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.     

Induction by Electric Currents of Ethylene Biosynthesis in Cucumber (Cucumis sativus L.) Fruit

The effects of an electric current on ethylene biosynthesis were investigated in cucumber (Cucumis sativus L.) fruit that were producing almost no ethylene. Direct currents at 0.5 to 3.0 milliamperes induced much ethylene synthesis, with a rapid continuous increase in the rate, which reached a peak within 5 to 6 hours and then decreased. The rate of production was greater with a stronger current. Ethylene production was not observed after the use of a sine-wave alternating current (60 hertz) at 3 milliamperes, the magnitude at which a direct current had the greatest effect. The activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ethylene forming enzyme (EFE) increased before the rise in ethylene production. ACC synthase and EFE were activated sixfold and fourfold, respectively, by 2 hours. The concentration of ACC increased linearly up to 6 hours and then decreased. Ethylene induction by an electric current was suppressed almost completely by the infiltration of the cucumbers with 5 millimolar aminooxyacetic acid, an inhibitor of ACC synthase, and was also suppressed 70% by 5 millimolar salicylic acid, an inhibitor of EFE. The results indicate that the ethylene induced by the direct current was synthesized via the ACC-ethylene pathway as a result of electrical stress, a new kind of stress to be identified.     

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     

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.     

Role of Aminocyclopropane-1-carboxylic Acid (ACC) in Control of Ethylene Production in Fresh and Cold-stored Rose Flowers

The relationships between ethylene production, aminocyclopropane-1-carboxylicacid (ACC) content and ethylene-forming-enzyme (EFE) activityduring ageing and cold storage of rose flower petals (Rose hybridaL. cv. Gabriella) were investigated. During flower ageing at20 °C there was a climacteric rise in petal ethylene production,a parallel increase in ACC content, but a continuous decreasein EFE activity. Applied ACC increased petal ethylene productionc. 200-fold. During cold storage of flowers at 1 °C therewere parallel increases in petal ethylene production and ACCcontent, to levels greater than those reached in fresh flowersheld at 20 °C. EFE activity decreased during storage. Immediatelyafter cold-stored flowers were transferred to 20 °C ethyleneproduction and ACC levels were c. four times greater than infreshly cut flowers. These levels increased to maximum valuesof two to four times the maximum values reached during ageingof fresh, unstored, flowers. It was concluded that in rose petalsethylene synthesis is probably regulated by ACC levels and thatcold storage stimulates ethylene synthesis because it increasesthe levels of ACC in the petals. Key words: Rose flower, senescence, ethylene     

Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits

Biggs, M. S., Woodson, W. R. and Handa, A. K. 1988. Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits. Physiol. Plant. 72: 572578
Incubation of fruits of tomato ( Lycopersicon esculentum Mill. cv. Rutgers) at 34°C or above resulted in a marked decrease in ripening-associated ethylene production. High temperature inhibition of ethylene biosynthesis was not associated with permanent tissue damage, since ethylene production recovered following transfer of fruits to a permissive temperature. Determination of pericarp enzyme activities involved in ethylene biosynthesis following transfer of fruits from 25°C to 35 or 40°C revealed that 1-aminocyclopropane-l-carboxylic acid (ACC) synthase (EC 4.4.1.14) activity declined rapidly while ethylene forming enzyme (EFE) activity declined slowly. Removal of high temperature stress resulted in more rapid recovery of ACC synthase activity relative to EFE activity. Levels of ACC in pericarp tissue reflected the activity of ACC synthase before, during, and after heat stress. Recovery of ethylene production following transfer of pericarp discs from high to permissive temperature was inhibited in the presence of cycloheximide, indicating the necessity for protein synthesis. Ethylene production by wounded tomato pericarp tissue was not as inhibited by high temperature as ripening-associated ethylene production by whole fruits.     

Relationship between the activity of the ethylene-forming enzyme and the level of intracellular 2,4-dichlorophenoxyacetic acid in pear cell culturesin vitro

Ethylene production by auxin-dependent pear cells culturedin vitro falls rapidly when they are deprived of 2,4-D. This phenomenon is associated with a decrease in ACC production. Readdition of 2,4-D causes a resumption of ACC production and ethylene synthesis. Ethylene-forming enzyme (EFE) activity, although never limiting, decreases sharply during 2,4-D depletion and rises again upon addition of 2,4-D. This increase in the EFE activity is not a rapid response to 2,4-D, since it requires several hours. Changes in EFE activity follow the same pattern as changes in 2,4-D concentration; the decrease in EFE activity is also concomitant with a decrease in the ability of 2,4-dinitrophenol to inhibit ethylene production. The possible role of auxins in the modulation of EFE activity is discussed.     

