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1-Methylcyclopropene alleviates chilling injury by regulating energy metabolism and fatty acid content in ‘Nanguo’ pears
Postharvest Biology and Technology 109 (2015) 130–136
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1-Methylcyclopropene alleviates chilling injury by regulating energy metabolism and fatty acid content in ‘Nanguo’ pears Shunchang Cheng1, Baodong Wei, Qian Zhou, Dehong Tan, Shujuan Ji* College of Food Science, Shenyang Agricultural University, Shenyang 110866, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2014 Received in revised form 13 May 2015 Accepted 23 May 2015 Available online xxx
This study investigated the effects of 1-methylcyclopropene (1-MCP) treatment on chilling injury (CI), energy metabolism, and membrane fatty acid content in harvested ‘Nanguo’ pears during the shelf life after cold storage at 0 C. 1-MCP treated fruit showed slower CI injury development, lower ion leakage and malondialdehyde accumulation, increased adenosine triphosphate content and energy charge during a 25 day shelf life period at 20 C. Activities of enzymes associated with energy metabolism, including H+-adenosine triphosphatase, Ca2+-adenosine triphosphatase, succinic dehydrogenase, and cytochrome C oxidase increased after 1-MCP treatment. The ratio of unsaturated to saturated fatty acids in 1-MCP-treated fruit was higher than that in control fruit. These results suggest that alleviation of CI after 1-MCP treatment might be due to enhanced energy metabolism-related enzyme activities and higher levels of energy and unsaturated to saturated fatty acid ratio. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Pear 1-Methylcyclopropene Chilling injury Energy metabolism Fatty acid
1. Introduction ‘Nanguo’ pear (Pyrus ussuriensis) is grown in the northeastern cold regions of China. This pear cultivar is preferred by consumers because of its good taste, fragrant flavor, and pleasant aroma. Pears are climacteric fruit that show increased ethylene production and respiration rates during ripening. Hence, they remain firm for a limited time after harvest due to ripening. Rapid ripening and softening makes them sensitive to mechanical injury and pathogen infection (Li et al., 2014a,b; Paul et al., 2012). While storage at low temperature delays fruit senescence and extends postharvest life of the fruit, pears are susceptible to development of chilling injury (CI)-associated disorders, including peel and core browning and decay (Wang, 1990). Ensuring sufficient cellular energy is an important factor in controlling fruit ripening and senescence after harvest. Physiological disorders and browning in harvested fruit might be related to inadequate supplies and reduced efficiency of cellular energy generation (Jiang et al., 2007). Cell membranes are thought to be the primary sites for the development of CI. A higher ratio of unsaturated/saturated fatty acids has been shown to improve tolerance to chilling temperature in various kinds of fruit such as
loquat (Cao et al., 2009), banana (Jiang et al., 2004), and mango (Li et al., 2014a,b). Cell membrane damage is thought to be associated with the lack of energy, and ATP is known to play an important role in fatty acid synthesis and membrane repair (Rawyler et al., 1999). 1-Methylcyclopropene (1-MCP), an inhibitor of ethylene perception, is thought to interact with ethylene receptors and thereby prevent ethylene-dependent responses (Watkins, 2006). 1-MCP has been shown to inhibit CI incidence in some horticultural products such as apple (Apollo Arquiza et al., 2005), avocado (Pesis et al., 2002), pineapple (Fan et al., 1999), persimmon (Salvador et al., 2004), ‘Fallgold’ tangerine and grapefruit (Dou et al., 2005), ‘Nova’ and ‘Ortanique’ mandarins (Salvador et al., 2006), plum (Candan et al., 2011), and loquat (Cao et al., 2010). On the other hand, CI symptoms were induced in banana (Jiang et al., 2004), ‘Shamouti’ orange (Porat et al., 1999), and peach (Fan et al., 2002) by 1-MCP. The effects of 1-MCP on energy metabolism and fatty acidrelated changes associated with CI in ‘Nanguo’ pear fruit have not been investigated. Thus, this study aimed to evaluate the effect of 1-MCP treatment on energy status, energy metabolism-related enzyme activities, and fatty acid composition in ‘Nanguo’ pear fruit during the shelf life after storage at low temperatures. 2. Materials and methods
* Corresponding author at: No. 120 Dongling Road, Shenyang, 110866, Liaoning Province, People’s Republic of China. Tel.: +86 24 88487161. E-mail addresses: [email protected] (S. Cheng), [email protected] (S. Ji). 1 Address: Food Science College, Shenyang Agricultural University, No.120 Dongling Road, Shenyang, Liaoning. 110161, People’s Republic of China. Tel.: +86 24 88487161; fax: +86 24 88487162. http://dx.doi.org/10.1016/j.postharvbio.2015.05.012 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
2.1. Fruit material and treatment Hard, mature (green skin and light cream color) ‘Nanguo’ pears were harvested from a commercial orchard located at Anshan City
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(longitude, 122.97E; latitude, 41.07N), Northeast China. The fruit were packed in fiberboard cartons and transferred to the laboratory at the Shenyang Agriculture University within 3 h. Fruit with uniform shape and size and free from damage and fungal infection were selected. The fruit were divided into two groups (150 fruit per group). Three replicates were used for treatment (50 fruit per replicate). Each replicate of treated fruit was exposed to 0.5 mL L 1 1-MCP (EthylBloc, Floralife Inc., USA) for 20 h at 20 C in an airtight 1 m3 plastic container. 1-MCP was released in the dissolved form in 1 mL distilled water in a glass dish to ensure the release of 1-MCP. For the control treatment, 150 fruit were placed in an identical plastic container without 1-MCP. Following treatment, the samples were placed in polyethylene bags (0.02 mm), secured with rubber bands, and stored under 80–90% relative humidity (RH) and at 0 C. After 90 days, fruit from both the groups were transferred from cold storage to ambient temperature (20 C) with 60–65% RH, and 10 fruit from each group were used for analysis at 5-day intervals during the remaining shelf life.
