Research Article Received: 21 October 2014
Revised: 28 April 2015
Accepted article published: 18 June 2015
Published online in Wiley Online Library: 14 July 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7310
Effects of nitric oxide on mitochondrial oxidative defence in postharvest peach fruits Guangqin Jing, Jie Zhou and Shuhua Zhu* Abstract BACKGROUND: It has been confirmed that the accumulation of reactive oxygen species (ROS) in fruit can cause oxidative damage and nitric oxide (NO) can regulate the accumulation of ROS and the antioxidative defence of fruit. However, little is known about the roles of NO on the antioxidant system in mitochondria of fruit. In this study, Feicheng peach fruits were dipped with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and NO solutions to explore the effects of NO on the membrane permeability transition and antioxidant system in mitochondria of peach fruit. RESULTS: Treatment with 15 𝛍mol L−1 NO solution could delay the decrease of mitochondrial permeability transition and decrease the content of ROS in mitochondria. Besides, when the endogenous NO was scavenged by c-PTIO, the ROS in mitochondria increased greatly and superoxide dismutase activity decreased, while the content and activities of peroxidase and catalase changed slightly. CONCLUSION: By delaying the decrease of mitochondrial permeability transition, 15 𝛍mol L−1 NO treatment could promote a more stable internal medium in mitochondria of Feicheng peach fruit. The increases in the activities of antioxidant enzymes in mitochondria caused by the remove of endogenous NO suggested that NO also plays an important role in the mitochondrial antioxidant system of Feicheng peach fruit. © 2015 Society of Chemical Industry Keywords: NO; mitochondrial permeability transition; mtDNA; ROS; antioxidant enzymes
INTRODUCTION
J Sci Food Agric 2016; 96: 1997–2003
The NO acting in cells should be of moderate concentration under normal circumstances; otherwise, it will become a potent toxin.12 In recent years, there have been increasing numbers of reports concerning NO in mitochondria. On one hand, NO can inhibit the electron transport chain (ETC) by affecting the activities of complexes of the respiratory chain;12 on the other hand, NO can also participate in uncompetitive inhibition by binding to the oxidized form of the enzyme, forming nitrite in the process and decreasing cyclooxygenase (COX) activity;13 besides, NO can react with O2 •− , forming ONOO− , and it was found that NO could interact with H2 O2 .14 Studies on ROS and NO have been carried out in recent years, and the results indicated that ROS and NO could interact functionally in cells of both mammals and plants.12,15,16 Additionally, in recent years, there have been reports about interaction of NO, ROS and antioxidant enzymes in postharvest fruits.16 – 18 Our previous study also found that exogenous NO treatment on postharvest peach fruit could relieve the damage caused by oxidative stress and regular ROS and its antioxidative system.19
∗
Correspondence to: Shuhua Zhu, Research Center for Food Safety and Assessment Engineering of Shandong Province, College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, China. E-mail: [email protected] Research Center for Food Safety and Assessment Engineering of Shandong Province, College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, China
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1997
The mitochondrion, a functional organelle with a double membrane, is the main producer of ATP and reactive oxygen species (ROS) such as superoxide (O2 •− ) and hydrogen peroxide (H2 O2 ).1 ROS can react with various intracellular targets, including lipids,2 proteins and DNA.3 Although ROS are generated during normal aerobic metabolism, their biological effects on these intracellular targets are dependent on the concentration, and increased levels of these species are present during oxidative stress.4 When levels of ROS increase, mitochondrial permeability transition (MPT) increases correspondingly. However, if the contents of ROS are too high, the MPT decreases as a result of oxidative damage.5 The intercellular levels of ROS depend on the balance between their production and consumption. The synthesis and scavenging of ROS in and around mitochondria are a complex network of events.6 There are several antioxidant enzymes in mitochondria, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), which can catalyse the decomposition of ROS to ensure its redox equilibrium.7 SOD in mitochondria is of vital importance as the first line of defence. It protects plants from the damage of accumulated ROS by metabolizing O2 •− into H2 O2 ; the H2 O2 thus generated will be converted into H2 O and O2 with the catalysis of CAT and other enzymes such as POD. Therefore, there are some overlaps in activities of POD and CAT. POD also catalyses the conversion of ROOH besides H2 O2 .8 Nitric oxide (NO) plays important roles in mammalian and plant cells, with well-known functions in signal transduction, enzyme regulation, immune response and anti-apoptotic response.9 – 11
www.soci.org Based on the facts that NO can react with ROS and most ROS are from mitochondria, it is suggest that NO plays a vital role in plant mitochondrial oxidative defence. However, research on ROS and NO in mitochondria, especially in plant mitochondria, are still rare and there is little data available for reference, to say nothing of the way NO mediates the antioxidative reactions in mitochondria of postharvest fruit. In order to study the role of NO in mitochondria of postharvest fruit, In this paper Feicheng peach fruits were treated with different concentrations of NO solutions and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide (c-PTIO, as NO scavenger) solution, and the mitochondrial permeability transition, mitochondrial ROS content and activities of mitochondrial SOD, CAT and POD were determined to explore the effects of NO on mitochondrial oxidative defence as well as to make a contribution to the practical application of exogenous NO in treatment of postharvest fruits and vegetables.
MATERIALS AND METHODS Plant material Fruits of peach (Prunus persica (L.) Batsch, cv. Feicheng) were harvested from a commercial orchard in Taian (Shandong, China) at mature stage with an average firmness of ∼80 N cm−2 and soluble sugar content (SSC) of ∼7.5 ∘ Brix. Fruits were selected to be of similar size and colour and free from defects and mechanical damage. The fruits were pre-cooled at 4 ∘ C for 24 h, and then soaked for 0.5 h in NO solutions of different concentrations (5, 15 and 30 μmol L−1 , respectively) as well as with distilled water as the control group and c-PTIO solution (5 μmol L−1 ) as NO scavenger. There were 10 units of peaches in each treatment. One unit comprised 30 peaches. The NO solutions were prepared according to the method of Lim et al.20 First, an NO aqueous solution (2.0 mmol L−1 , 20 ∘ C) was prepared by delivering NO gas (99.99%; Tianjin Saiteer Special Gases Co., Tianjin, China) into deoxygenated and deionized water at 20 ∘ C under an N2 atmosphere. The aqueous solution was then diluted with deoxygenated and deionized water to a final concentrations of 5, 15 and 30 μmol L−1 . The concentration of NO saturated solution was detected using an Apollo 1000 free radical analyser equipped with an NO probe (ISO-NOPF200; World Precision Instruments, Sarasota, FL, USA). After drying with cool air, all fruits were stored at 0 ∘ C. Sampling of the fruits was carried out every 2 days (2, 4, 6 and 8 days) during cold storage.
