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Effect of combined heat and 1-MCP treatment on the quality and antioxidant level of peach fruit during storage
Postharvest Biology and Technology 145 (2018) 193–202
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Effect of combined heat and 1-MCP treatment on the quality and antioxidant level of peach fruit during storage Chen Huana, Xiujuan Ana, Mingliang Yub, Li Jianga, Ruijuan Mab, Mingmei Tua, Zhifang Yua, a b
T
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College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Jiangsu, 210095, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Peach fruit Treatment Storage temperature Antioxidant enzyme Oxidative stress
Peach fruit undergo a fast ripening and senescence after harvest, which results in fruit quality deterioration. In this study, a combined treatment with hot water and 1-MCP (HM) was applied to peach fruit to investigate its effect on the quality and antioxidant level during room temperature (RT) storage and low temperature (LT) storage. HM treatment is effective in slowing fruit softening, increasing total soluble solids (TSS) concentration and delaying fruit senescence during RT storage. However, HM treatment has no positive effect on delaying fruit senescence during LT storage, and even decreases fruit sweetness during the late stage of LT storage. Moreover, HM treatment can induce a short-term oxidative stress on the first day of both storages. However, during the late stage of RT storage, HM treatment reduces electrolyte leakage (EL) and suppresses reactive oxygen species (ROS) accumulation by enhancing the antioxidant ability at enzymatic and transcriptional levels in peach fruit. In contrast, HM treatment causes higher EL and ROS level and induces antioxidant activity only at the enzymatic level during the late stage of LT storage. In conclusion, HM treatment is more effective at improving fruit quality and suppressing oxidative stress for fruit at RT, compared with fruit stored at LT.
1. Introduction
Watkins, 2006; Yang et al., 2014). However, the effect of 1-MCP on peach fruit is temporary and limited (Dal Cin et al., 2006). In contrast to single 1-MCP treatment, combined treatments such as 1-MCP combined heat or controlled atmosphere may be more effective on maintaining fruit quality and limiting fruit disorders (Leverentz et al., 2003; Lum et al., 2017). Heat treatment is another safe and environmentally friendly postharvest technology. It has been reported that heat treatment can inhibit fruit softening and TA loss, while maintaining high TSS, slowing respiration rate and ethylene production, and alleviating chilling injury in peach fruit (Zhou et al., 2015). Our preliminary study has shown that the application of 1-MCP, in combination with heat treatment, can have a synergistic effect that enhances the antioxidant potential and maintain fruit quality of peach fruit (Huan et al., 2016; Jiang et al., 2014). However, the underlying mechanism involved in the regulation of the antioxidant system, under the combination of 1-MCP and heat, remains largely unknown. Fruit ripening and senescence is an oxidative phenomenon accompanied by a pronounced increase in ROS, particularly H2O2 and O2– accumulation (Mondal et al., 2004; Tian et al., 2013). In normal conditions, ROS are rapidly scavenged by various cellular enzymatic and non-enzymatic mechanisms. Enzymatic antioxidant defense in plants
Peach (Prunus persica L.) is a worldwide stone fruit with a desirable flavor and high marketing value. However, peach deteriorates rapidly after harvest, leading to significant changes of fruit quality traits (flavor, weight, total soluble solids, titratable acidity and firmness) as well as physiological traits (respiration rate and ethylene production) and antioxidant activity (Ramina et al., 2008). Cold storage is a conventional and useful method for slowing fruit ripening and prolonging the storage life of peach. However, peach fruit are sensitive to low temperature and exhibit physiological disorders when stored for a long period. Therefore, developing an effective method to reduce postharvest loss and extent shelf life of peach fruit has been a long-standing goal for producers and traders. As a climacteric fruit, peach is characterized by a peak in ethylene production in concert with a burst of respiration during the ripening stage. Commercial ethylene inhibitor, 1-MCP, is widely used to improve storage potential and maintain fruit quality in apple (Watkins, 2008). In peach fruit, it has been reported that application of 1-MCP can delay fruit softening, inhibit respiratory rate decrease, maintain titratable acid concentration and alleviate chilling injury (Jin et al., 2011;
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Corresponding author. E-mail addresses: [email protected] (C. Huan), [email protected] (X. An), [email protected] (M. Yu), [email protected] (L. Jiang), [email protected] (R. Ma), [email protected] (M. Tu), [email protected] (Z. Yu). https://doi.org/10.1016/j.postharvbio.2018.07.013 Received 23 January 2018; Received in revised form 19 July 2018; Accepted 25 July 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Determination of respiration rate and ethylene production
includes enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX). The balance between ROS and antioxidant enzymes is closely related to the ripening and senescence of fruit, since an imbalance can cause oxidative stress, leading to cell death (Perotti et al., 2014). For example, Singh et al. (2012) reported that a faster decline in the enzymatic antioxidant system correlates with a faster rate of ripening and senescence in plums {Singh, 2012 #11}. Induction of antioxidant-related gene expression was reported to enhance stress tolerance in a range of fruits such as banana (Wu et al., 2014), kiwifruit (Xia et al., 2016) and peach (Spadoni et al., 2014). Recently, there is an increasing interest in understanding regulation of antioxidant genes in fruit ripening process (Manganaris et al., 2017; Mou et al., 2015). To date, little information exists regarding the effect of applying HM treatment in combination with cold storage on fruit quality and the antioxidant system of peach. The objective of this work is not only to investigate the efficiency of HM treatment on fruit quality stored under different temperatures, but also to further understand the mechanism of the antioxidant system in regulating oxidative stress in peach fruit. Our results may provide a guide for developing a new postharvest treatment to extend shelf life of peach fruit.
At each time point for each group, five fresh fruit were randomly selected from each biological replicate and enclosed in 4.7 L glass jars at 25 °C or 4 °C for 1 h. Respiration rate was directly measured by a CO2 gas analyzer (CheckMate 3, Dansensor, Denmark). For ethylene production analysis, 1 mL headspace gas was injected into a gas chromatograph (Agilent GC7890 A) equipped with a 2-m stainless-steel packed column (1.8 m × 2 mm) and a flame ionization detector (FID). The temperatures of injector, column, and detector were 50 °C, 50 °C and 150 °C, respectively. Respiration rate was expressed as mg kg−1 h−1 of CO2 and ethylene production was expressed as μg kg−1 h−1, respectively.
2.4. Determination of superoxide radical (O2−) and hydrogen peroxide (H2O2) concentrations Each fresh sample (2.0 g) was homogenized in 6 mL of 65 mM sodium phosphate buffer (pH 7.8) and centrifuged at 10,000×g at 4 °C for 15 min. The resulting supernatant was collected for O2− assays using the method of Huan et al. (2017). A standard curve with NaNO2 was used to calculate the O2− concentration. O2− concentration was expressed as micromoles per gram of fresh weight. To determine H2O2 concentration, flesh sample (2.0 g) was homogenized with 5 mL of chilled 100% acetone and then centrifuged at 10,000×g for 20 min at 4 °C. The supernatant was collected immediately for H2O2 analysis according to the method of Patterson et al. (1984). The H2O2 concentration was expressed as micromoles per kilogram of fresh weight.
