Pears with optimum commercial maturity (based on soluble solid and firmness) were harvested in the harvest season from commercial orchards located in Jinzhou city and Gaocheng city, Hebei Province, China. After harvest, pears were immediately transported to the laboratory at Shandong Agricultural University (Tai’an, China). Fruits with similar size, maturity and without physical injury or infection were selected and randomly divided into groups for further use.

Slow-released ethephon (5% solid ethephon powder/sachet, 0.3 g/sachet) and 1-MCP (0.045% 1-MCP cyclodextrin powder, 0.4 g/sachet) were supplied by Shandong Yingyangyuan Food Technology Co., Ltd (Shandong, China).

To investigate the effect of ethylene and 1-MCP on peel browning rate, pears harvested from Gaocheng city (Hebei Province, China) in 2012 were used. The fruits were packed in boxes with plastic liner, and 15 boxes of pears were randomly divided into five groups (three boxes of fruits for each group) with a total of 144 fruits (48 fruits per box as one repeat with three repeats, 48^∗3 = 144 fruits). The fruits immediately stored at 0°C served as control. For air, 1-MCP, ethylene, and 1-MCP + ethylene treatments, fruits were treated by air (served as air control), slow-released 1-MCP (a sachet/box), slow-released ethylene (a sachet/box), and slow-released 1-MCP (a sachet/box) and ethylene (a sachet/box) at 20°C for 8 h, then stored at 0°C for 30 days. During the 8 h treatment, the concentrations of ethylene and 1-MCP were detected at 0.70–1.28 and 1 μL/L, respectively. On sampling day, fruits were taken from each treatment (48 fruits per repeat with three biological repeats, total of 144 fruits).

Effect of ethylene on physiological functions of ‘Huangguan’ pear was investigated with fresh pears harvested from Jinzhou city (Hebei Province, China) in 2014. Pears were randomly divided into three groups with 168 fruits per group (eight fruits per repeat for each sampling day, 7 sampling days, three repeats) and placed in the containers as mentioned above. For control, pears were packed with plastic foam sleaves, placed into trays overwrapped with plastic film, and immediately stored at 0°C (rapid cooling). For ethylene treatment, ethylene (10000 μL/L, ethylene/nitrogen) was injected into the sealed containers, maintaining the final concentration of ethylene (5 μL/L) at 20°C for 8 h. For air treatment, air with equal volume to ethylene was injected into the sealed container. Treatments were carried out at 20°C. After treatment, the pears were repacked with plastic foam sleeves, placed into trays wrapped with plastic film and stored at 0°C for 30 days. In addition, during cold storage, 24 fruits from each group (eight fruits per repeat with three biological repeats) were sampled on 0, 5, 10, 15, 20, 25, and 30 days, hand-peeled and manually cut into slices, and immediately frozen in liquid nitrogen. Samples (peel and pulp) were ground to fine powder in a mill and stored at -80°C for further analysis.

Prior to above experiments, a preliminary test was conducted to investigate the influence of ethylene concentration on peel browning. In this test, fruits harvested from Gaocheng city, Hebei Province, China, in 2012, were treated by 0, 5, and 50 μL/L ethylene at 20°C for 8 h as mentioned above (40 fruits per repeat with 3 replicates per treatment, 40^∗3 = 120 fruits/treatment). Fruits immediately stored at 0°C were served as control (rapid cooling). Peel browning index and browning rate were determined 20 and 30 days post storage.

To examine long-term effects of ethylene treatments on the fruit quality, the firmness, titratable acidity (TA) and total soluble solids (TSS) were measured using the pears harvested in 2013. For detailed information, see Supplementary Table S1.

The degree of brown spot disorder was evaluated by the method previously described by Xing et al. (2013). This method was scored visually by the percentage of pear fruit surface covered by spots, with a scale from 0 to 4: 0 for no browning, 1 for 1–10%, 2 for 11–20%, 3 for 21–40% and 4 for 41–100%. Disorder index was calculated based on the formula of (fruit number × scale)/[total fruit number × 4 (the severest scale)].