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Mitochondria isolation Mitochondria were isolated from peach fruits and purified using sucrose density gradients at 0–4 ∘ C according to the method of Millar et al.21 Mesocarps of peach fruits (12 g) were ground into power with a grinder, and the power were homogenized with 24 mL of 25 mmol L−1 3-(N-morpholino)propanesulfonic acid (Mops)-KOH (pH 7.8; including 0.4 mol L−1 D-mannitol, 1 mmol L−1 (ethylene glycol tetraacetic acid (EGTA), 10 mmol L−1 tricine, 8 mmol L−1 L-cysteine, 1 g L−1 bovine serum albumin (BSA) and 10 g L−1 polyvinylpyrrolidone (PVP)) at 4 ∘ C for about 20 min. The homogenates were then filtered through six layers of cheesecloth. The filtrates were centrifuged at 3000 × g and 4 ∘ C for 5 min. The supernatants were then centrifuged at 16000 × g and 4 ∘ C for 28 min. The pellets were resuspended with 2 mL of 10 mmol L−1 Mops-KOH buffer (pH 7.2; including 0.4 mol L−1 D-mannitol, 1 mmol L−1 EGTA and 1 g L−1 BSA), and centrifuged at 16 000 × g and 4 ∘ C for 30 min. The pellets were collected as crude
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mitochondria. The crude mitochondria were resuspended with 2.5 mL of pre-cooled 0.1 mol L−1 Tris–HCl buffer (pH 8.5) for the following purification. Purification of the crude mitochondria was carried out using sucrose density-gradient centrifugation to remove pollution by the other cell compartments such as plastids. A two-layer 0.6/1.4 mol L−1 sucrose (including 0.6/1.4 mol L−1 sucrose, 1 mmol L−1 EGTA, 1 g L−1 BSA and 10 mmol L−1 Mops-KOH, pH 7.2) density gradient was set in a centrifuge tube. The upper layer was 5 mL of 0.6 mol L−1 sucrose solution and the lower layer was 5 mL of 1.4 mol L−1 sucrose solution. The crude mitochondrial solution was spread on the surface of the upper layer, and centrifuged at 16 000 × g and 4 ∘ C for 30 min. The band between the two layers was the purified mitochondria. The purified mitochondrial solution was then extracted from the middle of the sucrose density gradients and centrifuged at 16 000 × g and 4 ∘ C for 25 min. The pellets were collected and resuspended with 2.5 mL of pre-cooled 0.1 mol L−1 Tris–HCl buffer. The yield of purified mitochondria was about 0.4 mg mitochondrial protein per gram of fruit fresh weight, assayed by detecting the activity of cytochrome c oxidase21 before this experiment. The integrity of mitochondria was confirmed by determination of the breathing stage of mitochondria before this experiment. To confirm the purity of the mitochondria, the activity of catalase was determined before the mitochondria were disrupted using ultrasonication, and few peroxisomes were found in the purified mitochondrial solution. Only after the purity and integrity of mitochondria were confirmed were determinations of indexes concerning the antioxidative defence in mitochondria carried out in the following experiment. Measurement of mitochondrial membrane permeability transition The membrane permeability transition of mitochondria was measured following the method of Braidot et al.22 A mitochondrial suspension (200 μL) was mixed with 3 mL of 10 mmol L−1 Hepes-HCl buffer (pH 7.4; including 250 mmol L−1 sucrose, 0.1 mmol L−1 K-EGTA, 2 mmol L−1 MgCl2 , 4 mmol L−1 KH2 PO4 ) and incubated at 25 ∘ C for 5 min. After the incubation, 100 μL of 2 μg mL−1 rhodamine 123 were added. Instantly, fluorescence quenching of rhodamine 123 was monitored as fluorescence intensity changes using a Cary Eclipse spectrofluorimeter. The excitation and emission wavelengths were 500 and 520 nm, respectively. Fluorescence values calculated in the time range 0–60 s were normalised on the initial fluorescence (Fi). The membrane permeability transition of mitochondria was expressed as ΔF Fi−1 s−1 g−1 protein. Determination of ROS Content of ROS was determined according to the methods of Jambunathan23 and Esposti.24 First, 0.1 mL re suspended sample was diluted with 0.9 mL Tris–HCl buffer (10 mmol L−1 , pH 7.2). Then 10 μL of 2 mmol L−1 2′ ,7′ -dichlorofluorescein diacetate (DCF-DA) solution (prepared with dimethylsulfoxide (DMSO)) were mixed with the diluted sample. The mixture was incubated for 30 min in the dark. In the control group, the resuspended mitochondria were replaced by Tris–HCl buffer (10 mmol L−1 , pH 7.2). After incubation, the fluorescence value was measured on a Cary Eclipse spectrofluorimeter with a 10 nm slit. The excitation and emission wavelengths were 485 and 530 nm, respectively. Content of ROS was expressed as a.u. mg−1 protein.
© 2015 Society of Chemical Industry
J Sci Food Agric 2016; 96: 1997–2003
Effects of nitric oxide on mitochondrial oxidative defence in peach
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Figure 1. Changes in the mitochondrial membrane permeability transition of peach fruit after treatment during storage. Each value is presented as mean ± SE (n = 3).
