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.     

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.     

Involvement of wound and climacteric ethylene in ripening avocado discs

Avocado (Persea americana Mill. cv Hass) discs (3 mm thick) ripened in approximately 72 hours when maintained in a flow of moist air and resembled ripe fruit in texture and taste. Ethylene evolution by discs of early and midseason fruit was characterized by two distinct components, viz. wound ethylene, peaking at approximately 18 hours, and climacteric ethylene, rising to a peak at approximately 72 hours. A commensurate respiratory stimulation accompanied each ethylene peak. Aminoethoxyvinyl glycine (AVG) given consecutively, at once and at 24 hours following disc preparation, prevented wound and climacteric respiration peaks, virtually all ethylene production, and ripening. When AVG was administered for the first 24 hours only, respiratory stimulation and softening (ripening) were retarded by at least a day. When AVG was added solely after the first 24 hours, ripening proceeded as in untreated discs, although climacteric ethylene and respiration were diminished. Propylene given together with AVG led to ripening under all circumstances. 2,5-Norbornadiene given continuously stimulated wound ethylene production, and it inhibited climacteric ethylene evolution, the augmentation of ethylene-forming enzyme activity normally associated with climacteric ethylene, and ripening. 2,5-Norbornadiene given at 24 hours fully inhibited ripening. When intact fruit were pulsed with ethylene for 24 hours before discs were prepared therefrom, the respiration rate, ethylene-forming enzyme activity buildup, and rate of ethylene production were all subsequently enhanced. The evidence suggests that ethylene is involved in all phases of disc ripening. In this view, wound ethylene in discs accelerates events that normally take place over an extended period throughout the lag phase in intact fruit, and climacteric ethylene serves the same ripening function in discs and intact fruit alike.     

Role of Brassinosteroids, Ethylene, Abscisic Acid, and Indole-3-Acetic Acid in Mango Fruit Ripening

Rapid ripening of mango fruit limits its distribution to distant markets. To better understand and perhaps manipulate this process, we investigated the role of plant hormones in modulating climacteric ripening of ??Kensington Pride?? mango fruits. Changes in endogenous levels of brassinosteroids (BRs), abscisic acid (ABA), indole-3-acetic acid (IAA), and ethylene and the respiration rate, pulp firmness, and skin color were determined at 2-day intervals during an 8-day ripening period at ambient temperature (21?±?1°C). We also investigated the effects of exogenously applied epibrassinolide (Epi-BL), (+)-cis, trans-abscisic acid (ABA), and an inhibitor of ABA biosynthesis, nordihydroguaiaretic acid (NDGA), on fruit-ripening parameters such as respiration, ethylene production, fruit softening, and color. Climacteric ethylene production and the respiration peak occurred on the fourth day of ripening. Castasterone and brassinolide were present in only trace amounts in fruit pulp throughout the ripening period. However, the exogenous application of Epi-BL (45 and 60?ng?g^?1 FW) advanced the onset of the climacteric peaks of ethylene production and respiration rate by 2 and 1?day, respectively, and accelerated fruit color development and softening during the fruit-ripening period. The endogenous level of ABA rose during the climacteric rise stage on the second day of ripening and peaked on the fourth day of ripening. Exogenous ABA promoted fruit color development and softening during ripening compared with the control and the trend was reversed in NDGA-treated fruit. The endogenous IAA level in the fruit pulp was higher during the preclimacteric minimum stage and declined during the climacteric and postclimacteric stages. We speculate that higher levels of endogenous IAA in fruit pulp during the preclimacteric stage and the accumulation of ABA prior to the climacteric stage might switch on ethylene production that triggers fruit ripening. Whilst exogenous Epi-BL promoted fruit ripening, endogenous measurements suggest that changes in BRs levels are unlikely to modulate mango fruit ripening.     

Interrelationship of Gene Expression, Polysome Prevalence, and Respiration during Ripening of Ethylene and/or Cyanide-Treated Avocado Fruit

Upon initiation of ripening in avocado fruit (Persea americana Mill. cv Hass) with 10 microliters/liter ethylene, polysome prevalence and associated poly(A)⁺ mRNA increase approximately 3-fold early in the respiratory climacteric and drop off to preclimacteric levels at the peak of the respiratory climacteric. The increase in poly(A)⁺ mRNA on polysomes early in the respiratory climacteric constitutes a generic increase in constitutive mRNAs. New gene expression associated with ripening is minimal but evident after 10 hours of ethylene treatment and continues to increase relative to constitutive gene expression throughout the climacteric. The respiratory climacteric can be temporally separated into two phases. The first phase is associated with a general increase in protein synthesis, whereas the second phase reflects new gene expression and accumulation of corresponding proteins which may be responsible for softening and other ripening characteristics. A major new message on polysomes that arises concomitantly with the respiratory climacteric codes for an in vitro translation product of 53 kilodaltons which is immunoprecipitated by antiserum against avocado fruit cellulase.