2. Materials and methods 2.1. Fruit materials and storage condition Assays were conducted with peach fruit (Prunus persica L. cv. Xiahui 6) grown in the orchard of Jiangsu Academy of Agricultural Sciences in Nanjing, Jiangsu Province, China. “Xiahui 6” peach fruit is a typical melting flesh (MF) peach cultivar. Our previous study has revealed that ripe fruit is more sensitive to HM treatment compared with unripe fruit or half-ripe fruit (Huan et al., 2016). Therefore, in this study, approximately one thousand peach fruit, uniform in color, size and firmness were harvested at the ripe stage (110 days after full bloom; flesh firmness: 16–20 N). After 2 h of removing the field heat, selected fruit (day 0) were randomly divided into four groups (Table 1). At each time point for each group, 30 fruit samples in three biological replicates of 10 fruit each were taken for analysis of fruit quality, ROS level, and membrane condition. After the above determination, the pulp from the same fruit sample was collected, immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.
2.5. Determination of electrolyte leakage (EL) and malondialdehyde (MDA) EL was determined using 20 disks (5 mm diameter) of flesh tissue of 10 fruit, in three replicates. The disks were immersed in 50 mL of doubly distilled water in glass vials at 25 °C. One hour later, the conductivity was measured (C1) and the disks were boiled for 30 min to achieve 100% electrolyte leakage (C2). Relative electrolyte leakage was calculated as (C1/C2)×100. MDA was measured according to the method of Shah et al. (2001) and expressed as micromoles per gram of fresh weight.
2.2. Determination of fruit firmness, total soluble solids and titratable acid 2.6. Enzyme analysis
Flesh firmness was measured twice using a fruit hardness tester (FHM-1, Japan) on the opposite sides of each fruit equator after removal of a 1 mm-thick slice of skin. The results were expressed as Newton (N). TSS and TA of fruit flesh were measured from the pressed juice of each fruit sample using a Pocket Brix-Acidity Meter (PAL-BX|ACID 5, Atago, Japan). TSS and TA were expressed as percentage of Brix and malic acid, respectively.
Superoxide dismutase (SOD) activity was determined according to the method of Luo et al. (2012) and expressed as units (U) per milligram of protein. One unit of SOD activity was defined as the amount of enzyme that caused a 50% inhibition of nitro blue tetrazolium (NBT) at A560. Catalase (CAT) activity was determined according to the method of Change and Maehly (1955) and expressed as units (U) per milligram of protein. One unit was defined as a decrease at A240 in one minute. Ascorbate peroxidase (APX) activity was determined according to the method of Nakano and Asada (1981) and expressed as units (U) per milligram of protein. One unit was defined as a decrease at A290 in one minute. Glutathione peroxidase (GPX) activity was determined according to the method of Huan et al. (2017) and expressed as units (U) per kilogram of protein. One unit was defined as a decrease at A340 in one minute. The total soluble proteins of the enzyme extract were determined by the method of Bradford (1976) using bovine serum albumin as a standard.
Table 1 Treatments and storage conditions of peach fruit in four groups. Group RT LT HMR HML
Treatment No No Heat + 1-MCPa Heat + 1-MCPa
Storage condition b
RT LTc RTb LTc
Sample collection time Day Day Day Day
1, 1, 1, 1,
3, 3, 3, 3,
5 and 7 5, 7, 14, 21, 28 and 35 5 and 7 5, 7, 14, 21, 28 and 35
a Fruit were treated with hot water at 48 °C for 10 min and then with 10 μL L−1 1-MCP for 12 h. b Fruit were stored at 25 ± 1 °C with 85–90 % humidity. c Fruit were stored at 4 ± 0.5 °C with 85–90 % humidity.
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Fig. 1. Effect of HM treatment on (a) firmness, (b) total soluble solid (TSS) concentration and titratable acid (TA) concentration during peach fruit storage. RT: fruit were stored at ambient temperature (25 ± 1 °C) with 85–90% humidity; LT: fruit were stored at 4 ± 0.5 °C with 85–90 % humidity; HMR: fruit were treated with hot water at 48 ℃ for 10 min and 10 μL L−1 1-MCP for 12 h, and then were stored at ambient temperature (25 ± 1 °C) with 85–90 % humidity; HML: fruit were treated with hot water at 48 °C for 10 min and 10 μL L−1 1-MCP for 12 h, and then were stored at 4 ± 0.5 °C with 85–90 % humidity. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
(TEF 2) was selected as the reference gene for its high expression stability (Tong et al., 2009). The relative intensity was calculated according to the delta-delta-Ct method.
2.7. RNA isolation and expression analysis Basing on our previous researches about SOD, CAT, GPX and APX gene families in peach fruit, the main members of above gene families such as PpaSOD4, PpaSOD5, PpaCAT1, PpaCAT2, PpaGPX6, PpaGPX8, PpaAPX2 and PpaAPX4 were selected for expression analysis. The predicted properties of above genes in peach fruit were shown in Table S1 (Supporting information). Total RNA was isolated from peach fruit using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan). Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was performed with the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, USA) using the SYBR Premix Ex Taq™ (TaKaRa, Japan) and gene-specific primers (Supporting information Table S2) in a total volume of 20 μL. All experiments were performed in triplicate with identical thermal cycling conditions, which consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 1 min. Potential genomic DNA contamination was checked with reverse-transcribed negative control. The specificity of primers was verified by melting curve and gel-electrophoretic analysis. The amplification efficiency of primers, calculated by serial dilutions of cDNA samples, was between 95–105 %. Translation elongation factor 2
2.8. Statistical analysis All values were shown as the mean ± standard errors. Statistical analysis was performed with the SPSS 18.0 software (SPSS Inc, Chicago, IL, USA). Figures were made with Microsoft Excel 2010. Duncan’s test was used to compare the means at p ≤ 0.05. 3. Results 3.1. Firmness, TSS and TA HM treated fruit showed significantly higher firmness before day 5 of RT storage and before day 14 of LT storage compared to untreated fruit (Fig. 1a). The results indicate that HM treatment can effectively maintain fruit firmness during the early stage of fruit storage at both storage temperatures (Fig. 1a). TSS concentration in HM treated fruit was remarkably decreased on day 1 at both storage temperatures (Fig. 1b); however, an increase in 195
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Fig. 2. Effect of HM treatment on (a) respiration rate and (b) ethylene production during peach fruit storage. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
contrast, HM treatment showed no effect on the concentrations of O2− and H2O2 on the first day of RT storage. However, HM treatment significantly increased O2− concentration and H2O2 concentration after day 14 and day 3, respectively. It seems that HM treatment can transiently increase the ROS level in HM treated fruit on the first day of RT storage, but shows an opposite effect on the ROS level during the late stage of RT storage and LT storage.
TSS concentration was observed from day 1 to day 3. Afterword, HM treated fruit showed significantly higher TSS concentration during the late stage of RT storage, but significantly lower TSS concentration during the late stage of LT storage, compared with untreated fruit (Fig. 1b). Similar to TSS concentration, TA concentration in HM treated fruit was also significantly decreased on day 1 during the late stage of LT storage, but no significant difference was found during the late stage of RT storage (Fig. 1c). These data suggest that HM treatment can transiently decrease TSS and TA concentrations on day 1 at both storage temperatures, but shows a different effect on their concentrations during the late stage of RT storage and LT storage.
3.4. Membrane integrity EL and MDA are used to indirectly assess cell membrane integrity and reflect oxidative stress in fruit (Sharom et al., 1994). In this study, HM treated fruit showed lower EL during the whole RT storage and the early stage of LT storage, while higher EL remained during the late stage of LT storage, compared with untreated fruit (Fig. 4a). Unlike EL level, MDA concentration in HM treated fruit was significantly increased on day 1 at both storage temperatures, afterwards, no significant difference of MDA concentration was found between treated and untreated fruits (Fig. 4b). These results indicate that HM treatment helps to maintain membrane integrity during RT storage and the early stage of LT storage, but it may cause oxidative stress on day 1 at both storage temperatures and during the late stage of LT storage.