Enzyme assays Measurement of mitochondrial SOD activity Mitochondrial SOD activity was determined by measuring the ability of SOD to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm after exposure to light from a 10 W fluorescent lamp for 25 min.25 Before the illumination, 0.1 mL mitochondrial resuspension and 0.3 mL of 20 μmol L−1 riboflavin were added to 2.6 mL assay mixture (including 50 mmol L−1 Tris–HCl pH 8.5, 13 mmol L−1 Met, 75 μmol L−1 NBT, 10 μmol L−1 ethylenediaminetetraacetic acid) in a 5 mL centrifugal tube. In the control group the enzyme solution was replace by 0.1 mL Tris–HCl buffer (0.1 mol L−1 , pH 8.5). The centrifugal tubes were then placed under a 10 W fluorescent lamp for 25 min to initiate the reaction. After that, estimation of the mitochondrial SOD activity was carried out by UV spectroscopic analysis at 560 nm. One unit of SOD activity is defined as the amount of protein required to give half-maximal inhibition of NBT reduction, and SOD activity was expressed as U mg−1 protein. Measurement of CAT activity CAT activity was measured by monitoring the enzyme-catalysed decomposition of H2 O2 by UV spectroscopic kinetic analysis at 240 nm for 150 s on an ultraviolet (UV) spectrophotometer.26 The resuspended sample (0.1 mL) was mixed with 3 mL phosphate buffer (PBS, 50 mmol L−1 , pH 7.0). Then 0.1 mL of 1% H2 O2 was added to the mixture and the mitochondrial CAT activity of the mixture above was instantly estimated. The decay in absorbance at 240 nm was monitored for 150 s. One unit (1 U) of CAT activity was defined as the amount of enzyme that caused a 0.01 decrease in A240 in 1 s under the assay conditions. CAT activity was expressed as U mg−1 protein s−1 .
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Statistical analysis The experiments were conducted in a completely randomized design. Each experiment was repeated three times and the data were processed by analysis of variance (ANOVA), comparing treatments at a significance level of 0.05 according to the least significant difference (LSD) test. All data were expressed as mean ± SE (n = 3).
RESULTS Changes in mitochondrial MPT Treatment with 15 μmol L−1 NO significantly increased the MPTs of peach fruit during storage, which were about 10% higher than that of the control (Fig. 1). However, there were no significant differences in MPT among treatments with 5, 30 μmol L−1 NO and the control. Treatment with c-PTIO significantly decreased mitochondrial MPT of peach fruit during storage, which were only 90.0% of the control. Mitochondrial MPTs of peach fruit treated with 15 μmol L−1 NO were about 15% higher than that of fruit treated with c-PTIO. Changes in mitochondrial ROS content As shown in Fig. 2, the content of ROS in mitochondria of peach fruits increased during cold storage. Treatment with c-PTIO could increase the content of ROS by 56.3% on day 2 and then the content was kept at a stable value during the following 6 days, whereas NO appropriately decreased the ROS content in mitochondria slightly. In addition, during the first 2 days of storage,
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Measurement of mitochondrial POD activity The method of measurement of POD was according to the methods of Rahnama and Ebrahimzadeh27 and Sun.28 The resuspension of peach fruit mitochondria (1 mL) was added to 2 mL
benzidine–NaAc solution (0.005 mol L−1 ) and 1 mL of 3% H2 O2 was added to the above mixture. Immediately, the activity of mitochondrial POD was estimated by UV spectroscopic kinetic analysis at 580 nm for 150 s on a UV-2450 UV spectrophotometer. One unit (1 U) of POD activity was defined as the amount of enzyme that caused a 0.01 increase in A580 in 1 s under the assay conditions. POD activity was expressed as U mg−1 protein s−1 .
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Figure 2. Effects of different concentrations of NO solutions on ROS: each value is presented as mean ± SE (n = 3).
the ROS content of CK and NO treatment decreased respectively by 14.8% and 16.4%, then increased by 59.0% and 52.2%, respectively, on day 4. After that, the content of ROS of these two samples tended to a stable value. The stable value of ROS concentration of treatment of c-PTIO was then 13% higher than that of the control from day 4 to day 8, while the stable value of the 15 μmol L−1 NO treatment was 7.3% lower than that of the control. Effects of NO on antioxidant enzymes Effects of NO on mitochondrial SOD activity As shown in Fig. 3, during storage at low temperature SOD activity of the control group increased by about 16.98% overall. The sample treated with 15 μmol L−1 NO had lower SOD activity compared with the control on corresponding days. During cold storage (from day 2 to day 6), SOD activities of NO treatment were 4.43%, 17.84%, 9.44% and 33.62% lower than that of the control group on day 2, 4, 6 and 8, respectively. However, SOD activity decreased quickly in fruit treated with c-PTIO, which was 26.9% lower than that of the control and 12.6% lower than that of treatment with NO overall. Besides that, the variation trends of SOD activity of these three different treatments were similar to each other. All of them decreased on day 2 and then increased to a peak at day 6, before decreasing on day 8.
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Effects of NO on mitochondrial CAT activity When the samples were stored at low temperature, the mitochondrial CAT activity increased in a small range (Fig. 4). During storage, CAT activity of the control increased by 4.4%, CAT activity of c-PTIO treatment increased by 8.6% and CAT activity of the sample treated with 15 μmol L−1 NO solution increased by 3.8%. The sample treated with c-PTIO had the highest activity, whereas 15 μmol L−1 NO solution treatment led to a decrease in CAT activity (compared with CAT activity of the control on the corresponding day).
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CAT activities of fruits treated with c-PTIO were 16.86%, 5.27%, 7.12% and 3.90% higher than that of the control on day 2, 4, 6 and 8, respectively. However, CAT activity of fruits treated with 15 μmol L−1 NO solution were 10.39%, 8.91%, 3.28% and 7.09% lower than that of control on day 2, 4, 6 and 8, respectively. Effects of NO on mitochondrial POD activity As shown in Fig. 5, mitochondrial POD activity decreased slightly during low-temperature storage as a whole. Both the control and the 15 μmol L−1 NO solution treatment decreased by 5.4%, and POD activity of the c-PTIO treatment decreased the least. On day 2, POD activity of 15 μmol L−1 NO solution treatment was 15.3% higher than that of the c-PTIO treatment and 20.7% higher than that of the control. Then, on day 4, POD activity of 15 μmol L−1 NO solution treatment decreased to a value which was near to that of the control group. Therefore, POD activities of 15 μmol L−1 NO solution treatment and control group were about 6.1% lower than that of the c-PTIO group. On day 6, POD activity of the c-PTIO group increased sharply, then decreased to the average value on day 8.