Cyanide at 500 microliters/liter fails to affect the change in polysome prevalance or new gene expression associated with the ethylene-evoked climacteric in avocado fruit. Treatment of fruit with 500 microliters/liter cyanide alone initiates a respiratory increase within 4 hours, ethylene biosynthesis within 18 hours, and new gene expression akin to that educed by ethylene within 20 hours of exposure to cyanide.

     

Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene

The inhibitory effects of the cyclic olefin 2,5-norbornadiene (NBD) on ethylene action were tested in carnation (Dianthus caryophyllus L. cv White Sim) flowers. Treatment of flowers at anthesis with ethylene in the presence of 500 microliters per liter NBD increased the concentration of ethylene required to elicit a response (petal senescence), indicating that NBD behaves as a competitive inhibitor of ethylene action. Transfer of flowers producing autocatalytic ethylene and exhibiting evidence of senescence (petal in-rolling) to an atmosphere of NBD resulted in a rapid reduction in ethylene production, petal 1-aminocyclopropane-1-carboxylic acid synthase activity, 1-aminocyclopropane-1-carboxylic acid content, and ethylene forming enzyme activity. Removal of NBD resulted in recovery of ethylene biosynthesis. These results support the autocatalytic regulation of ethylene production during the climacteric stage of petal senescence and suggest that continued perception of ethylene is required for maintenance of ethylene biosynthesis. The inhibition of ethylene action by NBD after the flowers had reached the climacteric peak was associated with interruption of petal senescence as evidenced by reversal of senescence symptoms. This result is in contrast to the widely held belief that the rate of petal senescence is fixed and irreversible once petals enter into the ethylene climacteric.     

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.     

The climacteric-like behaviour of young, mature and wounded citrus leaves

Although leaves and other vegetative tissues are generally considered as non-climacteric, citrus leaves show a climacteric system II behaviour after detachment. Upon harvest, young, fully expanded 'Valencia' orange (Citrus sinensis) leaves ( approximately 60-d-old) exhibited two phases of ethylene production. The first phase, up to 6 d after detachment, was characterized by a low and constant ethylene production (system I pathway), associated with a constitutive expression of ACC synthase 2 (CsACS2), CsERS1, and CsETR1. ACC synthase 1 (CsACS1) was not expressed during this phase and autoinhibition of ethylene production was apparent following treatment with exogenous ethylene or propylene. The second phase, 7-12 d after detachment, was characterized by a climacteric rise in ethylene production, preceded by the induction of CsACS1 and ACC oxidase 1 (CsACO1) gene expression in the system II pathway. This induction was accelerated and augmented by exogenous ethylene or propylene, indicating an autocatalytic system II ethylene biosynthesis. Mature leaves (6-8-months-old) behaved similarly, except that the climacteric peak in ethylene production occurred earlier (day 5). Young and mature leaves varied in the timing of the climacteric ethylene rise and CsACS1 and CsACO1 induction. The two phases of ethylene production, system I and system II, were also detected in wounded leaf discs of both young and mature leaves. The first phase peaked 15 min after excision and the second phase peaked after 6 h.     

Effects of aminoethoxyvinylglycine and countereffects of ethylene on ripening of bartlett pear fruits

Pear fruits (Pyrus communis L. var. Bartlett) were treated with solutions containing aminoethoxyvinylglycine (AVG) using a modified vacuum infiltration method that introduced 4.3 milliliters solution per 100 grams tissue. At concentrations of 1 millimolar, AVG strongly inhibited ethylene production and delayed for 5 days the respiratory climacteric and accompanying ripening changes in skin color and flesh firmness. AVG was less effective in inhibiting the ripening of more mature fruits. Fruit infiltrated with 5 millimolar AVG had not begun to ripen 12 days after initiation of ripening in the controls. When treated with ethylene the inhibited fruit exhibited a climacteric rise in respiration, softened, and became yellow. Treatment of the AVG infiltrated fruits with ethyelne for 24 hours resulted in no recovery in endogenous ethylene production, but in a stimulation of protein synthesis measured as a 200% increase in leucine incorporation by excised tissue and a 74% increase in the percentage of ribosomes present as polysomes.     

The Relation of Ethylene Production and Respiration with Maturation and Senescence of Melon during Postharvest-Storage

There are three types of ethylene production and respiration during the postharvest storage of melon fruits: 1. with climacteric rise in both respiration and ethylene production; 2 with climacteric rise in ethylene production but respiration declined gradually; 3 with no appreciable change in both respiration and ethylene production. The last one has good storage quality.     

Effect of 2,5-Norbornadiene upon Ethylene Biosynthesis in Midclimacteric Carnation Flowers

The climacteric increase in ethylene production in carnation (Dianthus caryophyllus L. cv White Sim) flowers is known to be accompanied by an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase and ethylene forming enzyme (EFE) activities. When midclimacteric flowers were exposed to 2,5-norbornadiene, which blocks ethylene action, ethylene production began to decrease after 2 to 3 hours. ACC synthase activity was markedly reduced after 4 hours and the increase in EFE activity was blocked indicating that the autocatalytic signal associated with ethylene action stimulates both enzyme activities.     