2.2. CI index Ten fruit were cut equatorially to document the severity of CI. The CI index was determined using a rating scale ranging from 0 to 4 on the basis of common visual symptoms of water-soaked appearance of the flesh cross-section and the affected core area or flesh tissue. The following scale was used: 0 = no damage; 1 = very slight damage; 2 = slight damage; 3 = moderate damage, and 4 = severe damage. The CI index was calculated using the following P formula: [ (A B)/5C], where A is the injury score of individual fruit, B is the number of fruit affected, 5 is the total number of scores (0–4) used, and C is the total number of fruit recorded (Zaharah and Singh, 2011).
2.3. Electrolyte leakage and malondialdehyde content measurement Discs were excised from the fruit by using a 3-mm diameter cork borer. The discs were briefly rinsed with deionized water and blotted dry on a slightly moistened Whatman filter paper. Twelve discs were then incubated in 30 mL of 300 mM mannitol for 3 h in a 50-mL capped polypropylene centrifuge tube. The conductivity of the bathing solution was measured after 4 h of incubation at 25 C by using a DDS-307 conductivity meter (Shanghai Precise Science Instrument Co., Shanghai, China). Total electrolyte conductivity was determined after the discs and bathing solution were heated in a boiling water bath for 30 min. The discs and bathing solution were then cooled to room temperature, and the conductivity was measured once again. Electrolyte leakage was expressed as the percentage of the conductivity of total tissue electrolyte. Three replicates were used for each treatment (Luo et al., 2011). The content of malondialdehyde (MDA) was measured using the thiobarbituric acid (TBA) reaction method (Zhang et al., 2009), with some modifications. Tissue samples (1.0 g) were homogenized in 4.0 mL of 5% (w/v) trichloroacetic acid and centrifuged at 10,000 g for 20 min. The supernatant was mixed with 2.0 mL of 0.67% TBA, heated at 100 C for 30 min, and then immediately cooled on ice. After the supernatant was centrifuged at 5000 g for 10 min, its absorbance was measured using a spectrophotometer (TU-1810 DSPC; Beijing Puxi Instrument Co., Beijing, China) at 450, 532, and 600 nm. The MDA concentration was calculated according to the formula: 6.45 (A532 A600) 0.56 A450. Three replicates were used for each treatment.
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2.4. Measurement of activities of enzymes related to energy metabolism Mitochondria were isolated from pear fruit as described by Qin et al. (2009) with some modifications. In brief, approximately 60 g peeled pear tissue from 10 fruit was gently cut and homogenized in 300 mL ice-cold extraction buffer containing 250 mM sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% w/v polyvinylpyrrolidone (PVP), 0.1% w/v bovine serum albumin (BSA), 10 mM b-mercaptoethanol, and 50 mM Tris-HCl (pH 7.5). The homogenate was filtered through four layers of sterile cheesecloth, followed by centrifugation at 1200 g for 15 min. The supernatant was decanted and centrifuged at 16,000 g for 20 min. The pellets were re-suspended in washing buffer (250 mM sucrose, 0.1% w/v BSA, and 50 mM Tris-HCl, pH 7.5) by using a soft brush. The crude mitochondria were purified using density-gradient centrifugation with 28% (v/v) Percoll. The extraction buffer consisted of 250 mM sucrose, 1.0 mM EDTA, 0.5% PVP, 0.1% (w/v) BSA, 10 mM b-mercaptoethanol, and 50 mM Tris-HCl, pH 7.5. The washing buffer consisted of 10 mM Tris, 0.3 M mannitol, 1.0 mM EDTA, and 0.1% (w/ v) BSA, pH 7.2. The concentration of proteins was determined spectrophotometrically at 595 nm, as described by Bradford (1976) by using BSA as the standard. Respiratory activity was determined as described previously (Mazorra et al., 2013). Briefly, 1 mg of protein was used to determine the respiratory activity of mitochondria by using an oxygen electrode (Hansatech, Respire 1, UK) in 1.0 mL reaction medium [0.35 M mannitol, 10 mM phosphate buffer, 10 mM KCl, 5 mM MgCl2, 0.5% (w/v) BSA, pH 7.2] at 25 C. The entire assay was performed in the presence of 200 mM glutamate as the substrate for respiration. The addition of 100 nM ADP to the reaction medium induced the transition from phosphorylation (State 3) to resting (State 4) respiration, indicating the phosphorylation capacity of the mitochondria. The mitochondrial respiratory control rate was calculated as follows: (State 3)/(State 4). H+-ATPase and Ca2+-ATPase activities were determined by measuring the release of phosphorus. The reaction was initiated by the addition of 100 mL 0.03 M ATP-Tris (pH 8.0), and stopped with 5% (w/v) trichloroacetic acid after 20 min of incubation at 37 C. One unit of H+-ATPase and Ca2+-ATPase activities was defined as the release of 1 mM of phosphorus per second at 660 nm under the assay conditions (Jin et al., 2013). Succinic dehydrogenase (SDH) activity was determined according to the method of Ackrell et al. (1978). The assay medium contained 0.3 mL of the crude mitochondria extract, 3 mL 0.2 mM potassium phosphate buffer (pH 7.4), 1 mL 0.2 mM sodium succinate, 0.1 mL 1 mM dichlorophenylmethyl cardinal, and 0.1 mL 0.33% (w/v) methyl sulfenyl phenazine. One unit of SDH activity was defined as an increase of 0.01 in absorbance per second at 600 nm under the assay conditions. Cytochrome c oxidase (CCO) activity was assayed according to the method of Zhu et al. (2012). The assay medium contained 0.2 mL of the crude mitochondria extract, 0.2 mL 0.04% (w/v) cytochrome c solution, and 0.5 mL 0.4% (w/v) dimethyl phenylene diamine. One unit of CCO activity was defined as an increase of 0.1 in absorbance per second at 510 nm under the assay conditions. Specific activity of all the enzymes was expressed as units per kilogram protein. 2.5. ATP, ADP, and AMP contents and energy charge measurements ATP, ADP, and AMP contents were extracted and measured using the method of Yi et al. (2008). Flesh tissue (2 g) of pears was ground in 5 mL 0.6 M perchloric acid. The homogenate was then centrifuged at 20,000 g for 15 min at 4 C. Next, 3 mL of the
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supernatant was obtained, and its pH was rapidly adjusted to 6.5– 6.8 by using KOH solution, diluted to 4 mL, and passed through a 0.45-mm filter (Millipore Corp., Bedford, MA). ATP, ADP, and AMP levels were measured using an Agilent 1100 high-performance liquid chromatography (HPLC) instrument (Agilent Corp., Santa Clara, CA) by using a reserved-phase Nova-Pak C18 column (5 mm, 5 250 mm; Agilent Corp.) and an ultraviolet detector at 254 nm. Mobile phase A consisted of 0.06 M dipotassium hydrogen phosphate and 0.04 M potassium dihydrogen phosphate dissolved in deionized water and adjusted to pH 7.0 with 0.1 M KOH. Mobile phase B was pure acetonitrile. The elution was performed using a linear gradient program with 75–100% phase A and 0–25% phase B for 7 min. The flow rate was 0.02 mL per second. The concentrations of ATP, ADP, and AMP were determined by injecting 10 mL sample into the HPLC system according to the external standard program. The energy charge was calculated using the following equation: [ATP + 1/2 ADP]/[ATP + ADP + AMP].
