Ethylene Promotes the Capability To Malonylate 1-Aminocyclopropane-1-carboxylic Acid and d-Amino Acids in Preclimacteric Tomato Fruits

When whole unripe green tomato fruits (Lycopersicon esculentum Mill, cv T₃) were treated with ethylene (10 microliters per liter) for 18 hours, the fruit's ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to N-malonyl-ACC (MACC) increased markedly and such an effect was also observed in fruits of mutant nor, which cannot ripen normally. The promotion of the capability to malonylate ACC by ethylene increased with the increasing ethylene concentration from 0.1 to 100 microliters per liter and with increasing duration of ethylene treatment up to 8 hours; a longer duration of ethylene treatment did not further increase the malonylation capability. When ethylene was withdrawn, the promotion disappeared within 72 hours. Norbornadiene, a competitive inhibitor of ethylene action, effectively eliminated the promotive effect of ethylene. Ethylene treatment also promoted the fruits' capability to conjugate d-amino acids and α-amino-isobutyric acid. Since the increase in the tissue's capability to malonylate ACC was accompanied by an increase in the extractable activity of ACC and d-amino acid malonyltransferase, ethylene is thought to promote the development of ACC/d-amino acid malonyltransferase in unripe tomato fruits.     

Ethylene-promoted conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in peel of apple at various stages of fruit development

Internal ethylene concentration, ability to convert 1-amino-cyclopropane-1-carboxylic acid (ACC) to ethylene (ethylene-forming enzyme [EFE] activity) and ACC content in the peel of apples (Malus domestica Borkh., cv Golden Delicious) increased only slightly during fruit maturation on the tree. Treatment of immature apples with 100 microliters ethylene per liter for 24 hours increased EFE activity in the peel tissue, but did not induce an increase in ethylene production. This ability of apple peel tissue to respond to ethylene with elevated EFE activity increased exponentially during maturation on the tree. After harvest of mature preclimacteric apples previously treated with aminoethoxyvinyl-glycine, 0.05 microliter per liter ethylene did not immediately cause a rapid increase of development in EFE activity in peel tissue. However, 0.5 microliter per liter ethylene and higher concentrations did. The ethylene concentration for half-maximal promotion of EFE development was estimated to be approximately 0.9 microliter per liter. CO₂ partially inhibited the rapid increase of ethylene-promoted development of EFE activity. It is suggested that ethylene-promoted CO₂ production is involved in the regulation of autocatalytic ethylene production in apples.     

Regulation of Ethylene Biosynthesis in Avocado Fruit during Ripening

Preclimacteric avocado (Persea americana Mill.) fruits produced very little ethylene and had only a trace amount of l-aminocyclopropane-1-carboxylic acid (ACC) and a very low activity of ACC synthase. In contrast, a significant amount of l-(malonylamino)cyclopropane-1-carboxylic acid (MACC) was detected during the preclimacteric stage. In harvested fruits, both ACC synthase activity and the level of ACC increased markedly during the climacteric rise reaching a peak shortly before the climacteric peak. The level of MACC also increased at the climacteric stage. Cycloheximide and cordycepin inhibited the synthesis of ACC synthase in discs excised from preclimacteric fruits. A low but measurable ethylene forming enzyme (EFE) activity was detected during the preclimacteric stage. During ripening, EFE activity increased only at the beginning of the climacteric rise. ACC synthase and EFE activities and the ACC level declined rapidly after the climacteric peak. Application of ACC to attached or detached fruits resulted in increased ethylene production and ripening of the fruits. Exogenous ethylene stimulated EFE activity in intact fruits prior to the increase in ethylene production. The data suggest that conversion of S-adenosylmethionine to ACC is the major factor limiting ethylene production during the preclimacteric stage. ACC synthase is first synthesized during ripening and this leads to the production of ethylene which in turn induces an additional increase in ACC synthase activity. Only when ethylene reaches a certain level does it induce increased EFE activity.     

