Hypobaric Treatment Effects on Chilling Injury, Mitochondrial

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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

1

Hypobaric Treatment Effects on Chilling Injury, Mitochondrial Dysfunction, and the

2

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

ACS Paragon Plus Environment


23

Tel: 0086 (0)571 63740278

24

Fax: 0086 (0)571 63740809

25


26 27

***

28

Tel: 0086 (0)20 85280259

29

Fax: 0086 (0)20 85280259

30


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

47

dysfunction, and the ascorbate-glutathione (AsA-GSH) cycle in peach fruit stored at 0°C.

48

Internal browning of peaches was dramatically reduced by applying 10–20 kPa pressure.

49

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,

52

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

59

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

62

up-regulation of related enzymes.

63 64

Key words

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Peach; hypobaric treatment; chilling injury; mitochondrial dysfunction; AsA-GSH cycle

66



67 68

INTRODUCTION

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Peach (Prunus persica) is a fruit of high economic value and its juicy, sweet and

70

aromatic flesh and soft texture has earned it increasing popularity over the years. Since

71

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).

73

Subsequently, cold storage is employed as the main approach to inhibit these adverse

74

processes.

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Peaches tend to be vulnerable to chilling injury (CI), which manifests as a loss in ability

76

to maintain firmness, and accelerated progression of rotting and decay (3, 4). The damage

77

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).

80

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

82

primary generators of endogenous ROS and are particularly vulnerable to oxidative damage

83

(8). When oxidative injury under stress occurs, the superoxide anion (O2.-) is produced in the

84

mitochondrial electron transfer chain (ETC) and further reduced by dismutation to hydrogen

85

peroxide (H2O2), causing dysfunction of various mitochondrial components and finally

86

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

88

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).

95

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

98

has shown that the AsA-GSH cycle plays an important role in scavenging H2O2 (13), in which

99

ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate

100

reductase (MDHAR) are key enzymes (14). The AsA-GSH cycle includes a few relevant

101

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

104

generated from monodehydroascorbate (MDHA), while GSH is oxidized into GSSG by

105

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

108

the plant is undergoing senescence. Promoting the AsA-GSH cycle contributes to the increase

109

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

120

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

123

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).

125

Lima-Silva et al. (2012) (34) identified all five genes involved in AsA recycling using

126

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

130

ripening and senescence are well known. Therefore, this study aims to investigate the effects

131

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.

134 135

MATERIALS AND METHODS

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Plant material and physiological treatments

138 139

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

141

from a commercial orchard in Jiaxing, Zhejiang Province, China. Peach fruit were picked at

142

8:00–10:00 in the morning, taken to the laboratory within 3 h and pre-cooled overnight at

143

8–10°C. Individual fruits of similar shape and color with few blemishes were then selected.

144

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

147

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 ±

152

1°C and 90-95% relative humidity. A total of 18 fruit samples per treatment were taken from

153

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.



155

The chambers were opened for about 30 min and flushed with fresh air at 10-day intervals

156

during low-temperature storage in order to avoiding anoxic conditions.

157

To figure out the molecular mechanisms that control the effects of hypobaric treatment

158

on the harvested peaches, ten fruits each with similar appearance were collected before

159

hypobaric pressure treatment (BHP, 0 d), at normal atmospheric pressure stored for 30 days at

160

0°C (NAP, 30 d), and at hypobaric pressures of 10–20 kPa (HP, 30 d), and were then used to

161

extract RNA. Equal loads of high quality RNA from the harvested material were pooled

162

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,

169

no browning (excellent quality); 1, slight browning; 2, moderate browning (50%

171

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%.

173

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).

175

The penetration rate was 1 mm/s with an ultimate penetration depth of 10 mm. Measurements

176

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.

178 179

Determination of mitochondrial membrane fluidity and MPTP

180 181

Mitochondria purification

182 183

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

186

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

188

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

193

mitochondrial membrane fluidity and permeability transition pores (MPTP). The washing

194

buffer consisted of 25 mM of MOPS–HCl (pH 7.2), 400 mM of mannitol, 0.1% BSA and 1

195

mM EDTA.

196 197

Mitochondrial membrane fluidity

198



199

Mitochondrial membrane fluidity was monitored by determining the fluorescence

200

polarization of the mitochondria-bound dye, 8-anilino-1-naphthalenesulfonic acid (ANS)

201

according to a modification of the method proposed by Yang et al. (1983) (38). The

202

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,

206

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

209

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

214

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

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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).

218

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.

220


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Determination of SDH and CCO activities

222 223

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

226

(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

232

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

235

510 nm.

236 237

Determination of parameters associated with the AsA-GSH cycle

238 239

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



243

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

264


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Frozen tissue powder (2 g) from 10 peach fruit was homogenized with 5 mL of 0.05 M

266

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)

288

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


Page 15 of 49


309

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



Page 16 of 49

331

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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353

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

Hypobaric Treatment Effects on Chilling Injury, Mitochondrial

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