Fig. 1. Chilling injury (CI) index of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are means of three replicates.
2.6. Fatty acid quantification Total lipids in the flesh tissue were extracted according to the method of Cao et al. (2009). Briefly, 20 g of tissue was homogenized in 10 mL of chloroform–methanol: 0.1 M HCl: water (200:100:1), and then 10 mL 0.1 M HCl was added; the mixture was centrifuged at 4000 g for 10 min. The organic phase was collected and dried. The fatty acids were methylated by adding 1 mL of 140 mL L 1 boron trifluoride in methanol at boiling temperature for 10 min. Methylated fatty acids were extracted with hexane, dried, and redissolved in 200 mL chloroform before chromatography. Fatty acids were separated and quantified according to Mirdehghan et al. (2007) by gas chromatography by using CP-3800 GC instrument (Varian, USA) equipped with a flame ionization detector. An authentic methylated fatty acid (Sigma–Aldrich 47,801) was used as the external standard to identify and quantify the peaks, which were corrected for losses at this stage by using the C17:0 internal standard, added before methylation of the samples. The unsaturated to saturated fatty acid ration was calculated using the following equation: (18:1 + 18:2 + 18:3)/(16:0 + 18:0), where 16:0 is palmitic acid; 18:0 is stearic acid; 18:1 is oleic acid; 18:2 is linoleic acid; and 18:3 is linolenic acid. 2.7. Statistical analysis Experiments were performed using a completely randomized design. All statistical analyses were performed using SPSS Version 14.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed using oneway analysis of variance. The main effects and interactions were analyzed and means were compared using Duncan’s multiple range tests at the significance level of 0.05. 3. Results 3.1. CI index CI appeared in ‘Nanguo’ pears upon removal from 90 d of cold storage (at shelf life day 0), and, from day five onward, it increased rapidly throughout the remaining shelf life. 1-MCP treatment reduced the CI index of cold-stored pears. The CI index was 83.3% and 71.4% lower in 1-MCP-treated fruit than in the control fruit on the 10th and 20th day of storage, respectively (Fig. 1). 3.2. Electrolyte leakage and MDA content Both electrolyte leakage and MDA content of the control and 1-MCP-treated pears increased gradually with shelf life (Fig. 2). 1-MCP treatment remarkably inhibited the increase of electrolyte
Fig. 2. Electrolyte leakage (A) and MDA content (B) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
leakage and MDA content, and the pears treated with 1-MCP showed relatively lower ion leakage and MDA content in comparison with the control fruit during the shelf life after cold storage. The ATP content increased during the first ten days and decreased rapidly thereafter (Fig. 3A). 1-MCP treatment retarded the increase in ATP content prior to day ten and delayed the decline. ATP content was 18.13% and 31.79% higher in the 1-MCPtreated fruit than in the control fruit on the 15th and 20th day after storage, respectively. The energy charge increased in control fruit for the first 10 days of shelf life, then plateaued and decreased by
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Fig. 3. ATP content (A) and energy change (B) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
the day 20 day. 1-MCP treatment delayed increase in energy charge for the first ten days of shelf life, but thereafter it was higher than in control fruit (Fig. 3B). 1-MCP treatment maintained a higher energy charge compared with that in the control fruit on the 20th day after storage. H+-ATPase activity increased before the fifth day in fruit of both treatments, and declined thereafter in the control fruit (Fig. 4A). 1-MCP treatment increased the H+-ATPase activity even after the fifth day of storage and maintained higher levels thereafter compared with that in the control fruit. Ca2+-ATPase activity decreased gradually with shelf life. 1-MCP treated fruit had higher Ca2+-ATPase activity at removal from storage, and this higher activity was maintained during shelf life (Fig. 4B). SDH activity decreased remarkably before the tenth day and slightly thereafter in the control fruit (Fig. 4C). 1-MCP treatment retarded the decrease in SDH activity compared with that in the control fruit during the entire shelf life. In general, SDH activity in 1-MCPtreated fruit was higher than that in the control fruit. CCO activity increased slightly before the fifth day and decreased rapidly thereafter. 1-MCP treatment maintained higher CCO activity even after day fifteen (Fig. 4D). The mitochondrial protein content in both the control and 1-MCP-treated fruit increased rapidly during the first 5 days of shelf life and then declined gradually (Fig. 5A). The mitochondrial protein content in 1-MCP-treated fruit was higher than that in the control fruit during the entire shelf life and was higher during the first 5 days in the 1-MCP-treated fruit. Mitochondrial respiratory control rate increased rapidly with shelf life. 1-MCP treatment
Fig. 4. H+-ATPase activity (A), Ca2+-ATPase activity (B), SDH activity (C) and CCO activity (D) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
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Fig. 5. Mitochondrial protein content (A) and mitochondrial respiratory control rate (B) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
increased the respiratory control rate. Mitochondrial respiratory control rate was 34.15%, 36.70%, and 23.61% higher in the 1-MCPtreated fruit than in the control fruit on the 10th, 15th, and 20th day, respectively (Fig. 5B). Palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid were found to be the major membrane fatty acids in pear fruit (Fig. 6). Palmitic acid (16:0) and stearic acid (18:0), saturated fatty acids, increased gradually with shelf life. 1-MCP treatment inhibited the increase in palmitic acid and stearic acid. The content of oleic acid (18:1) increased slightly during the entire shelf life. 1-MCP treatment maintained the lower value of oleic acid compared with that in the control fruit. The levels of linoleic acid (18:2) and linolenic acid (18:3), unsaturated fatty acids, declined gradually during the shelf life. 1-MCP treatment delayed the decrease in these fatty acids. Therefore, 1-MCP-treated fruit had higher unsaturated to saturated fatty acid ratio than control fruit during the entire shelf life (Fig. 6F). 4. Discussion Cellular energy supply is an important factor that controls fruit ripening and senescence after harvest. Reduction of cellular energy can adversely affect ripening, senescence, pathogen infection, and