Electrolyte leakage rate was determined using the method described by Liu et al. (2013) with a slight modification. Twelve disks were collected from eight fruits with a cork-borer (diameter 8 mm) and washed in distilled water three times. The disks were soaked in a glass tube containing 20 mL distilled water and incubated in a water bath shaker at 25°C for 2 h. The initial electric conductivity (C₀) was measured using a conductivity meter. Then the glass tube was boiled for 30 min, cooled in room temperature and total electric conductivity (C₁) was taken. Three biological replications were used for each treatment. Electrolyte leakage rate was calculated using the following equation: electrolyte leakage (%) = (C₀/C₁) × 100%.

For each treatment, fruits were sealed in 4.5 L gas-tight jars at 0°C for 24 h. Gas samples were taken and then CO₂ concentration was measured using a gas analyser (PBI-940437B). Three biological replications were used with six fruits in each treatment (six fruits per repeat). The respiration rate was expressed as L/(kg⋅h).

The amount of total phenolics in pear peel was determined according to the Folin–Ciocalteu method described by Singleton and Rossi (1965). Two grams frozen pear peel tissues were extracted with 20 mL of 70% acetone and kept for 3 h in the dark. The extracts were centrifuged at 10000 × g for 20 min at 4°C. A 0.5 mL aliquot of supernatant was added to the reaction mixture (0.5 mL Folin–Ciocalteu and 0.5 mL of 10% Na₂CO₃). Then the mixture was diluted with distilled water to 10 mL and incubated in a water bath shaker at 25°C for 2 h. The absorbance was measured at 765 nm. The content of total phenolics was calculated from a standard curve developed with gallic acid and expressed as milligram gallic acid per gram fresh tissue (mg/g FW).

Polyphenol oxidase activity was assayed as previously described by Huang et al. (2009) with a slight modification. Peel (4 g) was extracted with 20 mL of 0.1 mol/L sodium phosphate buffer (pH 6.8) containing 5% polyvinylpolypyrrolidone (PVPP). Then the sample was homogenized and centrifuged at 10000 × g for 20 min at 4°C. The supernatant was used for analysis of PPO activity as well as POD activity (see below). Supernatants (0.4 mL) were mixed with 1 mL catechol (0.1 mol/L) and 2 mL of sodium phosphate buffer (0.1 mol/L, pH 6.8). The change in absorbance at 420 nm was measured every 30 s for 5 min. One unit of PPO activity was defined as the increase of 0.01 in absorbance per min under assay conditions. PPO activity was expressed as U/g FW.

The POD activity was determined according to Jiang et al. (2002) with some modifications. The reaction mixture contained 500 μL supernatant (see above), 2 mL 0.06% guaiacol in 0.1 mol/L sodium phosphate buffer (pH 6.0) and 1 mL 0.04% H₂O₂. One unit of POD activity was expressed as the change of 0.01 in absorbance at 470 nm per min. The result of POD was expressed as U/g FW.

To determine CAT activity, three grams of frozen tissue were ground with 10 mL sodium phosphate buffer (0.1 mol/L, pH 7.0) containing 5% PVPP. Then the sample was homogenized and centrifuged at 10000 × g for 20 min at 4°C. CAT activity was measured using the method of Beers and Sizer (1952) with some modification. 500 μL of supernatant was mixed with 2 mL sodium phosphate buffer (pH 7.0) and 500 μL H₂O₂ (0.1 mol/L). The absorbance was recorded every 30 s for 3 min. One unit of CAT activity was defined as the change of 0.01 in absorbance at 240 nm per min. The CAT activity was expressed as U/g FW.

Ascorbate peroxidase was determined according to Chen and Asada (1989) with modifications. APX was extracted from 3 g of the frozen pear peel tissue with 10 mL sodium phosphate buffer (50 mmol/L, pH 7.0, containing 0.1 mmol/L EDTA-Na2 and 5% PVPP) at 4°C. The homogenate was centrifuged at 10000 × g for 20 min at 4°C and the supernatant was used for the APX assay. Then, 500 μL supernatant was added to 3 mL reaction mixture containing 50 mmol/L sodium phosphate buffer (pH 7.0), 0.5 mmol/L ascorbate (extinction coefficient, 2.8 mM^-1 cm^-1) and 0.1 mmol/L H₂O₂. The absorbance of mixture was measured at 290 nm every 10 s for 1 min. One unit of APX was defined as the decrease of 0.01 in absorbance per min at assay conditions. The APX activity was expressed as U/g FW.

