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Hypobaric Treatment Effects on Chilling Injury, Mitochondrial Dysfunction, and the Ascorbate-glutathione (AsA-GSH) Cycle in Post-harvest Peach Fruit Lili Song, Jinhua Wang, Mohammad Shafi, Yuan Liu, Jie Wang, Jia Sheng Wu, and Aimin Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00623 • Publication Date (Web): 19 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Hypobaric Treatment Effects on Chilling Injury, Mitochondrial Dysfunction, and the
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Ascorbate-glutathione (AsA-GSH) Cycle in Post-harvest Peach Fruit
3 4
LILI SONG † *, JINHUA WANG † , MOHAMMAD SHAFI § , YUAN LIU † , JIE WANG † ,
5
JIASHENG WU†**, AND AIMIN WU‡***,
6 7
†
The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, School of
8
Forestry and Biotechnology, Zhejiang A & F University, Lin'an, 311300, Zhejiang Province,
9
People’s Republic of China
10
‡
State
Key
Laboratory
for
Conservation
and
Utilization
of
Subtropical
11
Agro-bioresources, South China Agricultural University;Guangdong Key Laboratory for
12
Innovative Development and Utilization of Forest Plant Germplasm;Guangdong Province
13
Research Center of woody forage engineering technology, South China Agricultural
14
University, Guangzhou, 510642, China
15
§
Department of Agronomy, The University of Agriculture, Peshawar, 25130, Pakistan
16 17
*
18
Tel: 0086 (0)571 63732766
19
Fax: 0086 (0)571 63740809
20
E-mail:
[email protected] The first corresponding author
21 22
**
The second corresponding author
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Tel: 0086 (0)571 63740278
24
Fax: 0086 (0)571 63740809
25
E-mail:
[email protected] 26 27
***
28
Tel: 0086 (0)20 85280259
29
Fax: 0086 (0)20 85280259
30
E-mail:
[email protected] The third corresponding author
31 32
Authorship of the paper Designing the work: L.L.S., A.M.W, J.S.W.; running the
33
experiments: J.H.W., J.S.W., J.W., Y.L.; data analysis and statistics: J.H.W., J.W., L.L.S.;
34
article writing and revising: J.H.W., L.L.S., A.M.W., M.S
35 36 37 38 39 40 41 42 43 44
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ABSTRACT
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In this study, hypobaric treatment effects were investigated on chilling injury, mitochondrial
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dysfunction, and the ascorbate-glutathione (AsA-GSH) cycle in peach fruit stored at 0°C.
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Internal browning of peaches was dramatically reduced by applying 10–20 kPa pressure.
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Hypobaric treatment markedly inhibited membrane fluidity increase, whereas it kept
50
mitochondrial permeability transition pores (MPTP) concentration and cytochrome C oxidase
51
(CCO) and succinic dehydrogenase (SDH) activity relatively high in mitochondria. Similarly,
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10–20 kPa pressure treatment reduced the level of decrease observed in AsA and GSH
53
concentrations, while it enhanced ascorbate peroxidase (APX), glutathione reductase (GR),
54
and monodehydroascorbate reductase (MDHAR) activities related to the AsA-GSH cycle.
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Furthermore, comparative transcriptomic analysis showed that differentially expressed genes
56
(DEGs) associated with the metabolism of glutathione, ascorbate and aldarate were
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up-regulated in peaches treated with 10–20 kPa for 30 days at 0°C. Genes encoding GR,
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MDHAR, and APX were identified, and exhibited higher expression in fruit treated with low
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pressure than in fruit treated with normal atmospheric pressure. Our findings indicate that the
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alleviation of chilling injury by hypobaric treatment was associated with preventing
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mitochondrial dysfunction and triggering the AsA-GSH cycle by the transcriptional
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up-regulation of related enzymes.
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Key words
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Peach; hypobaric treatment; chilling injury; mitochondrial dysfunction; AsA-GSH cycle
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INTRODUCTION
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Peach (Prunus persica) is a fruit of high economic value and its juicy, sweet and
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aromatic flesh and soft texture has earned it increasing popularity over the years. Since
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peaches mature in the hot and rainy season, the fruit deteriorates quickly after harvest at
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ambient temperature, causing softening of the flesh, tissue disruption, and rotting (2).