DISCUSSION In recent years, increasing evidence has shown that NO has an important role in the oxidative defence in plant cells.29 – 31 As a fat-soluble small molecule, NO could diffuse to different organelles, which makes it possible for exogenous NO to enter the inner part of mitochondria of plant cells. It is shown in this paper that during storage of peach fruits the decrease in MPT induced by low temperature could be delayed by treatment with 15 μmol L−1 NO and promoted by c-PTIO treatment. As shown in Fig. 1, no significant differences were observed in the changes in MPT among the treatments with 5 and 30 μmol L−1 NO and the control. These results suggest that the effects of NO on mitochondrial MPT depend on the dose of NO solution. NO at
© 2015 Society of Chemical Industry
J Sci Food Agric 2016; 96: 1997–2003
Effects of nitric oxide on mitochondrial oxidative defence in peach
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Figure 3. Effects of different concentrations of NO solutions on mitochondrial SOD activity: each value is presented as mean ± SE (n = 3).
Figure 4. Effects of different concentrations of NO solutions on mitochondrial CAT activity: each value is presented as mean ± SE (n = 3).
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when fruits were treated with c-PTIO (Fig. 2). This suggests that exogenous NO treatment could alleviate the increase in ROS in mitochondria. There exists crosstalk between NO and H2 O2 .14,33 NO could not only increase the expression of AOX, which can decrease the level of ROS in mitochondria,33 but also interact with H2 O2 ,14 to maintain homeostasis between NO and H2 O2 in mitochondria. As has been reported, SOD has been implicated as essentially the first line of defence against the potent toxicity of superoxide.34 In this study, the activity of SOD increased during storage, corresponding to the increase of ROS in mitochondria. However,
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5 μmol L−1 would be too low to trigger the changes in mitochondrial MPT of peach fruits, and NO at 30 μmol L−1 would be high and toxic to the mitochondrial membrane. The changes in mitochondrial MPT confirmed that treatment with 15 μmol L−1 NO could significantly improve the stability of mitochondrial permeability transition, which was consistent with our previous report.32 In this experiment, the content of ROS in mitochondria increased as a whole during storage at low temperature. ROS content of peaches treated with 15 μmol L−1 NO was much lower than that of the control, whereas the ROS content increased dramatically
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Figure 5. Effects of different concentrations of NO solution on mitochondrial POD activity: each value is presented as mean ± SE (n = 3).
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when the samples were treated with c-PTIO, their mitochondrial ROS content was highest, while their mitochondrial SOD activity was lowest; meanwhile, when the samples were treated with 15 μmol L−1 NO solution, their mitochondrial SOD activity was higher than that of the c-PTIO group. These phenomena may indicate that 15 μmol L−1 NO solution treatment could alleviate the increase in mitochondrial ROS content caused by the low temperature during storage by intervening in the synthesis of ROS and improving the activity of SOD in mitochondria. In mitochondria, CAT plays a role in promoting the catabolism of H2 O2 generated during aerobic metabolism and the process catalysed by SOD.35 Similar to CAT, POD is an oxidoreductase and its substrate is H2 O2 and some other peroxides. From Figs 4 and 5 it can be seen that the activities of CAT and POD in mitochondria were very low; this was especially true for CAT. Besides that, there were no significant distinctions between the three different treatments. This indicated that CAT and POD play much less important roles in mitochondria than does SOD. Figures 3 and 5 indicate that the activities of CAT correspond to the ROS contents to some extent, which explains the catalytic conversion mechanism of superoxide anion in mitochondria. However, the change in POD activity was not as significant compared with the change in CAT activity. Based on the fact that H2 O2 was the substrate of both CAT and POD, it is suggested that CAT and POD had cooperative effects in scavenging toxic ROS in peach fruit mitochondria. From the results of ROS content and antioxidative enzymes, it is suggested that exogenous NO treatment could alleviate oxidative damage from two aspects: on one hand, exogenous NO could regulate the synthesis of ROS in mitochondria by taking part in ETC; on the other hand, exogenous NO improved the activities of SOD and CAT in mitochondria, which promoted the scavenging of ROS in mitochondria. However, some studies have found that NO could also regulate the gene expression related to ROS36 in mitochondria. In this paper, studies concerning the relationship between exogenous NO and gene expression were not addressed, and further research needs to be carried out.
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CONCLUSIONS Treatment with 15 μmol L−1 NO solution could significantly relieve the decrease in mitochondrial permeability transition and the oxidative damage caused by low temperature during storage, and increased the activities of SOD. By scavenging parts roles of exogenous or endogenous NO, treatment with c-PTIO promoted the oxidative damage and the decrease in MPT of peach fruit with high ROS content and low activities of antioxidative enzymes. There were no significant changes in the activities of CAT and POD in mitochondria of peach fruit after treatment. It is suggested that moderate exogenous NO could be of benefit in improving the ability of the antioxidant system in Feicheng peach fruit mitochondria, as well as the mitochondrial permeability transition to some extent.
ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (31270723, 31370686, 31470686) and Science and Technology Development Planning of Shandong Province, China (2013GZX20109). We also appreciate Shunqing Hu and Bing Xie for their assistance in the extraction and purification of mitochondria, Ruirui Wang and Guanshuai Yi for their help in the determination of enzyme activities, and Lili Zhang for her guidance in the detection of NO concentration.
