S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Effects of 1-methylcyclopropene alone and in combination with polyethylene bags on the postharvest life of mango fruit

Experiments were conducted to determine how 1‐methylcyclopropene (1‐MCP) treatments influence ethylene‐stimulated ripening of harvested mango cv. Zihua fruit at 20°C. The ripening response of fungicide (prochloraz) treated fruit was characterised following various 1‐MCP treatments in sealed jars followed by storage in polyethylene bags and/or subsequent ethephon (ethylene) exposure. Exposure of fruit to increasing concentrations of 1‐MCP for 12 h resulted in the reduced softening of produce when subsequently held in air for 7 days after ethephon treatment. Application levels of between 1 and 100 μl litre^?1 1‐MCP had increasing impact, while 200 μl litre^?1 1‐MCP apparently began to approach response saturation. Exposure of fruit to 50 or 100 μl litre^?1 concentrations of 1‐MCP for periods from 1 to 24 h subsequently resulted in reduced softening of produce when held in air for 7 days after ethephon treatment. Increasing periods of exposure from 1 to 12 h had increasing impact, while exposure times greater that 12 h appeared to reach saturation. In the absence of ethephon‐stimulation, the natural ripening of mangoes held in polyethylene bags was delayed by prior exposure to 100 μl litre^?1 1‐MCP for 12 h. Extended holding of 1‐MCP treated and non‐1‐MCP treated control fruit in polyethyene bags encouraged physiological and pathological deterioration. Following exposure to 100 μl litre^?1 1‐MCP for 12 h, mango fruit held for 10 days in polyethylene bags showed a delay in the onset of ripening relative to bagged but non‐1‐MCP treated control fruit. Treatment with 1‐MCP allowed storage of mango fruit in plastic bags at 20°C for 30 days. Observations suggest that 1‐MCP treatments do not adversely influence the quality of the post‐storage ethephon‐ripened fruit. Thus, application of 1‐MCP in combination with the use of polyethylene bags can extend the postharvest life of mango fruit at ambient temperature. Treatments that extend postharvest life are important in developing countries, such as China, where the cold chain infrastructure is often lacking.     

Metabolic changes in 1-methylcyclopropene (1-MCP)-treated ‘Empire’ apple fruit during storage

??Empire?? apple fruit are more susceptible to flesh browning at 3.3°C if treated with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception. To better understand the metabolic changes associated with this browning, untargeted metabolic profiling with partial least squares analysis has been used to visualize changes in metabolic profile during hypoxic controlled atmosphere (CA) storage, ethylene insensitivity, and disorder development. Overall, most carbohydrates and organic acids were not appreciably affected, but the levels of amino acids and volatile metabolites were significantly affected, by 1-MCP treatment. Sorbitol and levels of some amino acids were elevated towards the end of storage in 1-MCP treated fruit. CA storage reduced the levels of many volatile components and 1-MCP reduced these levels further. Additionally multiple metabolites were associated with the development of flesh browning symptoms. Unlike other volatile compounds, methanol levels gradually increased with storage duration, regardless of 1-MCP treatment, while 1-MCP decreased ethanol production. Results reveal metabolic changes during storage that may be associated with development of flesh browning symptoms.     

Variability of responses to 1-methylcyclopropene by banana: influence of time of year at harvest and fruit position in the bunch

To examine the effect of early‐climacteric (postripening) 1‐methylcyclopropene (1‐MCP) exposure on the shelf‐life and quality of green Cavendish bananas (Musa acuminata cv. Williams) from the middle section of the bunch, bananas were harvested bimonthly and treated with 100 μL L^?1 ethylene for 2 consecutive days prior to exposure to 0, 100, 300, 1000, 3000 or 10 000 nL L^?1 1‐MCP for 24 h prior to storage at 22°C. 1‐MCP treatment at a concentration of 300 nL L^?1 or above increased banana shelf‐life significantly compared with the control, regardless of the month in which fruit were harvested except March where a higher concentration was needed (3000 nL L^?1). Fruit harvested in May were the most responsive with a greater than twofold increase in shelf‐life. To examine the effect of fruit position in the bunch on 1‐MCP efficacy, green fruit from the top or bottom of bunches were treated with 100 μL L^?1 ethylene for 2 consecutive days prior to early‐climacteric 1‐MCP (300 nL L^?1) exposure for 24 h at 22°C. In spring and autumn but not in summer, application of 1‐MCP to early‐climacteric fruit was more effective in fruit from the top than in those treated from the bottom of the bunch, increasing shelf‐life. Firmness of 1‐MCP‐treated fruit was up to 19% greater than that of the control across the year, except in fruit from the bottom of the bunch. Given that 1‐MCP is less effective in extending the shelf‐life of summer‐harvested fruit (particularly those from the bottom of the bunch), we conclude that preharvest conditions and fruit position in the bunch affect their responsiveness to ethylene and their behaviour during the ripening process.     