3.2. Respiration rate and ethylene production HM treatment was effective in decreasing the values of respiration rate until day 5 and delaying the respiration peak during RT storage; however, HM treatment had close to no effect on the values of respiration rate in the fruit during LT storage, except on day 1 (Fig. 2a). HM treatment also reduced the values of ethylene production in the fruit during RT storage, while having no effect on the ethylene peak. Similar to respiration rate, ethylene production in HM treated fruit significantly declined only on the first day of LT storage (Fig. 2b). These results suggest that HM treatment can stably inhibit the respiration rate and ethylene production in fruit stored at RT, but has only a transient effect on fruit stored at LT.
3.5. Activity of antioxidant enzymes During RT storage, the activity of SOD in HM treated fruit was significantly increased on day 1, afterwards, no significant difference can be found between treated and untreated fruits (Fig. 5a). In contrast, HM treatment had no obvious effect on SOD activity during the initial 5 days of LT storage. However, HM treated fruit showed higher SOD activity from day 5 to day 35 compared to untreated fruit. No significant difference in the CAT activity was observed between
3.3. Concentrations of O2− and H2O2 The concentrations of O2− and H2O2 in HM treated fruit were drastically increased on the first day of RT storage (Fig. 3). After day 1, their concentrations in HM treated fruit sharply declined and remained lower than untreated fruit during the late stage of RT storage. In 196
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Fig. 3. Effect of HM treatment on the concentrations of (a) superoxide radical (O2−) and (b) hydrogen peroxide (H2O2) during peach fruit storage. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
than that in untreated fruit on day 1 at both temperatures (Fig. 6c), but only PpaAPX4 in HM treated fruit exhibited a higher expression level on day 7 during RT storage. HM treatment significantly increased the expression of PpaGPXs throughout RT storage, apart from day 3 (Fig. 6d). During LT storage, HM treatment only transiently increased the expression of PpaGPXs on day 1. Above results suggest that HM treatment can transiently stimulate the expression of all the antioxidant genes on the first day of storage and induce higher expression of PpaSODs, PpaCAT1, PpaAPX4 and PpaGPXs during the late stage of RT storage, but has little obvious effect on the expression of antioxidant genes during LT storage.
HM treated and untreated fruits until day 3. However, the CAT activity in HM treated fruit was significantly higher than that in untreated fruit from day 5 to day 7 during RT storage and from day 7 to day 35 during LT storage (Fig. 5b). The APX activity in HM treated fruit was only elevated on day 7 during RT storage compared with untreated fruit (Fig. 5c). There was no significant difference between HM treated and untreated fruits during the whole LT storage. Similar to the CAT activity, there was no significant difference in the GPX activity between HM treated and untreated fruits until day 3. After day 3, HM treated fruit showed significantly higher GPX activity until the end of both RT storage and LT storage compared with untreated fruit (Fig. 5d). These results suggest that HM treatment can effectively induce the activity of CAT, APX and GPX during the late stage of RT storage and the activity of SOD, CAT and GPX after day 7 of LT storage.
4. Discussion Heat treatment and 1-MCP treatment have been widely studied in relation to their effect on postharvest peach fruit (Liu et al., 2012; Yu et al., 2017). However, nearly no information has been reported on the combined treatment between 1-MCP and heat. To further clarify the effect of HM treatment on peach fruit during storage, some quality and physiological parameters were evaluated. In the present study, HM treatment significantly decreased the values of respiration rate and ethylene production until day 5 and delayed the respiration peak during RT storage (Fig. 2). Similar results have been reported in previous studies using single 1-MCP treatment (Fan et al., 2002; Liu et al., 2015). These results indicate that HM treatment is also an effective method of delaying peach fruit ripening and senescence during RT storage. It has been reported that 1-MCP treatment has a stable effect on suppressing respiration rate and ethylene production in several fruits during long term cold storage (Koukounaras and Sfakiotakis, 2007; Larrigaudière et al., 2009), while heat treatment shows only a transient effect (D’Aquino et al., 2014). In this study, HM treatment transiently
3.6. Expression of antioxidant genes Fig. 6 showed expression patterns of antioxidant genes in peach fruit during storage. HM treatment had no significant effect on the expression of PpaSODs for most time of the storage. The expression of PpaSODs in HM treated fruit was only significantly increased on the first day of both storage conditions and on day 7 of RT storage (Fig. 6a). As shown in Fig. 6b, the expression of PpaCAT1 in HM treated fruit was about 7folds higher than that in untreated fruit on day 7 of RT storage (Fig. 6b). Unlike the expression of PpaCAT1, the expression of PpaCAT2 in HM treated fruit was significantly increased before day 5 at RT and before day 7 of LT storage. Moreover, nearly no expression of PpaCAT2 was detected during the late stage of LT storage. Similar to PpaSODs, the expression of PpaAPXs in HM treated fruit was also significantly higher 197
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Fig. 4. Effect of HM treatment on (a) electrolyte leakage (EL) and (b) malondialdehyde (MDA) concentration during peach fruit storage. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
1-MCP treatment (Liu et al., 2015). This result indicated that HM can improve the sweetness of peach fruit during the late stage of RT storage. In contrast, HM treated fruit showed significantly lower TSS and TA concentrations than untreated fruit during the late stage of LT storage (Fig. 1b, c), suggesting that HM treatment may cause a loss of fruit quality and a decrease of stress tolerance in peach fruit during long term LT storage. Fruit are subjected to biotic and abiotic stresses induced by different postharvest treatments and following storage conditions (Perotti et al., 2014). Parameters including EL, MDA concentration and ROS level are often used as measurements of membrane integrity and oxidative stress in fruits (Sevillano et al., 2009). In this study, HM treated fruit showed significantly lower EL than that in untreated fruit during RT storage and the early stage of LT storage (Fig. 4a). However, HM treatment significantly increased EL during the late stage of LT storage. These results suggest that HM treatment helps to stably maintain membrane integrity in peach fruit during RT storage. Moreover, the lower EL was associated with higher firmness in HM treated fruit before day 5 at RT and before day 14 at LT, suggesting that high membrane integrity may contribute to maintenance of fruit firmness during the early stage of storage. The concentrations of MDA, O2− and H2O2 in HM treated fruit significantly increased on day 1 (Figs. 3, 4b), which is consistent with the results reported in heat treated peach fruit (Huan et al., 2017; Wang et al., 2014). It is possible that HM treatment can induce a short-term oxidative stress in peach fruit. Moreover, we found that the lower concentrations of O2− and H2O2 in HM treated fruit coincide with the lower EL level during the late stage of RT storage; and the higher concentrations of O2− and H2O2 in HM treated fruit coincide with the higher EL level during the late stage of LT storage (Figs. 3, 4a). This finding suggests that HM treatment can effectively maintain membrane integrity by suppressing oxidative stress during the late stage of RT storage, but induces oxidative stress during the late stage of LT storage.
