Ethylene and Wound-Induced Gene Expression in the Preclimacteric Phase of Ripening Avocado Fruit and Mesocarp Discs

Whereas intact postharvest avocado (Persea americana Mill.) fruit may take 1 or more weeks to ripen, ripening is hastened by pulsing fruit for 24 h with ethylene or propylene and is initiated promptly by cutting slices, or discs, of mesocarp tissue. Because the preclimacteric lag period constitutes the extended and variable component of the ripening syndrome, we postulated that selective gene expression during the lag period leads to the triggering of the climacteric. Accordingly, we sought to identify genes that are expressed gradually in the course of the lag period in intact fruit, are turned on sooner in response to a pulse, and are induced promptly in response to wounding (i.e. slicing). To this end, a mixed cDNA library was constructed from mRNA from untreated fruit, pulsed fruit, and aged slices, and the library was screened for genes induced by wounding or by pulsing and/or wounding. The time course of induction of genes encoding selected clones was established by probing northern blots of mRNA from tissues variously treated over a period of time. Four previously identified ripening-associated genes encoding cellulase, polygalacturonase (PG), cytochrome P-450 oxidase (P-450), and ethylene-forming enzyme (EFE, or 1-aminocyclopropane-1-carboxylic acid synthase), respectively, were studied in the same way. Whereas cellulase, PG, and EFE were ruled out as having a role in the initiation of the climacteric, the time course of P-450 induction, as well as the response of same to pulsing and wounding met the criteria[mdash]together with several clones from the mixed library[mdash]for a gene potentially involved in preclimacteric events leading to the onset of the climacteric. Further, it was established that the continuous presence of ethylene is required for persisting induction, and it is suggested that in selected cases wounding may exert a synergistic effect on ethylene action.     

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage

Differential sereening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7°C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.AFRC Research Group in Plant Gene Regulation     

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.     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effect of phytohormones on pectate lyase activity in ripening Musa acuminata.

A differential activity peak of pectate lyase (PEL) was observed during ripening of banana fruits (Musa acuminata Harichhal) receiving different hormone treatments. Exposure of fruits to 25 ppm ethylene for 24 h, as well as dipping of M. acuminata fruits in 1 mM 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 h, hastened fruit ripening. Both PEL activity peak and climacteric peak were observed on the 4th and 10th days of treatment with ethylene and 2,4-D, respectively, compared to the 16th day in control fruits. Gibberellic acid (GA) treatment retarded fruit ripening and both PEL activity and climacteric peaks were observed on the 19th day. Treatment of fruits with ethylene or 2,4-D also advanced the appearance of a polygalacturonase (PG) peak and GA delayed its appearance, but the activity peaks always appeared in post-climacteric fruits, in contrast to PEL activity peaks coinciding with the respiratory peaks.     

PRO－LONG涂膜处理对后熟香蕉果实乙烯形成以及其它有关生理变化的影响

香蕉（ＭｕｓａａｃｕｍｉｎａｔａＣｏｌｌａｃｖ．ＤｗａｒｆＣａｖｅｎｄｉｓｈ）果实采后以商业上推荐使用的１．５％Ｐｒｏ－ｌｏｎｇ溶液处理,贮藏于２０℃和７５％相对湿度下,分别测定果实的ＡＣＣ含量、ＭＡＣＣ含量、ＥＦＥ酶活性、乙烯释放、叶绿素含量的变化和果实的硬度变化．结果表明,ＰＲＯ－ＬＯＮＧ处理延缓了香蕉果实果皮的叶绿素降解、硬度的下降以及乙烯释放的增加．在后熟过程中,处理果实的ＡＣＣ含量发生积累．ＡＣＣ含量的高峰在乙烯释放高峰和ＥＦＥ酶活性高峰之前出现．与对照比较,处理果实的ＡＣＣ含量和ＥＦＥ酶活性的高峰延迟了５ｄ出现．在后熟过程中,以Ｐｒｏ－ｌｏｎｇ处理果肉四片,其ＥＦＥ酶活性受部分抑制（抑制率为１９．４５％至４０．５１％）．果实ＭＡＣＣ含量在贮藏起初处于一个较显著水平,随着后熟的发展而逐步增加,但与ＡＣＣ含量的明显增加相比变化是微小的．我们的研究进一步阐明了ＰＲＯ－ＬＯＮＧ涂膜对香蕉果实后熟的影响主要是通过减少氧的供给,部分地抑制了ＥＦＥ酶活性,延缓了乙烯的形成和释放,从而延长了后熟过程．     