physiological disorder in various kinds of fruit after harvest (Jiang et al., 2007). Increasing evidence suggests that browning in pear (Liu et al., 2006) and litchi fruit (Saquet et al., 2003) results from the lack of cellular energy. In this study, 1-MCP treatment was found to effectively maintain high levels of ATP and cellular energy and reduce the development of CI in pear during cold storage. Similar results have been reported in mango fruit (Li et al., 2014a,b) and peach (Jin et al., 2014). The energy status of plant tissues is associated with the activities of energy metabolism enzymes, including H+-ATPase, Ca2+-ATPase, SDH, and CCO. ATPases are a class of enzymes that catalyze the decomposition of ATP to ADP and a free phosphate ion, involving the release of energy (Heyes and Townsend, 1992). SDH catalyzes the oxidation of succinate to fumarate and produces ATP, whereas CCO is the last enzyme in the respiratory electron transport chain and provides energy for mitochondrial oxidative phosphorylation (Millar et al., 1995). These energy metabolism enzymes are found in the mitochondrial inner membrane, which is involved in oxidative phosphorylation and ATP production. The inactivation of energy metabolism enzymes could adversely affect mitochondrial function, reduce energy supply, and cause cell death. Therefore, energy deficit and cell integrity are closely related to CI in fruit. The improvement of chilling tolerance in harvested fruit has been reported to be related to the enhancement of the activities of energy metabolism enzymes. Azevedo et al. (2008) reported that increase in H+-ATPase activity was a biochemical marker of ripening along with ethylene production and fruit softening in papaya. Zhu et al. (2012) suggested that energy charge was higher in low temperature-treated peaches than in the control fruit, and this resulted from the higher activities of SDH and CCO. Jin et al. (2013) indicated that methyl jasmonate contributes to the increased chilling tolerance of peach fruit during cold storage, and this beneficial effect was associated with the enhanced activities of H+-ATPase, Ca2+-ATPase, SDH, and CCO. In this study, the activities of energy metabolism enzymes were higher in the 1-MCP-treated pear fruit than in the control fruit (Fig. 4). This suggests that energy metabolism enzymes play positive roles in energy production, and decreased ATP level might be involved in the development of CI in pear fruit. Therefore, maintenance of mitochondrial enzyme activities is necessary to ensure sufficient cellular energy, which is crucial for cell survival under chilling stress. Damage to membrane structure and changes in lipid constituents are considered to lead to decompartmentalization and ion leakage increase (Wongsheree et al., 2009). The changes in the composition of membrane lipids due to a decrease in unsaturated fatty acid content might affect phase transition of membrane lipids. Cao et al. (2011) reported that the decrease of lipid unsaturation was associated with high levels of CI in loquat fruit. Zheng et al. (2007) found that increased unsaturated lipid content and membrane fluidity led to enhanced tolerance of pear fruit to chilling stress. In this study, the contents of linoleic acid and linolenic acid and ratio of unsaturated to saturated fatty acids were higher in 1-MCP-treated pears than in controls (Fig. 6). These results suggest that 1-MCP might help in maintaining the normal function of membranes and in reducing CI in pear fruit during cold storage. MDA is the product of membrane peroxidation and could damage the structure and integrity of membranes during CI. High ratio of unsaturated to saturated fatty acids in cell membranes could prevent ion leakage and MDA content increase, thereby preventing membrane peroxidation and damage. Cellular energy status has been reported to be crucial for maintaining membrane integrity, and energy depletion is associated with membrane damage (Jiangetal., 2007).Increasingevidence suggested that fruit browning, the main CI symptom, mainly resulted from membrane damage under chilling stress, which might be related to energy deficit (Su et al., 2005; Yi et al., 2008). Changes in ATP content
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Fig. 6. Palmtitic acid content (A), stearic acid content (B), oleic acid content (C), linoleic acid (D), linolenic acid content (E) and Uns/sat fatty acid ratio (F) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
and energy status directly affect the biosynthesis of membrane lipids and restoration of cell membrane. Thus, high levels of unsaturated fatty acids could maintain normal cellular function and prevent the interaction of polyphenol oxidase with phenyl substrates resulting in enzymatic browning (Liu et al., 2006). In conclusion, the results obtained in this study suggest that energy metabolism might be involved in the development of CI in ‘Nanguo’ pear fruit. 1-MCP treatment can effectively enhance chilling tolerance and alleviate CI in ‘Nanguo’ pears. The reduction of CI after 1-MCP treatment might be associated with the maintenance of higher levels of energy and unsaturated to saturated fatty acid ratio and the induction of enzymes related to energy metabolism. Acknowledgements This work was supported by National Nature Science Foundation of China (No. 31370685), the National Science Technology &
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