1-Aminocyclopropane-1-Carboxylic Acid Levels in Ripening Apple Fruits

1-Aminocyclopropane-1-carboxylic acid (ACC) in amino acid fractionsof apple fruits was assayed by chemical conversion to ethylene.The specificity of the assay was checked with other amino acids;homocysteine was the only naturally occurring compound foundto yield significant amounts of ethylene in the assay. Analysisof the thiol content of apples showed that homocysteine couldnot be a significant source of interference. Interference froman uncharacterized component of amino acid fractions was lessthan 20% of the ACC level in unripe fruit and insignificantin ripe fruit. Liquid chromatographic assay gave results inclose agreement with the standard assay. Higher apparent ACClevels were measured in unfractionated apple juice than in thestandard assay. Both of these methods and the liquid chromatographicassay were used on a number of apple samples during ripening.All three methods showed that ACC increased 30–40 foldwhereas ethylene production increased by a factor of 10⁴. Inindividual apples the ACC level increased about one day laterthan ethylene production. Key words: Apple fruit, 1-Aminocyclopropane-1-carboxylic acid, Analytical methods, Ethylene     

Effects of treatment with a composite preparation (2-chloroethylphosphonic acid and methacide) or butylated hydroxyanisole on ethylene release in apples

We studied the effect of a Russian composite preparation (2-chloroethylphosphonic acid and methacide) and butylated hydroxyanisole on ethylene release in whole fruit and peel disks of two apple cultivars, Antonovka obyknovennaya (Antonovka) and Simirenko's rennet (Simirenko). Treatment with the composite preparation was followed by an increase in ethylene release from whole apples and peel disks. The development of microbial infection (fruit rot) in whole apples became less pronounced after the treatment. Treatment of whole apples with the antioxidant butylated hydroxyanisole (BHA) increased the intensity of ethylene release during the first subsequent days; thereafter, ethylene release decreased and was 10-15% lower than in the control on days 10-12. In model experiments, BHA decreased ethylene release from apple peel disks below control levels as early as on day 1 after the treatment. Antonovka apples gave quick responses to the treatment. In the late-ripening Simirenko apples, the response persisted for a longer period. Our results suggest that treatment with physiologically active preparations affects ethylene release, ripening, and preservation of apples in storage.     

Storage of 'Gloster 69' apples in the preclimacteric state for up to 200 days after harvest

'Gloster 69' apples are unusual because they do not accumulate ethylene during storage at 2 kPa O₂ at 1.5–3.5°C with continuous ethylene removal. Their ethylene physiology and the extent of various ripening processes in storage were investigated. Ethylene production and l-aminocyclopropane-l-carboxylic acid (ACC) remained low for up to 200 days, and both increased on transfer of fruit to 15°C. The increase in ACC could be stimulated by ethylene treatment of apples after storage. In spite of this evidence that fruit remained preclimacteric, some softening and production of soluble pectin and volatile esters occurred at 3.5°C. These processes were suppressed at 1.5°C, but chlorophyll, starch, malate and sucrose losses and increases in glucose and fructose occurred at both temperatures.     