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2003
J Sci Food Agric 2016; 96: 1997–2003
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Revised: 28 April 2015
Accepted article published: 18 June 2015
Published online in Wiley Online Library: 14 July 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7310
Effects of nitric oxide on mitochondrial oxidative defence in postharvest peach fruits Guangqin Jing, Jie Zhou and Shuhua Zhu* Abstract BACKGROUND: It has been confirmed that the accumulation of reactive oxygen species (ROS) in fruit can cause oxidative damage and nitric oxide (NO) can regulate the accumulation of ROS and the antioxidative defence of fruit. However, little is known about the roles of NO on the antioxidant system in mitochondria of fruit. In this study, Feicheng peach fruits were dipped with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and NO solutions to explore the effects of NO on the membrane permeability transition and antioxidant system in mitochondria of peach fruit. RESULTS: Treatment with 15 𝛍mol L−1 NO solution could delay the decrease of mitochondrial permeability transition and decrease the content of ROS in mitochondria. Besides, when the endogenous NO was scavenged by c-PTIO, the ROS in mitochondria increased greatly and superoxide dismutase activity decreased, while the content and activities of peroxidase and catalase changed slightly. CONCLUSION: By delaying the decrease of mitochondrial permeability transition, 15 𝛍mol L−1 NO treatment could promote a more stable internal medium in mitochondria of Feicheng peach fruit. The increases in the activities of antioxidant enzymes in mitochondria caused by the remove of endogenous NO suggested that NO also plays an important role in the mitochondrial antioxidant system of Feicheng peach fruit. © 2015 Society of Chemical Industry Keywords: NO; mitochondrial permeability transition; mtDNA; ROS; antioxidant enzymes
INTRODUCTION
J Sci Food Agric 2016; 96: 1997–2003
The NO acting in cells should be of moderate concentration under normal circumstances; otherwise, it will become a potent toxin.12 In recent years, there have been increasing numbers of reports concerning NO in mitochondria. On one hand, NO can inhibit the electron transport chain (ETC) by affecting the activities of complexes of the respiratory chain;12 on the other hand, NO can also participate in uncompetitive inhibition by binding to the oxidized form of the enzyme, forming nitrite in the process and decreasing cyclooxygenase (COX) activity;13 besides, NO can react with O2 •− , forming ONOO− , and it was found that NO could interact with H2 O2 .14 Studies on ROS and NO have been carried out in recent years, and the results indicated that ROS and NO could interact functionally in cells of both mammals and plants.12,15,16 Additionally, in recent years, there have been reports about interaction of NO, ROS and antioxidant enzymes in postharvest fruits.16 – 18 Our previous study also found that exogenous NO treatment on postharvest peach fruit could relieve the damage caused by oxidative stress and regular ROS and its antioxidative system.19
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Correspondence to: Shuhua Zhu, Research Center for Food Safety and Assessment Engineering of Shandong Province, College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, China. E-mail: [email protected] Research Center for Food Safety and Assessment Engineering of Shandong Province, College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, China
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1997
The mitochondrion, a functional organelle with a double membrane, is the main producer of ATP and reactive oxygen species (ROS) such as superoxide (O2 •− ) and hydrogen peroxide (H2 O2 ).1 ROS can react with various intracellular targets, including lipids,2 proteins and DNA.3 Although ROS are generated during normal aerobic metabolism, their biological effects on these intracellular targets are dependent on the concentration, and increased levels of these species are present during oxidative stress.4 When levels of ROS increase, mitochondrial permeability transition (MPT) increases correspondingly. However, if the contents of ROS are too high, the MPT decreases as a result of oxidative damage.5 The intercellular levels of ROS depend on the balance between their production and consumption. The synthesis and scavenging of ROS in and around mitochondria are a complex network of events.6 There are several antioxidant enzymes in mitochondria, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), which can catalyse the decomposition of ROS to ensure its redox equilibrium.7 SOD in mitochondria is of vital importance as the first line of defence. It protects plants from the damage of accumulated ROS by metabolizing O2 •− into H2 O2 ; the H2 O2 thus generated will be converted into H2 O and O2 with the catalysis of CAT and other enzymes such as POD. Therefore, there are some overlaps in activities of POD and CAT. POD also catalyses the conversion of ROOH besides H2 O2 .8 Nitric oxide (NO) plays important roles in mammalian and plant cells, with well-known functions in signal transduction, enzyme regulation, immune response and anti-apoptotic response.9 – 11
www.soci.org Based on the facts that NO can react with ROS and most ROS are from mitochondria, it is suggest that NO plays a vital role in plant mitochondrial oxidative defence. However, research on ROS and NO in mitochondria, especially in plant mitochondria, are still rare and there is little data available for reference, to say nothing of the way NO mediates the antioxidative reactions in mitochondria of postharvest fruit. In order to study the role of NO in mitochondria of postharvest fruit, In this paper Feicheng peach fruits were treated with different concentrations of NO solutions and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide (c-PTIO, as NO scavenger) solution, and the mitochondrial permeability transition, mitochondrial ROS content and activities of mitochondrial SOD, CAT and POD were determined to explore the effects of NO on mitochondrial oxidative defence as well as to make a contribution to the practical application of exogenous NO in treatment of postharvest fruits and vegetables.
MATERIALS AND METHODS Plant material Fruits of peach (Prunus persica (L.) Batsch, cv. Feicheng) were harvested from a commercial orchard in Taian (Shandong, China) at mature stage with an average firmness of ∼80 N cm−2 and soluble sugar content (SSC) of ∼7.5 ∘ Brix. Fruits were selected to be of similar size and colour and free from defects and mechanical damage. The fruits were pre-cooled at 4 ∘ C for 24 h, and then soaked for 0.5 h in NO solutions of different concentrations (5, 15 and 30 μmol L−1 , respectively) as well as with distilled water as the control group and c-PTIO solution (5 μmol L−1 ) as NO scavenger. There were 10 units of peaches in each treatment. One unit comprised 30 peaches. The NO solutions were prepared according to the method of Lim et al.20 First, an NO aqueous solution (2.0 mmol L−1 , 20 ∘ C) was prepared by delivering NO gas (99.99%; Tianjin Saiteer Special Gases Co., Tianjin, China) into deoxygenated and deionized water at 20 ∘ C under an N2 atmosphere. The aqueous solution was then diluted with deoxygenated and deionized water to a final concentrations of 5, 15 and 30 μmol L−1 . The concentration of NO saturated solution was detected using an Apollo 1000 free radical analyser equipped with an NO probe (ISO-NOPF200; World Precision Instruments, Sarasota, FL, USA). After drying with cool air, all fruits were stored at 0 ∘ C. Sampling of the fruits was carried out every 2 days (2, 4, 6 and 8 days) during cold storage.