1-Methylcyclopropene alleviates chilling injury by regulating energy metabolism and fatty acid content in ‘Nanguo’ pears Shunchang Cheng1, Baodong Wei, Qian Zhou, Dehong Tan, Shujuan Ji* College of Food Science, Shenyang Agricultural University, Shenyang 110866, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2014 Received in revised form 13 May 2015 Accepted 23 May 2015 Available online xxx
This study investigated the effects of 1-methylcyclopropene (1-MCP) treatment on chilling injury (CI), energy metabolism, and membrane fatty acid content in harvested ‘Nanguo’ pears during the shelf life after cold storage at 0 C. 1-MCP treated fruit showed slower CI injury development, lower ion leakage and malondialdehyde accumulation, increased adenosine triphosphate content and energy charge during a 25 day shelf life period at 20 C. Activities of enzymes associated with energy metabolism, including H+-adenosine triphosphatase, Ca2+-adenosine triphosphatase, succinic dehydrogenase, and cytochrome C oxidase increased after 1-MCP treatment. The ratio of unsaturated to saturated fatty acids in 1-MCP-treated fruit was higher than that in control fruit. These results suggest that alleviation of CI after 1-MCP treatment might be due to enhanced energy metabolism-related enzyme activities and higher levels of energy and unsaturated to saturated fatty acid ratio. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Pear 1-Methylcyclopropene Chilling injury Energy metabolism Fatty acid
1. Introduction ‘Nanguo’ pear (Pyrus ussuriensis) is grown in the northeastern cold regions of China. This pear cultivar is preferred by consumers because of its good taste, fragrant flavor, and pleasant aroma. Pears are climacteric fruit that show increased ethylene production and respiration rates during ripening. Hence, they remain firm for a limited time after harvest due to ripening. Rapid ripening and softening makes them sensitive to mechanical injury and pathogen infection (Li et al., 2014a,b; Paul et al., 2012). While storage at low temperature delays fruit senescence and extends postharvest life of the fruit, pears are susceptible to development of chilling injury (CI)-associated disorders, including peel and core browning and decay (Wang, 1990). Ensuring sufficient cellular energy is an important factor in controlling fruit ripening and senescence after harvest. Physiological disorders and browning in harvested fruit might be related to inadequate supplies and reduced efficiency of cellular energy generation (Jiang et al., 2007). Cell membranes are thought to be the primary sites for the development of CI. A higher ratio of unsaturated/saturated fatty acids has been shown to improve tolerance to chilling temperature in various kinds of fruit such as
loquat (Cao et al., 2009), banana (Jiang et al., 2004), and mango (Li et al., 2014a,b). Cell membrane damage is thought to be associated with the lack of energy, and ATP is known to play an important role in fatty acid synthesis and membrane repair (Rawyler et al., 1999). 1-Methylcyclopropene (1-MCP), an inhibitor of ethylene perception, is thought to interact with ethylene receptors and thereby prevent ethylene-dependent responses (Watkins, 2006). 1-MCP has been shown to inhibit CI incidence in some horticultural products such as apple (Apollo Arquiza et al., 2005), avocado (Pesis et al., 2002), pineapple (Fan et al., 1999), persimmon (Salvador et al., 2004), ‘Fallgold’ tangerine and grapefruit (Dou et al., 2005), ‘Nova’ and ‘Ortanique’ mandarins (Salvador et al., 2006), plum (Candan et al., 2011), and loquat (Cao et al., 2010). On the other hand, CI symptoms were induced in banana (Jiang et al., 2004), ‘Shamouti’ orange (Porat et al., 1999), and peach (Fan et al., 2002) by 1-MCP. The effects of 1-MCP on energy metabolism and fatty acidrelated changes associated with CI in ‘Nanguo’ pear fruit have not been investigated. Thus, this study aimed to evaluate the effect of 1-MCP treatment on energy status, energy metabolism-related enzyme activities, and fatty acid composition in ‘Nanguo’ pear fruit during the shelf life after storage at low temperatures. 2. Materials and methods
* Corresponding author at: No. 120 Dongling Road, Shenyang, 110866, Liaoning Province, People’s Republic of China. Tel.: +86 24 88487161. E-mail addresses: [email protected] (S. Cheng), [email protected] (S. Ji). 1 Address: Food Science College, Shenyang Agricultural University, No.120 Dongling Road, Shenyang, Liaoning. 110161, People’s Republic of China. Tel.: +86 24 88487161; fax: +86 24 88487162. http://dx.doi.org/10.1016/j.postharvbio.2015.05.012 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
2.1. Fruit material and treatment Hard, mature (green skin and light cream color) ‘Nanguo’ pears were harvested from a commercial orchard located at Anshan City
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(longitude, 122.97E; latitude, 41.07N), Northeast China. The fruit were packed in fiberboard cartons and transferred to the laboratory at the Shenyang Agriculture University within 3 h. Fruit with uniform shape and size and free from damage and fungal infection were selected. The fruit were divided into two groups (150 fruit per group). Three replicates were used for treatment (50 fruit per replicate). Each replicate of treated fruit was exposed to 0.5 mL L 1 1-MCP (EthylBloc, Floralife Inc., USA) for 20 h at 20 C in an airtight 1 m3 plastic container. 1-MCP was released in the dissolved form in 1 mL distilled water in a glass dish to ensure the release of 1-MCP. For the control treatment, 150 fruit were placed in an identical plastic container without 1-MCP. Following treatment, the samples were placed in polyethylene bags (0.02 mm), secured with rubber bands, and stored under 80–90% relative humidity (RH) and at 0 C. After 90 days, fruit from both the groups were transferred from cold storage to ambient temperature (20 C) with 60–65% RH, and 10 fruit from each group were used for analysis at 5-day intervals during the remaining shelf life.