decreased the values of respiration rate and ethylene production on day 1 and had no effect on the values afterwards, during LT storage (Fig. 2). This result indicates that the effect of HM treatment on respiration rate and ethylene production during LT storage has a more consistent effect when compared with a single heat treatment. Fruit softening is related to bruising susceptibility and is one of the most important factors to determine consumer acceptance (Valero et al., 2007). Crisosto et al. (2004) reported that any fruit with firmness ≤14.0 N will likely be bruised during postharvest handling. In this study, HM treated fruit maintained higher firmness from day 1 to day 3 during RT storage and from day 3 to day7 during LT storage, compared with untreated fruit (Fig. 1a). This indicates that HM treatment can effectively enhance firmness and reduce bruising susceptibility of peach fruit during the early stage of storage. Moreover, we found that the sharp declined firmness in both treated and untreated fruit correlated with the accumulation of ethylene production during the late stage of RT storage (Fig. 1a, 2b), which is in agreement with previous reports that the ethylene climacteric is a late event occurring when the fruit have already softened to a low value (Tonutti et al., 1996). However, no direct relationship could be established between changes in firmness and in ethylene production during LT storage. This indicates that fruit softening does not exclusively depend on ethylene biosynthesis, especially during LT storage. Soluble solids and organic acids are important quality attributes which are involved in energy metabolism and stress tolerance in fruit (Borsani et al., 2009). In this study, TSS and TA concentrations in HM treated fruit was significantly lower than that in untreated fruit on day 1. Meanwhile, lower values of respiration rate and ethylene production were observed in HM treated fruit (Figs. 1b, 2). These results suggested that HM treatment may transiently inhibit energy metabolism of peach fruit. However, HM treated fruit showed significantly higher TSS concentration during the late stage of RT storage. Similar results have been reported in a previous study using single 198
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Fig. 5. Effect of HM treatment on the activity of (a) superoxide dismutase (SOD), (b) catalase (CAT), (c) ascorbate peroxidase (APX) and (d) glutathione peroxidase (GPX) during peach fruit storage. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
concentrations of MDA, O2− and H2O2 in HM treated fruit on day 1 (Fig. 3, 4b). These results indicate that the short-term oxidative stress caused by HM treatment, may induce transcription of antioxidant genes in peach fruit. We also found higher activity of CAT, APX and GPX in HM treated fruit was accompanied by the up-regulation of PpaCAT1, PpaAPX4 and PpaGPXs during the late stage of RT storage (Figs. 5, 6). Meanwhile, HM treated fruit showed significantly lower ROS level and EL during the late stage of RT storage (Fig. 3). Similar results have been demonstrated in various fruits using single 1-MCP or heat treatment (Li et al., 2016; Singh and Dwivedi, 2008; Ummarat et al., 2011). These findings suggest that HM treatment can effectively suppress oxidative stress by enhancing antioxidant ability at both enzymatic and transcript levels during the late stage of RT storage. In contrast, no direct relationship could be established between the gene expression and enzyme activity of antioxidant enzymes in HM treated fruit during LT storage. HM treated fruit showed higher activity of SOD, CAT and GPX after day 14, compared with untreated fruit (Figs. 3, 5, 6). However, HM treatment had almost no effect on the expression of related
This finding is consistent with some previous studies, which have revealed that 1-MCP treatment may induce a greater incidence of chillingrelated disorders in peach fruit (Fan et al., 2002; Girardi et al., 2005). However, Jin et al. (2011) reported that 1-MCP treatment markedly inhibited the increase of EL and the accumulation of ROS level in peach fruit during cold storage. The difference in effect of 1-MCP on EL and ROS level of peach fruit during LT storage may be attributed to species differences, 1-MCP concentration, treatment time or storage conditions. The development of oxidative stress is associated with a broken balance between ROS and antioxidant system (Mittler et al., 2004). In the present study, we analyzed activity and gene expression of some key antioxidant enzymes in response to HM treatment and elucidated their roles in regulating ROS level of peach fruit during storage. No significant difference in the activity of related antioxidant enzymes was observed between HM treated and untreated fruits on the first day of storage at both temperatures. However, the expression of all antioxidant genes in HM treated fruit was significantly increased on day 1 (Fig. 6), which coincided with the significantly increased 199
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Fig. 6. Effect of HM treatment on the relative expression profiles of (a) superoxide dismutase (PpaSOD4 and PpaSOD5), (b) catalase (PpaCAT1 and PpaCAT2), (c) ascorbate peroxidases (PpaAPX2 and PpaAPX4) and (d) glutathione peroxidase (PpaGPX6 and PpaGPX8) during peach fruit storage. Transcript levels are normalized with respect to translation elongation factor 2 (TEF 2) and are expressed relative to the value of each gene at day 0, which are set to 1. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
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Fig. 6. (continued)
induce the antioxidant function only at the enzymatic level in peach fruit. In conclusion, the results of this study reveal that novel heat combined 1-MCP treatment has a different effect on peach fruit under
antioxidant genes during LT storage. Moreover, the ROS level and EL in HM treated fruit were significantly higher than that in untreated fruit during the late stage of storage. These results suggest that after long term cold storage, HM treatment can increase the oxidative stress and 201
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different storage conditions. This technology is more effective in maintaining fruit quality and suppressing oxidative stress for the fruit stored at RT, compared with fruit stored at LT. During the late stage of RT storage, the enhanced antioxidant ability at both enzymatic and transcript levels, attributes to the lower oxidative damage in HM treated fruit.
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Impact of postharvest nitric oxide treatment on antioxidant enzymes and related genes in banana fruit in response to chilling tolerance. Postharvest Biol. Technol. 92, 157–163. Xia, Y., Chen, T., Qin, G., Li, B., Tian, S., 2016. Synergistic action of antioxidative systems contributes to the alleviation of senescence in kiwifruit. Postharvest Biol. Technol. 111, 15–24. Yang, X., Wei, W., Lv, P., Feng, J., 2014. Effectiveness of 1-methylcyclopropene treatment on peach fruit (Prunus Persica l.) for extending storage life. Adv. Mat. Res. 1089, 159–162. Yu, L., Shao, X., Wei, Y., Xu, F., Wang, H., 2017. Sucrose degradation is regulated by 1methycyclopropene treatment and is related to chilling tolerance in two peach cultivars. Postharvest Biol. Technol. 124, 25–34. Zhou, H.-j., Ye, Z.-w., Su, M.-s., Du, J.-h., Li, X.-w., 2015. Effect of heat treatment on protein content and the quality of ‘Hujingmilu’ peach [Prunus persica (L.) batsch]. HortScience 50, 1531–1536.