Superoxide dismutase was extracted from 3 g of the frozen pear peel tissue with 10 mL sodium phosphate buffer (50 mmol/L, containing 5% PVPP, pH 7.8) at 4°C. The homogenate was centrifuged at 10000 × g for 20 min at 4°C and the supernatant was used for the SOD assay. Then 500 μL enzyme extract was mixed with 1 mL sodium phosphate buffer (50 mmol/L, pH 7.8), 0.5 mL methionine (13 mmol/L), 0.5 mL nitroblue tetrazolium (NBT, 75 μmol/L), 10 μmol/L EDTA-Na₂ and 2 μmol/L riboflavin. The mixtures were illuminated under 4000 Lux light for 15 min at 25°C (Li et al., 2013). The absorbance was determined spectrophotometrically at 560 nm. Non-illuminated solutions held in the dark served as a control. One unit of SOD activity was defined as the amount of enzyme that gave half-maximal inhibition of NBT reduction. The SOD activity was expressed as U/g FW.

The extraction method for 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay was used according to Gou et al. (2010). Three grams of the frozen pear peel was mixed with 12 mL of 95% ethanol and ultrasonically shaken at 50°C for 30 min. Then, the homogenate was centrifuged at 10000 × g for 10 min, and 2 mL supernate was added to 2 mL DPPH methanolic solution (0.2 mmol/L). After incubation for 30 min at room temperature in the dark, the bleaching of DPPH was measured at 517 nm. The DPPH radical scavenging activity was calculated by the formula described by Salta et al. (2010).

All measurements were carried out in three biological replicates. Data were expressed as the mean ± standard deviation. A two-way ANOVA analysis for treatment and time of storage was performed. When the effect of treatment was compared with control, a one-way ANOVA analysis for the treatment effect as run at each time of storage. The mean values were separated using the Tukey HSD test (p < 0.05). The data were analyzed with the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).

Though ethylene was widely used for the ripening of fruits, the present research studied the effects of ethylene treatments on peel browning inhibition of ‘Huangguan’ pear. We first examined whether ethylene treatments affect fruit quality. Compared with control, ethylene treatments at 50 μL/L exhibited no significant influence on TA, TSS and firmness of fruits stored at 0°C for 100 and 200 days (Supplementary Table S1). Ethylene treatments at different concentrations (0, 5, and 50 μL/L) were performed for investigating its effect on skin browning of ‘Huangguan’ pear (Figure 1). We found that the peel browning mainly occurred at the early storage stage (data not shown), which is consistent with the investigations of many postharvest industries in the production area. Thereby, the browning rate and browning index were examined only on day 20 and 30 post storage. Peel browning index and rate were significantly affected by treatment, storage time and their interaction. Therefore the single effects were reported in Figure 1. The results showed that ethylene at various concentrations significantly reduced the browning compared with control when pears were stored at 0°C for 20 and 30 days. Ethylene treatments at 5 and 50 μL/L prevented the browning disorder during 200 days storage (data not shown). Interestingly, compared with control (rapid cooling at 0°C after harvest), 0 μL/L treatment (fruits were first placed at 20°C for 8 h and then stored at 0°C) also reduced the disorder.

As shown in Table 1, the browning index of pears stored at 0°C (rapid cooling) for 20 days reached 0.30. However, stored at 20°C for 8 h prior to cold storage (slow cooling) significantly decreased the browning index to 0.17. Nevertheless, this browning inhibition effect was no longer significant 30 days post storage at 0°C, compared to control. Though slow cooling significantly decreased the browning index, no differences were found in browning rate, indicating that slow cooling delayed the onset of browning, not the occurrence. 1-MCP slightly reduced the browning rate and index, however, no significant difference was existed when compared with control. Ethylene dramatically decreased the incidence of browning. The browning rates were only 6.80 and 16.59% after storage for 20 and 30 days, respectively. When ethylene was applied with 1-MCP, its inhibition efficiency was no longer remarkable (Table 1 and Supplementary Figure S1).