2.2. CI index Ten fruit were cut equatorially to document the severity of CI. The CI index was determined using a rating scale ranging from 0 to 4 on the basis of common visual symptoms of water-soaked appearance of the flesh cross-section and the affected core area or flesh tissue. The following scale was used: 0 = no damage; 1 = very slight damage; 2 = slight damage; 3 = moderate damage, and 4 = severe damage. The CI index was calculated using the following P formula: [ (A B)/5C], where A is the injury score of individual fruit, B is the number of fruit affected, 5 is the total number of scores (0–4) used, and C is the total number of fruit recorded (Zaharah and Singh, 2011).
2.3. Electrolyte leakage and malondialdehyde content measurement Discs were excised from the fruit by using a 3-mm diameter cork borer. The discs were briefly rinsed with deionized water and blotted dry on a slightly moistened Whatman filter paper. Twelve discs were then incubated in 30 mL of 300 mM mannitol for 3 h in a 50-mL capped polypropylene centrifuge tube. The conductivity of the bathing solution was measured after 4 h of incubation at 25 C by using a DDS-307 conductivity meter (Shanghai Precise Science Instrument Co., Shanghai, China). Total electrolyte conductivity was determined after the discs and bathing solution were heated in a boiling water bath for 30 min. The discs and bathing solution were then cooled to room temperature, and the conductivity was measured once again. Electrolyte leakage was expressed as the percentage of the conductivity of total tissue electrolyte. Three replicates were used for each treatment (Luo et al., 2011). The content of malondialdehyde (MDA) was measured using the thiobarbituric acid (TBA) reaction method (Zhang et al., 2009), with some modifications. Tissue samples (1.0 g) were homogenized in 4.0 mL of 5% (w/v) trichloroacetic acid and centrifuged at 10,000 g for 20 min. The supernatant was mixed with 2.0 mL of 0.67% TBA, heated at 100 C for 30 min, and then immediately cooled on ice. After the supernatant was centrifuged at 5000 g for 10 min, its absorbance was measured using a spectrophotometer (TU-1810 DSPC; Beijing Puxi Instrument Co., Beijing, China) at 450, 532, and 600 nm. The MDA concentration was calculated according to the formula: 6.45 (A532 A600) 0.56 A450. Three replicates were used for each treatment.
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2.4. Measurement of activities of enzymes related to energy metabolism Mitochondria were isolated from pear fruit as described by Qin et al. (2009) with some modifications. In brief, approximately 60 g peeled pear tissue from 10 fruit was gently cut and homogenized in 300 mL ice-cold extraction buffer containing 250 mM sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% w/v polyvinylpyrrolidone (PVP), 0.1% w/v bovine serum albumin (BSA), 10 mM b-mercaptoethanol, and 50 mM Tris-HCl (pH 7.5). The homogenate was filtered through four layers of sterile cheesecloth, followed by centrifugation at 1200 g for 15 min. The supernatant was decanted and centrifuged at 16,000 g for 20 min. The pellets were re-suspended in washing buffer (250 mM sucrose, 0.1% w/v BSA, and 50 mM Tris-HCl, pH 7.5) by using a soft brush. The crude mitochondria were purified using density-gradient centrifugation with 28% (v/v) Percoll. The extraction buffer consisted of 250 mM sucrose, 1.0 mM EDTA, 0.5% PVP, 0.1% (w/v) BSA, 10 mM b-mercaptoethanol, and 50 mM Tris-HCl, pH 7.5. The washing buffer consisted of 10 mM Tris, 0.3 M mannitol, 1.0 mM EDTA, and 0.1% (w/ v) BSA, pH 7.2. The concentration of proteins was determined spectrophotometrically at 595 nm, as described by Bradford (1976) by using BSA as the standard. Respiratory activity was determined as described previously (Mazorra et al., 2013). Briefly, 1 mg of protein was used to determine the respiratory activity of mitochondria by using an oxygen electrode (Hansatech, Respire 1, UK) in 1.0 mL reaction medium [0.35 M mannitol, 10 mM phosphate buffer, 10 mM KCl, 5 mM MgCl2, 0.5% (w/v) BSA, pH 7.2] at 25 C. The entire assay was performed in the presence of 200 mM glutamate as the substrate for respiration. The addition of 100 nM ADP to the reaction medium induced the transition from phosphorylation (State 3) to resting (State 4) respiration, indicating the phosphorylation capacity of the mitochondria. The mitochondrial respiratory control rate was calculated as follows: (State 3)/(State 4). H+-ATPase and Ca2+-ATPase activities were determined by measuring the release of phosphorus. The reaction was initiated by the addition of 100 mL 0.03 M ATP-Tris (pH 8.0), and stopped with 5% (w/v) trichloroacetic acid after 20 min of incubation at 37 C. One unit of H+-ATPase and Ca2+-ATPase activities was defined as the release of 1 mM of phosphorus per second at 660 nm under the assay conditions (Jin et al., 2013). Succinic dehydrogenase (SDH) activity was determined according to the method of Ackrell et al. (1978). The assay medium contained 0.3 mL of the crude mitochondria extract, 3 mL 0.2 mM potassium phosphate buffer (pH 7.4), 1 mL 0.2 mM sodium succinate, 0.1 mL 1 mM dichlorophenylmethyl cardinal, and 0.1 mL 0.33% (w/v) methyl sulfenyl phenazine. One unit of SDH activity was defined as an increase of 0.01 in absorbance per second at 600 nm under the assay conditions. Cytochrome c oxidase (CCO) activity was assayed according to the method of Zhu et al. (2012). The assay medium contained 0.2 mL of the crude mitochondria extract, 0.2 mL 0.04% (w/v) cytochrome c solution, and 0.5 mL 0.4% (w/v) dimethyl phenylene diamine. One unit of CCO activity was defined as an increase of 0.1 in absorbance per second at 510 nm under the assay conditions. Specific activity of all the enzymes was expressed as units per kilogram protein. 2.5. ATP, ADP, and AMP contents and energy charge measurements ATP, ADP, and AMP contents were extracted and measured using the method of Yi et al. (2008). Flesh tissue (2 g) of pears was ground in 5 mL 0.6 M perchloric acid. The homogenate was then centrifuged at 20,000 g for 15 min at 4 C. Next, 3 mL of the
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supernatant was obtained, and its pH was rapidly adjusted to 6.5– 6.8 by using KOH solution, diluted to 4 mL, and passed through a 0.45-mm filter (Millipore Corp., Bedford, MA). ATP, ADP, and AMP levels were measured using an Agilent 1100 high-performance liquid chromatography (HPLC) instrument (Agilent Corp., Santa Clara, CA) by using a reserved-phase Nova-Pak C18 column (5 mm, 5 250 mm; Agilent Corp.) and an ultraviolet detector at 254 nm. Mobile phase A consisted of 0.06 M dipotassium hydrogen phosphate and 0.04 M potassium dihydrogen phosphate dissolved in deionized water and adjusted to pH 7.0 with 0.1 M KOH. Mobile phase B was pure acetonitrile. The elution was performed using a linear gradient program with 75–100% phase A and 0–25% phase B for 7 min. The flow rate was 0.02 mL per second. The concentrations of ATP, ADP, and AMP were determined by injecting 10 mL sample into the HPLC system according to the external standard program. The energy charge was calculated using the following equation: [ATP + 1/2 ADP]/[ATP + ADP + AMP].