Acknowledgments This research was financially supported by the Innovation Project of Jiangsu Agricultural Science (CX (15) 1020) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.postharvbio.2018.07. 013. References Borsani, J., Budde, C.O., Porrini, L., Lauxmann, M.A., Lombardo, V.A., Murray, R., Andreo, C.S., Drincovich, M.F., Lara, M.V., 2009. Carbon metabolism of peach fruit after harvest: changes in enzymes involved in organic acid and sugar level modifications. J. Exp. Bot. 60, 1823–1837. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Change, B., Maehly, A., 1955. Assay of catalases and peroxidase. Methods Enzymol. 2, 764–775. Crisosto, C.H., Garner, D., Crisosto, G.M., Bowerman, E., 2004. Increasing ‘Blackamber’ plum (Prunus salicina Lindell) consumer acceptance. Postharvest Biol. Technol. 34, 237–244. D’Aquino, S., Chessa, I., Schirra, M., 2014. Heat treatment at 38 °C and 75–80% relative humidity ameliorates storability of cactus pear fruit (Opuntia ficus-indica cv “Gialla”). Food Bioprocess Technol. 7, 1066–1077. Dal Cin, V., Rizzini, F.M., Botton, A., Tonutti, P., 2006. The ethylene biosynthetic and signal transduction pathways are differently affected by 1-MCP in apple and peach fruit. Postharvest Biol. Technol. 42, 125–133. Fan, X., Argenta, L., Mattheis, J., 2002. Interactive effects of 1-MCP and temperature on’Elberta’peach quality. HortScience 37, 134–138. Girardi, C.L., Corrent, A.R., Lucchetta, L., Zanuzo, M.R., da Costa, T.S., Brackmann, A., Twyman, R.M., Nora, F.R., Nora, L., Silva, J.A., 2005. Effect of ethylene, intermittent warming and controlled atmosphere on postharvest quality and the occurrence of woolliness in peach (Prunus persica cv. Chiripá) during cold storage. Postharvest Biol. Technol. 38, 25–33. Huan, C., Jiang, L., An, X., Kang, R., Yu, M., Ma, R., Yu, Z., 2016. Potential role of glutathione peroxidase gene family in peach fruit ripening under combined postharvest treatment with heat and 1-MCP. Postharvest Biol. Technol. 111, 175–184. Huan, C., Han, S., Jiang, L., An, X., Yu, M., Xu, Y., Ma, R., Yu, Z., 2017. Postharvest hot air and hot water treatments affect the antioxidant system in peach fruit during refrigerated storage. Postharvest Biol. Technol. 126, 1–14. Jiang, L., Zhang, L., Shi, Y., Lu, Z., Yu, Z., 2014. Proteomic analysis of peach fruit during ripening upon post-harvest heat combined with 1-MCP treatment. J. Proteomics 98, 31–43. Jin, P., Shang, H., Chen, J., Zhu, H., Zhao, Y., Zheng, Y., 2011. Effect of 1Methylcyclopropene on chilling injury and quality of peach fruit during cold storage. J. Food Sci. 76, S485–S491. Koukounaras, A., Sfakiotakis, E., 2007. Effect of 1-MCP prestorage treatment on ethylene and CO2 production and quality of ‘Hayward’ kiwifruit during shelf-life after short, medium and long term cold storage. Postharvest Biol. Technol. 46, 174–180. Larrigaudière, C., Candan, A.P., Ubach, D., Graell, J., 2009. Physiological response of ‘Larry Ann’ plums to cold storage and 1-MCP treatment. Postharvest Biol. Technol. 51, 56–61. Leverentz, B., Conway, W.S., Janisiewicz, W.J., Saftner, R.A., Camp, M.J., 2003. Effect of combining MCP treatment, heat treatment, and biocontrol on the reduction of postharvest decay of ‘Golden Delicious’ apples. Postharvest Biol. Technol. 27, 221–233. Li, J., Yan, J., Ritenour, M.A., Wang, J., Cao, J., Jiang, W., 2016. Effects of 1-methylcyclopropene on the physiological response of Yali pears to bruise damage. Sci. Hortic. 200, 137–142. Liu, J., Sui, Y., Wisniewski, M., Droby, S., Tian, S., Norelli, J., Hershkovitz, V., 2012. Effect of heat treatment on inhibition of Monilinia fructicola and induction of disease resistance in peach fruit. Postharvest Biol. Technol. 65, 61–68. Liu, H., Cao, J., Jiang, W., 2015. Changes in phenolics and antioxidant property of peach fruit during ripening and responses to 1-methylcyclopropene. Postharvest Biol.
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Effect of combined heat and 1-MCP treatment on the quality and antioxidant level of peach fruit during storage Chen Huana, Xiujuan Ana, Mingliang Yub, Li Jianga, Ruijuan Mab, Mingmei Tua, Zhifang Yua, a b
T
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College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China Institute of Horticulture, Jiangsu Academy of Agricultural Sciences, Jiangsu, 210095, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Peach fruit Treatment Storage temperature Antioxidant enzyme Oxidative stress
Peach fruit undergo a fast ripening and senescence after harvest, which results in fruit quality deterioration. In this study, a combined treatment with hot water and 1-MCP (HM) was applied to peach fruit to investigate its effect on the quality and antioxidant level during room temperature (RT) storage and low temperature (LT) storage. HM treatment is effective in slowing fruit softening, increasing total soluble solids (TSS) concentration and delaying fruit senescence during RT storage. However, HM treatment has no positive effect on delaying fruit senescence during LT storage, and even decreases fruit sweetness during the late stage of LT storage. Moreover, HM treatment can induce a short-term oxidative stress on the first day of both storages. However, during the late stage of RT storage, HM treatment reduces electrolyte leakage (EL) and suppresses reactive oxygen species (ROS) accumulation by enhancing the antioxidant ability at enzymatic and transcriptional levels in peach fruit. In contrast, HM treatment causes higher EL and ROS level and induces antioxidant activity only at the enzymatic level during the late stage of LT storage. In conclusion, HM treatment is more effective at improving fruit quality and suppressing oxidative stress for fruit at RT, compared with fruit stored at LT.
1. Introduction
Watkins, 2006; Yang et al., 2014). However, the effect of 1-MCP on peach fruit is temporary and limited (Dal Cin et al., 2006). In contrast to single 1-MCP treatment, combined treatments such as 1-MCP combined heat or controlled atmosphere may be more effective on maintaining fruit quality and limiting fruit disorders (Leverentz et al., 2003; Lum et al., 2017). Heat treatment is another safe and environmentally friendly postharvest technology. It has been reported that heat treatment can inhibit fruit softening and TA loss, while maintaining high TSS, slowing respiration rate and ethylene production, and alleviating chilling injury in peach fruit (Zhou et al., 2015). Our preliminary study has shown that the application of 1-MCP, in combination with heat treatment, can have a synergistic effect that enhances the antioxidant potential and maintain fruit quality of peach fruit (Huan et al., 2016; Jiang et al., 2014). However, the underlying mechanism involved in the regulation of the antioxidant system, under the combination of 1-MCP and heat, remains largely unknown. Fruit ripening and senescence is an oxidative phenomenon accompanied by a pronounced increase in ROS, particularly H2O2 and O2– accumulation (Mondal et al., 2004; Tian et al., 2013). In normal conditions, ROS are rapidly scavenged by various cellular enzymatic and non-enzymatic mechanisms. Enzymatic antioxidant defense in plants
Peach (Prunus persica L.) is a worldwide stone fruit with a desirable flavor and high marketing value. However, peach deteriorates rapidly after harvest, leading to significant changes of fruit quality traits (flavor, weight, total soluble solids, titratable acidity and firmness) as well as physiological traits (respiration rate and ethylene production) and antioxidant activity (Ramina et al., 2008). Cold storage is a conventional and useful method for slowing fruit ripening and prolonging the storage life of peach. However, peach fruit are sensitive to low temperature and exhibit physiological disorders when stored for a long period. Therefore, developing an effective method to reduce postharvest loss and extent shelf life of peach fruit has been a long-standing goal for producers and traders. As a climacteric fruit, peach is characterized by a peak in ethylene production in concert with a burst of respiration during the ripening stage. Commercial ethylene inhibitor, 1-MCP, is widely used to improve storage potential and maintain fruit quality in apple (Watkins, 2008). In peach fruit, it has been reported that application of 1-MCP can delay fruit softening, inhibit respiratory rate decrease, maintain titratable acid concentration and alleviate chilling injury (Jin et al., 2011;
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Corresponding author. E-mail addresses: [email protected] (C. Huan), [email protected] (X. An), [email protected] (M. Yu), [email protected] (L. Jiang), [email protected] (R. Ma), [email protected] (M. Tu), [email protected] (Z. Yu). https://doi.org/10.1016/j.postharvbio.2018.07.013 Received 23 January 2018; Received in revised form 19 July 2018; Accepted 25 July 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Determination of respiration rate and ethylene production
includes enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX). The balance between ROS and antioxidant enzymes is closely related to the ripening and senescence of fruit, since an imbalance can cause oxidative stress, leading to cell death (Perotti et al., 2014). For example, Singh et al. (2012) reported that a faster decline in the enzymatic antioxidant system correlates with a faster rate of ripening and senescence in plums {Singh, 2012 #11}. Induction of antioxidant-related gene expression was reported to enhance stress tolerance in a range of fruits such as banana (Wu et al., 2014), kiwifruit (Xia et al., 2016) and peach (Spadoni et al., 2014). Recently, there is an increasing interest in understanding regulation of antioxidant genes in fruit ripening process (Manganaris et al., 2017; Mou et al., 2015). To date, little information exists regarding the effect of applying HM treatment in combination with cold storage on fruit quality and the antioxidant system of peach. The objective of this work is not only to investigate the efficiency of HM treatment on fruit quality stored under different temperatures, but also to further understand the mechanism of the antioxidant system in regulating oxidative stress in peach fruit. Our results may provide a guide for developing a new postharvest treatment to extend shelf life of peach fruit.