The effect of ethylene treatment on peel browning was verified (Figure 2). The results showed that ethylene treatment at 5 μL/L significantly inhibited the browning. Thereby, ethylene concentration at 5 μL/L was used in the following experiment to investigate the physiological functions of ethylene on ‘Huangguan’ pear.

To understand the physiological bases for the ethylene inhibition effects on the peel browning, we first measured electrolyte leakage, an indicator of the change of pear peel fruit in membrane permeability. Given that the effect of treatment, storage time as well as their interaction were significant for membrane permeability, therefore the single effects were reported in Figure 3. Electrolyte leakage in the control and treatments was sharply increased during the first 5 days of storage and then slightly decreased, which was still higher than 0 day (Figure 3). After 10 days storage, no significant difference was found between the control and air treatment in the electrolyte leakage. However, the electrolyte leakage in the pear fruit treated with ethylene changed slowly during the whole storage period. The difference in electronic conductivity among treatments after 5 days storage was attributed to the difference in treatment since the effect of storage time on electronic conductivity was slight. Compared with control and air treated fruits, ethylene-treated fruits exhibited much lower electronic conductivity, indicating that the electrolyte leakage caused by the disruption of membrane permeability was dramatically prevented by ethylene.

The respiration rate of fruits was sharply decreased during the first 5 days of storage, and the decreasing trend slowed afterward (Figure 4). The respiration rate was significantly affected by treatment and storage time as well as their interaction. Ethylene application significantly inhibited the respiration rate, compared to the control and air treatment.

Total phenolics content and PPO activity in pears during cold storage were significantly affected by treatment, storage time and their interaction (Figure 5). Significant differences were found between ethylene-treated and non-ethylene-treated pears in total phenolics content and PPO activity, except for total phenolics content in air-treated pears at day 5. Compared with control, ethylene at 0 μL/L showed no effect on preventing the loss of phenolics, except for day 10. Ethylene maintained higher content of phenolics during the 30 days storage, whereas the rapid cooling control and 0 μL/L ethylene (air) decreased the contents. Fruits in all three groups exhibited similar patterns in PPO activity, which was increased during the whole storage time. However, ethylene treatments significantly prevented the increase of PPO activity (Figure 5).

Peroxidase, CAT, APX and SOD are considered as antioxidant enzymes, participate in the defense of fruit against stresses through scavenging free radicals. The ANOVA result showed that treatment, storage time and their interaction were significant for POD, CAT and APX as well as SOD. The changes in the activity of these enzymes in fruits treated with ethylene and air were demonstrated in Figure 6. Peroxidase activity in all samples was increased during cold storage. However, ethylene treatment showed strong inhibition on the increase of POD activity (Figure 6A). Catalase activity was increased at the early stage of storage and then decreased thereafter. Fruits without ethylene treatment (rapid cooling) and treated with air (slow cooling) only showed distinctively lower levels of CAT activity than fruits treated with 5 μL/L ethylene (Figure 6B). Ascorbate peroxidase activity in the fruits treated with ethylene was increased at the early stage of storage and then slightly decrease (Figure 6C), and was remarkably higher than that in control and air-treated fruits. APX activity in untreated fruits was stable during the first 15 days storage and dramatically decreased thereafter. Ethylene treatments resulted in the induction of SOD activity (Figure 6D). In general, SOD activity in ethylene-treated fruit peel was remarkably higher than that in the control and air-treated fruit. Interestingly, SOD activity in air-treated fruits was also significantly higher than the control (p < 0.05).

The radical scavenging activity in the pear samples was decreased with the extension of storage (Figure 7). During the whole storage, the radical scavenging activity in the ethylene-treated fruits was over 90%, and remarkably higher than the control and air treatment. After 5 days storage, no significant difference in radical scavenging activity was observed between the control and air treatment (p > 0.05).