Metabolism of 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

In preclimacteric apple fruits ( Malus × domestica Borkh. cv. Golden Delicious) ethylene production is controlled by the rates of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis, and by its metabolism to ethylene by the ethylene-forming enzyme and to 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by malonyl CoA-ACC transferase. The onset of the climacteric in ethylene production is associated with an increase in the activity of the ethylene-forming enzyme in the pulp and with a rise in the activity of ACC synthase. Malonyl transferase activity is very high in the skin of immature fruit, decreases sharply before the onset of the climacteric, and remains nearly constant thereafter. More than 40% of the ACC synthesized in the skin and around 5% in the flesh, are diverted to MACC at early climacteric. At the climacteric peak there are substantial gradients in ethylene production between different portions of the tissue, the inner cortical tissues producing up to twice as much as the external tissues. This increased production is associated with, and apparently due to, increased content of ACC synthase. Less than 1% of the synthesized ACC is diverted to MACC in the flesh of climacteric apples. In contrast, the skin contains high activity of malonyl transferase, and correspondingly high levels [1000 nmol (g dry weight)⁻¹] of MACC.     

The Conversion of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid to 1-Aminocyclopropane-1-Carboxylic Acid in Plant Tissues

Since 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the major conjugate of 1-aminocyclopropane-1-carboxylic acid (ACC) in plant tissues, is a poor ethylene producer, it is generally thought that MACC is a biologically inactive end product of ACC. In the present study we have shown that the capability of watercress (Nasturtium officinale R. Br) stem sections and tobacco (Nicotiana tabacum L.) leaf discs to convert exogenously applied MACC to ACC increased with increasing MACC concentrations (0.2-5 millimolar) and duration (4-48 hours) of the treatment. The MACC-induced ethylene production was inhibited by CoCl₂ but not by aminoethoxyvinylglycin, suggesting that the ACC formed is derived from the MACC applied, and not from the methionine pathway. This was further confirmed by the observation that radioactive MACC released radioactive ACC and ethylene. A cell-free extract, which catalyzes the conversion of MACC to ACC, was prepared from watercress stems which were preincubated with 1 millimolar MACC for 24 hours. Neither fresh tissues nor aged tissues incubated without external MACC exhibited enzymic activity, confirming the view that the enzyme is induced by MACC. The enzyme had a K_m of 0.45 millimolar for MACC and showed maximal activity at pH 8.0 in the presence of 1 millimolar MnSO₄. The present study indicates that high MACC levels in the plant tissue can induce to some extent the capability to convert MACC to ACC.     

Chilling-Induced Ethylene Production in the Peel and Pulp of Banana

Ethylene production rates and 1-aminocyclopropane-1-carboxylic acid (ACC) synthetase activities were 0. 78,0.91 nl· g-l ·h-land 0.02,0.05 nmol·g-1·h-1 respectively in the peel and pulp of newly harvested banana fruits(Musa acuminata Colla “warf cavendish”),their ethylene-forming enzyme(EFE)activities were yet as high as 10.5 and 5.1 nl·g-1·h-1. When the fruits were chilled at 1.5℃ ,the ethylene production and EFE activities of the peel and pulp kept decreasing with the time course of chilling treatment. However, after these chilled fruits were transferred to 20℃ for 24 h,their ACC synthetase activities increased markedly,and ethylene production had separate peaks(1.75 and 2.45 nl·g-1 ·h-1) in the peel and pulp. In this case,the endogenous low content of S-adenosylmethionine (SAM)in vivo was insufficient for its ACC synthesis, The inhibitory effect of cycloheximide on ACC synthesis showed that chilling-induced ethylene production was mainly the result of activity of the resynthesized ACC synthetase induced by chilling treatment. The production of chilling-induced ethylene could be good indicator of chilling injury, but it is unlikely an indicator of chilling damage during ripening process in banana. In the severly chilling-injured fruits, both the peel and pulp still had the capability of converting ACC to ethylene.     