At each time point for each group, five fresh fruit were randomly selected from each biological replicate and enclosed in 4.7 L glass jars at 25 °C or 4 °C for 1 h. Respiration rate was directly measured by a CO2 gas analyzer (CheckMate 3, Dansensor, Denmark). For ethylene production analysis, 1 mL headspace gas was injected into a gas chromatograph (Agilent GC7890 A) equipped with a 2-m stainless-steel packed column (1.8 m × 2 mm) and a flame ionization detector (FID). The temperatures of injector, column, and detector were 50 °C, 50 °C and 150 °C, respectively. Respiration rate was expressed as mg kg−1 h−1 of CO2 and ethylene production was expressed as μg kg−1 h−1, respectively.
2.4. Determination of superoxide radical (O2−) and hydrogen peroxide (H2O2) concentrations Each fresh sample (2.0 g) was homogenized in 6 mL of 65 mM sodium phosphate buffer (pH 7.8) and centrifuged at 10,000×g at 4 °C for 15 min. The resulting supernatant was collected for O2− assays using the method of Huan et al. (2017). A standard curve with NaNO2 was used to calculate the O2− concentration. O2− concentration was expressed as micromoles per gram of fresh weight. To determine H2O2 concentration, flesh sample (2.0 g) was homogenized with 5 mL of chilled 100% acetone and then centrifuged at 10,000×g for 20 min at 4 °C. The supernatant was collected immediately for H2O2 analysis according to the method of Patterson et al. (1984). The H2O2 concentration was expressed as micromoles per kilogram of fresh weight.
2. Materials and methods 2.1. Fruit materials and storage condition Assays were conducted with peach fruit (Prunus persica L. cv. Xiahui 6) grown in the orchard of Jiangsu Academy of Agricultural Sciences in Nanjing, Jiangsu Province, China. “Xiahui 6” peach fruit is a typical melting flesh (MF) peach cultivar. Our previous study has revealed that ripe fruit is more sensitive to HM treatment compared with unripe fruit or half-ripe fruit (Huan et al., 2016). Therefore, in this study, approximately one thousand peach fruit, uniform in color, size and firmness were harvested at the ripe stage (110 days after full bloom; flesh firmness: 16–20 N). After 2 h of removing the field heat, selected fruit (day 0) were randomly divided into four groups (Table 1). At each time point for each group, 30 fruit samples in three biological replicates of 10 fruit each were taken for analysis of fruit quality, ROS level, and membrane condition. After the above determination, the pulp from the same fruit sample was collected, immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.
2.5. Determination of electrolyte leakage (EL) and malondialdehyde (MDA) EL was determined using 20 disks (5 mm diameter) of flesh tissue of 10 fruit, in three replicates. The disks were immersed in 50 mL of doubly distilled water in glass vials at 25 °C. One hour later, the conductivity was measured (C1) and the disks were boiled for 30 min to achieve 100% electrolyte leakage (C2). Relative electrolyte leakage was calculated as (C1/C2)×100. MDA was measured according to the method of Shah et al. (2001) and expressed as micromoles per gram of fresh weight.
2.2. Determination of fruit firmness, total soluble solids and titratable acid 2.6. Enzyme analysis
Flesh firmness was measured twice using a fruit hardness tester (FHM-1, Japan) on the opposite sides of each fruit equator after removal of a 1 mm-thick slice of skin. The results were expressed as Newton (N). TSS and TA of fruit flesh were measured from the pressed juice of each fruit sample using a Pocket Brix-Acidity Meter (PAL-BX|ACID 5, Atago, Japan). TSS and TA were expressed as percentage of Brix and malic acid, respectively.
Superoxide dismutase (SOD) activity was determined according to the method of Luo et al. (2012) and expressed as units (U) per milligram of protein. One unit of SOD activity was defined as the amount of enzyme that caused a 50% inhibition of nitro blue tetrazolium (NBT) at A560. Catalase (CAT) activity was determined according to the method of Change and Maehly (1955) and expressed as units (U) per milligram of protein. One unit was defined as a decrease at A240 in one minute. Ascorbate peroxidase (APX) activity was determined according to the method of Nakano and Asada (1981) and expressed as units (U) per milligram of protein. One unit was defined as a decrease at A290 in one minute. Glutathione peroxidase (GPX) activity was determined according to the method of Huan et al. (2017) and expressed as units (U) per kilogram of protein. One unit was defined as a decrease at A340 in one minute. The total soluble proteins of the enzyme extract were determined by the method of Bradford (1976) using bovine serum albumin as a standard.
Table 1 Treatments and storage conditions of peach fruit in four groups. Group RT LT HMR HML
Treatment No No Heat + 1-MCPa Heat + 1-MCPa
Storage condition b
RT LTc RTb LTc
Sample collection time Day Day Day Day
1, 1, 1, 1,
3, 3, 3, 3,
5 and 7 5, 7, 14, 21, 28 and 35 5 and 7 5, 7, 14, 21, 28 and 35
a Fruit were treated with hot water at 48 °C for 10 min and then with 10 μL L−1 1-MCP for 12 h. b Fruit were stored at 25 ± 1 °C with 85–90 % humidity. c Fruit were stored at 4 ± 0.5 °C with 85–90 % humidity.
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Fig. 1. Effect of HM treatment on (a) firmness, (b) total soluble solid (TSS) concentration and titratable acid (TA) concentration during peach fruit storage. RT: fruit were stored at ambient temperature (25 ± 1 °C) with 85–90% humidity; LT: fruit were stored at 4 ± 0.5 °C with 85–90 % humidity; HMR: fruit were treated with hot water at 48 ℃ for 10 min and 10 μL L−1 1-MCP for 12 h, and then were stored at ambient temperature (25 ± 1 °C) with 85–90 % humidity; HML: fruit were treated with hot water at 48 °C for 10 min and 10 μL L−1 1-MCP for 12 h, and then were stored at 4 ± 0.5 °C with 85–90 % humidity. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
(TEF 2) was selected as the reference gene for its high expression stability (Tong et al., 2009). The relative intensity was calculated according to the delta-delta-Ct method.