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     

Induction of 1-Aminocyclopropane-1-carboxylic Acid Synthase in Wounded Mesocarp Tissue of Winter Squash Fruit and the Effects of Ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase activityincreased rapidly after wounding of mesocarp tissue of wintersquash fruit (Cucurbita maxima Duch.) and reached a peak at16 h after excision and then declined sharply. The rise in ACCsynthase activity was followed by increases in the endogenousACC content and the rate of ethylene production. The activityof ethylene forming enzyme (EFE) also increased rapidly in theexcised discs of mesocarp of winter squash fruit. ACC synthase activity was strongly inhibited by aminoethoxyvinylglycinewith a Ki value of 2.1 µM. Michaelis-Menten constant ofACC synthase for S-adenosylmethionine was 13.3 µM. Ethylene suppressed the induction of ACC synthase in the woundedmesocarp tissue. The suppression by ethylene increased withthe increasing concentrations of applied ethylene and the maximumeffect was obtained at about 100 µl 1^–1 ethylene,at which point the induction was suppressed by 54%. Ethylenedid not inhibit ACC synthase activity, nor did it suppress theinduction of EFE, but rather it slightly enhanced the latter. (Received August 24, 1984; Accepted October 29, 1984)     

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.     

Autoinhibition of Ethylene Production in Citrus Peel Discs : SUPPRESSION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHESIS

Wound ethylene formation induced in flavede tissue of citrus fruit (Citrus paradisi MacFad. cv. Ruby Red) by slicing was almost completely inhibited by exogenous ethylene. The inhibition lasted for at least 6 hours after removal of exogenous ethylene and was then gradually relieved. The extent of inhibition was dependent upon the concentration of ethylene (1 to 10 microliters/liter) and the duration of treatment. The increase in wound ethylene production in control discs was paralleled by an increase in 1-aminocyclopropane-1-carboxylic acid (AAC) content, whereas in ethylene-treated discs there was little increase in ACC content. Application of ACC completely restored ethylene production in ethylene-pretreated discs, indicating that the conversion of ACC to ethylene is not impaired by the presence of ethylene. Thus, autoinhibition of ethylene synthesis was exerted by reducing the availability of ACC. Ethylene treatment resulted in a decrease in extractable ACC synthase activity, but this decrease was too small to account for the marked inhibition of ACC formation. The data indicate that autoinhibition of ethylene production in citrus flavede discs results from suppression of ACC formation through repression of the synthesis of ACC synthase and inhibition of its activity.     

2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers

2-Aminooxyisobutyric acid (AOIB) has a partial structure of aminooxyacetic acid (AOA) in its whole structure, and resembles 2-aminoisobutyric acid (AIB) in their tetrahedral structures. Both AOA and AIB are inhibitors of ethylene biosynthesis; AOA inhibits the action of 1-aminocyclopropane-1-carboxylate (ACC) synthase and AIB inhibits that of ACC oxidase. The present study showed that AOIB inhibited the in vitro activities of both ACC synthase and ACC oxidase, which were synthesized heterologously in E. coli cells from corresponding carnation cDNAs, and the magnitudes of inhibition were similar to those caused by AOA and AIB; AOIB and AOA at 0.1 mM inhibited ACC synthase action by 75%, and AOIB and AIB at 10 mM inhibited ACC oxidase action by 16.3 and 22.5%, respectively. AOIB at 1 mM caused 91.5% reduction of maximum ethylene production rate as compared to the control in cut ‘Excerea’ carnation flowers undergoing senescence, thereby lengthening their vase life to 7 d from 3 d of the control flowers. The inhibition by AOIB was probably caused by its action resembling AOA, but not AIB. AOIB also extended significantly the vase life of cut flowers of ‘Pax’ carnation, and tended to do so in ‘Primero Mango’ carnation. The present findings suggest the potential of AOIB as a new preservative for carnations and other ornamentals in which ethylene plays a key role in the induction of senescence.     

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     

Relationship between the activity of the ethylene-forming enzyme and the level of intracellular 2,4-dichlorophenoxyacetic acid in pear cell culturesin vitro

Ethylene production by auxin-dependent pear cells culturedin vitro falls rapidly when they are deprived of 2,4-D. This phenomenon is associated with a decrease in ACC production. Readdition of 2,4-D causes a resumption of ACC production and ethylene synthesis. Ethylene-forming enzyme (EFE) activity, although never limiting, decreases sharply during 2,4-D depletion and rises again upon addition of 2,4-D. This increase in the EFE activity is not a rapid response to 2,4-D, since it requires several hours. Changes in EFE activity follow the same pattern as changes in 2,4-D concentration; the decrease in EFE activity is also concomitant with a decrease in the ability of 2,4-dinitrophenol to inhibit ethylene production. The possible role of auxins in the modulation of EFE activity is discussed.     

Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence

Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity.Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.     

Inhibition of activity of the ethylene-forming enzyme by α(p-chlorophenoxy)isobutyric acid

(p-Chlorophenoxy)isobutyric acid (PCIB) inhibited indole-3-acetic acid (IAA)-induced ethylene production in etiolated mung bean hypocotyl sections. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC) was not significantly affected by PCIB, indicating that PCIB exerted its effect primarily by inhibiting the activity of the ethylene-forming enzyme (EFE). This conclusion was supported by the observations that PCIB inhibited the conversion of exogenously applied ACC to ethylene. The inhibitory effect of PCIB was already evident with 0.05 mM PCIB, and it increased with time after application of the inhibitor. PCIB also significantly inhibited ethylene production in apple fruit tissues, but it only slightly reduced the level of endogenous ACC. Similar to mung bean, EFE activity in apple tissue was significantly inhibited by PCIB. The possibility that PCIB also inhibits auxin-induced ACC synthase activity is discussed.     

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.     

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.