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Subsequently, cold storage is employed as the main approach to inhibit these adverse
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processes.
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Peaches tend to be vulnerable to chilling injury (CI), which manifests as a loss in ability
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to maintain firmness, and accelerated progression of rotting and decay (3, 4). The damage
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caused by chilling injury manifests after peaches are stored in a typical 0–1°C environment
78
for two weeks. Cultivar and maturity at harvest greatly influence the progression of chilling
79
injury-associated symptoms (5, 6).
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Injuries induced in plants following chilling usually involve an imbalance between the
81
generation and elimination of reactive oxygen species (ROS) (7). Mitochondria are the
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primary generators of endogenous ROS and are particularly vulnerable to oxidative damage
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(8). When oxidative injury under stress occurs, the superoxide anion (O2.-) is produced in the
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mitochondrial electron transfer chain (ETC) and further reduced by dismutation to hydrogen
85
peroxide (H2O2), causing dysfunction of various mitochondrial components and finally
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accelerating injury or aging (9). Yang et al. (2014) (10) reported that the integrity of
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mitochondrial membranes and the function of mitochondria in peach fruit can be adversely
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affected after harvest, as evidenced by the decrease in mitochondrial membrane fluidity and
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increase in the number of mitochondrial permeability transition pores (MPTP). In order to
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regulate ROS homoeostasis, plant mitochondria have evolved mechanisms for minimizing
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ROS production, including the use of alternative oxidase (AOX), enzymatic ROS-interactors
92
such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), and
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non-enzymatic antioxidant systems containing low molecular weight antioxidants including
94
glutathione, ascorbic acid and tocopherols (11).
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The AsA-GSH cycle is an antioxidant system of great importance that can regulate the
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oxidative and reductive environment by modulating glutathione/glutathione disulfide
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(GSH/GSSG) and ascorbate/dehydroascorbate (AsA/DHA) interconversion (12). Research
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has shown that the AsA-GSH cycle plays an important role in scavenging H2O2 (13), in which
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ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate
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reductase (MDHAR) are key enzymes (14). The AsA-GSH cycle includes a few relevant
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metabolic reactions (15). APX is the first enzyme to directly scavenge H2O2 into water, and
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AsA serves as an electron donor (16, 17). Dehydroascorbate reductase (DHAR) uses electrons
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generated by GSH to reduce dehydroascorbate (DHA) and provide them to AsA. DHA is
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generated from monodehydroascorbate (MDHA), while GSH is oxidized into GSSG by
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DHAR, and GSSG is subsequently reduced to GSH, which is, in turn, catalyzed by GR.
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Raseetha et al. (2013) (18) reported that the total glutathione and ascorbic acid in broccoli
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florets become degraded and that APX and GR are less active in the AsA-GSH cycle while
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the plant is undergoing senescence. Promoting the AsA-GSH cycle contributes to the increase
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in cold tolerance of plants (19). In addition, chilling tolerance enhancement of cold-stored
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peaches is related to the induction of important enzymes in the AsA-GSH cycle (20, 21).
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It has been shown that hypobaric storage dependent on cold storage and sub-atmospheric
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pressure has the potential for extending the shelf life of numerous horticultural crops (22, 23,
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24, 25). It has also been shown that hypobaric treatment has beneficial effects on the storage
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and shelf life of peaches. Chen et al. (2010) (26) found that hypobaric storage affected the
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shelf life and quality of peaches through the alleviation of membrane damage and
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enhancement of antioxidant enzyme activity during cold storage. Although hypobaric
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treatments have been acknowledged to alleviate the accumulation of ROS during cold storage
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(27), the regulatory mechanisms underlying hypobaric treatment and chilling injury in
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relation to the AsA-GSH cycle and mitochondrial oxidative stress in peaches remain largely
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unknown.
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Transcriptome analysis or transcriptomics is a powerful tool that can interpret functional
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aspects of the genome and reveal molecular components of tissues and cells (28, 29). As
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next-generation sequencing technologies are evolving, they have become widely used in
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expanding sequence databases in numerous model and non-model species (30, 31, 32, 33).