Metabolism of 1-Aminocyclopropane-1-Carboxylic Acid in Etiolated Maize Seedlings Grown under Mechanical Impedance

We investigated the metabolism of 1-aminocyclopropane-1-carboxylic acid (ACC) in etiolated maize (Zea mays L.) seedlings subjected to mechanical impedance by applying pressure to the growing medium. Total concentrations of ACC varied little in unimpeded seedlings, but impeded organs accumulated ACC. Roots had consistently higher concentrations of ACC than shoots or seeds, regardless of treatment. The concentration of ACC in the roots increased more than 100% during the first hour of treatment irrespective of the pressure applied; in shoots, total ACC concentration increased 46% at either low or high pressure during the first hour of treatment. The bulk of ACC synthesized under impeded and unimpeded conditions was present in a conjugated form, presumably, 1-(malonylamino)-cyclopropane-1-carboxylic acid. However, 1-(malonylamino)-cyclopropane-1-carboxylic acid increased 73% over controls after 10 hours at 25 kilopascals of pressure. Unimpeded tissue had about 77% ACC as the conjugate and 17% as free ACC, and less than 6% was used in ethylene production. Increased amounts of ACC were converted into ethylene under stress. In vivo ACC synthase activity in roots became six and seven times higher only 1 hour after initiation of treatment at 25 and 100 kilopascals of pressure, respectively, and remained high for at least 6 hours. However, the immediate and massive conjugation of mechanically induced ACC suggests that ACC N-malonyltransferase may play an important role in the regulation of mechanically induced ethylene production. After 8 hours, in vivo activity of the ethylene-forming enzyme complex increased 100 and 50% above normal level at 100 and 25 kilopascals, respectively. Furthermore, ethylene-forming enzyme complex activity was significantly greater at 100 kilopascals than in controls as early as 1 hour after treatment initiation. These data suggest that regulation of ethylene production under mechanical impedance involves the concerted action of ACC synthase, the ethylene-forming enzyme complex, and ACC N-malonyltransferase.     

Enhancement of wound-induced ethylene synthesis by ethylene in preclimacteric cantaloupe

Although intact fruits of unripe cantaloupe (Cucumis melo L.) produce very little ethylene, a massive increase in ethylene production occurred in response to excision. The evidence indicates that this wound ethylene is produced from methionine via 1-aminocyclopropanecarboxylic acid (ACC) as in ripening fruits. Excision induced an increase in both ACC synthase and the enzyme converting ACC to ethylene. Ethylene further increased the activity of the enzyme system converting ACC to ethylene. The induction by ethylene required a minimum exposure of 1 hour; longer exposure had increasingly larger effect. The response was saturated at approximately 3 microliters per liter ethylene and was inhibited by Ag⁺. Neither ethylene nor ACC had a promotive or inhibitory effect on ACC synthase beyond the effect attributable to wounding.     

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     

The Effect of Ethylene and Propylene Pulses on Respiration, Ripening Advancement, Ethylene-Forming Enzyme, and 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Avocado Fruit

When early-season avocado fruit (Persea americana Mill. cv Hass) were treated with ethylene or propylene for 24 hours immediately on picking, the time to the onset of the respiratory climacteric, i.e. the lag period, remained unchanged compared with that in untreated fruit. When fruit were pulsed 24 hours after picking, on the other hand, the lag period was shortened. In both cases, however, a 24 hour ethylene or propylene pulse induced a transient increase in respiration, called the pulse-peak, unaccompanied by ethylene production (IL Eaks [1980] Am Soc Hortic Sci 105: 744-747). The pulse also caused a sharp rise in ethylene-forming enzyme activity in both cases, without any increase in the low level of 1-aminocyclopropane-1-carboxylic acid synthase activity. Thus, the shortening of the lag period by an ethylene pulse is not due to an effect of ethylene on either of the two key enzymes in ethylene biosynthesis. A comparison of two-dimensional polyacrylamide gel electrophoresis polypeptide profiles of in vitro translation products of poly(A⁺) mRNA from control and ethylene-pulsed fruit showed both up- and down-regulation in response to ethylene pulsing of a number of genes expressed during the ripening syndrome. It is proposed that the pulse-peak or its underlying events reflect an intrinsic element in the ripening process that in late-season or continuously ethylene-treated fruit may be subsumed in the overall climacteric response. A computerized system that allows continuous readout of multiple samples has established that the continued presentation of exogeneous ethylene or propylene to preclimacteric fruit elicits a dual respiration response comprising the merged pulse-peak and climacteric peak in series. The sequential removal of cores from a single fruit has proven an unsatisfactory sampling procedure inasmuch as coring induces wound ethylene, evokes a positive respiration response, and advances ripening.     