Regulation of the expression of lipoxygenase genes in <Emphasis Type="Italic">Prunus persica</Emphasis> fruit ripening

Three genes of the lipoxygenase (LOX) family in peach (Prunus persica var. compressa cv. Ruipan 4) were cloned, and their expression patterns during fruit ripening were analyzed using real-time quantitative PCR. All of the three peach LOX genes had been expressed during fruit ripening; however, their expression patterns were significantly different. During the normal ripening of peach fruits, the expression levels of PpLox1, PpLox2 and PpLox3 increased in varying degrees accompanying upsurge of ethylene evolution. After treated by methyl jasmonic acid (MeJA), the peak of ethylene releasing occurred in advance, and the declining rate of fruit hardness was accelerated, the expression level of the three peach LOX genes in fruits markedly enhanced at the early stage of storage, but significantly decreased at the late storage stage. So, it could be suggested that all three LOXs relate to fruit ripening; however, their functions might be different. PpLox1 expression increase along with the upsurge of ethylene evolution in both control and MeJA-treated peach fruits suggested that PpLox1 probably played a major role in the peach fruit ripening. Expression peak of PpLox2 appeared at the 1 DAH (days after harvest) in both control and MeJA-treated peach fruits, while obvious changes in ethylene evolution and fruit hardness was not observed, which suggested that the rise of PpLox2 expression can be induced by certain stimulation related to ripening, such as harvesting stress and MeJA treatment. The expression of PpLox3 kept a lower level in the natural ripening fruits, whereas raced up at the early stage of storage in the fruits treated with MeJA, which indicated that PpLox3 was expressed inductively and had minor roles during the normal ripening of peach fruits, but when encountered with external stimulation, its expression level would rapidly enhance and accelerate the ripening of peach fruit.     

Acetic and propionic acids,inducers of ripening in pre-climacteric golden delicious apples

When treated with acetic or propionic acid vapours, intact pre-climacteric Golden Delicious apples started ripening prematurely. Experiments with [2?¹⁴C]propionic acid showed that a small part of the added acid is transformed into [¹⁴C]ethylene, and that ripening probably begins in these circumstances as a result of the artificial increase in the endogenous ethylene level. It may be that in unripe apples the small amount of evolved ethylene is mostly derived from simple organic acids, so that the moment at which its triggering concentration is reached depends on the available supply of acids. As this, in turn, is a function of the state of maturity of the fruit, there must be a direct relationship between the start of ripening and the degree of maturity. Thus, even in very unripe apples, catabolic processes are at work or potentially present because of the modified β–oxidation of [1?¹⁴C]propionic acid observed.     

The effects of ethylene concentration in controlled atmosphere storage of tomatoes

Previous studies have demonstrated that mature green tomatoes can be stored for up to 10 wk at 12. 5°C, 93–95% r.h. in a controlled atmosphere (CA) containing 5% CO ₂ , 5% O₂ and 90% N ₂ , and will then ripen satisfactorily in air at 20°C. The effects of different concentrations of ethylene between <0.1 and 30 μl/litre in this storage atmosphere on ripening changes and fruit quality after 5 wk CA storage and a further 8 or 9 days ripening in air were investigated using cv. Sonatine glasshouse tomatoes. Maintaining ethylene concentrations in the storage atmosphere to.1 plllitre resulted in poor and uneven ripening of the tomatoes after storage, and increased their susceptibility to infection by Botrytis cinerea and Penicillium spp. Fruit previously stored in atmospheres containing 5 to 30 μl/litre ethylene were significantly softer after ripening than tomatoes stored in lower ethylene concentrations. Overall, the best results in terms of fruit quality (colour, firmness) and a low incidence of fungal infection were achieved with 1–3 μl/litre ethylene. The practical problems in achieving and maintaining optimum conditions, including the correct ethylene level, in CA storage of tomatoes are also discussed.     

Effects of Treatment with a Composite Preparation (2-Chloroethylphosphonic Acid and Methacide) or Butylated Hydroxyanisole on Ethylene Release in Apples

We studied the effect of a Russian composite preparation (2-chloroethylphosphonic acid and methacide) and butylated hydroxyanisole on ethylene release in whole fruit and peel disks of two apple cultivars, Antonovka obyknovennaya (Antonovka) and Simirenko's rennet (Simirenko). Treatment with the composite preparation was followed by an increase in ethylene release from both the whole apples and peel disks. The development of microbial infection (fruit rot) in the whole apples became less pronounced after the treatment. Treatment of whole apples with the antioxidant butylated hydroxyanisole (BHA) increased the intensity of ethylene release during the first subsequent days; thereafter, ethylene release decreased and was 10–15% lower than in the control on days 10–12. In model experiments, BHA decreased ethylene release from apple peel disks below control levels as early as on the first day after treatment. Antonovka apples gave quick responses to the treatment. In the late-ripening Simirenko apples, the response persisted for a longer period. Our results suggest that treatment with physiologically active preparations affects the ethylene release, ripening, and preservation of apples in storage.     

Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples

Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO₂ production. The results with ripened, AVG-treated apples indicate (a) a dose-response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 μl/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO₂; (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene.

Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO₂, but not by 1.25% CO₂. However, each of these CO₂ concentrations reduced the rate of increase of ACC synthase activity.

     

Cloning of 9-cis-epoxycarotenoid dioxygenase (NCED) gene and the role of ABA on fruit ripening

In order to understand more details about the role of abscisic acid (ABA) in fruit ripening and senescence, six 740 bp cDNAs (LeNCED1, LeNCED2, PpNCED1, VVNCED1, DKNCED1 and CMNCED1) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) as a key enzyme in ABA biosynthesis, were cloned from fruits of tomato, peach, grape, persimmon and melon using an RT-PCR approach. A Blast homology search revealed a similarity of amino acid 85.76% between the NCEDs. A relationship between ABA and ethylene during ripening was also investigated. At the mature green stage, exogenous ABA treatment increased ABA content in flesh, and promoting ethylene synthesis and fruit ripening, while treatment with nordihydroguaiaretic acid (NDGA), inhibited them, delayed fruit ripening and softening. However, ABA inhibited the ethylene synthesis obviously while NDGA promoted them when treated the immature fruit with these chemicals. At the breaker, NDGA treatment cannot block ABA accumulation and ethylene synthesis. Based on the results obtained in this study, it was concluded that ABA plays different role in ethylene synthesis system in different stages of tomato fruit ripening.Key words: tomato, NCED gene, ABA, ethylene, fruit ripening, peach, grape, persimmon, melon     

Ozone-induced inhibition of kiwifruit ripening is amplified by 1-methylcyclopropene and reversed by exogenous ethylene

Background

Understanding the mechanisms involved in climacteric fruit ripening is key to improve fruit harvest quality and postharvest performance. Kiwifruit (Actinidia deliciosa cv. ‘Hayward’) ripening involves a series of metabolic changes regulated by ethylene. Although 1-methylcyclopropene (1-MCP, inhibitor of ethylene action) or ozone (O₃) exposure suppresses ethylene-related kiwifruit ripening, how these molecules interact during ripening is unknown.

Results

Harvested ‘Hayward’ kiwifruits were treated with 1-MCP and exposed to ethylene-free cold storage (0?°C, RH 95%) with ambient atmosphere (control) or atmosphere enriched with O₃ (0.3?μL?L^??1) for up to 6?months. Their subsequent ripening performance at 20?°C (90% RH) was characterized. Treatment with either 1-MCP or O₃ inhibited endogenous ethylene biosynthesis and delayed fruit ripening at 20?°C. 1-MCP and O₃ in combination severely inhibited kiwifruit ripening, significantly extending fruit storage potential. To characterize ethylene sensitivity of kiwifruit following 1-MCP and O₃ treatments, fruit were exposed to exogenous ethylene (100?μL?L^??1, 24?h) upon transfer to 20?°C following 4 and 6?months of cold storage. Exogenous ethylene treatment restored ethylene biosynthesis in fruit previously exposed in an O₃-enriched atmosphere. Comparative proteomics analysis showed separate kiwifruit ripening responses, unraveled common 1-MCP- and O₃-dependent metabolic pathways and identified specific proteins associated with these different ripening behaviors. Protein components that were differentially expressed following exogenous ethylene exposure after 1-MCP or O₃ treatment were identified and their protein-protein interaction networks were determined. The expression of several kiwifruit ripening related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1), ethylene receptor (ETR1), lipoxygenase (LOX1), geranylgeranyl diphosphate synthase (GGP1), and expansin (EXP2), was strongly affected by O₃, 1-MCP, their combination, and exogenously applied ethylene.

Conclusions

Our findings suggest that the combination of 1-MCP and O₃ functions as a robust repressive modulator of kiwifruit ripening and provide new insight into the metabolic events underlying ethylene-induced and ethylene-independent ripening outcomes.