2.7. RNA isolation and expression analysis Basing on our previous researches about SOD, CAT, GPX and APX gene families in peach fruit, the main members of above gene families such as PpaSOD4, PpaSOD5, PpaCAT1, PpaCAT2, PpaGPX6, PpaGPX8, PpaAPX2 and PpaAPX4 were selected for expression analysis. The predicted properties of above genes in peach fruit were shown in Table S1 (Supporting information). Total RNA was isolated from peach fruit using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan). Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was performed with the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, USA) using the SYBR Premix Ex Taq™ (TaKaRa, Japan) and gene-specific primers (Supporting information Table S2) in a total volume of 20 μL. All experiments were performed in triplicate with identical thermal cycling conditions, which consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 1 min. Potential genomic DNA contamination was checked with reverse-transcribed negative control. The specificity of primers was verified by melting curve and gel-electrophoretic analysis. The amplification efficiency of primers, calculated by serial dilutions of cDNA samples, was between 95–105 %. Translation elongation factor 2
2.8. Statistical analysis All values were shown as the mean ± standard errors. Statistical analysis was performed with the SPSS 18.0 software (SPSS Inc, Chicago, IL, USA). Figures were made with Microsoft Excel 2010. Duncan’s test was used to compare the means at p ≤ 0.05. 3. Results 3.1. Firmness, TSS and TA HM treated fruit showed significantly higher firmness before day 5 of RT storage and before day 14 of LT storage compared to untreated fruit (Fig. 1a). The results indicate that HM treatment can effectively maintain fruit firmness during the early stage of fruit storage at both storage temperatures (Fig. 1a). TSS concentration in HM treated fruit was remarkably decreased on day 1 at both storage temperatures (Fig. 1b); however, an increase in 195
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Fig. 2. Effect of HM treatment on (a) respiration rate and (b) ethylene production during peach fruit storage. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
contrast, HM treatment showed no effect on the concentrations of O2− and H2O2 on the first day of RT storage. However, HM treatment significantly increased O2− concentration and H2O2 concentration after day 14 and day 3, respectively. It seems that HM treatment can transiently increase the ROS level in HM treated fruit on the first day of RT storage, but shows an opposite effect on the ROS level during the late stage of RT storage and LT storage.
TSS concentration was observed from day 1 to day 3. Afterword, HM treated fruit showed significantly higher TSS concentration during the late stage of RT storage, but significantly lower TSS concentration during the late stage of LT storage, compared with untreated fruit (Fig. 1b). Similar to TSS concentration, TA concentration in HM treated fruit was also significantly decreased on day 1 during the late stage of LT storage, but no significant difference was found during the late stage of RT storage (Fig. 1c). These data suggest that HM treatment can transiently decrease TSS and TA concentrations on day 1 at both storage temperatures, but shows a different effect on their concentrations during the late stage of RT storage and LT storage.
3.4. Membrane integrity EL and MDA are used to indirectly assess cell membrane integrity and reflect oxidative stress in fruit (Sharom et al., 1994). In this study, HM treated fruit showed lower EL during the whole RT storage and the early stage of LT storage, while higher EL remained during the late stage of LT storage, compared with untreated fruit (Fig. 4a). Unlike EL level, MDA concentration in HM treated fruit was significantly increased on day 1 at both storage temperatures, afterwards, no significant difference of MDA concentration was found between treated and untreated fruits (Fig. 4b). These results indicate that HM treatment helps to maintain membrane integrity during RT storage and the early stage of LT storage, but it may cause oxidative stress on day 1 at both storage temperatures and during the late stage of LT storage.
3.2. Respiration rate and ethylene production HM treatment was effective in decreasing the values of respiration rate until day 5 and delaying the respiration peak during RT storage; however, HM treatment had close to no effect on the values of respiration rate in the fruit during LT storage, except on day 1 (Fig. 2a). HM treatment also reduced the values of ethylene production in the fruit during RT storage, while having no effect on the ethylene peak. Similar to respiration rate, ethylene production in HM treated fruit significantly declined only on the first day of LT storage (Fig. 2b). These results suggest that HM treatment can stably inhibit the respiration rate and ethylene production in fruit stored at RT, but has only a transient effect on fruit stored at LT.
3.5. Activity of antioxidant enzymes During RT storage, the activity of SOD in HM treated fruit was significantly increased on day 1, afterwards, no significant difference can be found between treated and untreated fruits (Fig. 5a). In contrast, HM treatment had no obvious effect on SOD activity during the initial 5 days of LT storage. However, HM treated fruit showed higher SOD activity from day 5 to day 35 compared to untreated fruit. No significant difference in the CAT activity was observed between
3.3. Concentrations of O2− and H2O2 The concentrations of O2− and H2O2 in HM treated fruit were drastically increased on the first day of RT storage (Fig. 3). After day 1, their concentrations in HM treated fruit sharply declined and remained lower than untreated fruit during the late stage of RT storage. In 196
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Fig. 3. Effect of HM treatment on the concentrations of (a) superoxide radical (O2−) and (b) hydrogen peroxide (H2O2) during peach fruit storage. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
than that in untreated fruit on day 1 at both temperatures (Fig. 6c), but only PpaAPX4 in HM treated fruit exhibited a higher expression level on day 7 during RT storage. HM treatment significantly increased the expression of PpaGPXs throughout RT storage, apart from day 3 (Fig. 6d). During LT storage, HM treatment only transiently increased the expression of PpaGPXs on day 1. Above results suggest that HM treatment can transiently stimulate the expression of all the antioxidant genes on the first day of storage and induce higher expression of PpaSODs, PpaCAT1, PpaAPX4 and PpaGPXs during the late stage of RT storage, but has little obvious effect on the expression of antioxidant genes during LT storage.
HM treated and untreated fruits until day 3. However, the CAT activity in HM treated fruit was significantly higher than that in untreated fruit from day 5 to day 7 during RT storage and from day 7 to day 35 during LT storage (Fig. 5b). The APX activity in HM treated fruit was only elevated on day 7 during RT storage compared with untreated fruit (Fig. 5c). There was no significant difference between HM treated and untreated fruits during the whole LT storage. Similar to the CAT activity, there was no significant difference in the GPX activity between HM treated and untreated fruits until day 3. After day 3, HM treated fruit showed significantly higher GPX activity until the end of both RT storage and LT storage compared with untreated fruit (Fig. 5d). These results suggest that HM treatment can effectively induce the activity of CAT, APX and GPX during the late stage of RT storage and the activity of SOD, CAT and GPX after day 7 of LT storage.