Fig. 1. Chilling injury (CI) index of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are means of three replicates.
2.6. Fatty acid quantification Total lipids in the flesh tissue were extracted according to the method of Cao et al. (2009). Briefly, 20 g of tissue was homogenized in 10 mL of chloroform–methanol: 0.1 M HCl: water (200:100:1), and then 10 mL 0.1 M HCl was added; the mixture was centrifuged at 4000 g for 10 min. The organic phase was collected and dried. The fatty acids were methylated by adding 1 mL of 140 mL L 1 boron trifluoride in methanol at boiling temperature for 10 min. Methylated fatty acids were extracted with hexane, dried, and redissolved in 200 mL chloroform before chromatography. Fatty acids were separated and quantified according to Mirdehghan et al. (2007) by gas chromatography by using CP-3800 GC instrument (Varian, USA) equipped with a flame ionization detector. An authentic methylated fatty acid (Sigma–Aldrich 47,801) was used as the external standard to identify and quantify the peaks, which were corrected for losses at this stage by using the C17:0 internal standard, added before methylation of the samples. The unsaturated to saturated fatty acid ration was calculated using the following equation: (18:1 + 18:2 + 18:3)/(16:0 + 18:0), where 16:0 is palmitic acid; 18:0 is stearic acid; 18:1 is oleic acid; 18:2 is linoleic acid; and 18:3 is linolenic acid. 2.7. Statistical analysis Experiments were performed using a completely randomized design. All statistical analyses were performed using SPSS Version 14.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed using oneway analysis of variance. The main effects and interactions were analyzed and means were compared using Duncan’s multiple range tests at the significance level of 0.05. 3. Results 3.1. CI index CI appeared in ‘Nanguo’ pears upon removal from 90 d of cold storage (at shelf life day 0), and, from day five onward, it increased rapidly throughout the remaining shelf life. 1-MCP treatment reduced the CI index of cold-stored pears. The CI index was 83.3% and 71.4% lower in 1-MCP-treated fruit than in the control fruit on the 10th and 20th day of storage, respectively (Fig. 1). 3.2. Electrolyte leakage and MDA content Both electrolyte leakage and MDA content of the control and 1-MCP-treated pears increased gradually with shelf life (Fig. 2). 1-MCP treatment remarkably inhibited the increase of electrolyte
Fig. 2. Electrolyte leakage (A) and MDA content (B) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
leakage and MDA content, and the pears treated with 1-MCP showed relatively lower ion leakage and MDA content in comparison with the control fruit during the shelf life after cold storage. The ATP content increased during the first ten days and decreased rapidly thereafter (Fig. 3A). 1-MCP treatment retarded the increase in ATP content prior to day ten and delayed the decline. ATP content was 18.13% and 31.79% higher in the 1-MCPtreated fruit than in the control fruit on the 15th and 20th day after storage, respectively. The energy charge increased in control fruit for the first 10 days of shelf life, then plateaued and decreased by
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Fig. 3. ATP content (A) and energy change (B) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
the day 20 day. 1-MCP treatment delayed increase in energy charge for the first ten days of shelf life, but thereafter it was higher than in control fruit (Fig. 3B). 1-MCP treatment maintained a higher energy charge compared with that in the control fruit on the 20th day after storage. H+-ATPase activity increased before the fifth day in fruit of both treatments, and declined thereafter in the control fruit (Fig. 4A). 1-MCP treatment increased the H+-ATPase activity even after the fifth day of storage and maintained higher levels thereafter compared with that in the control fruit. Ca2+-ATPase activity decreased gradually with shelf life. 1-MCP treated fruit had higher Ca2+-ATPase activity at removal from storage, and this higher activity was maintained during shelf life (Fig. 4B). SDH activity decreased remarkably before the tenth day and slightly thereafter in the control fruit (Fig. 4C). 1-MCP treatment retarded the decrease in SDH activity compared with that in the control fruit during the entire shelf life. In general, SDH activity in 1-MCPtreated fruit was higher than that in the control fruit. CCO activity increased slightly before the fifth day and decreased rapidly thereafter. 1-MCP treatment maintained higher CCO activity even after day fifteen (Fig. 4D). The mitochondrial protein content in both the control and 1-MCP-treated fruit increased rapidly during the first 5 days of shelf life and then declined gradually (Fig. 5A). The mitochondrial protein content in 1-MCP-treated fruit was higher than that in the control fruit during the entire shelf life and was higher during the first 5 days in the 1-MCP-treated fruit. Mitochondrial respiratory control rate increased rapidly with shelf life. 1-MCP treatment
Fig. 4. H+-ATPase activity (A), Ca2+-ATPase activity (B), SDH activity (C) and CCO activity (D) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
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Fig. 5. Mitochondrial protein content (A) and mitochondrial respiratory control rate (B) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
increased the respiratory control rate. Mitochondrial respiratory control rate was 34.15%, 36.70%, and 23.61% higher in the 1-MCPtreated fruit than in the control fruit on the 10th, 15th, and 20th day, respectively (Fig. 5B). Palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid were found to be the major membrane fatty acids in pear fruit (Fig. 6). Palmitic acid (16:0) and stearic acid (18:0), saturated fatty acids, increased gradually with shelf life. 1-MCP treatment inhibited the increase in palmitic acid and stearic acid. The content of oleic acid (18:1) increased slightly during the entire shelf life. 1-MCP treatment maintained the lower value of oleic acid compared with that in the control fruit. The levels of linoleic acid (18:2) and linolenic acid (18:3), unsaturated fatty acids, declined gradually during the shelf life. 1-MCP treatment delayed the decrease in these fatty acids. Therefore, 1-MCP-treated fruit had higher unsaturated to saturated fatty acid ratio than control fruit during the entire shelf life (Fig. 6F). 4. Discussion Cellular energy supply is an important factor that controls fruit ripening and senescence after harvest. Reduction of cellular energy can adversely affect ripening, senescence, pathogen infection, and
