Characterization of ethylene biosynthesis and its regulation during fruit ripening in kiwifruit,Actinidia chinensis ‘Sanuki Gold’

Ethylene biosynthesis in kiwifruit, Actinidia chinensis ‘Sanuki Gold’ was characterized using propylene, an ethylene analog, and 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception. In fruit harvested between a young stage (66 days after pollination) (DAP) and an early commercial harvesting stage (143 DAP), 2 days of exposure to propylene were sufficient to initiate ethylene biosynthesis while in fruit harvested at commercial harvesting stage (154 DAP), 4 days of propylene treatment were required. This observation suggests that response of ethylene biosynthesis to propylene treatment in kiwifruit declined with fruit maturity. Propylene treatment resulted in up-regulated expression of AC-ACO1, AC-ACO2, AC-SAM1 and AC-SAM2, prior to the induction of AC-ACS1 and ethylene production, confirming that AC-ACS1 is the rate limiting step in ethylene biosynthesis in kiwifruit. Treatment of fruit with more than 5 μL L^?1 of 1-MCP after the induction of ethylene production subsequently suppressed ethylene production and expression of ethylene biosynthesis genes. Treatment of fruit with 1-MCP at harvest followed with propylene treatment delayed the induction of ethylene production and AC-ACS1 expression for 5 days. These observations suggest that in ripening kiwifruit, ethylene biosynthesis is regulated by positive feedback mechanism and that 1-MCP treatment at harvest effectively delays ethylene production by 5 days.     

Ethylene synthesis,ripening capacity,and superficial scald inhibition in 1-MCP treated ‘d’Anjou’ pears are affected by storage temperature

A continuing challenge for commercializing 1-methylcyclopropene (1-MCP) to extend the storage life and control superficial scald of ‘d’Anjou’ pear (Pyrus communis L.) is how to initiate ripening in 1-MCP treated fruit. ‘D’Anjou’ pears harvested at commercial and late maturity were treated with 1-MCP at 0.15 μL L⁻¹ and stored either at the commercial storage temperature −1.1 °C (1-MCP@−1.1 °C), or at 1.1 °C (1-MCP@1.1 °C) or 2.2 °C (1-MCP@2.2 °C) for 8 months. Control fruit stored at −1.1 °C ripened and developed significant scald within 7 d at 20 °C following 3–5 months of storage. While 1-MCP@−1.1 °C fruit did not develop ripening capacity due to extremely low internal ethylene concentration (IEC) and ethylene production rate for 8 months, 1-MCP@1.1 °C fruit produced significant amounts of IEC during storage and developed ripening capacity with relatively low levels of scald within 7 d at 20 °C following 6–8 months of storage. 1-MCP@2.2 °C fruit lost quality quickly during storage. Compared to the control, the expression of ethylene synthesis (PcACS1, PcACO1) and signal (PcETR1, PcETR2) genes was stable at extremely low levels in 1-MCP@−1.1 °C fruit. In contrast, they increased expression after 4 or 5 months of storage in 1-MCP@1.1 °C fruit. Other genes (PcCTR1, PcACS2, PcACS4 and PcACS5) remained at very low expression regardless of fruit capacity to ripen. A storage temperature of 1.1 °C can facilitate initiation of ripening capacity in 1-MCP treated ‘d’Anjou’ pears with relatively low scald incidence following 6–8 months storage through recovering the expression of certain ethylene synthesis and signal genes.     

Influence of time from harvest to 1-MCP treatment on apple fruit quality and expression of genes for ethylene biosynthesis enzymes and ethylene receptors

A strong potent inhibitor of ethylene action, 1-methylcyclopropene (1-MCP) maintains apple fruit quality during storage. To understand the influence of time after harvest until 1-MCP treatment, we studied expression patterns of genes for ethylene biosynthesis enzymes and ethylene receptors in two apple cultivars, ‘Orin’ and ‘Fuji’, which differ in ethylene production. Ethylene production and expression of MdACS1, MdERS1, and MdERS2 were suppressed in all 1-MCP-treated ‘Fuji’ fruit, but in ‘Orin’, the later 1-MCP was applied after harvest, the less was the suppression of ethylene production and expression of these genes. In fruit in which 1-MCP had low efficacy (e.g., ‘Orin’ treated at 7 DAH), ethylene production and the level of MdERS1 were briefly reduced by 1-MCP treatment at 2 days after treatment, then began to increase. Since ethylene receptors negatively regulate the ethylene signalling pathway, the increased levels of ethylene production and ethylene receptors after 1-MCP treatment might reduce 1-MCP efficacy.     

The potential of ripening mutants for extending the storage life of the tomato fruit

Summary The effect of the fruit ripening mutants rin, gr, nor and Nr on storage life and pigmentation was investigated in homozygous material, in heterozygous F₁ combinations between the mutants with the colour mutant hp and with the normal cv. Kewalo. Crosses with nor showed a 3-to 5-fold increase in storage life in comparison with the normal cv. or with hp. Maximum pigmentation of the fruits of crosses with nor was pale-red with vineripened fruit and pink with fruit harvested at the breaker stage and ripened on the shelf. The ripening inhibitory effect of rin in the different F₁ combinations was less pronounced than that of nor, and the colour of the fruits was improved. Fruits of the F₁ cross between rin and nor showed greatly improved storage life and developed pink or pale red colour. Most heterozygotes with hp showed improved pigmentation. Problems anticipated in utilizing ripening mutants in breeding for improved keeping quality are discussed.     