Influence of Plant Hormones on Ethylene Production in Apple, Tomato, and Avocado Slices during Maturation and Senescence

Ethylene production by tissue slices from preclimacteric, climacteric, and postclimacteric apples was significantly reduced by isopentenyl adenosine (IPA), and by mixtures of IPA and indoleacetic acid, and of IPA, indoleacetic acid, and gibberellic acid after 4 hours of incubation. Ethylene production by apple (Pyrus malus L.) slices in abscisic acid was increased in preclimacteric tissues, decreased in climacteric peak tissues, and little affected in postclimacteric tissues. Indoleacetic acid suppressed ethylene production in tissues from preclimacteric apples but stimulated ethylene production in late climacteric rise, climacteric, and postclimacteric tissue slices. Gibberellic acid had less influence in suppressing ethylene production in preclimacteric peak tissue, and little influenced the production in late climacteric rise, climacteric peak, and postclimacteric tissues. IPA also suppressed ethylene production in pre- and postclimacteric tissue of tomatoes (Lycopersicon esculentum) and avocados (Persea gratissima). If ethylene production in tissue slices of ripening fruits is an index of aging, then IPA would appear to retard aging in ripening fruit, just as other cytokinins appear to retard aging in senescent leaf tissue.     

Activity of Ageing Carnation Flower Parts and the Effects of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid-Induced Ethylene

Peak levels of 1-aminocyclopropane-l-carboxylic acid (ACC) in flower parts of ageing carnations (Dianthus caryophyllus L. cv Scanea 3C) were detected 6 to 9 days after flower opening. The ethylene climacteric and the first visible sign of wilting was observed 7 days after opening. The concentration of conjugated ACC in these same tissues peaked at day three with reduction of 70% by day 4. From day 5 to day 9 all parts followed a diurnal pattern of increasing in conjugate levels 1 day and decreasing the next. Concentrations of conjugated ACC were significantly higher than those of ACC in all ageing parts. Preclimacteric petals treated with ACC or 1-(malonylamino)-cycloprane-1-carboxylic acid (MACC), started to senesce 30 to 36 hours after treatment. When petals were treated with MACC plus by 0.1 millimolar aminoethyoxyvinylglycine, premature senescence was induced, while ethylene production was suppressed relative to MACC-treated petals. Petals treated with MACC and silver complex produced ethylene, but did not senesce. The MACC-induced ethylene was inhibited by the addition of 1.0 millimolar CoC1₂. These results demonstrate MACC-induced senescence in preclimacteric petals. The patterns of ACC and MACC detected in the flower parts support the view that an individual part probably does not export an ethylene precursor to the remainder of the flower inducing senescence.     

Rapid Ethylene Production in Soybean in Response to the Cupric Ion

Very rapid accumulation of free 1-aminocyclopropane-1-carboxylicacid (ACC), followed by stimulation of ethylene production wereinduced by a Cu²⁺ in soybean cuttings. The accumulation mustbe attributed to an increase in ACC synthesis, because: (1)it was completely inhibited by aminoethoxyvinylglycine (AVG);and (2) the ethylene stimulation was inhibited by AVG, indicatingthat free ACC cannot be released from its conjugated form. Theactivity of the ethylene-forming enzyme slightly decreased followingthe Cu²⁺ pulse, and this event was accompanied by a slight increasein electrolyte leakage from the treated soybean tissues. Glycine max L., soybean, ethylene, cupric ion     

Role of the mitochondria in the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene in plant tissues

Intact plant mitochondria, isolated from climacteric (Lycopersicon esculentum, Mill., tomato) or non-climacteric (Solanum tuberosum, L., potato) tissues, and purified on Percoll density gradients, were unable to convert 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene. Energization or sonication did not enhance ethylene production. For both tissues, the low activity of ACC conversion found in crude mitochondrial fractions from both tissues was increased by sonication. After mitochondrial purification, this activity was located on top of the gradient together with the microsomal membrane fraction containing a high lipoxygenase activity. Addition of exogenous lipoxygenase and linoleic acid to isolated tomato or potato mitochondria greatly enhanced ACC conversion (to approx. 300 pmol h⁻¹ mg⁻¹ protein). Direct measurements of ACC uptake by mitochondria indicated that ACC uptake is not dependent on energization.     