1998
Mitochondria isolation Mitochondria were isolated from peach fruits and purified using sucrose density gradients at 0–4 ∘ C according to the method of Millar et al.21 Mesocarps of peach fruits (12 g) were ground into power with a grinder, and the power were homogenized with 24 mL of 25 mmol L−1 3-(N-morpholino)propanesulfonic acid (Mops)-KOH (pH 7.8; including 0.4 mol L−1 D-mannitol, 1 mmol L−1 (ethylene glycol tetraacetic acid (EGTA), 10 mmol L−1 tricine, 8 mmol L−1 L-cysteine, 1 g L−1 bovine serum albumin (BSA) and 10 g L−1 polyvinylpyrrolidone (PVP)) at 4 ∘ C for about 20 min. The homogenates were then filtered through six layers of cheesecloth. The filtrates were centrifuged at 3000 × g and 4 ∘ C for 5 min. The supernatants were then centrifuged at 16000 × g and 4 ∘ C for 28 min. The pellets were resuspended with 2 mL of 10 mmol L−1 Mops-KOH buffer (pH 7.2; including 0.4 mol L−1 D-mannitol, 1 mmol L−1 EGTA and 1 g L−1 BSA), and centrifuged at 16 000 × g and 4 ∘ C for 30 min. The pellets were collected as crude
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G Jing, J Zhou, S Zhu
mitochondria. The crude mitochondria were resuspended with 2.5 mL of pre-cooled 0.1 mol L−1 Tris–HCl buffer (pH 8.5) for the following purification. Purification of the crude mitochondria was carried out using sucrose density-gradient centrifugation to remove pollution by the other cell compartments such as plastids. A two-layer 0.6/1.4 mol L−1 sucrose (including 0.6/1.4 mol L−1 sucrose, 1 mmol L−1 EGTA, 1 g L−1 BSA and 10 mmol L−1 Mops-KOH, pH 7.2) density gradient was set in a centrifuge tube. The upper layer was 5 mL of 0.6 mol L−1 sucrose solution and the lower layer was 5 mL of 1.4 mol L−1 sucrose solution. The crude mitochondrial solution was spread on the surface of the upper layer, and centrifuged at 16 000 × g and 4 ∘ C for 30 min. The band between the two layers was the purified mitochondria. The purified mitochondrial solution was then extracted from the middle of the sucrose density gradients and centrifuged at 16 000 × g and 4 ∘ C for 25 min. The pellets were collected and resuspended with 2.5 mL of pre-cooled 0.1 mol L−1 Tris–HCl buffer. The yield of purified mitochondria was about 0.4 mg mitochondrial protein per gram of fruit fresh weight, assayed by detecting the activity of cytochrome c oxidase21 before this experiment. The integrity of mitochondria was confirmed by determination of the breathing stage of mitochondria before this experiment. To confirm the purity of the mitochondria, the activity of catalase was determined before the mitochondria were disrupted using ultrasonication, and few peroxisomes were found in the purified mitochondrial solution. Only after the purity and integrity of mitochondria were confirmed were determinations of indexes concerning the antioxidative defence in mitochondria carried out in the following experiment. Measurement of mitochondrial membrane permeability transition The membrane permeability transition of mitochondria was measured following the method of Braidot et al.22 A mitochondrial suspension (200 μL) was mixed with 3 mL of 10 mmol L−1 Hepes-HCl buffer (pH 7.4; including 250 mmol L−1 sucrose, 0.1 mmol L−1 K-EGTA, 2 mmol L−1 MgCl2 , 4 mmol L−1 KH2 PO4 ) and incubated at 25 ∘ C for 5 min. After the incubation, 100 μL of 2 μg mL−1 rhodamine 123 were added. Instantly, fluorescence quenching of rhodamine 123 was monitored as fluorescence intensity changes using a Cary Eclipse spectrofluorimeter. The excitation and emission wavelengths were 500 and 520 nm, respectively. Fluorescence values calculated in the time range 0–60 s were normalised on the initial fluorescence (Fi). The membrane permeability transition of mitochondria was expressed as ΔF Fi−1 s−1 g−1 protein. Determination of ROS Content of ROS was determined according to the methods of Jambunathan23 and Esposti.24 First, 0.1 mL re suspended sample was diluted with 0.9 mL Tris–HCl buffer (10 mmol L−1 , pH 7.2). Then 10 μL of 2 mmol L−1 2′ ,7′ -dichlorofluorescein diacetate (DCF-DA) solution (prepared with dimethylsulfoxide (DMSO)) were mixed with the diluted sample. The mixture was incubated for 30 min in the dark. In the control group, the resuspended mitochondria were replaced by Tris–HCl buffer (10 mmol L−1 , pH 7.2). After incubation, the fluorescence value was measured on a Cary Eclipse spectrofluorimeter with a 10 nm slit. The excitation and emission wavelengths were 485 and 530 nm, respectively. Content of ROS was expressed as a.u. mg−1 protein.
© 2015 Society of Chemical Industry
J Sci Food Agric 2016; 96: 1997–2003
Effects of nitric oxide on mitochondrial oxidative defence in peach
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Figure 1. Changes in the mitochondrial membrane permeability transition of peach fruit after treatment during storage. Each value is presented as mean ± SE (n = 3).