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Lima-Silva et al. (2012) (34) identified all five genes involved in AsA recycling using
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microarray technology, but did not find MDHR3 to be involved in AsA-GSH recycling or in
127
regulating AsA content. Therefore, the role of the AsA-GSH cycle antioxidant system in
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protecting the fruit from oxidative stress during chilling injury remains to be elucidated,
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especially at the molecular level, although physiological changes that occur during fruit
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ripening and senescence are well known. Therefore, this study aims to investigate the effects
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of hypobaric treatment on the physiological and molecular control of chilling injury, including
132
the effect on mitochondrial oxidative stress and the AsA-GSH cycle in peach fruit that are
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kept in cold storage.
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MATERIALS AND METHODS
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Plant material and physiological treatments
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Peach fruit (Prunus persica L. Batsch cv. Hujingmilu) at maturity levels of 80–90%
140
(turning stage to red stage, according to the standards for maturity scale (35)) were collected
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from a commercial orchard in Jiaxing, Zhejiang Province, China. Peach fruit were picked at
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8:00–10:00 in the morning, taken to the laboratory within 3 h and pre-cooled overnight at
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8–10°C. Individual fruits of similar shape and color with few blemishes were then selected.
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Treatments were performed in a storage system consisting of 90 L chambers in which
145
independent manipulation of pressure is possible (Model XL-5, Xianlü Low-pressure Fresh
146
Keeping Equipment Co., Ltd., Shanghai, China). A total of 108 peach fruit were selected and
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divided in two treatment groups: one group received 10–20 kPa in a hypobaric chamber,
148
which was set as optimal pressure for preservation based on previous research (27), and the
149
second group received 101.3 kPa as control treatment, which was equivalent of atmospheric
150
conditions. There were three technical replicates per treatment and 6 fruits per replicate. The
151
entire experiment was performed twice. Fruits were stored for a duration of 30 days at 0 ±
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1°C and 90-95% relative humidity. A total of 18 fruit samples per treatment were taken from
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chambers at 10-day intervals and measured to determine various physiological indexes in
154
terms of chilling injury, fruit firmness, mitochondrial dysfunction, and the AsA-GSH cycle.
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The chambers were opened for about 30 min and flushed with fresh air at 10-day intervals
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during low-temperature storage in order to avoiding anoxic conditions.
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To figure out the molecular mechanisms that control the effects of hypobaric treatment
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on the harvested peaches, ten fruits each with similar appearance were collected before
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hypobaric pressure treatment (BHP, 0 d), at normal atmospheric pressure stored for 30 days at
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0°C (NAP, 30 d), and at hypobaric pressures of 10–20 kPa (HP, 30 d), and were then used to
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extract RNA. Equal loads of high quality RNA from the harvested material were pooled
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for high-throughput sequencing (RNA-Seq). All samples for PCR analysis were flash frozen
163
in liquid nitrogen and stored at −80°C for future use.
164 165
Chilling injury (CI) index and the firmness of fruit
166 167
Since internal browning (IB) is the primary symptom of CI in peaches, CI was assessed
168
by calculating the total browned area on the flesh of 15 fruit using the following scale (3): 0,
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no browning (excellent quality); 1, slight browning; 2, moderate browning (50%
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browning). Results are shown as IB that is calculated with the following formula: IB = ∑ (IB
172
scale) × (number of fruit at that IB)/(4 × total number of fruit in each treatment) × 100%.
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Fruit firmness was determined with a TA-XT2i texture analyzer (Stable Micro Systems,
174
UK) fitted with a 5 mm diameter probe using the method reported by Song et al. (2009) (36).
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The penetration rate was 1 mm/s with an ultimate penetration depth of 10 mm. Measurements
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were taken on opposite sides of each fruit after a small piece of peel was removed. Data are
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shown as kg/cm2.
