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     

Effects of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on carnation flower longevity

The effects of a novel preservative for cut carnation flowers, 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS), were investigated. DPSS extended the vase life of cut carnation flowers not only by continuous treatment but pulse treatment as well. This inhibition of senescence by DPSS appeared to depend on that of ethylene production in carnation flowers. DPSS provided no protection from the action of ethylene nor did it inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. It did inhibit ACC-dependent ethylene production in carnation petal discs, suggesting possible potential for inhibiting ACC oxidase.     

Changes in 1-aminocyclopropane-1-carboxylic-acid content of cut carnation flowers in relation to their senescence

The rise in ethylene production accompanying the respiration climacteric and senescence of cut carnation flowers (Dianthus caryophyllus L. cv. White Sim) was associated with a 30-fold increase in the concentration of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petals (initial content 0.3 nmol/g fresh weight). Pretreatment of the flowers with silver thiosulfate (STS) retarded flower senescence and prevented the increase in ACC concentration in the petals. An increase in ACC in the remaining flower parts, which appeared to precede the increase in the petals, was only partially prevented by the STS pretreatment. Addition of aminoxyacetic acid (2 mM) to the solution in which the flowers were kept completely inhibited accumulation of ACC in all flower parts.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA -aminoxyacetic acid - STS silver thiosulfate complex     

Bicarbonate/CO(2)-Facilitated Conversion of 1-Amino-cyclopropane-1-carboxylic Acid to Ethylene in Model Systems and Intact Tissues

Bicarbonate markedly enhances ethylene production from 1-aminocyclopropane-1-carboxylic acid (ACC) in model chemical systems where the conversion is free radical-mediated, in thylakoid membrane suspensions of Phaseolus vulgaris L. cv Kinghorn where the reaction is light-dependent, and in microsomal membrane suspensions and intact tissues where the reaction is enzymically mediated. In two model systems generating free radicals—the Fenton reaction and a reaction mixture containing xanthine/xanthine oxidase, NaHCO₃ (200 millimolar) increased the formation of ethylene from ACC by 84-fold and 54-fold, respectively. Isolated thylakoid membranes also proved capable of ACC-dependent ethylene production, but only upon illumination, and this too was enhanced by added NaHCO₃. As well, light-induced inhibition of ACC-dependent ethylene production by leaf discs was relieved by adding 200 millimolar NaHCO₃. Finally, NaHCO₃ (200 millimolar) augmented ACC-dependent ethylene production from young carnation flowers by about 4-fold, and the conversions of ACC to ethylene by microsomes isolated from carnation flowers and etiolated pea epicotyls were higher by 1900 and 62%, respectively, in the presence of 200 millimolar NaHCO₃.

This increased production of ethylene appears not to be due to bicarbonate or CO₂-induced release of the gas from putative receptor sites, since the addition of NaHCO₃ to sealed reaction mixtures after the ACC to ethylene conversion had been terminated had no effect. Spin-trapping studies have confirmed that bicarbonate does not facilitate the formation of free radicals thought to be involved in the conversion of ACC to ethylene. Nor did bicarbonate alter the physical properties of the membrane bilayer, which might indirectly modulate the activity of the membrane-associated enzyme capable of converting ACC to ethylene. Rather, bicarbonate appears to directly facilitate the conversion of ACC to ethylene, and the data are consistent with the view that CO₂ derived from bicarbonate is the active molecular species.

     

Enhancement of petunia and dendrobium flower senescence by jasmonic acid methyl ester is via the promotion of ethylene production

Methyl jasmonate (JA-Me), applied to dendrobium and petunia flowers either as an aqueous solution through the cut stem or stigma, or as a gas, accelerated senescence. The rate of appearance of wilting symptoms was directly related to the amount of JA-Me applied to the flowers. JA-Me increased ethylene production by the flowers, irrespective of application method, and this effect was also proportional to the dose of the compound. In both dendrobium and petunia flowers, the JA-Me induced increases in ethylene production and 1-aminocyclopropane-1-carboxylic acid content followed similar patterns. Aminooxyacetic acid, an inhibitor of ACC-synthase, and silver-thiosulfate, an inhibitor of ethylene action, completely inhibited the effects of JA-Me. It is concluded that JA-Me enhances petunia and dendrobium flower senescence via the promotion of ACC and ethylene production.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA aminooxyacetic acid - Fl flower - JA jasmonic acid - JA-Me jasmonic acid methyl ester - LOX lipoxygenase - PLase A A-type phospholipase - STS silver-thiosulfate     

1-aminocyclopropane-l-carboxylic acid (ACC)—The transmitted stimulus in pollinated flowers?

Ethylene production and senescence of petals of pollinated carnation flowers were not prevented by removal of the ethylene produced by the gynoecium, suggesting that these events are a response to movement from the gynoecium of some stimulus other than ethylene gas. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) to the stigmas caused an initial increase in gynoecium and petal ethylene production similar to that reported for pollinated flowers. This response was not seen in flowers whose stigmas were treated with indoleacetic acid (IAA). When [2-¹⁴C]ACC was applied to the stigmas of carnation flowers, radioactive ethylene was produced both by the gynoecia and by the petals. The possibility that ACC, transported from the stigmas to the petals, is responsible for the postpollination changes in carnation flowers is discussed.     

