 5% CO 2 , but increased with higher CO 2 levels. In Granny Smith fruit, EFE increased gradually as CO 2 increased from 5 to 30%. EFE developed with storage time at 1±1°C in both fruits. Treating discs with cycloheximide showed that CO 2 induced EFE biosynthesis in Granny Smith apple stored for a short time, but not in Hosui fruit. Kinetic data from the interaction of CO 2 and 1-aminocyclopropane-1-carboxylic acid (ACC) on EFE activity indicated that CO 2 increased the maximum reaction rate ( V max ) of EFE in discs of both fruit without changing the apparent Michaelis-Menton constant ( K m ) value. EFE activity in Hosui fruit discs was stimulated by CO 2 only when ACC was added exogenously. It is suggested that the stimulatory effect of CO 2 on EFE activity may be caused by a CO 2 -EFE-ACC or EFE-ACC-CO 2 complex which enhances the maximum reaction rate of conversion of ACC to ethylene.

Effects of inhibitors on the carbon dioxide-stimulation of ethylene-forming enzyme activity in fruit of Japanese pear and apple

Abstract Cobalt ions (Co 2+ ) at 1 mM inhibited ethylene-forming enzyme (EFE) activity and ethylene production in apple (Granny Smith) discs. Low concentrations (0.01 mM) of Co 2+ ions stimulated, but higher concentrations (>0.02 mM) inhibited EFE activity in discs of both Japanese pear (Hosui) and apple. CO 2 stimulated EFE activity but did not reverse the inhibition caused by Co 2+ ions. AgNO 3 inhibited ethylene production of fruit discs in Granny Smith apple. Low concentrations ( + stimulated, but higher levels (0.5 and 1 mM) inhibited EFE activity in discs of both fruits. CO 2 did not reverse the inhibitory effect of Ag + (1 mM). 2,5-Norbornadiene (NDE) at 0.5% (v/v) inhibited EFE activity in discs from both fruits. CO 2 partially reversed the NDE inhibition in discs of Hosui and Granny Smith fruit stored at 1 ± 1°C for a short period. The possible mechanisms of the interactions between CO 2 and the inhibitors on EFE activity and ethylene receptors are discussed.

Genotype difference in rice postgermination growth under hypoxia

Rice ( Oryza sativa L.) genotypes that can establish seedlings in flooded anaerobic soil had longer coleoptile in postgermination growth than other genotypes (controls) in a gas flow of N 2 and air, or under various degrees of hypoxia created in closed flasks in the dark. The tolerant genotypes produced as much or more ethylene than the control genotypes, although both had the same activity of ethylene-forming enzyme. Ethylene stimulated coleoptile elongation of both genotypes to the same degree. The coleoptile elongation of the tolerant genotypes remained greater than that of the controlseven in the presence of Co 2+ and l-α-(2-aminoethixyvinyl)glycine. Thus, the longer coleoptile of the tolerant genotypes might be explained by a mechanism that is independent of ethylene and O 2. Mesocotyl and shoot elongation of the tolerant genotypes was more stimulated by ethylene than that of control genotypes. These developmental characteristics of tolerant genotypes may accelerate seedling establishment in flooded anaerobic soil.

Chromium‐induced inhibition of ethylene evolution in bean (Phaseolus vulgaris) leaves

The influence of chromium concentration on ethylene production in bean plants (Phaseolus vulgaris L. cv. Contender) was investigated. A Cr ion‐induced inhibition of ethylene synthesis from endogenous 1‐aminocyclopropane‐1‐carboxylic acid (ACC) was observed within both leaf discs floated on 2 mM CrO2−4 or Cr3+ and leaf discs from plants cultured in nutrient solutions containing 10, 20 or 40 μM CrO2−4. However, Cr ions supplied either to plants with the nutrient solution or to discs with the incubation medium rather increased the conversion of exogenous ACC to ethylene. Primary leaves of plants exposed to CrO2−4‐containing nutrient solutions showed a statistically insignificant decrease of ACC‐synthase activity. In the trifoliolate leaves of plants exposed to 10 μM CrO2−4, in which a significant decrease of ethylene production from endogenous ACC was observed, a substantial increase of ACC synthase was found. These results indicate that Cr ion‐induced inhibition of ethylene production is not due to a breakdown of membrane integrity, which is necessary for ethylene forming enzyme activity, but caused by metabolic alterations leading to decreased ACC availability. Chromium ions may act by inhibiting ACC synthase activity or by diverting a metabolic step prior to the ACC synthase catalyzed reaction.

Cold-induced activation of 1-aminocyclopropane-1-carboxylic acid metabolism in rewarmed ‘Granny Smith’ apples: Consequences on ripening

Changes in respiratory rate, 1-aminocyclopropane-1-carboxylic acid (ACC) metabolism and ripening in apples ( Malus domestica Borkh. L., cultivar ‘Granny Smith’) were studied in relation to cold storage. Fruit were held at 4°C or 22°C for 31 days (4°C controls and 22°C controls, respectively) or at 4°C for periods of 2, 5 or 10 days before transfer to 22°C for the rest of the 31 day period. Ethylene and CO 2 were produced at a low rate in 22°C controls. In 4°C controls, ACC levels increased greatly, showing an inhibition of both ethylene-forming enzyme (EFE) and malonyl transferase activities. In chilled fruit, ethylene, malonyl ACC (MACC) levels and EFE activity increased sharply after transfer to 22°C. These increases were accompanied by a significant rise in the respiratory rate, similar to an activation of a climacteric process. Chilled fruit also showed a loss of pulp firmness and acidity compared with fruit kept continuously at 22°C.