physiological disorder in various kinds of fruit after harvest (Jiang et al., 2007). Increasing evidence suggests that browning in pear (Liu et al., 2006) and litchi fruit (Saquet et al., 2003) results from the lack of cellular energy. In this study, 1-MCP treatment was found to effectively maintain high levels of ATP and cellular energy and reduce the development of CI in pear during cold storage. Similar results have been reported in mango fruit (Li et al., 2014a,b) and peach (Jin et al., 2014). The energy status of plant tissues is associated with the activities of energy metabolism enzymes, including H+-ATPase, Ca2+-ATPase, SDH, and CCO. ATPases are a class of enzymes that catalyze the decomposition of ATP to ADP and a free phosphate ion, involving the release of energy (Heyes and Townsend, 1992). SDH catalyzes the oxidation of succinate to fumarate and produces ATP, whereas CCO is the last enzyme in the respiratory electron transport chain and provides energy for mitochondrial oxidative phosphorylation (Millar et al., 1995). These energy metabolism enzymes are found in the mitochondrial inner membrane, which is involved in oxidative phosphorylation and ATP production. The inactivation of energy metabolism enzymes could adversely affect mitochondrial function, reduce energy supply, and cause cell death. Therefore, energy deficit and cell integrity are closely related to CI in fruit. The improvement of chilling tolerance in harvested fruit has been reported to be related to the enhancement of the activities of energy metabolism enzymes. Azevedo et al. (2008) reported that increase in H+-ATPase activity was a biochemical marker of ripening along with ethylene production and fruit softening in papaya. Zhu et al. (2012) suggested that energy charge was higher in low temperature-treated peaches than in the control fruit, and this resulted from the higher activities of SDH and CCO. Jin et al. (2013) indicated that methyl jasmonate contributes to the increased chilling tolerance of peach fruit during cold storage, and this beneficial effect was associated with the enhanced activities of H+-ATPase, Ca2+-ATPase, SDH, and CCO. In this study, the activities of energy metabolism enzymes were higher in the 1-MCP-treated pear fruit than in the control fruit (Fig. 4). This suggests that energy metabolism enzymes play positive roles in energy production, and decreased ATP level might be involved in the development of CI in pear fruit. Therefore, maintenance of mitochondrial enzyme activities is necessary to ensure sufficient cellular energy, which is crucial for cell survival under chilling stress. Damage to membrane structure and changes in lipid constituents are considered to lead to decompartmentalization and ion leakage increase (Wongsheree et al., 2009). The changes in the composition of membrane lipids due to a decrease in unsaturated fatty acid content might affect phase transition of membrane lipids. Cao et al. (2011) reported that the decrease of lipid unsaturation was associated with high levels of CI in loquat fruit. Zheng et al. (2007) found that increased unsaturated lipid content and membrane fluidity led to enhanced tolerance of pear fruit to chilling stress. In this study, the contents of linoleic acid and linolenic acid and ratio of unsaturated to saturated fatty acids were higher in 1-MCP-treated pears than in controls (Fig. 6). These results suggest that 1-MCP might help in maintaining the normal function of membranes and in reducing CI in pear fruit during cold storage. MDA is the product of membrane peroxidation and could damage the structure and integrity of membranes during CI. High ratio of unsaturated to saturated fatty acids in cell membranes could prevent ion leakage and MDA content increase, thereby preventing membrane peroxidation and damage. Cellular energy status has been reported to be crucial for maintaining membrane integrity, and energy depletion is associated with membrane damage (Jiangetal., 2007).Increasingevidence suggested that fruit browning, the main CI symptom, mainly resulted from membrane damage under chilling stress, which might be related to energy deficit (Su et al., 2005; Yi et al., 2008). Changes in ATP content
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Fig. 6. Palmtitic acid content (A), stearic acid content (B), oleic acid content (C), linoleic acid (D), linolenic acid content (E) and Uns/sat fatty acid ratio (F) of pears during shelf life at 20 C following 90 days of cold storage at 0 C. Values are the means standard error of triplicate assays.
and energy status directly affect the biosynthesis of membrane lipids and restoration of cell membrane. Thus, high levels of unsaturated fatty acids could maintain normal cellular function and prevent the interaction of polyphenol oxidase with phenyl substrates resulting in enzymatic browning (Liu et al., 2006). In conclusion, the results obtained in this study suggest that energy metabolism might be involved in the development of CI in ‘Nanguo’ pear fruit. 1-MCP treatment can effectively enhance chilling tolerance and alleviate CI in ‘Nanguo’ pears. The reduction of CI after 1-MCP treatment might be associated with the maintenance of higher levels of energy and unsaturated to saturated fatty acid ratio and the induction of enzymes related to energy metabolism. Acknowledgements This work was supported by National Nature Science Foundation of China (No. 31370685), the National Science Technology &
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