Ethylene production and β-cyanoalanine synthase activity in carnation flowers

The relationship between ethylene production and the CN^--assimilating enzyme -cyanoalanine synthase (CAS; EC 4.4.1.9) was examined in the carnation (Dianthus caryophyllus L.) flower. In petals from cut flowers aged naturally or treated with ethylene to accelerate senescence the several hundred-fold increase in ethylene production which occurred during irreversible wilting was accompanied by a one- to twofold increase in CAS activity. The basal parts of the petal, which produced the most ethylene, had the highest CAS activity. Studies of flower parts (styles, ovaries, receptacles, petals) showed that the styles had a high level of CAS together with the ethylene-forming enzyme (EFE) system for converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The close association between CAS and EFE found in styles could also be observed in detached petals after induction by ACC or ethylene. Treatment of the cut flowers with cycloheximide reduced synthesis of CAS and EFE. The data indicate that CAS and ethylene production are associated, and are discussed in relation to the hypothesis that CN^- is formed during the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglyoine - CAS -cyanoalanine synthase - CHI cycloheximide - EFE ethylene-forming enzyme     

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

Effect of gibberellic acid on ACC content,EFE activity and ethylene release by floral parts of the senescing carnation flower

The application of gibberellic acid via the stem of intact preclimacteric carnation flowers inhibited the climacteric surge of ethylene evolution by the flowers. Gibberellic acid also inhibited the rate of ethylene production by all individual floral parts during both the early preclimacteric (low basal level of ethylene production) and the later climacteric stages of flower development. The extent of inhibition did however, vary from one floral part to another. The most pronounced inhibition was recorded in the petal bases between the preclimacteric and senescing stages. This suggests that the petal base is an important regulatory site for ethylene production and therefore may be involved in controlling the onset and degree of petal inrolling. In all floral parts endogenous levels of ACC were reduced with GA₃ treatment, being more pronounced in the petal bases. The potential of the flowers to convert applied ACC to ethylene was not deminished by gibberellic acid.Abbreviations GA₃ gibberellic acid - ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme     

Suppression by Citrus Exocortis Viroid Infection of the Naturally Occurring Inhibitor of the Conversion of 1-aminocyclopropane-1-carboxylic Acid to Ethylene by Tomato Microsomes

Citrus exocortis viroid (CEVd) infection of tomato cell cultures suppresses the constitutive inhibitor which blocks the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by tomato microsomes. The inhibitor is associated to microsomal membranes and is also found in the water soluble fraction co-isolated from the cells. The inhibitory effect is concentration dependent, heat stable and could be removed from solution by dialysis. Its possible relationship with regulation of the viroid-induced ethylene production is discussed.     

Cell-free ethylene-forming systems lack stereochemical fidelity

In-vitro systems for the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene have been reported using pea supernatants, carnation petal microsomes, olive leaf protein and, most recently, pea mitochondria. It has also been shown, in intact tissues of apple, mung bean and pea, that the system responsible for conversion of ACC to ethylene can produce 1-butene from isomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC). This conversion shows a high degree of steroselectivity, and isomer discrimination is therefore a valuable criterion by which to judge the validity of subcellular systems. It is shown here that all in-vitro ethylene-forming systems so far described fail by a wide margin to match the AEC-isomer preference of the corresponding intact tissues with respect to 1-butene generation. This work supports and extends recent reports by McKeon and Yang (1984, Planta 160, 84–87) and by Guy and Kende (1984, Planta 160, 281–287) on the characteristics of ethylene formation by pea homogenates. The vacuolar conversion described by the latter authors is the simplest system yet described that retains appropriate sterochemical fidelity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - DEAE diethylaminoethyl - IAA indole-3-acetic acid     

The effect of plant-hormone pretreatments on ethylene production and synthesis of 1-aminocyclopropane-1-carboxylic acid in water-stressed wheat leaves

Excised wheat (Triticum aestivum L.) leaves, when subjected to drought stress, increased ethylene production as a result of an increased synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and an increased activity of the ethyleneforming enzyme (EFE), which catalyzes the conversion of ACC to ethylene. The rise in EFE activity was maximal within 2 h after the stress period, while rehydration to relieve water stress reduced EFE activity within 3 h to levels similar to those in nonstressed tissue. Pretreatment of the leaves with benzyladenine or indole-3-acetic acid prior to water stress caused further increase in ethylene production and in endogenous ACC level. Conversely, pretreatment of wheat leaves with abscisic acid reduced ethylene production to levels produced by nonstressed leaves; this reduction in ethylene production was accompanied by a decrease in ACC content. However, none of these hormone pretreatments significantly affected the EFE level in stressed or nonstressed leaves. These data indicate that the plant hormones participate in regulation of water-stress ethylene production primarily by modulating the level of ACC.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - BA N⁶-benzyladenine - EFE ethylene-forming enzyme - IAA indole-3-acetic acid     