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Determination of mitochondrial membrane fluidity and MPTP
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Mitochondria purification
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Mitochondria were extracted using a modified version of a method previously described
184
by Liang et al. (2003) (37). Briefly, approximately 50 g of peeled frozen peach tissue
185
obtained from ten fruits were carefully cut and homogenized in 100 ml of ice-cold extraction
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buffer containing 0.4 M mannitol, 1 mM EDTA, 8 mM cysteine, 1% polyvinylpyrrolidone,
187
0.1% bovine serum albumin (BSA), 10 mM tricine, and 25 mM MOPS–HCl (pH 7.5). The
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homogenate was filtered through six layers of sterile cheesecloth and subsequently
189
centrifuged at 1200 x g for 10 min at 4°C. Supernatants were then decanted and further
190
centrifuged at 14,000 x g for 20 min. Next, the pellets were resuspended in 20 mL of the
191
isolation medium and centrifuged at 5,000 x g for 10 min at 4°C. The purified mitochondrial
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pellet was then resuspended in 2 ml of washing buffer with a soft brush for measurements of
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mitochondrial membrane fluidity and permeability transition pores (MPTP). The washing
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buffer consisted of 25 mM of MOPS–HCl (pH 7.2), 400 mM of mannitol, 0.1% BSA and 1
195
mM EDTA.
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Mitochondrial membrane fluidity
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Mitochondrial membrane fluidity was monitored by determining the fluorescence
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polarization of the mitochondria-bound dye, 8-anilino-1-naphthalenesulfonic acid (ANS)
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according to a modification of the method proposed by Yang et al. (1983) (38). The
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polarization value of the probe fluorescence was used to measure membrane fluidity. Briefly,
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60 µl of ANS solution (5 mM) was added to 0.5 ml mixtures of mitochondrial suspensions
204
and 5.65 ml of mannitol solution (0.3 M). After incubation at 25°C for 5 min, membrane
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fluorescence polarization was calculated using an RF-540 spectrofluorometer (Shimadu,
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Kyoto, Japan) fitted with polarizers at wavelengths of 400 nm for excitation and 480 nm for
207
emission. The fluorescence intensity (F) positively correlated with membrane fluidity, and
208
was determined according to the method developed by Shi et al. (2013) (39). Membrane
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fluidity is expressed in F g-1 protein.
210 211
MPTP assay
212 213
MPTP, which is the positive swelling of mitochondrial suspensions, was measured by
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tracking a decrease in absorbance at 540 nm with a UV-1750 spectrophotometer (Shimadu,
215
Kyoto, Japan) and by generating absorbance measurements at 20 s intervals (40). The mixture
216
was initially incubated at 25°C for 5 min by adding mitochondria (5 mg protein) to 10 ml of 5
217
mM HEPES buffer (220 mM mannitol, 70 mM sucrose, 5 mM sodium succinate, pH 7.2).
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The reaction was started by adding 30 µl of 30% H2O2 to the reaction and changes in MPTP
219
were determined as the ∆OD540 nm min-1 g-1 FW.
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Determination of SDH and CCO activities
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SDH activity was assayed based on a slightly modified version of the method proposed by
224
Ackrell et al. (1984) (41). The reaction was performed at 30°C for 5 min in a reaction mixture
225
that contained 1 ml of mitochondria extract, 0.5 ml of 0.2 mM potassium phosphate buffer
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(pH
227
di-p-chlorophenylmethylcarbinol (DCPIP), and 0.5 ml of 10 mM methyl sulfenylphenazine
228
(PMS). One unit of SDH activity is defined as the change in absorbance at 600 nm per
229
minute.
7.4),
0.5
ml
of
0.2
mM
sodium
succinate,
0.2
ml
of
1
mM
230
CCO activity was assayed according to a slightly modified version of the method
231
proposed by Errede et al. (1978) (42). The reaction was performed at 37°C for 3 min in a
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reaction mixture containing 1 ml of mitochondria extract, 1.75 ml of 0.2 mM potassium
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phosphate buffer (pH 7.4), 0.5 ml of 0.3 mM cytochrome C solution, and 0.5 ml of 2%
234
TritonX-100. One unit of CCO activity is defined as the change in absorbance per minute at
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510 nm.