Enzyme assays Measurement of mitochondrial SOD activity Mitochondrial SOD activity was determined by measuring the ability of SOD to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm after exposure to light from a 10 W fluorescent lamp for 25 min.25 Before the illumination, 0.1 mL mitochondrial resuspension and 0.3 mL of 20 μmol L−1 riboflavin were added to 2.6 mL assay mixture (including 50 mmol L−1 Tris–HCl pH 8.5, 13 mmol L−1 Met, 75 μmol L−1 NBT, 10 μmol L−1 ethylenediaminetetraacetic acid) in a 5 mL centrifugal tube. In the control group the enzyme solution was replace by 0.1 mL Tris–HCl buffer (0.1 mol L−1 , pH 8.5). The centrifugal tubes were then placed under a 10 W fluorescent lamp for 25 min to initiate the reaction. After that, estimation of the mitochondrial SOD activity was carried out by UV spectroscopic analysis at 560 nm. One unit of SOD activity is defined as the amount of protein required to give half-maximal inhibition of NBT reduction, and SOD activity was expressed as U mg−1 protein. Measurement of CAT activity CAT activity was measured by monitoring the enzyme-catalysed decomposition of H2 O2 by UV spectroscopic kinetic analysis at 240 nm for 150 s on an ultraviolet (UV) spectrophotometer.26 The resuspended sample (0.1 mL) was mixed with 3 mL phosphate buffer (PBS, 50 mmol L−1 , pH 7.0). Then 0.1 mL of 1% H2 O2 was added to the mixture and the mitochondrial CAT activity of the mixture above was instantly estimated. The decay in absorbance at 240 nm was monitored for 150 s. One unit (1 U) of CAT activity was defined as the amount of enzyme that caused a 0.01 decrease in A240 in 1 s under the assay conditions. CAT activity was expressed as U mg−1 protein s−1 .
J Sci Food Agric 2016; 96: 1997–2003
Statistical analysis The experiments were conducted in a completely randomized design. Each experiment was repeated three times and the data were processed by analysis of variance (ANOVA), comparing treatments at a significance level of 0.05 according to the least significant difference (LSD) test. All data were expressed as mean ± SE (n = 3).
RESULTS Changes in mitochondrial MPT Treatment with 15 μmol L−1 NO significantly increased the MPTs of peach fruit during storage, which were about 10% higher than that of the control (Fig. 1). However, there were no significant differences in MPT among treatments with 5, 30 μmol L−1 NO and the control. Treatment with c-PTIO significantly decreased mitochondrial MPT of peach fruit during storage, which were only 90.0% of the control. Mitochondrial MPTs of peach fruit treated with 15 μmol L−1 NO were about 15% higher than that of fruit treated with c-PTIO. Changes in mitochondrial ROS content As shown in Fig. 2, the content of ROS in mitochondria of peach fruits increased during cold storage. Treatment with c-PTIO could increase the content of ROS by 56.3% on day 2 and then the content was kept at a stable value during the following 6 days, whereas NO appropriately decreased the ROS content in mitochondria slightly. In addition, during the first 2 days of storage,
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1999
Measurement of mitochondrial POD activity The method of measurement of POD was according to the methods of Rahnama and Ebrahimzadeh27 and Sun.28 The resuspension of peach fruit mitochondria (1 mL) was added to 2 mL
benzidine–NaAc solution (0.005 mol L−1 ) and 1 mL of 3% H2 O2 was added to the above mixture. Immediately, the activity of mitochondrial POD was estimated by UV spectroscopic kinetic analysis at 580 nm for 150 s on a UV-2450 UV spectrophotometer. One unit (1 U) of POD activity was defined as the amount of enzyme that caused a 0.01 increase in A580 in 1 s under the assay conditions. POD activity was expressed as U mg−1 protein s−1 .
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G Jing, J Zhou, S Zhu
Figure 2. Effects of different concentrations of NO solutions on ROS: each value is presented as mean ± SE (n = 3).
the ROS content of CK and NO treatment decreased respectively by 14.8% and 16.4%, then increased by 59.0% and 52.2%, respectively, on day 4. After that, the content of ROS of these two samples tended to a stable value. The stable value of ROS concentration of treatment of c-PTIO was then 13% higher than that of the control from day 4 to day 8, while the stable value of the 15 μmol L−1 NO treatment was 7.3% lower than that of the control. Effects of NO on antioxidant enzymes Effects of NO on mitochondrial SOD activity As shown in Fig. 3, during storage at low temperature SOD activity of the control group increased by about 16.98% overall. The sample treated with 15 μmol L−1 NO had lower SOD activity compared with the control on corresponding days. During cold storage (from day 2 to day 6), SOD activities of NO treatment were 4.43%, 17.84%, 9.44% and 33.62% lower than that of the control group on day 2, 4, 6 and 8, respectively. However, SOD activity decreased quickly in fruit treated with c-PTIO, which was 26.9% lower than that of the control and 12.6% lower than that of treatment with NO overall. Besides that, the variation trends of SOD activity of these three different treatments were similar to each other. All of them decreased on day 2 and then increased to a peak at day 6, before decreasing on day 8.
2000
Effects of NO on mitochondrial CAT activity When the samples were stored at low temperature, the mitochondrial CAT activity increased in a small range (Fig. 4). During storage, CAT activity of the control increased by 4.4%, CAT activity of c-PTIO treatment increased by 8.6% and CAT activity of the sample treated with 15 μmol L−1 NO solution increased by 3.8%. The sample treated with c-PTIO had the highest activity, whereas 15 μmol L−1 NO solution treatment led to a decrease in CAT activity (compared with CAT activity of the control on the corresponding day).