The role of ethylene in the senescence of carnation flowers, a review

The senescence of flower petals is a highly regulated developmental process which requires active gene expression and protein synthesis. The biochemical changes associated with petal senescence in carnation flowers include an increase in hydrolytic enzymes, degradation of macro-molecules, increased respiratory activity and a climacteric-like increase in ethylene production. It is clear that the gaseous phytohormone ethylene plays a critical role in the regulation and coordination of senescence processes. Many reviews on physiology and mode of action of ethylene are available. Molecular cloning led to the isolation of genes involved in ethylene biosynthesis and action. This review describes the current status of the studies on regulation of ethylene biosynthesis and ethylene response in carnation flowers. An overview is given of studies on senescence-related gene expression and possibilities to improve postharvest longevity by genetic engineering.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AIB -amino-isobutyric acid - AOA amino oxyacetic acid - AVG aminoathoxyvinyl glycine - DACP diazocyclopentadiene - EFE ethylene forming enzyme - MACC malonyl 1-aminocyclopropane-1-carboxylic acid - MTA 5-methylthio-adenosine - NBD 2,5 norbornadiene - ppb parts per billion - SAM S-adenosyl-methionine - STS silver thiosulphate     

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     

Light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in leaves is mediated through carbon dioxide

The mechanism of light-inhibited ethylene production in excised rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L.) leaves was examined. In segments of rice leaves light substantially inhibited the endogenous ethylene production, but when CO₂ was added into the incubation flask, the rate of endogenous ethylene production in the light increased markedly, to a level which was even higher than that produced in the dark. Carbon dioxide, however, had no appreciable effect of leaf segments incubated in the dark. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, was not significantly affected by lightdark or CO₂ treatment, indicating that dark treatment or CO₂exerted its effect by promoting the conversion of ACC to ethylene. This conclusion was supported by the observations that the rate of conversion of exogenously applied ACC to ethylene was similarly inhibited by light, and this inhibition was relieved in the presence of CO₂. Similar results were obtained with tobacco leaf discs. The concentrations of CO₂ giving half-maximal activity was about 0.06%, which was only slightly above the ambient level of 0.03%. The modulation of ACC conversion to ethylene by CO₂ or light in detached leaves of both rice and tobacco was rapid and fully reversible, indicating that CO₂ regulates the activity, but not the synthesis, of the enzyme converting ACC to ethylene. Our results indicate that light inhibition of ethylene production in detached leaves is mediated through the internal level of CO₂, which directly modulates the activity of the enzyme converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid Recipient of a Republic of China National Science Council Fellowship     

Synergistic effect of 1-aminocyclopropane-1-carboxylic acid and ethylene during senescence of isolated carnation petals

The effects of ethylene (C₂H₄), (2-chloroethyl)phosphonic acid (ethefon) and 1-aminocyclopropane-1-carboxylic acid (ACC) on senescence of isolated intact petals and of upper petal parts of carnation flowers ( Dianthus caryophyllus L. cv. White Sim) were investigated.
Isolated upper petal parts did not respond to treatment with ethefon or ACC. These tissues did, however, show severe wilting in intact petals that were treated with ethefon or ACC. When isolated upper petal parts were simultaneously treated with ACC and ethefon or ACC and ethylene, a marked synergistic effect on senescence was found. Treatment of isolated petals with radiolabeled ACC led to the accumulation of radiolabeled ACC and N-malonyl-ACC (MACC) in the upper parts. The formation of ethylene and the malonylation of ACC were inhibited by pretreatment of the flower with the inhibitor of ethylene action, silver thiosulphate (STS), which indicates that both were induced by endogenously produced ethylene. Treatment of isolated upper parts with ACC slightly increased their ethylene production. However, when these petal parts were simultaneously treated with ethylene and ACC, the conversion of ACC to ethylene was markedly stimulated.
The results indicate that, in intact petals, ethylene may be translocated from the basal to the upper part where it stimulates the activity of the ethylene-forming enzyme (EFE), thereby making the tissue receptive to ACC.
In addition, it was found that upon incubation of petal portions in radiolabeled ACC, both the petal tissue and the incubation solutions produced radiolabeled carbon dioxide. This was shown to be due to microorganisms that were able to metabolize the carbon atoms in the 2 and 3 position of ACC into carbon dioxide.     

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     

Salicylic acid inhibits the biosynthesis of ethylene in detached rice leaves

The effects of salicylic acid (SA) on ethylene biosynthesis in detached rice leaves were investigated. SA at pH 3.5 effectively inhibited ethylene production within 2 h of its application. It inhibited the conversion of ACC to ethylene, but did not affect the levels of ACC and conjugated ACC. Thus, the inhibitory effect of SA resulted from the inhibition of both synthesis of ACC and the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - SA salicylic acid