Ethanol reduces sensitivity to ethylene and delays petal senescence in cut Tweedia caerulea flowers

Tweedia caerulea flowers are sensitive to ethylene and the closing of the flowers, a characteristic of senescence, is accelerated by exposure to ethylene. T. caerulea flowers were continuously treated with ethanol at concentrations of 0, 2, 4, 6, 8, 10 or 12 %, and treatment levels at 4 % and above showed delayed closing. Ethanol accelerated climacteric increase in ethylene production from flowers. Although ethylene production was higher in gynoecium than in petals, ethanol treatment accelerated ethylene production by both organs. Exposure to ethylene increased autocatalytic ethylene production, and production was further accelerated by ethanol treatment. When flowers treated with ethanol were exposed to ethylene, senescence was delayed compared to that for untreated flowers, suggesting that ethanol reduces the sensitivity of flowers to ethylene. These results indicate that treatment with ethanol delays petal senescence in cut T. caerulea flowers, possibly through reduced sensitivity to ethylene.     

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.     

Promotion by Ethylene of the Capability to Convert 1-Aminocyclopropane-1-carboxylic Acid to Ethylene in Preclimacteric Tomato and Cantaloupe Fruits

The intact fruits of preclimacteric tomato (Lycopersicon esculentum Mill) or cantaloupe (Cucumis melo L.) produced very little ethylene and had low capability of converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. When these unripe tomato or cantaloupe fruits were treated with ethylene for 16 hours there was no increase in ACC content or in ethylene production rate, but the tissue's capability to convert ACC to ethylene increased markedly. Such an effect was also observed in fruits of tomato mutants rin and nor, which do not undergo ripening and the climacteric increase in ethylene production during the senescence. The development of this ethylene-forming capability induced by ethylene increased with increasing ethylene concentration (from 0.1 to 100 microliters per liter) and duration (1 to 24 hours); when ethylene was removed this capability remained high for sometime (more than 24 hours). Norbornadiene, a competitive inhibitor of ethylene action, effectively eliminated the promotive effect of ethylene in tomato fruit. These data indicate that the development of the capability to convert ACC to ethylene in preclimacteric tomato and cantaloupe fruits are sensitive to ethylene treatment and that when these fruits are exposed to exogenous ethylene, the increase in ethylene-forming enzyme precedes the increase in ACC synthase.     

Inhibition of apple ripening by succinic Acid, 2,2-dimethyl hydrazide and its reversal by ethylene

Ripening of McIntosh apples (Malus sylvestris L.) was delayed by treatments of succinic acid 2,2-dimethyl hydrazide (B-9) applied 2 weeks after bloom. The extent of retardation was dependent on concentration to 7.4 × 10⁻² m, at which level complete inhibition of ripening occurred under the conditions applied. The onset of the respiratory climacteric and fruit firmness changes were factors used to assess ripening.     

Effect of phenolic compounds in Lycopersicon esculeutum on the synthesis of ethylene

Caffeic, coumaric, sinapic and ferulic acids and naringenin were found in green tomato fruit, Chlorogenic acid accounted for 75% of the total phenolics in mature green fruit but only 35% in ripe fruit. There was very little change in the phenolic composition of the flesh of the fruit during ripening, whereas in the skin, naringenin increased markedly at the onset of the climacteric and three unidentified compounds increased during the climacteric rise. The increase in the concentration of naringenin was accompanied by an increase in the production of ethylene in the skin. Investigation of three systems producing ethylene from 4-methylmercapto-2-oxobutyric acid in the presence of peroxidase, showed that only p`-coumaric acid or naringenin were capable of acting as phenolic substrates, the other phenolic compounds being inhibitory.     

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.     

Ethylene biosynthesis in oilseed rape pods in relation to pod shatter

Ethylene production was studied during the development and senescence of seeds and pericarp tissues of oilseed rape (Brassica napus L.) pods (siliquae). In the course of the rise to a pre-senescence climacteric, little change in 1-aminocyclopropane-1-carboxylic acid (ACC) was recorded in the seeds, indicating a rapid conversion to ethylene. In contrast, very small amounts of ethylene were produced by the pod wall (PW) tissues, which included the dehiscence zone (DZ), while levels of free and conjugated ACC in the PW increased consistently. As climacteric thylene production by the seeds declined, biosynthesis of ethylene by the PW increased. Effects of reducing ethylene production by various means were examined in relation to cell separation in the dehiscence zone. Aminoethoxyvinylglycine (AVG) applied during the pre-senescence climacteric reduced ACC levels and ethylene production by the seeds, but did not affect subsequent values in the PW. The production of -1,4-glucanase and the separation of the cells of the DZ were delayed for 3-4 d by AVG, but the force required to open fully mature pods was unaltered. In parthenocarpic (seedless) pods, ethylene was produced during senescence. Cell separation in the DZ took place as in seeded pods, although it was also delayed by 3-4 d. The results are related to changes in indole-3-acetic acid (IAA) levels in oilseed rape pods which decline in PW and DZ tissues during senescence. It is concluded that separation in the cells of the dehiscence zone requires only small amounts of ethylene to trigger the process when IAA levels are low.