Cold pre-treatment is effective for 1-MCP efficacy in ‘Tsugaru’ apple fruit

1-Methylcyclopropene (1-MCP) treatment maintains apple fruit quality during storage, but its efficacy is dependent on a number of conditions. ‘Tsugaru’ apples are a major early season cultivar in Japan, but because ‘Tsugaru’ fruit produce abundant ethylene, they have a short shelf-life, and efficacy of 1-MCP is not as high with ‘Tsugaru’ as with other cultivars. To improve 1-MCP efficacy, ‘Tsugaru’ fruit were pre-cooled at −1 °C or −3 °C for 24 h before 1-MCP treatment. Ethylene production decreased with the cold treatment, resulting in better storage after 1-MCP treatment. Although ethylene production was low at the end of 24 h of the cold pre-treatment, expression of ACS1, the ethylene receptor genes ERS1, ETR1(a), ETR1b, ETR2 and ETR5, and the cell wall degradation-related gene PG1 all increased with a 24 h cold treatment. It is assumed that these elevated gene expression levels were not caused by ethylene, but more directly by cold stimulus. Thus, a short period of cold stimulus suppresses ethylene production, but induces expression of some genes. 1-MCP treatment was more effective with some initial fruit chilling.     

Ethylene biosynthesis in apricot: Identification of a ripening-related 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene

Apricots are climacteric fruits with a high susceptibility to flesh softening and loss of flavor during postharvest storage, and most of the ripening processes are regulated by ethylene, which also has an effect on its own biosynthesis. To understand this process in apricot, inhibition of ethylene biosynthesis and perception was performed for studying key genes involved in the ethylene biosynthetic pathway. Apricots, cv. “Patterson”, were harvested with yellow-green ground color and immediately treated with either the ethylene perception inhibitor 1-methyl cyclopropene (1-MCP) at 10 μL L⁻¹ or the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) at 1 g L⁻¹. After treatment, quality and physiological attributes such as firmness, color, total soluble solids, acidity, fruit weight, ethylene production and respiration rates were evaluated every 2 d until they ripened at 20 °C. Gene expression analysis was performed by quantitative polymerase chain reaction (qPCR). Both ethylene inhibitors were effective in reducing ethylene production, respiration rate and fruit softening. Three 1-aminocyclopropane-1-carboxylic-acid synthase (ACS) genes were characterized, but only the expression of ACS2 was highly reduced by ethylene inhibition, suggesting a key role in ethylene synthesis at ripening. Contrarily, ACS1 and ACS3 showed a higher expression under ethylene inhibition suggesting that the corresponding genes are individually regulated in a specific mode as observed in other climacteric fruits. Finally, changes in 1-aminocyclopropane-1-carboxylic-acid oxidase genes did not show a consistent pattern of ethylene modulation.     

Rhodosporidium paludigenum induced resistance in Ponkan mandarin against Penicillium digitatum requires ethylene-dependent signaling pathway

This work determined if the ethylene dependent signal pathway was required for antagonist-mediated fruit defense mechanisms through investigation of disease resistance against Penicillium digitatum in Ponkan mandarin induced by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, and Rhodosporidium paludigenum. Blocking ethylene perception with 1-MCP resulted in an increase in ACS1, ACS2 and ACO expression, and consequently an increase in ethylene production during mechanical wounding and resistance induction. The expression of the ethylene receptors ETR1, ETR2 and ETR4 as well as ethylene response factor (ERF) were observed with similar responses to yeast and 1-MCP stimuli, with ETR3 mRNA accumulation being the most sensitive to yeast application while ERS1 was the least sensitive. When applied at concentrations greater than 500 nL L⁻¹, 1-MCP pre-fumigation significantly reduced the fruit's natural protection and R. paludigenum induced disease resistance to Penicillium decay, indicating that ethylene perception was involved in inducting disease resistance. Moreover, expression of the defensive genes CHI, β-1,3-glucanase, PAL and CIN up-regulated by yeast was inhibited to different degrees by the 1-MCP pre-treatment. This study provides evidence that the biocontrol yeast R. paludigenum increased disease resistance in Ponkan mandarin against P. digitatum infection due to ethylene and signaling pathway dependent mechanisms.     