Suppression of Ripening-Associated Gene Expression in Tomato Fruits Subjected to a High CO2 Concentration

High concentrations of CO2 block or delay the ripening of fruits. In this study we investigated the effects of high CO2 on ripening and on the expression of stress- and ripening-inducible genes in cherry tomato (Lycopersicon esculentum Mill.) fruit. Mature-green tomato fruits were submitted to a high CO2 concentration (20%) for 3 d and then transferred to air. These conditions effectively inhibited ripening-associated color changes and ethylene production, and reduced the protein content. No clear-cut effect was observed on the expression of two proteolysis-related genes, encoding polyubiquitin and ubiquitin-conjugating enzyme E2, respectively. Exposure of fruit to high CO2 also resulted in the strong induction of two genes encoding stress-related proteins: a ripening-regulated heat-shock protein and glutamate decarboxylase. Induction of these two genes indicated that high CO2 had a stress effect, most likely through cytosolic acidification. In addition, high CO2 blocked the accumulation of mRNAs for genes involved in the main ripening-related changes: ethylene synthesis (1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase), color (phytoene synthase), firmness (polygalacturonase), and sugar accumulation (acid invertase). The expression of ripening-specific genes was affected by CO2 regardless of whether their induction was ethylene- or development-dependent. It is proposed that the inhibition of tomato fruit ripening by high CO2 is due, in part, to the suppression of the expression of ripening-associated genes, which is probably related to the stress effect exerted by high CO2.     

Exo- and Endo-Cellular Cellulase and Polygalacturonase in Abscission Zones of Developing Orange Fruits

The activity of cellulase, cellulase-isoenzymes and polygalacturonase (PG) in the shoot/peduncle and calyx abscission zones (AZ-A and AZ-C, respectively) of young and mature Shamouti orange (Citrus sinensis (L.) Osbeck) fruit explants was tested after extraction of total enzymes from either exo- or endo-cellular fractions from fruits treated with ethylene or 2,4-D. Ethylene enhanced and 2,4-D delayed both abscission and the activity of exo- and endo-cellular cellulase and PG. When tested separately in the exo- and endo-cellular fraction, the effects of both growth regulators on the activity of almost all cellulase isoenzymes were similar, irrespective of their location in the tissue. In mature fruits no abscission occurred in AZ-A, and yet the activity of cellulase and PG was regulated by the hormones as in abscising AZs. This was also true for total activity of exo- and endo-cellular cellulase and PG. Similar effects were observed when the activity of cellulase isoenzymes was tested in AZ-A of non-abscising mature fruits. It is suggested that whenever the increase in activity of the hydrolytic enzymes, and especially cellulase, is not followed by abscission, the substrate is either immune or not available to the enzymes.     

The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening

Tomato mRNA was extracted from individual fruits at different stages of development and ripening, translated in a rabbit reticulocyte lysate and the protein products analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicate that there are at least two classes of mRNA under separate developmental control. One group of approximately six mRNAs is present during fruit growth and then declines at the mature-green stage. Another group of between four and eight mRNAs increases substantially in amount at the onset of ripening, after the start of enhanced ethylene synthesis by the fruit, and continues to accumulate as ripening progresses. Studies of protein synthesis in vivo show that several new proteins are synthesised by ripening fruits including the fruit-softening enzyme polygalacturonase. One of the ripening-related mRNAs is shown to code for polygalacturonase, by immunoprecipitation with serum from rabbits immunised against the purified tomato enzyme. Polygalacturonase mRNA is not detectable in green fruit but accumulates during ripening. It is proposed that the ripening-related mRNAs are the products of a group of genes that code for enzymes important in the ripening process.Abbreviation SDS sodium dodecyl sulfate     

