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.     

Identification and genetic characterization of an ethylene-dependent polygalacturonase from apricot fruit

During fruit ripening a loss of firmness occurs, which is a key factor limiting postharvest life. In apricot, Prunus armeniaca L., a wide range of fruit firmness at commercial maturity has been observed in different cultivars. Endopolygalacturonase (endoPG) activity has been reported to be associated with differences in firmness in many fruit species, but never in apricot. In this paper, we reported the identification of an apricot cDNA (PaPG) coding for an endoPG-like protein with 393 amino acids. Protein sequence comparison with known polygalacturonases (PGs) revealed that multiple features as conserved domains and functional residues and a predicted signal peptide were present in PaPG. Moreover, a phylogenetic analysis of this and other plant PGs placed PaPG into a clade containing endoPGs expressed in fruit, abscission and dehiscence zones without a propeptide sequence, very close to PRF5 from peach (Prunus persica L. Batsch). PaPG gene expression increased during postharvest storage of the fruit, correlating with fruit softening and ethylene release, and it responded to exogenous ethylene treatments. We localized the PaPG gene in apricot linkage group 4 after genetic mapping based on SNP analysis, in a position apparently syntenic to the PRF5 locus from peach. Results obtained offer genetic evidence supporting the hypothesis that PaPG and PRF5 are orthologous genes, and consequently position PaPG as a gene of interest for studies on fruit softening in apricot, and contribute to the development of molecular tools for breeding apricots with longer shelf life.     

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.     

甜瓜ACS基因家族成员的鉴定及其表达特性分析

乙烯在高等植物生长发育等多个方面都具有重要的调控作用,而ACS(1-aminocyclopropane-1-carboxylic acid(ACC)synthase,1-氨基环丙烷-1羧酸合成酶)是乙烯生物合成途径的限速酶,由多基因家族编码,不同的发育、环境、激素刺激等信号诱导不同的ACS基因表达.本研究以甜瓜品种'河...     

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.     

Molecular analysis of softening and ethylene synthesis and signaling pathways in a non-softening apple cultivar, ‘Honeycrisp’ and a rapidly softening cultivar, ‘McIntosh’

A feature of ‘Honeycrisp’ apples [Malus sylvestris (L.) Mill var. domestica (Borkh.) Mansf.] is that they maintain flesh firmness over extended storage. The objective of this study was to elucidate molecular mechanisms that are responsible for slow softening of ‘Honeycrisp’ as compared with a rapidly softening cultivar, ‘McIntosh’. Fruit from both cultivars were picked during the normal harvest period and stored at 20 °C for 10 d. Internal ethylene concentrations (IECs) in ‘Honeycrisp’ fruit were lower than in ‘McIntosh’, but at climacteric levels of ethylene ‘Honeycrisp’ fruit maintained their firmness over this period, while ‘McIntosh’ softened rapidly. Concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were higher in ‘Honeycrisp’ than in ‘McIntosh’ apples. qRT-PCR analysis was carried out for genes involved in ethylene biosynthesis, perception and signaling [ACC synthase (MdACS); ACC oxidase (MdACO); ethylene receptors (MdETR and MdERS); constitutive triple response (MdCTR); ethylene response factor (MdERF)], as well as those involved in cell wall metabolism [polygalacturonase (MdPG); xyloglucan endotransglucosylase (MdXTH); expansin (MdEXP); β-galactosidase (Md β-GS); arabinofuranosidase (MdAFase); pectate lyase (MdPL)]. At comparable IECs, the expression of genes involved in ethylene synthesis, ethylene perception and signal transduction was generally much higher in ‘Honeycrisp’ than in ‘McIntosh’ fruit. However, the expression of MdAFase and MdEXP3 was generally lower in ‘Honeycrisp’ than in ‘McIntosh’, while that of MdPG and MdPL was extremely low in ‘Honeycrisp’. Expression of MdPG1 was very low, even though IECs were at climacteric levels. Absence of fruit softening in ‘Honeycrisp’ is probably associated with restricted cell wall enzyme activity. The lower maximum IECs found in ‘Honeycrisp’ compared with ‘McIntosh’ do not appear to be related to expression of genes involved in ethylene biosynthesis.     

1-MCP regulates ethylene biosynthesis and fruit softening during ripening of ‘Tegan Blue’ plum

To investigate the effects of postharvest application of 1-MCP on ethylene production and fruit softening, activities of ethylene biosynthesis and fruit softening enzymes were measured during postharvest ripening of plum (Prunus salicina Lindl. cv. Tegan Blue) fruit after being exposed to 1-MCP (0, 0.5, 1.0 or 2.0 μL L⁻¹) at 20 ± 1 °C for 24 h. Following the treatments, fruit were allowed to ripen at ambient temperature (20 ± 1 °C), and ethylene production in fruit, activities of ACS and ACO, ACC content and fruit softening enzymes (PE, EGase, exo-PG and endo-PG) in fruit skin and pulp were recorded at different intervals. Postharvest application of 1-MCP significantly delayed and suppressed the climacteric ethylene production with reduction in the activities of ethylene biosynthesis enzymes (ACS, ACO) and ACC content, and fruit softening enzymes (PE, EGase, exo-PG and endo-PG) in the skin as well as in pulp tissues. The reduction was more pronounced with increased concentrations of 1-MCP. 1-MCP treated fruit showed different rates of fruit softening and activities of ethylene biosynthesis enzymes in the skin and pulp tissues which warrant further investigation on regulation of gene expression related to these enzymes with the inhibitory effect of 1-MCP.     

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.     

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.     

Ethanol vapor treatment maintains postharvest storage quality and inhibits internal ethylene biosynthesis during storage of oriental sweet melons

In order to evaluate the effect of ethanol vapor treatments (0.5 mL/kg and 3 mL/kg) on postharvest storage at 23 °C, quality of oriental sweet melons, and to clarify the mechanism of the inhibition of senescence, we investigated physiological and quality changes induced by ethanol vapor, decay incidence, internal ethylene concentration (IEC) and ethylene-related enzymes activities as well as gene expression. Both ethanol vapor treatments, irrespective of concentration, significantly (P < 0.5) delayed skin color changes, retarded softening and suppressed fruit decay in ethanol vapor-treated fruit. Between the two treatments, 0.5 mL/kg of ethanol vapor maintained better quality in storage than that of 3 mL/kg. Compared with the control, both ethanol vapor treatments resulted in different profiles and composition of aromatic volatile compounds of fruit during storage, and a significant increase of ethyl esters, including ethyl acetate, ethyl butanoate, ethyl hexanoate, ethyl 2-methylbutanoate, 3-(methylthio) propionate and 2-phenethyl acetate, and five new ethyl esters were also detected. Both treatments increased alcohol acyl-transferase (AAT) activity levels, which peaked earlier than in the control, but there were no significant differences in activities of alcohol dehydrogenase (ADH). Both treatments significantly (P < 0.5) suppressed internal ethylene concentrations (IEC) during storage at 23 °C, which was evident from reducing 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) activities, and inhibiting ACC biosynthesis, and the effect of the 0.5 mL/kg treatment was better than that of 3 mL/kg. Real-time quantitative PCR (Q-PCR) analysis showed that the expression patterns of CM-ACO1, CM-ACO2, CM-ACS1 and CM-ACS2 were consistent with ethylene production during storage. These results suggest that postharvest ethanol vapor treatments markedly delayed the senescence of harvested oriental sweet melons, maintained better quality in storage and improved levels of volatile aroma compounds, especially the ethyl esters, through suppressing the expression of particular members of ethylene-forming enzyme gene families as well as ethylene biosynthesis, and the effect is dose dependent.