Unusual ethylene‐related behavior in senescing flowers of the carnation Sandrosa

The time course of ethylene production by senescing carnation (Dianthus caryophyllus L. cv. Sandrosa) flowers was studied. These flowers are unusual in that they do not exhibit an autocatalytic increase in ethylene production nor do they develop petal in‐rolling. Exposure of the flowers to exogenous ethylene resulted in a rise in their ethylene‐forming enzyme (EFE) activity and ethylene production, and at the same time a marked decline in their fresh weight. Natural senescence was also accompanied by a rise in EFE activity, but with no concomitant rise in 1‐amino cyclopropane carboxylic acid synthase activity nor in ethylene production. A shift in responsiveness to ethylene was observed, with young flowers more responsive to exogenous ethylene than older flowers. The results are discussed in terms of a proposed mechanism allowing for the decline in competence of this cultivar to respond to ethylene during senescence.

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.

Effect of methyl jasmonate on ethylene biosynthesis and stomatal closure in olive leaves

The influence of methyl jasmonate (MeJA) on ethylene production and 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-ACC (MACC) contents in olive leaf discs and detached leaves was investigated. In leaf discs, both ACC content and ethylene-forming enzyme (EFE) activity were clearly stimulated by 45 μM MEJA. In detached leaves MeJA treatment increased ACC content with no clear increase in ethylene production. When detached leaves were incubated with MeJA (45 μM) and ACC (1 mM), the total endogenous ACC content was significantly lower than that of leaves incubated only with ACC (1 mM). These data could be explained on the basis that MeJA caused stomatal closure, and hence reduced ACC uptake.

Roles of Ethylene Production and Sensitivity in Senescence of Carnation Flower ( Dianthus caryophyllus) Cultivars White Sim, Chinera and Epomeo

Summary Longevity of carnation (Dianthus caryophyllus) cultivar Chinera was much longer than that of cultivar White Sim. In ‹Chinera›, the increases in 1-aminocyclopropane-1-carboxylic acid synthase (EC 4.4.1.14) activity, ethylene-forming enzyme activity, ethylene production and membrane permeability, associated with the start of the senescence process, occurred later, and the maximum levels during senescence were much lower than in ‹White Sim›. Ethylene treatment stimulated the activity of the ethylene biosynthetic pathway and associateted enzymes but stimulation was less in ‹Chinera› than in ‹White Sim›. Ethylene dose-response experiments showed lower sensitivity m ‹Chinera› while binding site characteristics were supposedly similar in the two cultivars. The reduced sensitivity of ‹Chinera› to ethylene, therefore, is apparently determined at a point beyond the receptor, presumably m the signal transduction chain. In cultivar Epomeo which is the progeny of ‹Chinera› and a non-commercial breeding line «9775», ethylene production and sensitivity showed the same trends as in ‹Chinera›, showing that the decreased sensitivity and the associate longer vase life is an inheritable feature.

Roles of pollination and short-chain saturated fatty acids in flower senescence

Pollination induced an immediate increase in ethylene production in Dianthus caryophyllus and Petunia hybrida. In Cymbidium, a lag of several hours was observed. In all three species, pollination induced premature flower senescence. Treatment of the stigmatic surface with aminoethoxyvinylglycine prior to pollination effectively blocked the increase in ethylene production and alleviated the detrimental effect of pollination on flower life. In all three tested species, octanoic and decanoic acids, when applied to the stigmatic surface, had no effect on ethylene production and flower life. In isolated Cymbidium lips placed with their cut base in solutions containing these fatty acids, no effects on red colouration, ethylene production, and ethylene forming enzyme activity were observed. In addition, ethylene sensitivity of isolated lips was not affected. The putative regulatory role of short-chain saturated fatty acids in (pollination-induced) flower senescence is discussed.

キウイフルーツの収穫後における自己触媒的なエチレン生成の特徴について

The mechanism of autocatalytic ethylene production was studied in relation to kiwifruit ripening.1. Ethylene production by kiwifruit was induced by 18- and 24-hr ethylene treatments at 20 °C. On the other hand, the other physiological changes associated with ripening were accelerated even by 15-hr or less.2. The physiological changes such as flesh softening, decrease in acid content and increase in respiration rate were appreciably accelerated by 0.1 to 10 ppm of ethylene at 5 °C, while ethylene production, ACC (1-aminocyclopropane-l-carboxylic acid) accumulation and EFE (ethylene forming enzyme) activity were not stimulated by those conditions.Hence, it is concluded that autocatalytic ethylene production in kiwifruit is less inducible with ethylene treatment than most physiological changes relating ripening.