Comparison of Propylene-induced Responses of Immature Fruit of Normal and rin Mutant Tomatoes

Continuous application of propylene to 40 to 80% mature fruits of normal tomato strains (Lycopersicon esculentum Mill.) advanced ripening in fruits of all ages by at least 50%. Although preclimacteric respiration was stimulated by propylene treatment, there was no concomitant increase in ethylene production. Once ripening commenced, the rates of endogenous ethylene production were similar in both propylene-treated and untreated fruits. Continuous exposure to propylene also stimulated respiration in immature fruits of rin, a nonripening mutant. Although respiration reached rates similar to those during the climacteric of comparable normal fruits there was no change in endogenous ethylene production which remained at a low level. Internal ethylene concentrations in attached 45 to 75% mature fruits of rin and a normal strain were similar. It is suggested that the onset of ripening in normal tomato fruit is not controlled by endogenous ethylene, although increased ethylene production is probably an integral part of the ripening processes.     

Xylem Transport of 1-Aminocyclopropane-1-carboxylic Acid, an Ethylene Precursor, in Waterlogged Tomato Plants

Waterlogging is known to cause an increase in ethylene synthesis in the shoot which results in petiole epinasty. Evidence has suggested that a signal is synthesized in the anaerobic roots and transported to the shoot where it stimulates ethylene synthesis. Experimental data are presented showing that 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, serves as the signal. Xylem sap was collected from detopped tomato plants (Lycopersicon esculentum Mill. cv. VFN8). ACC in the sap was quantitated by a sensitive and specific assay, and its tentative chemical identity verified by paper chromatography. ACC levels in both roots and xylem sap increased markedly in response to waterlogging or root anaerobiosis. The appearance of ACC in the xylem sap of flooded plants preceded both the increase in ethylene production and epinastic growth, which were closely correlated. Plants flooded and then drained showed a rapid, simultaneous drop in ACC flux and ethylene synthesis rate. ACC supplied through the cut stem of tomato shoots at concentrations comparable to those found in xylem sap caused epinasty and increased ethylene production. These data indicate that ACC is synthesized in the anaerobic root and transported to the shoot where it is readily converted to ethylene.     

Effect of low oxygen and high carbon dioxide on the levels of ethylene and 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

The association of the level of ACC and the ethylene concentration in ripening apple fruit (Malus sylvestris Mill, var. Ben Davis) was studied. Preclimacteric apple contained small amounts of ACC and ethylene. With the onset of the climacteric and a concomitant decrease in flesh firmness, the level of ACC and ethylene concentration both increased markedly. During the postclimacteric period, ethylene concentration started to decline, but the level of ACC continued to increase. Ethylene production and loss of flesh firmness of fruits during ripening were greatly suppressed by treatments with low O₂ (O₂ 1–3%, CO₂ O%) or high CO₂ (CO₂ 20–30%, O₂ 15–20%) at the preclimacteric stage. However, after 4 weeks an accumulation of ACC was observed in treated fruits when control fruit was at the postclimacteric stage. Treatment of fruit with either low O₂ or high CO₂ at the climacteric stage resulted in a decrease of ethylene production. However, the ACC level in fruit treated with low O₂ was much higher than both control and high CO₂ treated fruit; it appears that low O₂ inhibits only the conversion of ACC to ethylene, resulting in an accumulation of ACC. Since CO₂ inhibits ethylene production but does not result in an accumulation of ACC, it appears that high CO₂ inhibits both the conversion of ACC to ethylene and the formation of ACC.