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CAT activities of fruits treated with c-PTIO were 16.86%, 5.27%, 7.12% and 3.90% higher than that of the control on day 2, 4, 6 and 8, respectively. However, CAT activity of fruits treated with 15 μmol L−1 NO solution were 10.39%, 8.91%, 3.28% and 7.09% lower than that of control on day 2, 4, 6 and 8, respectively. Effects of NO on mitochondrial POD activity As shown in Fig. 5, mitochondrial POD activity decreased slightly during low-temperature storage as a whole. Both the control and the 15 μmol L−1 NO solution treatment decreased by 5.4%, and POD activity of the c-PTIO treatment decreased the least. On day 2, POD activity of 15 μmol L−1 NO solution treatment was 15.3% higher than that of the c-PTIO treatment and 20.7% higher than that of the control. Then, on day 4, POD activity of 15 μmol L−1 NO solution treatment decreased to a value which was near to that of the control group. Therefore, POD activities of 15 μmol L−1 NO solution treatment and control group were about 6.1% lower than that of the c-PTIO group. On day 6, POD activity of the c-PTIO group increased sharply, then decreased to the average value on day 8.
DISCUSSION In recent years, increasing evidence has shown that NO has an important role in the oxidative defence in plant cells.29 – 31 As a fat-soluble small molecule, NO could diffuse to different organelles, which makes it possible for exogenous NO to enter the inner part of mitochondria of plant cells. It is shown in this paper that during storage of peach fruits the decrease in MPT induced by low temperature could be delayed by treatment with 15 μmol L−1 NO and promoted by c-PTIO treatment. As shown in Fig. 1, no significant differences were observed in the changes in MPT among the treatments with 5 and 30 μmol L−1 NO and the control. These results suggest that the effects of NO on mitochondrial MPT depend on the dose of NO solution. NO at
© 2015 Society of Chemical Industry
J Sci Food Agric 2016; 96: 1997–2003
Effects of nitric oxide on mitochondrial oxidative defence in peach
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Figure 3. Effects of different concentrations of NO solutions on mitochondrial SOD activity: each value is presented as mean ± SE (n = 3).
Figure 4. Effects of different concentrations of NO solutions on mitochondrial CAT activity: each value is presented as mean ± SE (n = 3).
J Sci Food Agric 2016; 96: 1997–2003
when fruits were treated with c-PTIO (Fig. 2). This suggests that exogenous NO treatment could alleviate the increase in ROS in mitochondria. There exists crosstalk between NO and H2 O2 .14,33 NO could not only increase the expression of AOX, which can decrease the level of ROS in mitochondria,33 but also interact with H2 O2 ,14 to maintain homeostasis between NO and H2 O2 in mitochondria. As has been reported, SOD has been implicated as essentially the first line of defence against the potent toxicity of superoxide.34 In this study, the activity of SOD increased during storage, corresponding to the increase of ROS in mitochondria. However,
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2001
5 μmol L−1 would be too low to trigger the changes in mitochondrial MPT of peach fruits, and NO at 30 μmol L−1 would be high and toxic to the mitochondrial membrane. The changes in mitochondrial MPT confirmed that treatment with 15 μmol L−1 NO could significantly improve the stability of mitochondrial permeability transition, which was consistent with our previous report.32 In this experiment, the content of ROS in mitochondria increased as a whole during storage at low temperature. ROS content of peaches treated with 15 μmol L−1 NO was much lower than that of the control, whereas the ROS content increased dramatically
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G Jing, J Zhou, S Zhu
Figure 5. Effects of different concentrations of NO solution on mitochondrial POD activity: each value is presented as mean ± SE (n = 3).
2002
when the samples were treated with c-PTIO, their mitochondrial ROS content was highest, while their mitochondrial SOD activity was lowest; meanwhile, when the samples were treated with 15 μmol L−1 NO solution, their mitochondrial SOD activity was higher than that of the c-PTIO group. These phenomena may indicate that 15 μmol L−1 NO solution treatment could alleviate the increase in mitochondrial ROS content caused by the low temperature during storage by intervening in the synthesis of ROS and improving the activity of SOD in mitochondria. In mitochondria, CAT plays a role in promoting the catabolism of H2 O2 generated during aerobic metabolism and the process catalysed by SOD.35 Similar to CAT, POD is an oxidoreductase and its substrate is H2 O2 and some other peroxides. From Figs 4 and 5 it can be seen that the activities of CAT and POD in mitochondria were very low; this was especially true for CAT. Besides that, there were no significant distinctions between the three different treatments. This indicated that CAT and POD play much less important roles in mitochondria than does SOD. Figures 3 and 5 indicate that the activities of CAT correspond to the ROS contents to some extent, which explains the catalytic conversion mechanism of superoxide anion in mitochondria. However, the change in POD activity was not as significant compared with the change in CAT activity. Based on the fact that H2 O2 was the substrate of both CAT and POD, it is suggested that CAT and POD had cooperative effects in scavenging toxic ROS in peach fruit mitochondria. From the results of ROS content and antioxidative enzymes, it is suggested that exogenous NO treatment could alleviate oxidative damage from two aspects: on one hand, exogenous NO could regulate the synthesis of ROS in mitochondria by taking part in ETC; on the other hand, exogenous NO improved the activities of SOD and CAT in mitochondria, which promoted the scavenging of ROS in mitochondria. However, some studies have found that NO could also regulate the gene expression related to ROS36 in mitochondria. In this paper, studies concerning the relationship between exogenous NO and gene expression were not addressed, and further research needs to be carried out.
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CONCLUSIONS Treatment with 15 μmol L−1 NO solution could significantly relieve the decrease in mitochondrial permeability transition and the oxidative damage caused by low temperature during storage, and increased the activities of SOD. By scavenging parts roles of exogenous or endogenous NO, treatment with c-PTIO promoted the oxidative damage and the decrease in MPT of peach fruit with high ROS content and low activities of antioxidative enzymes. There were no significant changes in the activities of CAT and POD in mitochondria of peach fruit after treatment. It is suggested that moderate exogenous NO could be of benefit in improving the ability of the antioxidant system in Feicheng peach fruit mitochondria, as well as the mitochondrial permeability transition to some extent.
ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (31270723, 31370686, 31470686) and Science and Technology Development Planning of Shandong Province, China (2013GZX20109). We also appreciate Shunqing Hu and Bing Xie for their assistance in the extraction and purification of mitochondria, Ruirui Wang and Guanshuai Yi for their help in the determination of enzyme activities, and Lili Zhang for her guidance in the detection of NO concentration.
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