Reversible Inhibition of Tomato Fruit Gene Expression at High Temperature (Effects on Tomato Fruit Ripening)

The reversible inhibition of three ripening-related processes by high-temperature treatment (38[deg]C) was examined in tomato (Lycopersicon esculentum L. cv Daniella) fruit. Ethylene production, color development, and softening were inhibited during heating and recovered afterward, whether recovery took place at 20[deg]C or fruit were first held at chilling temperature (2[deg]C) after heating and then placed at 20[deg]C. Ethylene production and color development proceeded normally in heated fruit after 14 d of chilling, whereas the unheated fruit had delayed ethylene production and uneven color development. Levels of mRNA for 1-aminocyclopropane-1-carboxylic acid oxidase, phytoene synthase, and polygalacturonase decreased dramatically during the heat treatment but recovered afterward, whereas the mRNA for HSP17 increased during the high-temperature treatment and then decreased when fruit were removed from heat. As monitored by western blots, the HSP17 protein disappeared from fruit tissue after 3 d at 20[deg]C but remained when fruit were held at 2[deg]C. The persistence of heat-shock proteins at low temperature may be relevant to the protection against chilling injury provided by the heat treatment. Protein levels of 1-aminocyclopropane-1-carboxylic acid oxidase and polygalacturonase also did not closely follow the changes in their respective mRNAs. This implied both differences in relative stability and turnover rates of mRNA compared to protein and nontranslation of the message that accumulated in low temperature. The results suggest that high temperature inhibits ripening by inhibiting the accumulation of ripening-related mRNAs. Ripening processes that depend on continuous protein synthesis including ethylene production, lycopene accumulation, and cell-wall dissolution are thereby diminished.     

Foliar application of aminoethoxyvinylglycine (AVG) delays fruit ripening and reduces pre-harvest fruit drop and ethylene production of bagged “Kogetsu” apples

Covering apple fruits with double layer waterproof bags to enhance fruit quality and evenness of blush colour is typical on many cultivars in Korea and Japan. Aminoethoxyvinylglycine (AVG) applied to unbagged apple fruits at 3–4 weeks before commercial harvest reduces ethylene production in the fruit, delays fruit ripening and reduces pre-harvest fruit drop. Spray application of AVG to trees of bagged apples should have no effect on apple ripening as there is␣no direct contact with the fruit and the translocation of AVG in apple trees is regarded as negligible. However, preliminary experiments suggested that AVG applied to trees of bagged apples reduced pre-harvest fruit drop in “Kotgetsu” apples. This study investigated the effect of spray treatments of 125 ppm of AVG on fruit drop, fruit ripening (firmness, starch conversion and soluble solids) and ethylene production to whole trees with bagged or unbagged “Kogetsu” fruit, as well as sprays of only the bagged or unbagged fruit on trees on two orchards. AVG applied to whole trees with unbagged apples reduced fruit drop from an average of 58.9% to 10.4%, delayed starch conversion and decreased ethylene production. AVG applied to whole trees with bagged fruit was equally effective in reducing pre-harvest drop, delaying fruit ripening and reducing ethylene production. Application of AVG to unbagged fruit only was nearly as effective as application to whole trees with unbagged fruit but application to bagged fruit only had no effect on fruit ripening or ethylene production. Application of AVG to bagged fruit only did reduce fruit drop to an average of 42.5% but this was not as effective as spraying unbagged fruit only or whole trees with bagged fruit. Possible mechanisms for this effect are discussed.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Induction of ethylene synthesis and lipid peroxidation in damaged or TMV-infected tobacco leaf tissues by light