236 237
Determination of parameters associated with the AsA-GSH cycle
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AsA and GSH content
240 241
AsA content was assayed using a method proposed by Roe et al. (1948) (43). GSH was
242
extracted and assayed according to a slightly modified version of the method proposed by
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Bernt and Bergmeyer (1974) (44). Briefly, 2.0 g of fruit flesh was ground finely in 20 ml of 1
244
M HClO4, filtered through Miracloth, and the homogenate was centrifuged at 15,000 × g for
245
15 min at 4°C. The supernatant was adjusted to pH 7.0 with 1.75 M tripotassium phosphate
246
and centrifuged again for 15 min at 4°C. GSH content was assayed spectrophotometrically at
247
240 nm using methylglyoxal and glyoxalase I (Sigma). Oxidized glutathione (GSSG) content
248
was assayed spectrophotometrically at 340 nm by adding 10 mM NADPH and GR (Sigma) to
249
the reaction mixture. Concentrations are expressed as milligrams per gram of fresh tissue (mg
250
g−1).
251 252
APX and GR activity
253 254
Frozen tissue powder (2 g) from ten peach fruit was homogenized with 5 mL of 0.1 M
255
potassium phosphate buffer (pH 7.8) containing 2 mM EDTA-Na2 and 2 mM DTT. The
256
homogenate was centrifuged at 12,000 rpm for 30 min at 4°C, and supernatants were
257
collected for enzyme activity measurements. GR activity was measured by monitoring the
258
oxidization of NADPH based on the method reported by Edwards et al. (1990) (45). Briefly,
259
the absorbance decrease per minute at 340 nm was measured. APX activity was determined
260
on the basis of oxidization of AsA (extinction coefficient of 2.8/mM/cm) by assaying the
261
decrease in absorbance at 290 nm (46).
262 263
MDHAR and DHAR activity
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Frozen tissue powder (2 g) from 10 peach fruit was homogenized with 5 mL of 0.05 M
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Tris-Hcl buffer (pH 7.2) containing 1 mM EDTA-Na2, 0.3 mM D-mannose, 0.1% (w/v)
267
bovine albumin, 0.05% (w/v) L(+)-cysteine, and 2% (w/v) PVP. The homogenate was
268
centrifuged at 16,000 rpm for 20 min at 4°C and the supernatant was collected for enzyme
269
activity measurements. MDHAR activity was calculated on the basis of oxidization of
270
nicotinamide adenine dinucleotide (NAD) (extinction coefficient of 6.22/mM/cm) by
271
monitoring the decrease in absorbance at 340 nm (47). DHAR activity was measured by
272
monitoring the reduction of DHA as described by Arrigoni et al. (1992) (48). Briefly, the
273
increase in absorbance per minute at 265 nm was measured.
274
Protein content of the enzyme extracts was measured by the Bradford method (49), using
275
bovine serum albumin (BSA) as a standard. Specific enzyme activity was expressed as units
276
per milligram of protein.
277 278
RNA extraction, library construction, and RNA-seq
279 280
RNA exaction
281 282
Total RNA from each sample was isolated separately using the RNAprep Pure Plant Plus
283
Kit (Tiangen Biotech, Beijing, China). The mRNA-seq library was constructed using
284
Illumina’s TruSeq RNA Sample Preparation Kit (Illumina Inc, San Diego, CA, USA). The
285
isolation of mRNA, fragment interruption, cDNA synthesis, adapter addition, PCR
286
amplification and RNA-Seq were performed at Beijing BioMarker Technologies (Beijing,
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China). In brief, poly (A+) mRNA was isolated using Magnetic Oligo (dT) Beads (Illumina)
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and broken into smaller pieces using an RNA fragmentation kit (Ambion, Austin, TX, USA).
289
Cleaved RNA fragments were then copied into first strand cDNA with Super Script III reverse
290
transcriptase and random hexamer primers (Invitrogen, Carlsbad, CA, USA). Next,
291
second-strand cDNA synthesis was performed using DNA polymerase I (New England
292
BioLabs, Ipswich, MA, USA) (NEB). A single A base was ligated to short fragments after
293
purification using a MinElute PCR Purification Kit (Qiagen), preparing the fragments for
294
ligation to the sequencing adapters. Fragments (200 bp ± 25 bp) were next separated by 1.8%
295
agarose gel electrophoresis and selected for PCR amplification as sequencing templates.
296
Finally, the mRNA-seq library was constructed for sequencing on the Illumina HiSeqTM 2000
297
sequencing platform.