4. Discussion Heat treatment and 1-MCP treatment have been widely studied in relation to their effect on postharvest peach fruit (Liu et al., 2012; Yu et al., 2017). However, nearly no information has been reported on the combined treatment between 1-MCP and heat. To further clarify the effect of HM treatment on peach fruit during storage, some quality and physiological parameters were evaluated. In the present study, HM treatment significantly decreased the values of respiration rate and ethylene production until day 5 and delayed the respiration peak during RT storage (Fig. 2). Similar results have been reported in previous studies using single 1-MCP treatment (Fan et al., 2002; Liu et al., 2015). These results indicate that HM treatment is also an effective method of delaying peach fruit ripening and senescence during RT storage. It has been reported that 1-MCP treatment has a stable effect on suppressing respiration rate and ethylene production in several fruits during long term cold storage (Koukounaras and Sfakiotakis, 2007; Larrigaudière et al., 2009), while heat treatment shows only a transient effect (D’Aquino et al., 2014). In this study, HM treatment transiently
3.6. Expression of antioxidant genes Fig. 6 showed expression patterns of antioxidant genes in peach fruit during storage. HM treatment had no significant effect on the expression of PpaSODs for most time of the storage. The expression of PpaSODs in HM treated fruit was only significantly increased on the first day of both storage conditions and on day 7 of RT storage (Fig. 6a). As shown in Fig. 6b, the expression of PpaCAT1 in HM treated fruit was about 7folds higher than that in untreated fruit on day 7 of RT storage (Fig. 6b). Unlike the expression of PpaCAT1, the expression of PpaCAT2 in HM treated fruit was significantly increased before day 5 at RT and before day 7 of LT storage. Moreover, nearly no expression of PpaCAT2 was detected during the late stage of LT storage. Similar to PpaSODs, the expression of PpaAPXs in HM treated fruit was also significantly higher 197
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Fig. 4. Effect of HM treatment on (a) electrolyte leakage (EL) and (b) malondialdehyde (MDA) concentration during peach fruit storage. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
1-MCP treatment (Liu et al., 2015). This result indicated that HM can improve the sweetness of peach fruit during the late stage of RT storage. In contrast, HM treated fruit showed significantly lower TSS and TA concentrations than untreated fruit during the late stage of LT storage (Fig. 1b, c), suggesting that HM treatment may cause a loss of fruit quality and a decrease of stress tolerance in peach fruit during long term LT storage. Fruit are subjected to biotic and abiotic stresses induced by different postharvest treatments and following storage conditions (Perotti et al., 2014). Parameters including EL, MDA concentration and ROS level are often used as measurements of membrane integrity and oxidative stress in fruits (Sevillano et al., 2009). In this study, HM treated fruit showed significantly lower EL than that in untreated fruit during RT storage and the early stage of LT storage (Fig. 4a). However, HM treatment significantly increased EL during the late stage of LT storage. These results suggest that HM treatment helps to stably maintain membrane integrity in peach fruit during RT storage. Moreover, the lower EL was associated with higher firmness in HM treated fruit before day 5 at RT and before day 14 at LT, suggesting that high membrane integrity may contribute to maintenance of fruit firmness during the early stage of storage. The concentrations of MDA, O2− and H2O2 in HM treated fruit significantly increased on day 1 (Figs. 3, 4b), which is consistent with the results reported in heat treated peach fruit (Huan et al., 2017; Wang et al., 2014). It is possible that HM treatment can induce a short-term oxidative stress in peach fruit. Moreover, we found that the lower concentrations of O2− and H2O2 in HM treated fruit coincide with the lower EL level during the late stage of RT storage; and the higher concentrations of O2− and H2O2 in HM treated fruit coincide with the higher EL level during the late stage of LT storage (Figs. 3, 4a). This finding suggests that HM treatment can effectively maintain membrane integrity by suppressing oxidative stress during the late stage of RT storage, but induces oxidative stress during the late stage of LT storage.
decreased the values of respiration rate and ethylene production on day 1 and had no effect on the values afterwards, during LT storage (Fig. 2). This result indicates that the effect of HM treatment on respiration rate and ethylene production during LT storage has a more consistent effect when compared with a single heat treatment. Fruit softening is related to bruising susceptibility and is one of the most important factors to determine consumer acceptance (Valero et al., 2007). Crisosto et al. (2004) reported that any fruit with firmness ≤14.0 N will likely be bruised during postharvest handling. In this study, HM treated fruit maintained higher firmness from day 1 to day 3 during RT storage and from day 3 to day7 during LT storage, compared with untreated fruit (Fig. 1a). This indicates that HM treatment can effectively enhance firmness and reduce bruising susceptibility of peach fruit during the early stage of storage. Moreover, we found that the sharp declined firmness in both treated and untreated fruit correlated with the accumulation of ethylene production during the late stage of RT storage (Fig. 1a, 2b), which is in agreement with previous reports that the ethylene climacteric is a late event occurring when the fruit have already softened to a low value (Tonutti et al., 1996). However, no direct relationship could be established between changes in firmness and in ethylene production during LT storage. This indicates that fruit softening does not exclusively depend on ethylene biosynthesis, especially during LT storage. Soluble solids and organic acids are important quality attributes which are involved in energy metabolism and stress tolerance in fruit (Borsani et al., 2009). In this study, TSS and TA concentrations in HM treated fruit was significantly lower than that in untreated fruit on day 1. Meanwhile, lower values of respiration rate and ethylene production were observed in HM treated fruit (Figs. 1b, 2). These results suggested that HM treatment may transiently inhibit energy metabolism of peach fruit. However, HM treated fruit showed significantly higher TSS concentration during the late stage of RT storage. Similar results have been reported in a previous study using single 198
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Fig. 5. Effect of HM treatment on the activity of (a) superoxide dismutase (SOD), (b) catalase (CAT), (c) ascorbate peroxidase (APX) and (d) glutathione peroxidase (GPX) during peach fruit storage. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
concentrations of MDA, O2− and H2O2 in HM treated fruit on day 1 (Fig. 3, 4b). These results indicate that the short-term oxidative stress caused by HM treatment, may induce transcription of antioxidant genes in peach fruit. We also found higher activity of CAT, APX and GPX in HM treated fruit was accompanied by the up-regulation of PpaCAT1, PpaAPX4 and PpaGPXs during the late stage of RT storage (Figs. 5, 6). Meanwhile, HM treated fruit showed significantly lower ROS level and EL during the late stage of RT storage (Fig. 3). Similar results have been demonstrated in various fruits using single 1-MCP or heat treatment (Li et al., 2016; Singh and Dwivedi, 2008; Ummarat et al., 2011). These findings suggest that HM treatment can effectively suppress oxidative stress by enhancing antioxidant ability at both enzymatic and transcript levels during the late stage of RT storage. In contrast, no direct relationship could be established between the gene expression and enzyme activity of antioxidant enzymes in HM treated fruit during LT storage. HM treated fruit showed higher activity of SOD, CAT and GPX after day 14, compared with untreated fruit (Figs. 3, 5, 6). However, HM treatment had almost no effect on the expression of related
This finding is consistent with some previous studies, which have revealed that 1-MCP treatment may induce a greater incidence of chillingrelated disorders in peach fruit (Fan et al., 2002; Girardi et al., 2005). However, Jin et al. (2011) reported that 1-MCP treatment markedly inhibited the increase of EL and the accumulation of ROS level in peach fruit during cold storage. The difference in effect of 1-MCP on EL and ROS level of peach fruit during LT storage may be attributed to species differences, 1-MCP concentration, treatment time or storage conditions. The development of oxidative stress is associated with a broken balance between ROS and antioxidant system (Mittler et al., 2004). In the present study, we analyzed activity and gene expression of some key antioxidant enzymes in response to HM treatment and elucidated their roles in regulating ROS level of peach fruit during storage. No significant difference in the activity of related antioxidant enzymes was observed between HM treated and untreated fruits on the first day of storage at both temperatures. However, the expression of all antioxidant genes in HM treated fruit was significantly increased on day 1 (Fig. 6), which coincided with the significantly increased 199
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Fig. 6. Effect of HM treatment on the relative expression profiles of (a) superoxide dismutase (PpaSOD4 and PpaSOD5), (b) catalase (PpaCAT1 and PpaCAT2), (c) ascorbate peroxidases (PpaAPX2 and PpaAPX4) and (d) glutathione peroxidase (PpaGPX6 and PpaGPX8) during peach fruit storage. Transcript levels are normalized with respect to translation elongation factor 2 (TEF 2) and are expressed relative to the value of each gene at day 0, which are set to 1. Each value is the mean for three replicates, with vertical bars indicating standard errors. The different lower-case letters at each time point indicate significant difference at p ≤ 0.05 by Duncan’s multiple range tests.
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Fig. 6. (continued)
induce the antioxidant function only at the enzymatic level in peach fruit. In conclusion, the results of this study reveal that novel heat combined 1-MCP treatment has a different effect on peach fruit under
antioxidant genes during LT storage. Moreover, the ROS level and EL in HM treated fruit were significantly higher than that in untreated fruit during the late stage of storage. These results suggest that after long term cold storage, HM treatment can increase the oxidative stress and 201
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different storage conditions. This technology is more effective in maintaining fruit quality and suppressing oxidative stress for the fruit stored at RT, compared with fruit stored at LT. During the late stage of RT storage, the enhanced antioxidant ability at both enzymatic and transcript levels, attributes to the lower oxidative damage in HM treated fruit.
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