The effect of light on ethylene and ethane production in damaged leaf tissues was investigated. When whole leaves of tobacco cv. Samsun NN were damaged with liquid nitrogen, the ethylene formation was the highest, if 100?% of leaves were injured and were kept in the light, the lowest when leaves after 100?% injury were kept in darkness. Ethane production (lipid peroxidation) could be detected only in damaged, but not in control leaves, and was much higher in light than in darkness. In addition, there was a strong degradation of chlorophyll of damaged leaves kept in light. In light aminoethoxy-vinylglycine (AVG) inhibited ethylene formation in control, non-damaged whole leaves effectively, but in leaves with 100?% damage the inhibitory effect was much weaker and similar to the effect of propyl gallate (PG), a free radical scavenger. Both AVG and PG treatments decreased ethylene formation by control leaf discs and discs with 100?% damage. Ethane production was significantly inhibited by PG and slightly by AVG in the case of 100?% damage. Tiron, another free radical scavenger gave similar results on leaf discs as PG did. Paraquat (methylviologen, Pq), as a photosynthesis inhibiting and reactive oxygen species (ROS) producing herbicide produced a large amount of ethylene and ethane in light but very small amount in darkness. In accordance, tobacco mosaic virus (TMV) infection on the necrotic host resulted in significantly larger amount of ethylene and ethane formation in light than in darkness. We conclude that ethylene and ethane production of damaged plant tissues is strongly induced by light and ROS that are involved in this induction.     

Inhibition of expression of tomato-ripening genes at high temperature

Abstract. Ripening tomato fruits incubated at 35°C fail to achieve normal pigmentation, soften little and show a marked decline in ethylene evolution. Labelling studies in vivo indicate that protein synthesis continues throughout incubation at 35°C although the spectrum of labelled proteins is different to that observed at 25°C. Translation of mRNAs in vitro shows traces of several 'heat-shock' mRNAs at 35°C and the loss of several others normally found in fruit ripened at 25°C. Using ripening-related cDNA clones as hybridization probes the expression of 12 ripening-related genes was followed during incubation at 25°C and 35°C. In general, there was a marked decline in the amounts of these mRNAs following incubation of ripening fruit at 35°C. In particular, mRNA homologous to pTOM 6, a cDNA clone coding for polygalacturonase, a major cell wall degrading enzyme, showed a rapid decline following incubation at 35°C and after 72-h at elevated temperature was undetectable. There was no recovery of expression during 120 h at 35°C and the application of exogenous ethylene did not overcome the inhibition of ripening or lead to the renewed accumulation of polygalacturonase mRNA. It is proposed that the failure to soften normally at elevated temperature is due, in part, to the suppression of polygalacturonase mRNA and that the inhibition of other facets of ripening at 35°C is due to the inhibition or reduced expression of other, as yet unidentified, ripening-related genes.     

Ethylene is required for both the initiation and progression of softening in pear (Pyrus communis L.) fruit

In order to investigate the physiological role of ethylene in the initiation and subsequent progression of softening, pear fruit were treated with propylene, an analogue of ethylene or 1-methylcyclopropene (1-MCP), a gaseous inhibitor of ethylene action at the preclimacteric or ripening stages. The propylene treatment at the pre-ripe stage stimulated ethylene production and flesh softening while the 1-MCP treatment at the same stage markedly retarded the initiation of the ripening-related events. Moreover, 1-MCP treatment after the initiation of ripening markedly suppressed the subsequent flesh softening and ethylene production. These results clearly indicate that ethylene is not merely a by-product, but plays a crucial role in both the initiation and maintenance of regulating the softening process during ripening. The observations also suggest that ethylene in ripening is regulated entirely in an autocatalytic manner. The mRNA accumulation of pear polygalacturonases (PG) genes, PC-PG1 and PC-PG2, was in parallel with the pattern of fruit softening in both propylene and 1-MCP treatments. However, the expression pattern of pear endo-1,4-beta-D-glucanases (EGase) genes, PC-EG1 and PC-EG2, was not affected in both treatments. The results suggest that ethylene is required for PGs expression even in the late ripening stage, but not for EGases.     

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.