298 299
Mapping and analysis of Illumina reads
300 301
By eliminating adapters and low-quality sequences the raw reads were first cleaned
302
(reads with ambiguous bases ‘N’), and reads with greater than 10% Q, 20 bases (those with a
303
base quality less than 20) were selected. The high-quality sequences were annotated based on
304
the
305
http://www.rosaceae.org/species/prunus/prunus_persica) using BLAST Tophat (50). Gene
306
names were assigned to sequences according to the best hit (highest score).
International
Peach
Genome
Initiative
(IPGI;
307
Genes were also evaluated against the NCBI Nt database (BLASTn), Swiss-Prot, NCBI
308
Nr databases (BLASTx) with an E-value of 1e-5 (51). Functional annotation by gene
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ontology terms (GO, http://www.geneontology.org) was determined by the Blast2 GO
310
software (BLASTx, E-value cutoff of 1e-5). Sequences were aligned to the Clusters of
311
Orthologous Groups (COG) database (http://www.ncbi.nlm.nih.gov/COG/) to predict and
312
classify functions (BLASTx, E-value cutoff of 1e-5). Kyoto Encyclopedia of Genes and
313
Genomes (KEGG) pathways were assigned to the sequences using the online KEGG
314
Automatic Annotation Server (KAAS) (http://www.genome.jp/kegg/kaas/) (BLASTx, E-value
315
cutoff of 1e-5). The bi-directional best hit (BBH) method was employed to determine KEGG
316
Orthology (KO) assignment (52). KEGG analysis output includes KO assignments and
317
KEGG pathways populated with KO assignments.
318
The RPKM measure of read density indicates the molar concentration of a transcript in
319
the starting sample by normalizing for RNA length and for the total read number in the
320
measurement. Differential gene expression significance was calculated using the chi-squared
321
test integrated in IDEG6 software (http://telethon.bio.unipd.it/bioinfo/IDEG6/). P values of
322
findings made using this method were modified to account for multiple testing by using the
323
false discovery rate (FDR).
324
statistically significant result. Genes with an FDR ≤ 0.01 and the absolute value of expression
325
fold change ≥ 2 were deemed differentially expressed.
Here, an FDR-adjusted P value ≤ 0.01 was considered a
326 327
Real-time quantitative PCR analysis (RT-PCR)
328 329
RT-PCR experiments were conducted on genes related to the AsA-GSH cycle, selected
330
based on RNA-Seq data. First-strand cDNA synthesis was carried out with 3 µg of purified
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total RNA using PrimeScript® RT reagent Kit Perfect Real Time (Takara Biotech, Japan).
332
Forward and reverse specific primers were designed for four differentially expressed genes
333
(ppa004673m,
334
precursor-specific oligonucleotide primers are listed in additional file 1. The ACTB gene was
335
used as a control in the describedd experiments. PCR amplifications were done in a 20 µl
336
reaction volume with the SYBR® Premix Ex TapTM II Tli Rnase H Plus (Takara Biotech,
337
Japan) using the Roche LightCyler 480 System (Roche). PCR involved a 95°C step hold for
338
30 s, followed by 40 cycles at 95°C for 5 s, melting temperature (depending on primer Tm
339
value) for 10 s, 72°C for 20 s and 72°C for 2 min. All RT-PCR experiments described in this
340
study were performed at least three independent times.
ppa005081m,
ppa005968m,
ppa009538m).
Fusion-specific
and
341 342
Statistical analysis
343 344
All experiments in this study were done employing completely randomized designs. The
345
data was tested by analysis of variance (ANOVA) using SPSS Version 11.0. Least
346
significance differences (LSDs) were calculated to compare significant effects at the 5% level.
347 348
RESULTS
349 350
Hypobaric treatment, chilling injury, and firmness
351 352
Compared with peach fruit before storage, CI symptoms were clearly observed in fruit
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kept at a normal atmosphere at 0°C (control), but this was not evident in samples treated with
354
10–20 kPa pressure after 30 days at 0°C (Figure 1). The IB index showed no significant
355
changes in fruits during the first 10 days, but increased swiftly as cold storage was continued.
356
Hypobaric treatment dramatically reduced the IB index of cold-stored peach fruit (P