Antioxidant systems of ripening avocado (Persea americana Mill.) fruit following treatment at the preclimacteric stage with aqueous 1-methylcyclopropene

Preclimacteric avocado (Persea americana Mill. cv. Booth 7) fruit were treated with aqueous 1-methylcyclopropene (1-MCP) at 0.93 and 9.3 mmol m⁻³ and then stored at 20 °C to investigate the effect of 1-MCP on antioxidant systems of mesocarp tissue during ripening. Exposure to 1-MCP concentrations significantly delayed softening and peak ethylene production. 1-MCP significantly delayed accumulation of total soluble phenolics, flavonoids, and total antioxidant capacity although levels eventually reached control fruit maxima. The influence of 1-MCP was more pronounced at the higher concentration. Activities of peroxidase [POD (EC 1.11.1.7)], superoxide dismutase [SOD (EC 1.15.1.1)], catalase [CAT (1.11.1.6)] and l-ascorbate peroxidase [APX (EC 1.11.1.11)] increased during early ripening of control fruit followed by slight (CAT) or significant (POD, APX) declines with further ripening. Increases in activities of all enzymes were delayed in proportion to 1-MCP concentration, and maximum activities attained during ripening were largely unaffected by 1-MCP. Postclimacteric declines in POD and APX were not observed at the higher 1-MCP concentration, possibly reflecting incomplete ripening. The results indicate that changes in antioxidant parameters of avocado fruit are not markedly influenced by 1-MCP but are delayed or altered in proportion to the general suppression of ripening as indicated by ethylene production and fruit softening trends. Together with previously published reports, the data also indicate that the effects of ethylene-action suppression on antioxidant parameters during ripening vary considerably among different fruits. Relationships between antioxidant systems, ethylene and ripening are discussed.     

Expression of genes encoding xyloglucan endotransglycosylase/hydrolase in ‘Saijo’ persimmon fruit during softening after deastringency treatment

Persimmon (Diospyros kaki Thunb.) fruit undergoes intensive cell wall modification during postharvest fruit softening. Xyloglucan metabolism is important in cell wall disassembly. We cloned cDNAs for two xyloglucan endotransglycosylase/hydrolase genes (DkXTH1 and DkXTH2) from ‘Saijo’ persimmon fruit treated with dry ice to remove astringency. In order to determine the ethylene dependence of XTH gene expression, fruit were exposed to 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, prior to removal of astringency. Ethylene production increased in mature control and 1-MCP-pretreated fruit after dry-ice treatment, and flesh firmness decreased to the same extent during dry-ice treatment in the control and 1-MCP-pretreated fruit. After dry-ice treatment, control fruit softened completely, but fruit firmness was maintained in 1-MCP-pretreated fruit. Accumulation of DkXTH1 mRNA was induced simultaneously with commencement of ethylene production in mature control fruit. Pretreatment with 1-MCP delayed accumulation of DkXTH1 mRNA. DkXTH2 expression also coincided with fruit softening but was intensified by 1-MCP treatment during the deastringency treatment. These results indicate that fruit softening was related to both DkXTH1 and DkXTH2 expression in ‘Saijo’ persimmons.     

Treatment with 1-MCP and the role of ethylene in aroma development of mountain papaya fruit

Mountain or highland papaya (Vasconcellea pubescens) is a climacteric fruit which develops a strong and characteristic aroma during ripening. The dynamics of aroma volatile production during ripening of whole papaya fruit were analysed by headspace-SPME. The main compounds produced by the fruit were esters (aliphatic and branched) and alcohols: the most abundant esters were ethyl acetate, ethyl butanoate, methyl butanoate and butyl acetate, comprising 88% of the volatiles in fully ripe fruit; butanol was the most abundant alcohol. Among the volatiles produced, ethyl butanoate, ethyl acetate, ethyl hexanoate and ethyl 2-methylbutanoate were found to be the most potent odour compounds. During ripening of mountain papaya fruit there was an increase in the total content of both esters and alcohols. In order to clarify the role of ethylene in aroma formation, mature fruit were treated with 0.3 μL L⁻¹ of 1-MCP (16 h at 20 °C) or with 2 g L⁻¹ Ethrel, and then allowed to ripen at 20 °C. The treatment of the fruit with 1-MCP inhibited the rise in ethylene production in the fruit, while Ethrel advanced the development of the climacteric phase. Most esters identified in mountain papaya were dependent on ethylene, showing an increase in production during ripening and in response to Ethrel treatment, and a strong reduction in response to 1-MCP treatment. The data presented provide evidence that most esters produced by mountain papaya are derived from fatty acids and amino acid metabolic pathways, both of them being affected by ethylene.     

Molecular characterization of two banana ethylene signaling component MaEBFs during fruit ripening

EIN3 Binding F-box protein (EBF) is an essential signaling component necessary for ethylene response. However, little information is available on EBF genes during banana fruit ripening. Two EBF genes designated MaEBF1 and MaEBF2 were isolated and characterized from banana fruit. Subcellular localization analysis showed that MaEBF1 and MaEBF2 were both nuclear proteins. Expression of MaEBF1 and MaEBF2 in fruit with four ripening characteristics revealed that MaEBF2 was enhanced by ethylene during fruit ripening, while MaEBF1 changed only slightly. Moreover, the MaEBF2 promoter was activated after ethylene treatment, further supporting its involvement in fruit ripening. More importantly, MaEBF2 was shown to physically interact with MaEIL5, using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Together, these results suggest that MaEBF may be involved in banana fruit ripening, at least partly via interaction with MaEIL5. Our findings expand our understanding of the regulatory network of ethylene signaling cascade in banana fruit ripening.